COMPLEMENTARITY AND CONTRARIETY in Proteomics
Majid Ali, M.D.
II. Structure and Function of Amino Acids
III. Structure of Proteins
IV. Oxidative Protein Folding
V. Functions of Proteins
VI. Ion Channel Proteins
VII. Autoantibodies: Mr. Hyde Proteins
IX. Amino Acids Self-Organize Into Proteins
X. Genes: Products of Evolution
XI. Protein Protection of DNA
XII. Protein Chaperons of mRNA
XIII. Protein Regulation of Genes
XIV. The Web of G- Proteins
XV. Turncoat Antibodies
XVI. Antibodies That Burn Water
XVII. Protein Protection of Blood Glucose Level
XVIII. The Web and Kaleidoscope of PPARs
XIX. Proteins as Molecular Switches
XX. Protein Catalysts
XXI. Protein Proton Pumps
XXII. Hot Proteins, Cool Proteins
XXIII. Targeted Protein Blockade
XXIV. NF- B: The Master Transcription Factor
XXV. Of Cowboys, Ubiquitination, and Oxygen
XXVI. Protein Misfolding Disorders
XXVII. Now An Anti-Cancer Protein, Now a Pro-Cancer Protein
XXVIII.Proteins in Cellular Energetics
XXIX. Obliterating Lines Between Structural and Functional Proteins
XXX. Protein-Based Therapeutics
XXXI. Concluding Comments
Proteins in the human body form an enormous web. The range of estimates of the number of proteins is disconcertingly large; the number often suggested is about 100,000. Proteins are considered responsible for cellular development and differentiation (Dr. Jekyll roles) as well as cellular demise (Mr. Hyde roles in apoptosis). Indeed, proteins belonging to the same family both promote and inhibit apoptosis. The subject of cellular death is discussed at length in the chapter entitled “Apoptosis: The Oxidative-Dysoxygenative Perspective.”
Proteins in the body also form an immense
kaleidoscope. Their structures and functions change readily in response to changes in their molecular environment. Oxidants and free radicals inactivate and denature enzymes (proteins) under some circumstances and lead to increased production of those enzymes under others. For example, as indicated elsewhere in this volume, catalase breaks down hydrogen peroxide into water and oxygen, and its production is also stimulated by hydrogen peroxide. Hydrogen peroxide also triggers the expression of genes for glutathione peroxidase, a potent antioxidant enzyme.
In textbooks of biochemistry and medicine, the chapters on amino acids and proteins often begin by stating that those two families of molecules are the primary molecular species involved in cellular developmental, differentiative, modeling, energetic, and apoptotic functions. That view is challenged here. Oxygen, singlet oxygen, superoxide, nitric oxide, and hydrogen peroxide—the “primal molecules” of the oxygen and redox homeostasis—are here designated as primarily responsible for all fundamental aspects of cellular and matrix dynamics. In the oxidative-dysoxygenative model of the health/dis-ease/disease continuum presented in this volume, the protein dynamics in health and disease are also considered to be subservient to the workings of those primal molecules. As in the case of chapters on glycomics, lipidomics, genenomics, and epigenomics, a full discussion of the structures and functions of proteins is clearly outside the scope of this book. Chemistry texts, such as Medical Biochemistry1 by Baynes and Dominiczak, may be used for that purpose. For discussion of oxidative phenomena concerning proteins, I suggest Oxidology2 by Bradford and Allen. In this chapter, I focus on those aspects of proteins that illuminate the themes of molecular complementarity and contrariety in human biology.
XXXI. CONCLUDING COMMENTS
Structure and function of amino acids and proteins are reviewed. The essentials of proteins of cell membrane receptors, ion channels, and ligands are presented. Other aspects of protein dynamics include: self-assembly of amino acids into proteins, the roles of fibronectin in matrix homeostasis, protein protection of DNA, protein regulation of genes, the web of G-proteins, turncoat proteins, and glucoregulatory aspects of proteins in clinical medicine. The case of chloride channel protein is presented to illustrate the enormous range of biologic roles of proteins. Outlines of G-protein and PPAR webs are shown to further underscore the twin themes of molecular complementarity and contrariety.
Proteins are generally regarded as products of genes. The regulatory influences of protein on genes, by contrast, generally receive scant attention. Some aspects of protein regulation of DNA are included to provide a counterbalance. Mr. Hyde roles of proteins—in autoantibodies, allergen-specific IgE antibodies, and turncoat antibodies—are discussed. All autoimmune and atopic disorders are mediated by turncoat proteins that turn on the body’s own defense mechanisms and create diverse pathophysiologic pathways.
Proteins form many webs within webs of human energetic, developmental, differentiative, and detoxificative kaleidoscopes. Those aspects of protein dynamics are included to demonstrate that the twin themes of molecular complementarity and contrariety are as relevant to proteomics as to lipidomics, glycomics, and redonics. Finally, the relevance of oxidative phenomena involving proteins—folding and others—to the other twin themes in this volume of spontaneity of oxidation in nature and dysoxygenosis is underscored. A brief note about the probable role free radicals seemed to have played in the evolutionary biology of proteins is included.
Intensive work being currently conducted to develop agents for protein-based therapeutics will undoubtedly yield valuable drugs for clinical use. I consider it safe to predict that all such agents will prove to be of limited benefit unless vigorous attempts are made concurrently to restore oxygen homeostasis, redox equilibrium, and acid-base balance. The basis of that statement should be self evident in view of the information concerning Nature’s preoccupation with complementarity and contrariety presented in this chapter.
OF AMINO ACIDS
Every amino acid molecule carries a central carbon atom—designated as the á-carbon—to which is attached the following four groupings schematically shown below:
1. A basic amino acid (-NH2);
2. An acidic carboxylic group (-COOH);
3. A hydrogen atom (-H); and
4. A distinctive side chain (-R).
Figure 1. Structure of an Amino Acid
NH2 — C — COOH
(Amino group) (Carboxyl group)
Side chains (-R) primarily determine the biologic properties of amino acids as well as of proteins that contain them. Thus the major aspects of protein structural conformation—and hence their functionalities as well as electrical charge—are established by the side chains. Amino acids with charged, polar, or hydrophilic side chains are generally exposed on the surfaces of proteins, while nonpolar hydrophobic residues are buried in the molecular interiors, outside direct contact with water. Amino acids are designated as aliphatic when their side chain carbohydrates are saturated. All aliphatic amino acids are hydrophobic. Glycine, alanine, valine, and isoleucine are included in this category. Phenylalanine, tyrosine, and tryptophan have aromatic side chains. In some instances, tyrosine and tryptophan are located on the surface because of their limited polarity.
Aromatic amino acids are involved in the absorption of ultraviolet radiation by most proteins. Nonpolar amino acids belonging to both aliphatic and aromatic amino acids contribute to maintenance of hydrophobic interiors of the protein molecules. In the case of tyrosine in some enzyme molecules, reversible phosphorylation of its hydroxyl group is critically important for the enzymatic activities and regulation of metabolic pathways in which they play roles.
Of the twenty known amino acids, all amino acids except glycine contain at least one asymmetric carbon atom yielding two optically active isomers that can rotate plane-polarized light. The two isoforms are called dextro (or D for right) and levo (or L for left). All amino acids in proteins are of the L-configuration, since proteins are synthesized by enzymes that insert only L-amino acids into the protein chains. Table 1 summarizes some important aspects of amino acids.
Table 1. Summary of the Functional Groups of Amino Acids and Their Polarity
Hydro-philic (polar) or hydro-phobic (apolar)
Ala, Val, Leu
Phe, Trp, Tyr
Following are the nine essential (not produced in the body) amino acids: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In the past, arginine was also sometimes listed as an essential amino acid. However, it is produced in the body from citrulline in the urea cycle and may not be included in that list. It may be mentioned here that some clinically significant amino acids are not found in proteins. Three examples of such amino acids are creatinine, glycine, and citrulline. The blood levels of creatinine are used for diagnosing renal failure, whereas urinary excretion of glycine reflects hepatic detoxification processes. Citrulline is a metabolite of arginine produced by the action of nitric oxide synthase.
The universal genome encodes for twenty amino acids. That evidently was not happenstance. Rather, it was the result of mutations and selections that occurred over a long period of time.
III. STRUCTURE OF PROTEINS
Proteins are thread-like molecules composed of amino acids, the sequence of which in the polypeptide chain forms the primary structure of the protein. The hydrogen-bond interactions within contiguous stretches of polypeptides, composed of amino acids, impart either á helice or â sheet configurations called the protein’s secondary structure. Most protein molecules exhibit combinations of second and third folding that define the tertiary structure of that protein. Finally, an assembly of subunits create yet another level of complexity of the protein structure called quaternary structure. While the basic organization of small proteins, such as 58-amino acid basic pancreatic trypsin inhibitor, can be displayed in simple three-dimensional schema, the structure of larger proteins may require resolution at several levels of organization.1,2
Combinations of á helices or â sheets within sections of the polypeptide chain (usually containing 50 to 350 amino acids) pack together to form domains. Small proteins may contain one or two domains, while larger proteins may contain several domains linked to each other by relatively open polypeptide chains. For example, cytochrome b562 is a single domain protein composed almost entirely of á helices, while the NAD-binding domain of lactic dehydrogenase is composed of á helices or â sheets. The variable domain of an immunoglobulin light chain is composed of a sandwich of two â sheets. In general, polypeptide chains traverse back and forth across the entire domain, making sharp turns only at the protein.
Proteins can form stable surfaces by undergoing configurational changes in response to local molecular ecologic conditions. Further levels of complexity are created when proteins form new surfaces with different binding properties by joining with other proteins through noncovalent interactions. In some large proteins, parts of the polypeptide chains fold independently into discrete globular domains and specific functions.
Proteins serve structural, cytoskeletal, metabolic, replicative, transcriptive, translational, secretory, digestive, absorptive, and a host of other functions.3-11 To serve those roles, proteins are phosphorylated, sulfated, glycosylated, acetylated, ubiquinolated, farnesylated, hooked onto glycophosphatidylinositol, and enhanced or degraded in yet other ways. Proteins respond to their altered molecular environment by changing their intracellular relocations, exiting from cells, undergoing cleavage, adjusting their stability, and changing their partners in service, and are embellished in yet other ways.
IV. OXIDATIVE PROTEIN FOLDING
Most secretory proteins fold by disulfide bond formation in specialized compartments. For example, the endoplasmic reticulum in eukaryotes and periplasm of Gram-negative bacteria are the site of such bond generations. In the past, this process was deemed as the simple joining of two cysteine residues in a molecular environment in which oxidizing equivalents are provided by molecular oxygen or some other suitable molecules, such as oxidized glutathione. Later it was recognized that disulfide bond formation requires oxidative enzyme catalysts in both prokaryotes and eukaryotes. Recent studies have revealed extraordinary complexity in electron transfer events in such bond formation in which a host of cellular proteins and several small molecules participate.12 For example, at least four proteins assist the process of disulfide bond formation in E. coli. First, DsbA, a periplasmic protein with a thioredoxin-like fold and a CXXC motif, serves as the primary oxidant of secretory proteins. It transfers its disulfide bond to thiols in the target protein. Second, DsbB, a membrane protein, uses its four essential cysteine residues to maintain DsbA in the active oxidized state, passing electrons on to quinones during the process. The redox state of quinone is then reversed—reduced quinones are reoxidized—by terminal oxidases with molecular oxygen. Under anaerobic conditions, dimethyl sulfoxide (DMSO) or electron-accepting nitrates are used. E. coli has yet another reducing pathway by which disulfide bonds can be produced. In that pathway, periplasmic DsbC reduces proteins with incorrectly paired cysteine residues. Next, to maintain DsbC in the reduced state, DsbD, a membrane protein with six essential cysteine residues, acts to transfer electrons from cytoplasmic thioredoxin to oxidized DsbC in periplasm.
V. FUNCTIONS OF PROTEINS
The names of proteins and peptides usually reflect their functions when first discovered. Later studies generally reveal biologic activities entirely distinct from those first recognized. That, of course, calls into question conclusions drawn from the initial observations. New hypotheses are then advanced in light of new findings. That cycle repeats itself continually. Science, we are told, advances by trial and error and is self-correcting. This should be fully acknowledged and heeded in clinical medicine, but rarely is. I underscore this point in this chapter by presenting the cases of many individual protein molecules that play multiple Dr. Jekyll/Mr. Hyde roles under varying conditions. It will also become evident how rapidly our notions of protein structures and function are evolving in view of recent knowledge. In this section, I present some basic information about proteins to further emphasize their structural and functional diversity.
A single protein may subserve different roles under varying conditions. Conversely, many different proteins may assume similar functions. Proteomics include identification, characterization, quantification, localization, modification, interaction, and delineation of their functionalities.
Protein actions in the past were generally presented in terms of static structures. However, in the protein world, as elsewhere, function depends on motion. The core events in signal transduction involve movements of domain(s) of a protein, switching from inactive to active forms of that protein. That switching process may be facilitated or blocked by other domains, ligands, other proteins, or covalent modifications. The best-studied example of the last is phosphorylation.
Earlier investigators advanced two models to explain how such effectors bring about the molecular motion and change in activity. In the first model, the effector induces a new structure. Phosphorylation that regulates signal transduction is generally thought to trigger a conformational switch, the so-called induced-fit mechanism. This model was based on the notion that unique folds in the molecules are dictated by the effector. The second model, the so-called allosteric model, proposed a new equilibrium, shifting the protein molecule from the relaxed (R) to the tense (T) state. Thus, the second model was based on the notion of a population shift involving the R and T molecular configurations and was favored for changes in multidomain proteins. Later researchers put forth concepts that emphasized molecular geography (protein-folding tunnels) and energetics (energy landscapes). According to those later models, protein functions are not determined merely by their static structures. Rather, functions are created and served by redistributions of existing molecular populations in response to changing local ecologic conditions. Protein functions, in such models, are then seen as workings of assemblages of conformational substrates.
The molecular dynamics of shifting roles of proteins are now being explored on exponential scales. It seems safe to predict that when those areas of protein structure and function are elaborated, oxygenative and oxidative events will be found to provide the underpinning of protein complementarity and contrariety.
Total antioxidant activity of serum proteins can significantly change after repeated oxidative stress.13 Proteins fold and unfold all the time. This flexibility in structure is made possible by rotating hinge-like bonds among many of the atoms in their structure. An average protein containing 500 amino acids has about 5,000 atoms, many of which participate in such rotation. It is a marvel of biology that any given protein flexes and unflexes in such determined way that its functions are preserved, whether it is to provide a firm scaffold for other molecules or to wander around ready to spring into action as an enzyme. Enter oxygen.
Two Types of Molecular Motions
The first evidence that proteins are dynamic structures came from nuclear magnetic experiments that revealed ring-flipping motion of aromatic side chains. Subsequent spectroscopic, time-resolved cystallographic and computational studies revealed complex side chain and backbone thermal motions over time scales ranging from picoseconds to seconds. Additional studies of hydrogen exchange and disulfide trapping employed to probe thermal motions on longer time scales of microseconds to hours demonstrated motional amplitude as large as 1.5 nm on the millisecond scale. It is now recognized that a protein in solution undergoes constant random thermal motions within a stable equilibrium structure. Not unexpectedly, such motions involve displacements of individual atoms, bonds, side chains, local regions of the backbone, secondary structure elements, complete folded domains, and functional groups. It is also known that many proteins undergo thermally driven transitions—designated conformational changes—between two or more equilibrium structures.
Teleologically, one would expect that such motions of protein molecules must have some functional consequences. That, indeed, is the case. The first type of molecular motion (the random type) serves as molecular “lubricants” that permit proteins to sample conformational space. Both types of motions—random and conformational—change substantially when the protein molecule comes under the spell of other influences in its environment, such as substrates, ligands, docking regions of macromolecules, and covalent modification (such as phosphorylation). All such changes profoundly affect the “fine-tuning” of the binding affinities of proteins, as well as their switching on and off functions. In the case of proteins with enzymatic activities, the molecular motion dynamics and the catalytic functions are linked. The substrate binding generally induces a conformational change within the enzyme so that it creates a guarded space or a “cavity” on its surface for the substrate to be enveloped and protected from the solvent. In some cases, such changes place the catalytic residues near the substrate.
A special class of proteins called allosteric proteins can take two or more stable forms. Such proteins shift back and forth between their states, thus serving as molecular switches. However, to perform that switch function, allosteric proteins need a partner that brings forth its energy field (weak or strong electrostatic forces, hydrophobic interactions and others). Several substances can act as partners to activate the hinges of protein molecules (such as methyl, acetyl, or phosphoryl groups). Of considerable significance to the subject of molecular complementarity and contrariety is the fact that certain oxygen atoms in such proteins can bind to calcium and change the structure—and function—of that protein.10
VI. ION CHANNEL PROTEINS
Sodium, potassium, calcium, magnesium, and chloride ions are employed by living organisms in most, if not all, fundamental cellular tasks.14-18 Ion channels occur both in cell membranes as well as in biomembranes of cellular organelles—not surprising in view of functional compartmentation that exists within cells. For those metabolic and homeostatic tasks, sharp ionic gradients across biomembranes must be produced and preserved which:
1. Mediate fluid and electrolyte transport;
2. Regulate volume in intra- and extracellular compartments;
3. Produce electrical signals;
4. Activate signal transduction pathways; and through those basic functions,
5. Exert myriad redox, acid-base, hormonal, and nonhormonal homeostatic influences.
Minimal changes in the structure and function of ion channels can have profound clinical effects. For example, epilepsy, cystic fibrosis, and cardiac arrhythmia can result from malfunction of the calcium, chloride, and potassium ion channels respectively.
It is estimated that the potassium channel can shuttle up to 100 million potassium ions across a cell membrane in a single second, while keeping sodium ions out. For every 1,000 or so potassium ions, the channel protein barely admits one sodium ion. This phenomena becomes even more astounding when it is realized that sodium ions are not only similarly charged but of smaller size. So how can a single protein molecule allow the larger potassium ion to go through rapidly while it holds back the smaller sodium ion? Enter the mysteries of oxygen. It turns out that the wide exterior opening of the KcsA potassium channel is lined by oxygen, which serves as a selectivity filter through which potassium ions (stripped of water that normally surrounds them when dissolved in body fluids) fit well into the oxygen-lined tunnel structure. Sodium ions, by contrast, carry a shell of water that keeps them from entering the channel.
Living organisms expend large fractions of their system energy for creating and perpetuating those ionic gradients. This is where proteins enter the picture. If it were not for highly specialized protein-designated ion channels, ionic gradients essential for the above tasks could not be preserved even if all of the system energy was allocated to creation and preservation of those gradients.
Proteins forming the ion channels are highly specific for particular types of ions, permit extremely high transport rates, and yet are capable of regulating ionic flow by switching themselves off and on. Channel proteins form specially designed hydrophilic pores through which ions flow down their electrochemical gradients. The electrical currents carried by ions moving through membrane channels generate and sustain fundamental cellular phenomena, such as resting membrane potential in all cells, action potential in neurons, and neurotransmission at synapses. Biomembrane channels, however, do not merely provide pores for passive flow of ions through them. The channel molecules can alter their configurations at extremely high speeds, shifting back and forth between nonconducting (closed) and conducting (open) states—a process called gating. Many channel proteins are gated by voltage—electrical differences across the biomembranes which house them.
Ion flux through biomembranes, not unexpectedly, alters the intracellular ion concentrations. The most intensively investigated among such concentration changes are those involving Ca2+, which, in turn, are notable for their widespread and profound influences on the cellular signaling pathways.
Calcium Is a Vasoconstrictor;
Calcium Is a Vasodilator
Calcium-channel blocking agents constitute a major family of drugs employed for control of hypertension. Those drugs have also been held responsible for many serious adverse effects, including congestive heart failure, myocardial infarction, and death. The following quote from Time magazine19 partially defines the problem:
Many patients suffering from high blood pressure were probably surprised last week to hear that one of the most popular classes of drugs for treating their condition, calcium channel blockers, was being blamed for some 85,000 avoidable heart attacks and heart failures a year.
I briefly outline below some dynamics of calcium and potassium channels that shed light on molecular underpinnings of the clinical paradox of beneficial effects of channel blockers and the serious consequences caused by them. Also, these comments further add to the splendor of the molecular webs and kaleidoscopes in human biology. Arterial tone is regulated by myriad regulatory influences. Calcium plays an interesting Dr. Jekyll/Mr. Hyde role in the maintenance of that tone. Intra-arterial pressure—blood pressure, in the common parlance—affects the arterial tone by a complex process involving a graded membrane depolarization and increased calcium influx through dihydropyridine-sensitive, voltage-dependent calcium channels.20,23 Calcium influx increases cytoplasmic calcium in a global fashion which, in turn, causes vasoconstriction. Calcium influx also evokes localized “calcium sparks”—a phenomenon involving localized calcium release events related to ryanodine receptors. Those sparks then activate calcium-activated potassium channels in the vicinity which, in turn, cause hyperpolarization and vasodilatation. Thus, the vascular tone, in the context of calcium dynamics, is regulated by a complex interplay of opposing calcium-dependent mechanisms: vasoconstriction is driven by global cytosolic calcium increase, whereas vasodilatation is driven by localized calcium concentrations in vicinity of certain receptors.21-23
But the effects of the calcium-potassium channel dynamics are not likely to be limited to only vascular tone. Targeted deletion of the gene for the â1 subunit of the calcium-activated potassium channels (BK channels) results in: (1) diminished calcium sensitivity of the BK channels; (2) reduction in functional coupling of calcium sparks to BK channel activation; (3) increase in vascular tone; and (4) effects on the translation of calcium signals in the central nervous system.23
Calmodulin Bifurcates Calcium Signals
Calmodulin (CaM)—a regulatory protein that controls the activities of the calcium ion channel—is an elegant example of molecular autoregulation. The two lobes of this molecule at its two extremities provide a counterbalance to their effects on calcium channels. The two structural lobes of CaM are: (1) the C-terminal lobe that responds preferentially to the spike-like component of local Ca2+ concentration attributable to local channel activity; and (2) the N-terminal lobe that detects the slow component of the signals resulting from aggregate cellular Ca2+ signaling.23a Calmodulin promotes (facilitates) channel opening under one set of conditions and inhibits (inactivates) channel activity under another. Some evidence suggests that biochemical precedents exist for lobe-specific CaM signaling.23b The structural and functional discreteness of the two lobes of calmodulin represent important molecular design advantages that allow the protein molecule to play both sides of the field, exerting two opposing—and kinetically disparate—effects on a type of calcium ion channel called the P/Q-type calcium channel.
The bifunctional capability of this regulatory protein also provides a window to Nature’s preoccupation with contrariety. Experiments with mutant CaMs with selective impairment of Ca2+-binding to either the N-terminal (CaM12) or C-terminal lobe (CaM1234) have revealed an interesting paradox. Ca2+-binding to the N-terminal lobe of CaM selectively inactivates, whereas Ca2+-binding to the C-terminal lobe selectively triggers facilitation. Thus, a lobe-specific detection system of CaM creates a means to decode local Ca2+signals in two distinct ways and to separately elicit two distinct and opposing responses from a single molecular complex.
The functional duality of CaM is thought to enrich the neurocomputational capabilities of the brain. It is unlikely that the benefits of such duality are confined only to neurons. It seems to me that the advantages of such molecular design will also be found in other tissues in time.
The chloride anion channel facilitates the selective flow of Cl– ions across biomembranes, thereby regulating the flow of salt and water across epithelial barriers. In the skeletal muscle, these channels regulate electrical excitation of cell membranes. Several families of genes are involved in producing channel proteins. For example, by 2002, nine different genes were known to exist for the ClC group of chloride anion channel proteins.17 In the past decades, most attention was drawn by cation channels, in part due to their roles in generating electrical signals in nerve cells, as well as their potential for developing pharmacologic agents for blocking those channels in pathologic states. Recently, there has been similar success in deciphering the structure and gating mechanisms of anion channels. Genetic defects in chloride channels cause several familial renal and muscle disorders. X-ray structural studies of chloride ion channels have revealed two identical pores in each channel molecule, each pore being formed by a discrete subunit contained within a homodimeric membrane protein. Both subunits comprise two roughly repeated halves that span the cell membrane with opposite orientations. The antiparallel architecture so created defines a selectivity filter in which the chloride ion is stabilized by electrostatic interactions with á-helix dipoles as well as by chemical coordination with nitrogen atoms and hydroxyl groups.15
MR. HYDE ROLES OF PROTEINS
Proteins were the first bioactive molecules to confuse researchers and clinicians by their range of molecular complementarity and contrariety. Indeed, long before their identification and characterization, the “ways of proteins” confounded physicians as well as observers of nature. To provide a framework for presenting the subjects of multifunctionalities and Mr. Hyde roles of proteins, I reproduce below some text regarding smallpox from the chapter on the history of medicine in the second volume of The Principles and Practice of Integrative Medicine:
Yet it was with those who had recovered from the disease that the sick and the dying found most compassion. These knew that it was from experience, and had now no fear for themselves; for the same man was never attacked twice—never at least fatally.
The above is the earliest written record of a knowledge of acquired immunity. The nineteenth century was a period of exponential growth in knowledge of the mechanisms of immunity, most of which involved identification of antibodies and study of their dynamics (presented at length in the second volume of this book). The Mr. Hyde-side of antibody function was first conceived with considerable difficulty and presented tentatively. The two quotes given below show the early struggle with the notion of functional contrariety of proteins.
The conception that antibodies, which should protect against disease, are also responsible for disease, sounds at first absurd.
Clemens von Pirquet25
Paul Ehrlich, one of the four most-celebrated men of medicine of the nineteenth century, shows how much consternation it caused him.
It would be exceedingly dysteleologic, if in this situation self-poisons, autotoxins, were formed.
I include below brief comments about the glycoprotein fibronectin to illustrate some of the complexities of protein structure and function presented above. Fibronectin is a major protein in the extracellular matrix (ECM) and also occurs in the plasma in soluble form. Its versatility and multifunctionality are often not fully appreciated. It plays a central role in binding and holding together many components of the ECM, such as collagen and proteoglycan, as well as cell surfaces. Its other biologic activities include important roles in embryonic differentiation, cytoskeletal organization, cell morphology, and cell migration.27
Twenty different tissue-specific fibronectin isoforms have been identified. All are produced by alternative splicing of a single precursor mRNA. It is a dimer of two identical subunits joined by a pair of disulfide bonds at their C-terminal. Each subunit has discrete domains called type I, II, and III. Each subunit is folded to form a ‘string of beads’ configuration. Fibronectin has several functions recognizable by their binding affinity for other ECM components. For instance, type I modules bind fibrin, heparin, and collagen. Through those affinities, type I modules exert regulatory influences both on the coagulative and fibrinolytic pathways. Type II modules bind with collagen, whereas type III binds heparin and the members of the integrin family of proteins which, in turn, bind extracellular proteins on the cell surface to cytoskeletal protein, such as actin, on the inside of the cell. Thus, fibronectin is involved in communication between the intracellular and extracellular environments.
Malignant cells show loss of fibronectin. This phenomenon has been implicated in formation of metastases. Fibronectin loss is thought to allow tumor cells to penetrate the gel-forming components of the ECM produced by proteoglycans.
Specific enzyme systems break down various components of ECM. Collagen, the dominant family of proteins in ECM, forms rigid thermostable fibrils that withstand oxidative stress well in health. In collagen disorders, however, oxidative injury that leads to autoimmune response inflicts injury on all the ECM components. The subject has been reviewed.2
The ECM also undergoes significant change in malignant neoplasms. Specifically, there is: (1) increased activity of prolyl hydroxylase, which leads to a desmoplastic (fibroproliferative) response; (2) diminished activity of hyaluronidase and reduction in the amount of hyaluronic acid; and (3) changes in the concentration of laminin.28-30 Varying degrees of oxidative coagulopathy are nearly always present in disseminated malignant tumors (personal unpublished observations). It seems highly probable that normal and altered fibronectin play some roles in ECM changes in cancer since, as pointed out earlier, this ECM protein binds both fibrin and heparin.
IX . AMINO ACIDS SELF-ORGANIZE
In their famous 1953 experiment, Stanley Miller and Harold Urey of the University of Chicago subjected simple molecules such as ammonia, methane, hydrogen and water in a flask to repeated cycles of heating, electrification and cooling—conditions that are thought to have existed when life began on planet Earth. The experiment produced a crimson-colored primordial soup, rich in amino acids. Amino acids, of course, are the building blocks of proteins, including enzymes.
In subsequent years, origin-of-life research has been focused on the fundamental chemical mechanisms of prebiological molecules. Notable among these studies are those of Sidney Fox, who clearly demonstrated that under primordial conditions, amino acids could self-assemble into proteins—without the intervention of genes. The next important step was to demonstrate whether or not proteins so formed could assemble themselves into tiny protein microspheres—the precursors of cells as we know them today. Experiments clearly showed that this assemblage does occur. Biologist Aristotle Pappelis put that evolutionary perspective succinctly in the following words:
If the reactions within a sphere help make macromolecules, then we’re talking about a cage in which thermal proteins could help build nucleic acids like RNA or DNA. Perhaps thermal proteins can also help synthesize true proteins…through this type of mechanism, cellular evolution could have arisen from microspheres.31
Where does cancer begin? What are the initial energetic-molecular events that turn a healthy cell into a cancer cell? How does a cancer cell go into a nonoxygen-utilizing mode, becoming an oxyphobe? How does it begin to produce prodigious amounts of hydrogen peroxide? How does it accumulate electrons on its surface and acquire a strong negative charge? Those essential questions have not been answered yet. However, it seems safe to predict that the answers will one day be found in the workings of the protein web and kaleidoscope.
I include the above brief comments about the origin-of-life research to support my view that molecules have their own “minds”—their own sense of natural order of things. Simple molecules (such as ammonia and water), larger molecules (such as amino acids), and complex molecules (such as proteins, including enzymes), can assemble and disassemble themselves without any organizing influence of genes. Such observations further the range of complementarity and contrariety in nature.
X. GENES: PRODUCTS OF EVOLUTION
It is sometimes stated that genes drive evolution. In that view, genes not only create new biologic characteristics in organisms, but also assure their perpetuation. Thus, genes are considered responsible for bringing forth diversity in nature. However, available evidence clearly shows that genes are the products of evolutionary changes. If DNA was copied flawlessly each time a cell divided—and its DNA split—there would have been no evolution. To evolve is to change, and no change can be maintained for a long time except through a change in DNA configuration. Without DNA alterations, today we would have the same DNA as our genetic ancestors over three billion years ago—and the same physical characteristics as single-celled microbes that existed then. When DNA is copied with a mistake, each copying error carries the risk of fatal structural or functional derangements.
Every second that passes, the DNA in each cell of your body is being damaged. Chemical bonds are breaking, DNA strands are snapping, and nucleotide bases are flying off. Each cell loses more than 10,000 bases per day just from spontaneous breakdown of DNA at body temperature. Meanwhile, many cells are dividing and therefore copying DNA, and each copy introduces the possibility of error.32
DNA copying occurs with an astounding accuracy—on the average, only three base pair mistakes are thought to occur when copying three billion base pairs of the human genome. This mind-numbing copying accuracy is assured by the most extraordinary DNA repair system composed of several enzymes. Thus, the reason that everyone does not develop clinicopathologic disorders (resulting from damaged genes) and cancer at all times is the enormous capacity of our DNA repair enzyme systems.
XI . PROTEIN PROTECTION OF DNA
Proteins protect DNA directly by putting four major locks on it. The first lock is put on it by a special class of proteins that hold it in its double-helix state by providing an attractant force from within. The second lock is provided by protein molecules that wrap around the DNA to create a strong protective sheath to produce a structure called a nucleosome. Electron microscopy of DNA reveals two higher orders of structure—the 10-nm fibril and the 25-30 nm chromatin fiber—beyond the nucleosome, each providing an additional lock. There are other indirect ways in which proteins create microecologic conditions (of redox and acid-balance homeostasis) that preserve the structural and functional integrity of DNA.
The DNA in a human cell nucleus in the usual state measures a few cubic microns in volume. If simply extended, it is estimated to be nearly one meter long. In the past, it was believed that DNA was so “packaged” by a class of closely related basic proteins called histones.33-38 In that model, these proteins merely form positively charged spools around which negatively charged strands of DNA—about 146 nucleotides long—are wound to provide a neat fit into the tiny cell nucleus. It is now clear that histones do not merely provide mechanical support for DNA strands, but actively protect them from nefarious influences of various kinds as well as regulate the behavior of genes formed by them. There are five basic forms of histones: H1, H2A, H2B, H3, and H4.
Nucleosomes are ultrastructural “beads” along the “necklace” of DNA formed when DNA strands wrap roughly twice around the histone complex—an octamer polymer composed of two copies of histone H3, H4, H2A, and H2B each. Within that complex, H2A and H2B flank a tetramer formed by H3 and H4. One end of each histone is thought to protrude from the central core of the complex located within the double helix of DNA to interact with other molecules. The DNA within a nucleosome is further anchored—and protected—by an outer coat formed of histone A. Thus, the nuclear chromatin is made up of the beads of nucleosomes connected by the thread of histone-free DNA. Histone H1 participates in keeping DNA to coil into 30-nanometer fibers. The genes housed within those fibers are considered to be silent until such time that the coils are unfolded and the contained DNA is exposed to the influences of various inductive and regulatory factors. More importantly in the present context, DNA within the coils is shielded from free radical injury and untimely interactions with various proteins. Histones are remarkably conserved, varying little from plant species to mammals. From an evolutionary perspective, such lack of diversity among amino acid sequences in histones is indicative of their biologic essentiality. If the only purpose of histones in chromatin were DNA folding—it may be stated—any group of proteins with positively charged amino acids would have sufficed.
XII. PROTEIN CHAPERONS OF mRNA
It takes a village to raise a child, so goes an African saying. It takes a large family of proteins to chaperon messenger RNA (mRNA). Transcription of genes into their respective mRNA is a highly complex process in which protein coactivator complexes actively participate and broadly regulate the catalytic functions of protein enzymes. Specifically, initiation of transcription among eukaryotes is sustained and regulated at multiple levels—with mechanisms involving activators, pre-initiation complexes, core promoter recognition complexes, and chromatin modifying factors. To give the reader an idea about the enormous complexity of the transcription processes, following is an incomplete list of molecules and/or factors that have been implicated:
1. RNApolymerase II;
2. Transfer factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH);
3. Activators (VP16, SREBP); and
4. Coactivators (ARC/DRIP, TRAP/SMCC, hMed, NAT, CRSP, and PC2).
True to its preoccupation with contrariety, Nature has also assigned contrarian roles to the above molecular species. For example, in vitro transcription experiments have demonstrated repressive activities of NAT and SMCC. I italicize in vitro in the preceding sentence to emphasize that the incomplete list shown above is derived from laboratory experiments. Undoubtedly, many additional initiators, activators, and coactivators will be found with time; most, if not all, will be found to subserve complementarian and contrarian roles. Those are Nature’s ways. Chemistry is about controlled conditions in the laboratory, while biology is about uncontrolled in vivo conditions. I return to this subject often, because it forms my core argument for subordinating all experimental findings to true-to-life empirical observations in clinical medicine.
Returning to the subject of protein chaperons, Mediator, a large coactivator complex discovered in the early 1990s, plays a central role in the transcriptional machinery of yeast. In humans, Mediator-like coactivator complexes participate in switching on transcription by binding to activators as well as RNA polymerase II itself.39,40 Two major human Mediator-like coactivator complexes—the large ACR-L and the small CRSP—have been purified and characterized, and their interactions with various activators of transcription have been delineated. Not unexpectedly, some subunit homologs of yeast Mediator are preserved in metazoan complexes. Transcription activators, including VP16 and SREBP, modulate the structure of CRSP and alter its conformation. The change in conformation of CRSP alters its biologic activity and influences its interactions with a host of coactivators. The transcriptionally inactive ARC-L appears to both serve as the docking site of RNA polymerase and exert a negative effect on transcription by competing with CRSP for transcriptional activators as well as coactivator complexes.39,40
XIII. PROTEIN REGULATION OF GENES
Genes are recognized to control the developmental, replicative, differentiative, and apoptotic roles of proteins. But the regulatory influences of proteins over the structural and functional integrity of genes are usually not given full consideration. In reality, proteins shape and alter the structure of genes and fundamentally change their activities at every step in the highly complex processes that are involved in the protein-DNA dynamics.
Genes of eukaryotic cells contain some highly specialized domains, including the core coding region, which specifies the amino acid sequence of individual proteins. Other important domains include those that determine or influence whether the coding domain is copied into mRNA and the number of RNA molecules generated. Specific sets of proteins are needed to switch on individual genes. Such gene-protein dynamics require the protein molecules to assemble on a regulatory domain of the gene designated the proximal promoter. As the first step in that interaction, one specific protein binds to a segment of the proximal promoter called the TATA box*—so named because it includes some approximation of the nucleotide sequence TATAAATA.
In the next step, other proteins join the first protein to produce a protein-DNA complex known as the preinitiation complex. One member protein of that complex, the enzyme RNA polymerase, is then positioned at a spot within the proximal promoter designated as the transcription initiation site. The messenger RNA is then synthesized by the precisely positioned polymerase that moves up and down the entire length of the coding zone, using the sequences in that zone as the template. Under basal in vitro conditions, the proteins of the preinitiation complex recognize the DNA and initiate a low level of transcription, hence the designation basal factors for the components of that complex. The transcription process proceeds by binding yet additional proteins—designated activators—to a second regulatory site called the upstream activator sequence (also referred to as enhancers). These dynamics speed up RNA generation to a maximal level.
There are other important aspects of gene regulation by proteins. Gene activation requires unwinding of DNA associated with TATA boxes to make it accessible to operant inductive factors. Here, nuclease enzymes that cleave DNA serve essential regulatory functions. To make the matter of complementarity in protein regulation of genes
*The letter T in this context stands for the nucleotide base thymine, whereas A represents the base adenine. The remaining two bases, guanine and cytosine, are referred to with letters G and C respectively. Base pairing is a phenomenon of fundamental importance in considerations of the structure and function of DNA. Adenine and thymine always pair by hydrogen bonding, as do guanine and cytosine. Such complementarity explains the observation that the guanine content of any fragment of a double-stranded DNA always equals its cytosine content. The same holds for adenine and thymine.
yet more interesting, histone H3 and histone H4 are very similar to each other in many of their biologic roles, including patterns of acetylation and participation in the formation of nucleosomes. Yet, histone H3 is required for suppression of some of the same genes that require histone H4 for activation.
Proteins regulate genes which, in turn, regulate proteins. Localization of messenger RNA (mRNA) in diverse cells—from an ovum to a neuron—is asymmetric and determines the expression and spatial distribution of proteins.41,42 That localization is, in turn, determined by special signals that arise from within mRNA molecules themselves (‘cis-acting’ signal)—an elegant example of molecular autoregulation—as well as from those on the RNA-binding proteins (‘trans-acting’ factors). The RNA-protein complexes formed by those dynamics interact with machinery that targets mRNA molecules to specific subcellular compartments. For example, in Drosophila, Egalitarian (Egl) and Bicaudal-D (BicD) are two maternal protein components of a selective dynein motor complex that drive transcript localization in various tissues.
XIV. THE WEB OF G-PROTEINS
Among the more fascinating “webs within webs” of human biology is that formed by G-proteins—a superfamily of catalytic active regulatory molecules with kaleidoscopic responsibilities.43 The members of this family bind to nucleotide guanine, hence the designation G-proteins. The range of regulatory functions is extraordinarily broad and includes the following:
1. Intracellular trafficking (targeted delivery of substances to biomembranes as well as cellular organelles);
2. Exocytosis (exclusion of bioactive and waste substances from within the cells);
3. Signal transduction;
4. Protein synthesis;
5. Cellular movement;
6. Cellular growth;
7. Cellular proliferation;
8. Cellular differentiation; and
9. Self-regulation and self-policing.
The G-protein web—like other wide biologic webs and kaleidoscopes—is a skillful self-regulator. For teleologic consideration it would be expected that proteins with such broad responsibilities must have capabilities for turning themselves off to prevent cellular chaos and mayhem. That, indeed, is the case.44
There are two dominant subfamilies of G-proteins: a smaller subfamily of monomeric Ras-like G-proteins and a larger one that includes heterotrimeric G-proteins. The proteins in the latter subfamily consist of three distinct classes of subunits: á (39-46 kDa), â (37 kDa), and ã (8 kDa). On the basis of their cDNA homology, four principal subfamilies of á-subunit genes have been recognized: G12, Gq, Gs (stimulatory), and Gi (inhibitory). Many Gá subunits are ubiquitous in mammalian systems—especially at the mRNA level—but others serve tissue-restricted roles.
In general, the á-subunit contains the guanosine triphosphate (GTP)-binding site and confers effector specificity on the protein molecule. This subunit also includes an intrinsic GTPase activity—thus assuming a self-policing role as well. The âã complexes, under certain conditions, can also exert regulatory influences on effector molecules, such as phospholipase A2 (PLA2), phospholipase C-â (PLC-â) isoforms, adenylate cyclase, and ion channels in mammalian species. In yeast, âã complexes also influence cellular responses such as mating-factor receptor pathways.
None of the subunits of G-proteins are an integral part of cell membranes. However, the proteins are anchored to biomembranes by lipid modification of the ã-subunits (prenylation) and some of the á-subunits (myristoylation). In the inactive state, guanosine diphosphate (GDP) is bound tightly to the á-subunit. Ligation of the receptor drives the exchange of GDP for GTP, thus inducing a conformational change in Gá which, in turn, causes a decrease in its affinity for the âã complexes as well as for receptors, and facilitates dissociation of the receptor-G-protein complex. Thus, the G-protein molecule regulates itself. The activated Gá and released âã complexes—singly or in concert—are then free to interact with other effector molecules to generate intracellular second messengers which, in turn, activate myriad downstream signaling cascades.
The G-Protein-Coupled â-Adrenergic Receptor Dynamics
G-protein-coupled membrane receptors constitute another superfamily of structurally related receptors for neurotransmitters, hormones, proteinases, mediators of inflammation, highly specialized molecules for taste and odor functions as well as for response to light photons. Brief comments are included below the G-protein-coupled â-adrenergic receptor dynamics to provide an illustration of complexity as well as complementarity of energetic-molecular dynamics.
A â-adrenergic receptor is a ligand for epinephrine. Like other G-protein-coupled receptors, â-adrenergic receptor is an integral part of the cell membrane and is composed of seven transmembrane-spanning helices (each containing 20-28 hydrophobic amino acids), an extracellular N-terminus, and an intracellular C-terminal tail. The epinephrine molecule binds with the â-adrenergic receptor by occupying a shallow pocket-like domain on the receptor. The generation of cAMP is one of the earliest consequences of ligation of the receptor by epinephrine which, in turn, regulates the intracellular signal transduction. To cite an example, cAMP—derived from ATP by the catalytic action of the signaling enzyme, adenylate cyclase—is involved in the conversion of glycogen into glucose. The cAMP activity is terminated by its hydrolysis to 5′-AMP by specific cAMP phosphodiesterases. Parenthetically, I might add that hormones that activate adenylate cyclase in adipocytes also stimulate glycogen breakdown.
XV. TURNCOAT ANTIBODIES
Autoimmune disorders are diverse clinicopathologic entities caused by antibodies that turn on the body’s own tissues. Another major class of turncoat antibodies is IgE antibodies, which mediate atopic reactions and cause serious chronic disorders, including asthma, eczema, sinusitis, otitis media, and others.45-48 Those two subjects of autoimmunity and atopy are discussed at length in the second volume of this textbook. Here I briefly describe some lesser-known examples of turncoat antibodies to show yet other dimensions of the functional web of proteins.
Malaria is usually more severe during pregnancy. Plasmodium falciparum employs ingenious methods to protect itself from the host defenses of the pregnant woman. The first of those mechanisms to be recognized was the binding of malaria-infected erythrocytes to chondroitin sulfate and hyaluronic acid produced by syncytiotrophoblasts. Another mechanism involves adsorption of nonimmune immunoglobulin G on the surface of infected erythrocytes, which then becomes anchored to syncytiotrophoblasts. Thus, human nonimmune IgG protects the invading protozoa rather than defending the body against them. Similarly, the helminth Schistosoma mansoni adsorbs host immunoglobulins to avoid recognition by specific antibodies that are produced to facilitate the killing of the parasite.49,50
XVI. ANTIBODIES THAT BURN WATER
A detailed presentation of the subject of structure and functions of proteins is clearly outside the scope of this volume. I include below brief comments about some of the recently recognized roles of proteins to underscore the theme of Nature’s preoccupation with complementarity and contrariety.
Among the lesser-known roles of proteins is the participation of some of them in hydrogen peroxide chemistry. Specifically, antibodies produce hydrogen peroxide through the photogeneration of singlet oxygen.50,51 This process is catalytic in nature in that it derives electrons from water—and not from non-photo-oxidizable residues, such as tryptophan, metal ions, and chloride, as was previously suspected. This reaction has considerable chemical and biologic importance. First, this provides yet another defense mechanism against toxicity of singlet oxygen. Second, it creates a pathway by which antibodies can serve both recognition and microbe-killing functions. Third, this redox-restorative function of antibodies suggests that singlet oxygen might have played an important role in the evolution of the immunoglobulin molecule.
The discovery of an antibody-mediated photo-oxidative pathway supports the hypothesis that the interaction between water and singlet oxygen results in the formation of H2O3 as the first intermediate molecule. Formation of H2O3 appears to represent a special constellation of organized water molecules at the active site conditioned by antibody-specific “molecular environment.”
It may be added here that besides antibodies, áâ T receptor—which shares a similar arrangement of immunoglobulin fold domains—is also capable of catalytically converting singlet oxygen into hydrogen peroxide. It is likely that additional proteins will be found to share this function as well.
XVII. PROTEIN PROTECTION OF BLOOD GLUCOSE LEVEL
Proteins exert a powerful glucoregulatory influence that is regularly put to use by nutritionists but is not fully appreciated by diabetologists and glycomics researchers. The glucoregulatory dynamics are presented at length in the chapters entitled “Complementarity and Contrariety in Glycomics” and “Beyond Insulin Resistance and Syndrome X: The Oxidative-Dysoxygenative Insulin Dysfunction (ODID) Model.” Here, I include brief comments about my personal perspective on the clinical efficacy of protein formulations containing approximately 90% of the calories in proteins. Rapid hyperglycemic-hypoglycemic shifts, as indicated in a preceding chapter, are extremely common in patients with diverse nutritional, ecologic, and autoimmune disorders. Nearly all such patients receiving protein supplementation report improvement in symptoms of unexplained irritability and mood swings, headache, episodes of heart palpitations, lightheadedness, and undue fatigue.52 Another theoretical approach to the problem of rapid hyperglycemic-hypoglycemic shifts is essential oil supplements. However, my clinical experience has not shown that to be a generally effective strategy.
XVIII. THE WEB AND KALEIDOSCOPE
Peroxisome proliferator activator receptors (PPARs) form an extraordinary molecular family that is another superb example of nature’s preoccupation with complementarity and contrariety.53-58 It is now certain that the members of this family serve pivotal roles in the pathogenesis of obesity, diabetes, heart disease, and cancer. Also PPARs are intricately involved in apoptotic pathways.
The peroxisomes are a class of membrane-limited organelles, morphologically similar but functionally discrete from lysosomes. Enzymes of peroxisomes degrade amino acids and fatty acids, producing large quantities of hydrogen peroxide—a potent oxidizer to which amino acids with side chains, such as methionine, are especially vulnerable. Not unexpectedly, peroxisomes contain copious amounts of catalase to protect amino acids and fatty acids from hydrogen peroxide. Some mystery has surrounded peroxisomes, since amino acids and fatty acids are metabolized by other organelles that do not produce large amounts of hydrogen peroxide.49 It has been speculated that these organelles are sites of preferential generation of heat, rather than ATP production.
PPAR proteins occur directly on DNA molecules in the cell. They associate with other proteins and smaller molecules to initiate and perpetuate a wide range of biologic responses which, in turn, serve as molecular switches for regulating gene activities—yet another example of a family of proteins that regulate the regulators.
However, it seems to me that peroxisomes, by producing much hydrogen peroxide, play wider and more significant roles in redox homeostasis and regulation of genes (see chapter on the pathophysiologic roles of hydrogen peroxide). It is most likely that PPARs will eventually be found to play pivotal roles in the pathogenesis of all degenerative and immune disorders. That may seem far-fetched to some. But PPARs are found in a wide array of cell types, including leukocytes, endothelial cells, adipocytes, the bowel, and the retina. Thus, my proposed universality of their biologic roles may not be seen as merely speculative. Indeed, I consider it a good example of my safe predictions regarding energetic-molecular events that take place in the health/dis-ease/disease continuum but have not yet been experimentally validated to date.
PPAR-Gamma Prevents Diabetes,
PPAR-Gamma Promotes Diabetes
In the adipocyte, PPAR-gamma regulates fat storage. PPAR-alpha is involved with lipolysis for energy release. The roles of a third member called PPAR-beta (sometimes also called delta) remain elusive. The dynamics of PPAR-gamma have been extensively investigated in the hope of finding novel hypoglycemic agents for clinical use. Thiazolidinediones (TZDs) form such a class of drugs that act by binding to PPAR-gamma-protein complexes and turning on PPAR-gamma which, in turn, exerts an insulin-sensitizing effect and lowers blood sugar levels. Three drugs in this class that have been introduced in clinical practice are troglitazone (Rezulin), rosiglitazone (Avandia), and pioglitazone (Actos).
Another proposed mechanism of action of PPAR-gamma involves its influence on the release of energy from fats and sugars. There is some evidence that this factor spurs adipocyte growth with increased uptake of fats from the blood. The lower concentrations of lipids in the blood then prompt myocytes, hepatocytes, and other cells to shift their energy pathways away from lipids and toward sugars.
In the context of diabetes, PPAR-gamma also serves Dr. Jeykill/Mr. Hyde roles. One common variant of its gene has been associated with increased risk of diabetes, whereas a second variant actually protects from diabetes. PPAR-gamma dynamics also appear to be linked with a mRNA that codes for resistin, a polypeptide specifically expressed and released by adipocytes and so named because it causes insulin resistance.55 Genes for both resistin and PPAR-gamma are activated during adipocyte differentiation and downregulated in mature fat cells. The roles of PPAR-gamma and resistin in the pathogenesis of insulin resistance and diabetes are further discussed in the chapter titled “Beyond Insulin Resistance and Syndrome X: The Oxidative-Dysoxygenative Insulin Dysfunction (ODID) Model.”
PPAR-Gamma Prevents Atherogenesis,
PPAR-Gamma Promotes Atherogenesis
Another example of Dr. Jekyll/Mr. Hyde roles of PPAR-gamma involve the pathogenesis of atherosclerosis. PPAR-gamma coaxes macrophages to scoop up oxidized LDL in the circulating blood as well as tissues. Some PPARs trigger biochemical cascades that govern the cellular maturation. Through those functions, PPAR-gamma actively participates in the pathogenesis of arteriosclerosis. Here PPAR-gamma plays a well-defined Dr. Jekyll role—facilitation of removal of oxidized LDL cholesterol from the circulating blood. Next, it turns around and plays a Mr. Hyde role and facilitates deposition of LDL in atherosclerotic plaques. Thus, damaged molecules are cleared by PPAR-gamma, only to be delivered to a wrong location.
In atherogenesis, the story of PPAR-gamma takes yet another fascinating turn. Surprisingly, thiozolidinediones—which bind with and spur PPAR-gamma—appear to offer some protection against atherosclerosis in diabetes as well as lower blood pressure. As for the former, some evidence suggests that PPAR-gamma converts—or participates in conversion of — oxidized LDL to HDL. That has excited some, who see a possibility of developing more desirable drugs than statins that only lower LDL. The need for caution in that is self-evident. As for the effect on blood pressure, PPAR-gamma seems to trigger release of as-yet undefined vasoregulatory factors.
PPAR-Gamma Prevents Carcinogenesis,
PPAR-Gamma Promotes Carcinogenesis
In carcinogenesis, PPAR-gamma plays yet other Dr. Jekyll/Mr. Hyde roles. Limited studies concerning the administration of troglitazone (Rezulin) to persons with prostatic carcinoma show stabilization of blood levels of prostatic-specific antigen and lack of new symptoms attributable to the tumor. Furthermore, it was discovered that transgenic mice (which produce about half the normal amount of PPAR-gamma are more likely to develop colon cancer than the wild-type mice. Such initial data brought forth much enthusiasm about the possibility of a novel pharmacologic approach to the treatment of cancer. However, that enthusiasm was dampened soon after by the discovery that drugs that turn of PPAR-gamma production promote the formation of cancer in experimental animals.
PPAR-Gamma Prevents Inflammation,
PPAR-Gamma Promotes Inflammation
It is predictable, in view of the recognized roles of PPARs in cell development and differentiation, that those proteins will be found to play roles in inflammatory disorders. Preliminary studies have revealed antiinflammatory roles of proteins in this family. The possibility of pharmacologic stimulation of PPARs for controlling diverse inflammatory disorders (including rheumatoid arthritis, inflammatory bowel disease, and psoriasis) are being explored.46,50
It is also predictable that PPARs play proinflammatory roles. All known “pro-obesity” molecules are also proinflammatory. Since PPAR-gamma is clearly a pro-obesity molecule, it is safe to predict that it will be found to be a potent proinflammatory molecule. Thus, there is reason to suspect that unrestrained PPAR activity, without physiologic counterbalances, will be implicated in the pathogenesis of most inflammatory disorders. Some aspects of the proinflammatory roles of pro-obesity molecules are presented in the chapter titled “Beyond Insulin Resistance and Syndrome X: The Oxidative-Dysoxygenative Insulin Dysfunction (ODID) Model.”
PPAR-Gamma Prevents Obesity,
PPAR-Gamma Promotes Obesity
PPAR-gamma is involved in optimizing metabolism of both sugars and lipids. Specifically, it exerts an insulin-sensitizing role, which is the basis of the clinical use of thiazolidinediones in diabetes. Insulin-sensitization, of course, facilitates cellular glucose uptake and so prevents rising serum concentration of insulin. Persistent hyperinsulinemia is known to lead to beta cell exhaustion—”pancreatic burnout,” so to speak—and sets the stage for insulin resistance as well as diabetes. Both states are strongly associated with obesity (see the chapter on insulin resistance for further discussion). Thus, PPAR-gamma—by preventing insulin wastage, insulin resistance, and diabetes—serves an anti-obesity role.
PPAR-gamma is also a fat-storing protein. It promotes the development and differentiation of adipocytes. Indeed, it has been assigned an evolutionary role in the development of fat cells that served as energy storage sites in times of scarcity of food. But the conditions in the developed countries have changed—some studies put the incidence of obesity in the United States at 60% of the total population—and fat-rich foods are consumed in large amounts. However, adipocytes once formed in excess in obesity do not disappear, but only become “empty cells” when the food becomes scarce during caloric restriction in weight control programs. Such empty cells fill up very quickly when fats or fat-precursor sugars become available. This is one of the major causes of rebound weight gain that is the bane of dieters. The search for PPAR-gamma-suppressor drugs is on for prevention of rebound weight gain. Again, it seems unlikely to me that an agent will be discovered that will yield good long-term results in view of the enormous diversity of roles of PPAR-gamma.
PPAR-gamma also promotes fat deposition in excess in abnormal locations, such as the heart, liver, and other organs in which fat turnover ordinarily is limited. That raises the specter of anatomic alterations and functional derangement in those organs due to “organ obesity” caused by pharmacologic PPAR-gamma promoters.
Tinkering With the Tinkerers
Native molecular tinkerers do not take kindly to alien molecular tinkerers. PPARs are native tinkerers par excellence. So it would not be surprising if PPARs were found to be especially aroused by external molecular tinkerers. Such teleologic considerations call for caution in predicting the potential clinical benefits of pharmacologic tinkerers designed to tweak PPARs. Thus, the enthusiastic hype generated by the theoretical possibilities of using drugs that block or enhance biologic activities of PPARs to cure many chronic diseases may turn out to be just that: possibilities for drugs that may yield good short-term results in acute illness, but miserably fail to produce sustained beneficial effects in chronic disorders, including heart disease, cancer, and autoimmune entities.
The hazards of tinkering with biologic tinkerers are not of merely theoretical interest. Indeed, troglitazone (Rezulin), the first PPAR-gamma enhancer used on a large scale as an insulin-sensitizing agent, was withdrawn soon after introduction due to its hepatotoxicity. Two other members of the thiazolidinedione class of “PPAR drugs”—rosiglitazone (Avandia) and pioglitazone (Actos)—are still in clinical use as insulin-sensitizing agents at this time. However, the long-term consequences of altered PPAR physiology associated with the use of those drugs are not known. The essential points here are: (1) The web and kaleidoscope of the PPAR family of proteins are vast; and (2) Notwithstanding the benefits of “PPAR-active” pharmacologic agents for acute illnesses, it is unlikely that such drugs will provide long-term clinical benefits for chronic autoimmune, degenerative, metabolic, and ecologic disorders.
XIX. PROTEINS AS MOLECULAR SWITCHES
Many families of proteins serve as molecular switches. One of the most investigated families is the Ras family. By their switch functions, Ras proteins regulate diverse cellular processes, including information pathways by which intelligence at the cell surface is passed on to its innards.59-61 When bound to guanine nucleotide triphosphate (GTP), the protein serves as a switch in an ‘on’ position, whereas the protein functions as an ‘off’ switch when bound to guanine nucleotide diphosphate (GDP)—the energetically less advantaged form of the molecule. The Ras proteins are anchored to the cytoplasmic aspect of biomembranes through a covalently bound lipid group. When examined with the technique of fluorescent resonance energy transfer (FRET), Ras protein itself—the eponymous member of the family—is switched on at a location near the cell membrane. By contrast, Rap1—a close cousin protein—is triggered at a site near the nucleus.
Generally Ras proteins are switched on in response to activation of biomembrane receptor molecules. For instance, receptors for growth factors situated at biomembrane surfaces detect the presence of those factors outside the cell. In most instances, those receptors span the biomembranes and their inner extremities are connected to guanine nucleotide exchange factors. Once activated, those factors replace GDP with GTP in Ras proteins, thus energizing the protein switch and turning into an ‘on’ position.
The autoregulatory and complementarian dimensions of Ras molecular switches are recognized in the ability of Ras proteins to switch themselves off hydrolyzing bound GTP to GDP. Since that is an enzymatic function, Ras proteins function as—and are called—GTP-hydrolysing enzymes (GTPases). Such functional duality allows the molecules to circumvent problems of the domain interference with other cellular GTPases. It may be added here that the regulator molecules themselves are regulated by other proteins as well as lipids, so adding yet another dimension to the web of Ras proteins. For example, the cycling of G proteins of the Ras family between the GTP-bound active and GDP-bound inactive forms is regulated by guanine nucleotide exchange factor (GEF), the activator, GTPase activating protein (GAP), and the inactivator. In addition, it must be recognized that many internal and external factors converge on G proteins of Ras family through different types of GEF and GAP proteins. With such massive input, how the signal specificity of Ras proteins is preserved remains to be elucidated.
XX. PROTEIN CATALYSTS
All energetic, metabolic, developmental, differentiative, detoxificative and cellular demise pathways require catalysts to oil their machinery. Next to reactive oxygen species, protein molecules of enzymes comprise the most important and ubiquitous catalysts. The subject of enzymology is vast and its various aspects are presented throughout the volumes of this textbook. Still, below I include brief comment about some enzymes to provide a glimpse into their functional diversities.
In view of the twin themes of molecular complementarity and contrariety presented in the chapter “Molecular Steps and Countersteps of Primordial Self-Organization,” enzymes would not be expected to have evolved merely to serve discrete enzymatic roles, as we are used to thinking in classical enzymology. One might predict that some members of the two major families of proteins—for example, ion channel proteins and protein kinases capable of phosphorylating channel proteins—would have evolved along different axes and yet acquired functionalities of more than one axis. To be specific, some proteins would eventually be found to play both sides of the field; i.e., function as an ion channel under one set of conditions and as a phosphorylating kinase under another set of conditions. That, indeed, is true.
Molecular Evolution, Diversity, and Self-Organization
TRP (transient receptor potential) proteins constitute a family of cation channel proteins with diverse functionalities, including influencing the calcium influx system. On the basis of their sequence similarities, TRP family has been subdivided into three subfamilies: long (LTRP), short (STRP), and osm-9-like (OTRP). In humans, the three members of the long TRP family are: LTRPC1, LTRPC2, and LTRPC5. Recent studies have revealed that two members of the LTRP serve not only as ion channels, but also as a kinase capable of phosphorylating the channel itself.61,62 What a fascinating example of molecular evolution creating diversity, then turning back to create a sublime example of molecular self-organization! It may be mentioned here that the first member of the TRP family was discovered in Drosophila photoreceptors. It is a Ca2+-permeable channel that brings about depolarization of the cell in response to light.
The case of RNase L is equally fascinating. For years, the primary role of RNase L—an enzyme that degraded viral and cellular RNA—was thought to be in the immune defenses against viral infection. The enzyme suppresses viral replication and evidently prevents rapid build-up of viral load. A second defense role involved induction of apoptosis of virally infected cells. Then in early 2002, an altogether new role of this enzyme was recognized. It was found that the enzyme is involved in protection against malignant change in certain types of prostatic carcinomas.63 Specifically, men with prostate cancer in some families with high incidence of that type of cancer had inherited mutations in one of their two copies of the gene that codes for RNase. A subsequent spontaneous mutation in the prostate cell was thought to have deactivated the other copy, thus allowing the cell to escape the expected apoptotic influence of the gene and develop cancer. What fascinated me most in that report was that it seemed to be the first recognized case of cancer in which RNA turnover was implicated in tumor suppression.
Proteins facilitate the travels of other molecular species. One might predict that proteins themselves would also need some assistance in their own travels. That simply should be considered predictable from our consideration of the phenomena of molecular steps and countersteps in evolutionary biology. That, indeed, is also true. To date, eleven distinct translocases have been identified in eukaryotic cells. These protein molecules reside in biomembranes and facilitate traffic of newly formed proteins into or through those membranes.64 Even lowly gram-negative bacteria bounded by two membranes have six distinct translocases.
The ATP synthase (ATPase) is an enzyme that serves pivotal roles in cellular energetics of plants, bacteria, and animals.65-66 Employing energy derived from proton gradients produced during photosynthesis or respiration, it synthesizes the high-energy ATP (adenosine triphosphate) molecule—the main form of cellular energy storage—from low-energy ADP (adenosine diphosphate) and inorganic phosphate.67,68 ATP is a rotary enzyme with two rotary motors designated F0 and F1, which are rotationally coupled through their drive shafts. The enzyme captures energy as protons flow through biomembranes. Energy flow in the enzyme molecule—electrochemical to mechanical to chemical—occurs with molecular motion, in the form of 120o rotary steps by F1 as an intermediate. Each rotary step consists of two smaller substeps. Once the rotational process begins, the time elapsed before the 120o is completed depends on the frictional drag imposed by the F1 filament, and thus on the length of the filament. In vitro experiments show that at saturating ATP concentrations, 120o steps begin nearly every two milliseconds and the entire process is completed within about 0.25 milliseconds.
As for molecular complementarity, ATPase has long been known for its remarkable cooperative interactions between its catalytic sites. ATP binding to F1 provides a good example of molecular machineries that use ATP binding at one catalytic site to induce products release at another through large mechanical movements. (Two other examples of such cooperative interactions are the motor protein kinesin and the chaperon molecule GroEL, which facilitate correct folding of newly formed protein chains.)
As for the inherent molecular contrariety of ATPase enzyme, isolated F1, in the absence of torque from F0, hydrolyses ATP and is sometimes called F1-ATPase. In that role, F1 powers reverse rotation. As for the functionalities of F0, to date there is only indirect evidence that it can drive rotation.
XXI. PROTEIN PROTON PUMPS
Transport of substances across biomembrane can be passive or active. In passive transport, substances move into or out of the cells and cellular organelles by simple diffusion according to their concentration gradients or facilitated by carrier proteins (ionophores) that move passively across the membrane together with ions bound to them. Active transport against the concentration is carried by specialized ATP-driven proteins called the pump ATPases. There are four groups of such pumps:
1. F-ATPase (coupling factor ATPase). One member of the group is H+-ATPase at mitochondrial inner membrane;
2. P-ATPase (phosphorylation ATPase). H+/ K+ -ATPase in parietal cells of the stomach and Na+/ K+-ATPase present ubiquitously—and especially abundantly in the heart and kidney—are two members of this group;
3. V-ATPase (vacuolar). H+-ATPase in cytoplasmic vesicles and plasma membranes is a member of this group; and
4. ABC transporter (ATP binding cassette). P-glycoprotein and MRP in plasma membranes are two members of this group.
Four Proteins for Gastric Acid Production
The production of hydrochloric acid in the stomach is an excellent model to illustrate the fascinating functional integration of one ATPase with two channel proteins and an anion antiport protein to achieve an astounding pH gradient across the gastric lining epithelium. The pH on the apical membrane (luminal surface) of parietal cells during peak acid production can be as low as 1, while that on the opposite basolateral membrane (the cell surface that rests on the basement membrane) remains as high as 7.4. The ATPase in this case is H+/ K+-ATPase, channel proteins are the K+ and Cl– channel proteins, and the antiport protein is the anion antiport protein. The process of acid production involves the following steps:
1. Delivery of CO2 (by diffusion) from the capillaries to the adjacent gastric parietal cells;
2. Hydration of CO2,facilitated by carbonic anhydrase inside the cells to produce H+ and HCO3– ions;
3. Delivery of HCO3– ions from within the cell to the capillary blood by the anion antiport protein;
4. Delivery of H+ ions to the luminal surface of the apical membrane by H+/ K+-ATPase;
5. Delivery of Cl– ions by the anion antiport protein from the capillaries to the adjacent gastric parietal cells, and then across the apical membrane of the cell into the gastric lumen by the Cl– channel protein;
6. Combining of the H+ and Cl– ions to form hydrochloric acid; and
7. Delivery of K+ ions from the gastric lumen to the parietal cells by H+/ K+-ATPase to restore ionic equilibrium across the apical membrane of parietal cells.
The H+/K+ ATPase pump actually exchanges two cytosolic H+/ ions for every K+ ion. The K+ ion channel protein maintains the potassium ion equilibrium by permitting bi-directional flow of the ions. Thus, the transport of Cl– ions requires two proteins—the anion antiport protein and the Cl– channel protein, while that of H+ ions requires the single H+/ K+-ATPase, which also conducts the H+/ K+ exchange for preserving ionic equilibrium.
The H+/ K+ -ATPase proton pump creates the highly acidic (pH=1) environment at the gastric lumen necessary for proteolysis, both physiological for breakdown of dietary proteins and pathophysiological for denaturing proteins of microbial species included in the diet. The gastric proton pump is situated intracellularly in vesicles in the resting state. It is activated by stimuli such as gastrin and histamine. The pump antiports two protons from the cytoplasm of parietal cells and two potassium ions from the extracellular space. One molecule of ATP is hydrolysed to energetically drive the pump. Concurrently, chloride channel secretes Cl– to produce hydrochloric acid in the lumen.
Omeprazole (Prilosec) inhibits acid secretion directly by covalently modifying cysteine residue located in the extracytoplasmic domain of the proton pump and inactivating them. By contrast, H2 receptor blockers (such as cimetidine [Tagamet] and ranitidine [Zantac]) inhibit acid secretion by competing with histamine for its receptors.
The precise workings of the proton (and other ATPase) pumps have not been fully delineated. I cite the case of bacteriorhodopsin to provide a glimpse into the extraordinary complexity of involved molecular dynamics. Bacteriorhodopsin serves as a powerful proton pump for a salt-loving microorganism. It is driven by light. It is composed of seven membrane-spanning helical structures—designated A to G—which are linked by short loops on either side of the biomembrane. The bacteriorhodopsin molecule also contains one molecule of a linear pigment called retinal. Visible light alters the structure of retinal, which traps light energy. The protein molecule harnesses that energy to push a single proton through the seven-helix bundle, from the interior to the exterior.69 A single, large conformational change in the protein scaffold has been recognized during the pump cycle. Undoubtedly, experimental observations concerning bacteriorhodopsin will pave the way for delineation of similar molecular conformational changes undergone by enzymes and pumps of great clinical significance, such as Ca-ATPase, transporters that regulate quantities of neurotransmitters in the brain, and G-protein-coupled drug receptors.
XXII. HOT PROTEINS, COOL PROTEINS
For decades, warm and cold temperatures were thought to be sensed by ‘thermoreceptor’ proteins. However, the molecular biology of thermosensation remained elusive. During the last five years, this field has experienced a renaissance since the cloning and characterization of two ion channels of somatosensory neurons that serve as principal heat sensors.70-72 The two channels are designated vanilloid receptor subtype 1 (VR1) and vanilloid-receptor-like type 1 (VRL-1). VR1 responds to temperatures above 43o C. and by vanilloid compounds, such as capsaicin, while VRL-1 is activated by temperature above 50o C. Both types of proteins also serve as the first molecular mediators in pain perception.
The mechanism proposed to explain cold detection are: (1) activation of Na+-selective channels called degenerins; (2) inhibition of Na+/K+-ATPase—the channel that pumps K+ into cells and Na+ out; (3) differential modulation of Na+ and K+ channels; and (4) inhibition of K+ channels.73
XXIII. TARGETED PROTEIN BLOCKADE
I present the subject of blockade of protein products of normal and mutated genes in other volumes of this book. Here I include brief comments about one such example.
Protein kinases transfer phosphate from ATP to specific amino acids—energizing them, so to speak. Phosphorylation of proteins containing those amino acids results in activation of signal transduction pathways involved in cellular development, differentiation, and demise. There are two families of protein kinases: tyrosine kinases and serine-threonine kinases. Several kinases are dysregulated — overexpressed or impaired—in human cancers. The best known example is the BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML).
The Philadelphia chromosome is found in nearly 95% of hematogenous cells in CML. It is a truncated chromosome 22 constituted abnormally as a result of a reciprocal exchange of nucleotide sequences between the long arms of chromosome 9 and 22, hence the designation t(9;22). That translocation leads to the juxtaposition of 3/ sequences derived from the Abelson (ABL) proto-oncogene normally located on chromosome 9 with 5/ sequences of the breakpoint cluster region (BCR) A gene on chromosome 22.74,75
The t(9;22) translocation of regions of BCR and ABL genes creates a BCR-ABL-fusion gene. That translocation also produces Philadelphia chromosome. The fused gene encodes an abnormal tyrosine kinase, which has been linked to the pathogenesis of leukemia.76,77 Those experimental observations opened the possibility of developing a drug that might inhibit (or stabilize) the abnormal protein BCR/ABL to bring about remission of CML. Imatinib mesylate (Gleevec) is one such agent that has been clinically used with promising results.78-80 Positive hematologic and cytogenetic responses have been reported with this orally bioavailable agent in many patients with limited adverse effects. The putative safety and long-term efficacy of the drug, however, is open to question. For example, the manufacturer of imatinib reported three cases of splenic rupture among more than 10,000 patients with CML who were administered the drug. By contrast, one group of investigators reported two cases of splenic rupture in three patients with CML and a third case of rupture among 23 patients with myeloid metaplasia who were given the drug.81
XXIV. NF-êB: THE MASTER TRANSCRIPTION FACTOR
In this section, I include a thumbnail sketch of the transcription factor NF-êB for three reasons: (1) to provide yet another look at the enormous, ever-changing kaleidoscopic mosaics of proteomics by outlining the enormously complex functionalities of this master transcription factor; (2) to provide basic information about a transcription factor which is currently in sharp focus in the pharmacotherapeutics; and (3) to dampen the enthusiasm of those infatuated with ‘blockade medicine.’ Enormous funds are presently allocated to the development and clinical uses of agents that bind NF-êB (such as etanercept [Enbrel]) and antibodies that block it (such as infliximab [Remicade] and adalimumab [Humira]). It is crucially important to recognize that notwithstanding the short-term benefits of such agents, the notion that chronic immune disorders can be treated with long-term good clinical outcome with blockade of this master transcription factor is pure Alice-in-Wonderland.
The cellular components of both the innate and the adaptive immune systems employ a diverse array of cell surface receptors to recognize and respond to adverse influences. These receptors have one common function: activation of the transcription factor NF-êB, hence the designation of the ‘master transcription factor.’82-85 NF-êB is activated when its natural inhibitor, IêB, is removed through phosphorylation and ubiquitin-mediated degradation. The first phosphorylation step of that process is carried out by a large kinase complex termed IKK, which integrates signals from multiple pathways. This is where the plot thickens and complexity builds up.
The pathways involving IKK activation are diverse and include such mechanisms as T cell activation pathway.86 T cells express the cell surface receptor designated the T cell receptor (TCR). Twelve or more proteins now are known to be involved in the functional links between TCR and IKK. TCR also has an ability to recognize foreign peptides embedded in host major histocompatibility complex (MHC) molecules, and so implicates that crucially important system in this widening web.
The TCR/peptide-MHC dynamics activate a sleuth of intracellular protein tyrosine phosphorylation events, including those coordinated by three families of protein tyrosine kinases designated Src, Syk, and Tec.87 As for Src (Lck in T cells), upon TCR ligation it selectively phosphorylates two tyrosine residues present in the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR subunits. In a biphosphorylated state, the ITAMs are bound in a complex with the tandem Src homology 2 domains of a protein called ZAP-70, which is one member of the Syk protein tyrosine kinase group. It turns out that catalytic activation of ZAP-70 occurs not only by its autophosphorylation, but also by transphosphorylation by Lck, thus providing another link. To move the story further, activated ZAP-70 then phosphorylates tyrosine residues on another adaptor protein that serves as the linker of activated T cells (hence designated LAT). The LAT protein is a type II transmembrane moiety localized in the plasma membrane by addition of palmitoyl groups to its two cysteine residues. The phosphorylated tyrosine residues in LAT offer docking sites for a number of other SH2-containing adaptor and effector proteins, including phospholipase C, SLP76, and guanine nucleotide exchange factors (GEFs, such as one called Vav).
Stimulation of a TCR by a peptide-MHC complex also involves costimulatory factors.88,89 One such factor, CD28, present on antigen-presenting cells, activates yet another cascade of tyrosine phosphorylation, resulting in the activation of PI3K, PDK1, and PKC. Of those, PDK1 recruits IKK to the lipid rafts of the plasma membrane through PKC and BCL10 and MALT1 factors through CARMA1. The role of MALT1 involves polyubiquitination of the IKK subunit NEMO directly or indirectly through the TRAF6 ubiquitin ligase. Another factor required for IKK activation is caspase-8. Membrane translocation and ubiquitination of IKK lead to its activation, the activated IKK then phosphorylates IêB and targets this inhibitor of NF-êB for ubiquitination and subsequent degradation by the proteasome. Degradation of IêB then allows NF-êB to enter the nucleus and switch on expression of genes that are essential for the proliferation and function of activated T cells.
The roles of caspase-8 in TCR pathways require further comments.90-92 Caspase-8 is a cysteine protease that initiates apoptosis through clipping and activating various other proteins. The signaling functions of caspase-8 seem to be an evolutionarily conserved mechanism for playing multiple, sometimes contrarian, roles in innate and adaptive immunity. For example, the Drosophila caspase-8 homolog Dredd is needed for the antibacterial response mediated by the NF-êB homolog Relish.92 In Dredd-deficient cells or those exposed to caspase inhibitors, Relish cannot be cleaved into a mature subunit in response to bacterial challenge. There is also evidence that Dredd mediates the activation of Drosophila IKK, which phosphorylates and targets Relish for cleavage.93 Thus, the caspase-8 proteins appear to have evolved to serve multiple—sometimes complementarian, sometimes contrarian—roles in cellular development, differentiation, and demise.
Caspase-8 binds to CBM and IKK upon TCR stimulation. Interestingly, T cells from patients with caspase-8 mutations not only have impaired apoptosis, but also fail to activate NF-êB after TCR stimulation.90 It is noteworthy that the enzymatic activity, but not autoprocessing, of caspase-8 is required for restoring NF-êB functionalities in a caspase-8-deficient cell line. In cells lacking caspase-8, BCL10 still binds to MALT1, but this complex is unable to recruit IKK in response to signals. Another protein of interest in caspase/TCR dynamics is an adaptor protein called FADD. It binds BCL10 and MALT1 in a signal-dependent manner.
XXV. OF COWBOYS, UBIQUITINATION, AND OXYGEN
A cowboy scans his herd to choose a cow for sale for beef. He makes a judgment call and selects an animal, He casts his lasso, and ropes her gently closer to him. Then he loads her on his truck, removes the lasso, and hauls her to a meat packing company. There a butcher takes over. He looks her over and goads her to enter the door of the slaughter house. Next, the butcher’s team begins its work with knives. At the end, the cow ends up in cellophane packets. A nearly identical process occurs within a cell when the protein molecules are selected for sacrifice.
To be optimally functional, proteins in a cell must be at the right place, at the right time, and in the right concentration. That, of course, requires a communal balancing act among several families of proteins. In the prevailing heuristic models, proteins are considered either as structural units or molecules with specific functions. The focus is on their production and seldom on their ‘cycling.’ That is rapidly changing with recent unravelling of the enormous complexity of protein degradation.
The proteasome is considered as the cellular protein executioner. It is a molecular tribe with two major functional units composed of two multiprotein components: (1) a central cylinder that churns and chews up the protein molecules drawn in; and (2) a regulatory component that provides a lid for portal of entry for the cylinder as well as a base.94,95 The regulatory particle of proteasome traps ubiquininated proteins, unfolds them, removes the ubiquitin attachment, and the ‘feeds’ the target protein into the death chamber (cylinder) within the proteasome. Polyubinquininated proteins are deubiquintinated not only to preserve and recycle ubiquitin molecules but also because the opening of the regulatory particle of the proteasome is too narrow for the larger-sized ubinquitinated molecules. Within the cylinder, proteins are broken down with hydrolysis.
Ubiquitination is a sophisticated mechanism involving a protein ubiquitous in eukaryotes—hence the designation ubiquitin—that sticks to the protein molecules either to modify their behavior or to mark them for sacrifice. The marked proteins are then coaxed into the death chamber (cylinder) within the proteasome. The attachment of one molecule of ubiquitin—monoubiquitination of a protein—is not necessarily fatal for the target protein and appears to be a reversible phenomenon. By contrast, when several molecules of ubiquitin gang up on a protein moleculae—the process of polyubiquitinination—the marked protein faces sure demise. Two de-ubiquitinating enzymes, tighlty associated with the proteasome closely have been identified that contain thiol (sulfhydryl) group in their active sites. One of those enzyme is sensitive to ubiquitin aldehyde while the other is not. It appears that both are metalloproteins that employ a metal-ion-dependent protein-cleaving mechanism.
How does the cowboy make his judgment call for the right animal to be given away? I suppose the answer is that he has a ‘sense’ of things. How does a ubiquitin molecule make its judgment call for the right protein molecule to be done away with? I suppose the ubiquitin also has a ‘sense’ of its herd of proteins. But what might be the molecular basis of such ‘sense’? The experimental data concerning such ubiquitin selection does not furnish a clear answer. Based on my clinical ‘sense’ of oxytherapeutics, I offer a hypothesis. I suggest that a cell’s decision concerning what proteins need to be conserved and what proteins might be given away to ubiquitins at any given time is primarily influenced by the prevailing oxygen conditions within that cell as well as by the oxygen microecologic conditions in its environment. In support of that suggestion, I offer this: the 19S regulatory complex of 26S proteasome is composed of two subcomplexes, one of which (the base particle) includes Rpn1 and Rpn2 debiquitinases as well as six ATPases. I recognize that whereever one sees the play of ATPases, the hand of oxygen is not too far. So, it does not seem an unreasonable stretch to me to see the role cellular oxygen conditions in cellular protein cycling. It also seems likely to me such a relationship between oxygen and ubiquitination of proteins will be revealed when it is scrutinized with experiemntal work in the futures.
XXVI. PROTEIN MISFOLDING DISORDERS
During the early 1980s, taking lead from Linus Pauling — he first used the term molecular medicine when he establsihed hemoglobin S to be the cause of sickle cell anemia — I embarked upon a personal journey of ‘molecular thinking.’ During the mid-1980s, in a series of monograph published in the Syllabi of The American Ascademy of Environmental Medicine, I recognized a need for such thinking in coping with environmentally-induced illnesses.96,97 In 1990, in The Cortical Monkey and Healing, 98 I introduced the terms life span molecules for moieties that function primarily to preserve the full life span of the species and aging-oxidant molecules for those that provide a counterforce to assure that no one lives for ever. In 1993, I chose ‘Molecular Medicine’ as the name of our laboratory at the Institute to keep a sharp focus on molecular basis of the health/dis-ease/disease continuum. It was clear to me then that molecular disfigurements — produced by oxidative-dysoxygenative injuries — will be proven to be the root of an ever-increasing number of clinicopathologic disorders. What I did not realize then was how rapid would be the advances in technology that allow us to dissect molecular pathways to precisely delineate the energetic-molecular basis of the health/dis-ease/disease continuum. Here are I include brief comments on the subject of the so-called protein folding disorders.
A growing number of sporadic and genetic disorders are now thought to be caused by what may be properly designated as ‘molecular lesions of proteins’ caused by misfolding or other structural abnormalities involving the protein molecules.99-103 Among the best examples are childhood diseases (cystic fibrosis and others), diosrders of the elderly (Alzheimer’s disease and others), and oof age groups in between thgose extremes (Parkinson’s disease, alpha-1-trypsin deficiency, and others). It seems safe to predict that the list of such disorders will grow rapidly.
Accuracy in protein folding is crucial for the functional integrity of the protein. In health, such folding occurs both in the cytoplasm and within the secretory pathway in the endoplasmic reticulum (ER) under the regulatory influences of ATP-dependent chaperones. If physiologic folding is unsuccessful, the protein is herded to the proteasome — a complex multisubunit protease — for the degradative process described in the section on ubiquitination. This is being recognized as the primary molecular lesion in growing number of disorders in which a loss of protein function occurs when misfolded proteins are degraded by the proteasome. Two good examples of such disorders are the well-known heritable diseases cystic fibrosis and alpha 1-antitrypsin deficiency. In other disorders, protein misfolding is coupled to the aggregation of misfolded proteins outside the cell — and hence beyond the influence of intracellular quality-control mechanisms. Various forms of amyloidosis are the best known examples of this category of misfolding disorders. As well-illustrated by kidney failure caused by renal amyloidosis, protein aggregation in the extracellular space in this category is associated with clinicopathologic consequences.104,1056
Conformational changes in the protein structure — physiologic folding in health and pathologic misfolding in disease — are influenced primarily by the protein amino-acid sequence and the cellular microecologic conditions. Proteins that fold in the ER and then proceed through the secretory pathway to their final destination are over-represented in protein-misfolding diseases. The micrpecologic conditions for such proteins can vary considerably during their journey. For instance, lysosomal enzymes fold at neutral pH in the ER and undergo further folding optimization and stability for their functionalities at the lysosomal pH of 5. Mutations in lysosomal proteins can adversely alter folding in the ER, leading to canabilization by the ubiquitinative quality-control machinery. It has been speculated that many of the variants that cause heritable lysosomal storage diseases caused by abnormal folding might fold into functional enzymes in the lysosome, if only they could reach there un-degraded.104,105 The case with amyloidogenic proteins seem to be different since they fold efficiently in the ER but are degraded — misfolded and misassembled in a process known as amyloidogenesis — in their destination environments.
Under physiologic conditions, many native (wild-type) proteins fold inefficiently in the ER, even under the protective influences of the heat shock protein chaperone system — hsp40, hsp70 and hsp90 — that prevents misfolding and/or facilitates the refolding or retrotranslocation of misfolded proteins back into the cytoplasm for degradation in the proteasome.106,107 The energetics of optimal folding are put in jeopardy by mutations in the chaperone system that diminish the folding efficiency. There is considerable interest at present in chemical chaperones that bind to a protein, stabilize the folded state and thereby reduce protein misfolding. Specifically, there is active search for small-molecule ligands that have the potential for binding such mutant proteins, and for facilitating stabilization of the native state or destabilization of the transition states. For instance, it is known that selective V2-receptor antagonists — SR121463A and VPA985 — can increase 15-fold the expression of functional V2 vasopressin receptor at the cell surface, rescuing the receptor proteins from proteasomic degradation. Furthermore, non-cell-permeable antagonists are ineffective in such “protein-protective functions”, indicating the crucial importance of intercepting the misfolded protein in the cell and most likely in the ER.
Protein Folding in Microseconds to nanoseconds
Under experimental conditions, proteins collapse in a microsecond to surrender intermediate stages with generally well preserved native helical secondary structure. Such collapsed structures are more vulnerable to further molecular folding. Attempts to delineate the structures and the folding pathways of folded proteins have been made with combined experimental and simulation data. The results so far have not been conclusive. One reason for that is that computer simulation have been done at atomic resolution limited to about a microsecond or less. However there are indications that ultrafast-folding proteins fold and unfold on timescales of nanoseconds. With such experimental designs, it appears that certain mutant protein contain dynamic, native-like helices with unstructured side chains. In the transition state between such and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion–collision model. Molecular dynamics simulations have yielded rate constants and structural details that are highly consistent, thus allowing completion of the description of folding at atomic resolution.
Since protein misfolding is considered as the primary molecular lesion in such disorders, there is much interest in explorinmg the possibility of preventing and reversing such disorders by employing agents that prevent protein misfolding.108-111 Furthermore, such efforts are being expanded to develop integrated models of the molecular and cellular pathogenesis of those disorders that can lead to the search for molecular chaperones that can arrest, slow, or revert disease progression.
XXVII. NOW A N ANTI-CANCER PROTEIN, NOW A PRO-CANCER PROTEIN
The protein p27, also known as Kip1, is a Dr. Jekyll/Mr. Hyde protein par excellence. p27 operates at the very heart of the cell cycle and is a key player in crucial cell fate decisions, including those in cellular proliferation, differentiation, de-differentiation, and demise.112-114 During the early years of its study, this cell cycle regulator was primarily seen as a brake paddle in cell cycle progression, which role it serves by inhibiting cyclin/cyclin-dependent kinase complexes. It soon spiked the interest of researchers investigating diverse disorders involving aberrations in cellular proliferation, differentiation, and other cell fates. In that role, p27 also emerged as an important tumor suppressor protein.
Evidence for that role included the following: (1) p27 expression is frequently downregulated in diverse human cancers; (2) Reduced p27 expression generally correlates well with poor prognosis; (3) Studies with murine and tissue culture models have revealed that p27 gene is a potent tumor suppressor for multiple epithelially derived neoplasias; (4) Loss of p27 cooperates with mutations in several oncogenes and tumor suppressor genes to facilitate tumor growth.; and (5) Tumor suppression by p27 is critically dependent on the absolute level of p27 expression.
For all of the above reasons, p27 has earned the distinction of being the “nodal point” for tumor suppression. It is noteworthy that p27 is haploinsufficient for tumor suppression — by contrast, most tumor suppressor genes investiagted to date are recessive at the cellular level — and is considered to act as a rheostat rather than as an on/off switch to regulate cellular proliferation and cell cycle arrest.
p27Kip1 binds to, and regulates the activity of, a family of proteins called Rho proteins. Specifically, p27Kip1 inhibits RhoA activation by interfering with its interaction with GEFs.112 (Besson). This family includes Cdc42, Rac, and RhoA, all of which serve as molecular switches in signalling pathways for gene transcription and control of the filaments skeletal proteins, such as actin. 113 (Ridley, A. J. et al. Science 302, 1704-1709 (2003). Rho proteins are switched on when bound to guanosine triphosphate (GTP), and off when bound to guanosine diphosphate (GDP). When activated, Rho proteins regulate and coordinate the cytoskeletal remodelling of crucial importance to cell adhesion and migration. In addition, Rho protiens interact with many different effector proteins that evoke a number of different downstream responses. Various other proteins control the on/off state; guanine-nucleotide-exchange factors (GEFs), for instance, activate Rho proteins by promoting the replacement of GDP with GTP.
The motility of p27Kip1-deficient fibroblast cell types is diminished markedly in comparison with that of wild-type cells. Re-expression of wild-type p27Kip1 — or a truncated mutant that cannot bind to cyclin-CDK complexes — restores migration, indicating that p27Kip1 regulation of motility is independent of its effects on proliferation. It is noteworthy that the region of p27Kip1 that binds RhoA and is required for migration is different from the region that binds cyclin-CDKs. Furthermore, the
non-migratory, p27Kip1-deficient cells contain more actin stress fibres and focal adhesions than wild-type cells. ROCK enzyme is a downstream effector of RhoA. It is also known that the inhibition of ROCK, restores the migration of p27Kip1-deficient cells in response to growth factors.
Simply stated, Rho activity increases in fibroblasts with decreased p27Kip1 concentration, and this, in turn, increases ROCK activity which impairs cell migration. This is one of the mechanism by which p27Kip1acts as a pro-cancer protein.
XXVIII. PROTEINS IN CELLULAR ENERGETICS
In this section, I include brief comments about how proteins drive cellular energetics. For that I have chosen four examples: cytochrome c oxidase, P-type ATPases, mechanosensitive channel (MscL), and neurotropin channel proteins.
In 1925, Keillin at Cambridge University discovered cytochrome c oxidase and ushered in the modern age of cellular energetics involving a multitude of electron transport events. E1 Keillin systematically examined 86 different eukaryote and prokaryote, and characterized a series of pigments in cells and tissues that were visible with a hand spectroscope and sensitive to light. He coined the term cytochrome for his bands of colors. Subsequent work over a period of 13 years led to the recognition that Keillin color bands associated with cytochrome a3 was the elusive Warburg’s “Atmungsferment.” e2,e3
Now we know cytochrome c oxidase to be the terminal enzyme in the respiratory electron transfer chain. Located in the inner mitochondrial membrane in eukaryotes, cytochrome c oxidase functions as a proton pump. In that capacity, it primarily functions as a cellular energy conserver. Specifically, it uses redox chemistry to drive protons from the mitochondrial matrix across the membrane. The electrochemical gradient so generated causes protons to flow back into the matrix via the F1F 0 ATP synthase. Thus, the inward electron flow is coupled to ATP synthesis (from ADP).
Electron transfer into cytochrome c oxidase begins with its binding to subunit II on the external membrane of mitochondria. The subunit contains the CuA center which occurs at the bimetallic Cu-Cu site. Electron transfer appears to follow linear sequence of events — electron proceed from cytochrome c to CuA , then to heme a, and on to heme a3-CuB center. Oxygen binds to heme a3 and is reduced to water.
P-type ATPases are ion pump proteins that utilize ATP energy to transport cations through the cell membrane against a concentration gradient. For inmstance, in yeast and plants these membrane proton pump proteins (`100 kD) maintain intracellular pH and membrane potentials, thus supplying energey requirements for the uptake of nutrients and exchange of ions by secondary transporters. These pumps cycle between states — called E1 and E2 — that have different binding affinities for nucleotides as well as for the transported ions. The functions of such proteins are associated with configurational changes. For instance, the nucleotide-binding domain of the palsma membrane H+-ATPase rotates by 70o to deliver ATP to phosphorylation sites, and shed light on the path of protons through the membrane.M1
Mechanoreceptor channels act as membrane-embedded mechano-electrical switches which respond to mechanical membrane deformations. When activated, these channels open a large water-filled pore in the lipid bilayer in response. Complex structural rearrangements underly such channel responses, and involve the large prokaryotic mechanosensitive channel (MscL) and two transmembrane helices (TM1 and TM2). The open state of the channel is largely lined by TM1. M2
In many types of human cellular energetics, sodium ions drive the propogation of action potential. In the case of neuronal transmissions, such ions are temporarily permitted to enter cells through channels in their membranes that open in response to a transmembrane voltage change. To cite one example, the a brain protein designated brain-derived neurotrophic factor (BDNF) causes membrane deploarization in milliseconds. A recent surprise finding is that BDNF secreted by neurons also plays a critical role in the opening of a particular type of sodium channel.
XXIX. OBLITERATING LINES BETWEEN STRUCTURAL AND FUNCTIONAL PROTEINS
I began by presenting the generally accepted notions of protein structure and function. However, the twin themes of this volume—molecular complementarity and contrariety—would dictate that the sharp distinctions generally made between protein structure and function, notwithstanding their didactic merit, are not likely to stand close scrutiny. Indeed, it should have been evident from the preceding presentations of protein webs and kaleidoscopes that what passes for structural proteins must have diverse functions beyond being mere structural scaffolds. I cite the example of the newly discovered ability of TAB1 scaffolding protein to mediate autophosphorylation of p38á—an enzyme belonging to the family of mitogen-activated protein kinases (MAPKs). 123 These enzymes are critical components of diverse signaling pathways that regulate cell division, cellular mobility, gene expression, and death. In the past, the activation of MAPKs was thought to be caused by: (1) assembly or disassembly of oligomeric protein complexes regulated by guanosine triphosphatases; and (2) covalent protein modifications such as phosphorylation, ubiquitation, sumoylation, and proteolysis. The discovery that TAB1—a protein previously believed to be a mere scaffold molecule—can activate a MAPK enzyme reveals an altogether novel mechanism by which structural proteins can profoundly influence cellular processes. The TAB1 story led some to emphasize the need for defining all protein interactions in cells to ensure that no unsuspected regulatory responses remain uncovered.124 That view, to me, seems overly simplistic.
XXX. PROTEIN-BASED THERAPEUTICS
The subject of ‘protein-based’ therapeutics — treatments designed to suppress, enhance, or otherwise modify protein functions — to address putatively ‘specific’ molecular lesions is drawing much attention these days. The rising interest in treating protein misfolding disorders with ‘chemical chaperone’ molecules has been mentioned in an earlier section. In this section, I include brief additional comments about therapies designed to alter the structure and function of intgrins.
Integrins are adhesion molecules that serve many roles in maintain mechanical stability to interactions between cells and their microecologic conditions. Beyond that, integrins act as sensors and signaling molecules.125-127 All integrins are composed of noncovalently linked á and â chains. The á4 chain dimerizes with either â1 chain or â7 chain. The á4â1 integrin is also referred to as very late antigen or CD49d-CD29 while á4â7 is also called lamina propria-associated molecule 1. The probable mechanisms of action of antibody against á4 integrin involve blockade of binding of integrin molecules to their respective endothelial counter-receptors, vascular wall adhesion molecules I (VCAM-1) and mucosal addressin-cell adhesion molecule. These molecular interactions regulate lymphocytic traffic, and specifically entry of lymphocytes into the CNS. The rationale for the clinical use of natalizumab in MS and Crohn’s colitis is interruption of á4 integrin-mediated migration of lymphocytes to extravascular sites. The treatment leads to peripheral blood lymphocytosis that is regarded as evidence of the interruption of that pathway. Not unexpectedly, antibodies develop against the agent, however no serious effects were encountered in the early trials.
Natalizumab is a recombinant monoclonal antibody against á4 integrin which has been tried to treat multiple sclerosis and Crohn’s colitis. Fewer (a reduction of about 90%) new inflammatroy lesions of the CNS and nearly half as many clinical relapses were observed in MS in one study.125 In a study of clinical benefits of the agent in Crohn’s colitis, responses and remission rates were nearly half as low but no difference in adverse events.126 But if my basic hypothesis concerning Nature’s preoccupation with complementarity and contrariety is correct, one can safely predict that with increasing experience natalizumab and similar agents will not be found to have lasting benefits with minimal adverse effects. I doubt that the oxidative-dysoxygenative phenomena that are at the root of the pathogenesis of multiple sclerosis, Crohn’s colitis, and other clinicopathologic entities can be effectively addressed by simply blocking the binding of one or more moities to their respective ligands.
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