Regulatory Molecular Biology Arthur B. Pardee Dana-Farber Cancer Institute; Boston, Massachusetts USA

1. Regulatory Molecular BiologyArthur B. PardeeDana-Farber Cancer Institute; Boston, Massachusetts USA

2. INTRODUCTIONNumerous molecular mechanisms regulate normal and cancer cells’ biological machinery.These processes operate at multiple levels to produce coordinated and economically functioning biological activities and structures. The cells in a multi-cellular organism have essentially the same genes but differ in functions, and their genes are expressed differently.Thus the genotype does not alone determine phenotype, and life depends on both Nature and Nurture, interplay of heredity with environment, selecting expressions of hereditary information from genes and mRNAs, activities of enzymes, and specificity of membrane transport. These regulations act by different biochemistries and in different time frames. They control transit between cell quiescence and proliferation, and between stages of the cell cycle. The theme of this article is briefly to summarize innovative discoveries that continue to provide paradigms of regulatory processes.

3. Much of what we now take for granted was then unknown. Methods were comparatively primitive. Chromatography and spectrophotometry came in the early 1940’s. Radioactive organic compounds became available after World War II. There were no biochemical supply houses and no kits. Nucleic acids were not in the main picture; their status was like that of carbohydrates and fats. Around 1950 major interlocking developments of biochemistry with chemistry and genetics turned research from metabolism and enzymes toward macromolecules.1 The field now called Molecular Biology was born. Pinnacles are studies on organic structures and nature of the chemical bond by Linus Pauling, the first sequencing of a protein (insulin) by Frederick Sanger, and of 3-dimensional protein structures by MaxPerutz and John Kendrew.

4. Molecular-biochemical regulation is an enormous subject. It is summarized here historically as discoveries and functions, as I remember them in a scientific path that has led across unexploredterrain and along byways toward the goal of learning about the defects of molecular regulation that lie at the heart of cancer. 2References are limited to pioneering articles and germinal reviews to indicate thinking at the time, and to updating reviews. More canbe readily found by searching the Internet (PubMed) for reviews on any topic.

5. MAJOR MECHANISMS OF REGULATIONMutation.Genetic changes are now recognized to be the origins of cancer. Although genetics and biochemistry were separate disciplines in the 1950s, mutation was known to change enzyme activities dramatically, per the one gene-one enzyme model of George Beadle and Edward Tatum, And added genetic material changes metabolism; nine enzyme activities are quickly altered by additional genetic information provided by infection of Escherichia coli with a DNAbacteriophage. Strikingly, a completely novel enzyme involved in synthesizing hydroxymethyl-cytosine appears, discovered by Seymour Cohen in 1954. These include deoxyribonuclease, consistent with a role of DNA in virus replication, and many mutant progeny are produced after replacement of thymidine by bromodeoxyuridine in the phage DNA, as shown by Rose Litman in 1956. These experimentsare forerunners of genetic engineering, involving introduction of normal or specifically modified DNAs.

6. Control of metabolic pathways. The great achievement of biochemistry is to connect most metabolites into the now familiar pathways catalyzed by enzymes. Approaching its apex in the 1950s,most biochemists were very busy successfully creating this map. All its roads were of the same intensity, although traffic along some is far greater than on others. Questions about regulatory mechanisms were not posed. But it was noticed that metabolism is precisely regulatedand is not wasteful; intermediary metabolites are not overproduced and do not accumulate in the medium. 3,4 Living organisms usually produce their constituent molecules in amounts only sufficient to meet their needs, neither more nor less. It was also noticed that these balanced internal events respond to extracellular conditions. This tight control of metabolism is important for efficient and economical cell functioning. This focuses a cell’s resources.

7. Feedback inhibition. A mechanism for adjustments to both environmental metabolites and to prevent excessive intracellular end products is by economically shutting down their synthesis when unneeded. A breakthrough that established a ‘Root’ of molecularbiology was discovery of the general Feedback Inhibition mechanism. The end product of a biosynthetic pathway blocks production of an intermediate molecule in that pathway by inhibiting an enzyme’s activity, see ref. 5. Initial indications made in 1954 are rapid inhibitionby added tryptophan of biosynthesis of an intermediate in its pathway, reported by Aaron Novick6 and Richard Yates and Arthur Pardee.7 stated “ added uracil blocks an enzyme step between aspartate and ureidosuccinate formation”; “this block may be an important regulatorymechanism in the cell” .

8. Feedback inhibition immediately created the problem if its molecular basis. How can ATCase be inhibited by uracil that is structurally very dissimilar from the substrates aspartate and carbamyl phosphate? Enzyme catalysis was described in the 1940’s as a three-step process in which substrate(s) specifically bind to the catalytic site of an enzyme, are then converted to product(s), and are released. The enzyme is then free for another catalysis. Inhibitors were seen tocompete specifically for the enzyme’s catalytic site, thereby excluding substrate. This was quantitatively described in 1913 by the equation of Lenor Michaelis and Maude Menten: the velocity of the reaction (v) depends on the maximal rate (VM), concentrations of enzyme (E), substrate (S), and inhibitor (I), and their affinities (KM) and (KI).v = VM E S______S + [KM (I + KI) ]/ KI.

9. The key demonstration by John Gerhart and Pardee of independent catalytic and regulatory sites came from an unexpected observation made to establish the basis for the feedback control of activity. Variable results of inhibition of the pure enzyme by CTP wererepeatedly obtained. Frozen enzyme thawed at the beginning of a week was strongly inhibited. But thereafter, inhibition was lost during storage in the refrigerator. Furthermore the activity actually increased, and kinetics changed from the subunit-cooperative S-shapeto the classical Michaelis-Menten shape. Hypothesizing that ATCase must change, even at zero degrees, systematic warming showed that five minute exposure to 65° C abolishes its inhibition by CTP but not its catalytic activity.

10. That enzymes are often complexes rather than single proteins, as was then the general biochemical concept is major development arising from feedback inhibition, now well established. Hemoglobin and the b-galactosidase repressor are tetramers of identical subunits,ribonucleotide reductase has catalytic and regulatory subunits, and there are many other multi-protein complexes. Examples are cyclins that activate cdks. And more than a dozen B proteins differently control properties of the pleiotropically functioning and ubiquitous PP2A phosphatase; one regulates degradation of oncogenicmyc.22 An early extreme example is the ribosome, a multi-protein complex that catalyzes protein synthesis. And DNA synthesis is catalyzed by a Replitase complex that contains enzymes for both precursor synthesis and polymerase, as found by Prem Reddy.23

11. ACTIVATION BY COVALENT MODIFICATION.After a protein is synthesized it may not have enzymatic activity, which can be produced by a subsequent covalent modification. A major mode of changing activity (plus or minus) in higher organisms is produced by covalent phosphorylation of proteins by the kinases, discovered by Eugene Kennedy in 1954, which can be reversed by phosphatases. Edwin Krebs and Edward Fisher in the 1950s discovered that this covalent protein phosphorylation is a mechanism for enzyme activity regulation; ATP level controls glycogen phosphorylase which provides metabolic energy.24 The human genome contains 518 kinases (the kinome), each of which is regulated to phosphorylate a distinct set of substrates.25 Kinases, and also proteases, are often organized into sequentially activating cascades that catalyze rapid,exponential-like amplifications of downstream activity. Examples are the kinase cascades activated by binding of growth factors to their receptors on the mammalian cell’s surface.

12. CONTROL OF GENE ACTIVITYThe rate of a reaction depends upon the amount of its enzyme as well as upon its activity, as seen in the Michealis-Menten equation. Amounts (maximal activities) of some enzymes in bacteria knownbefore 1950 to be change by environmental molecules. They “adapted” as a function of extracellular nutrients, dramatically increasing in amount when their substrate is provided. Jacques Monod, the outstanding investigator of this problem, performedelegant experiments on the dependence of b-galactosidase production in E. coli as a function of availability of -galactoside sugars which were proposed to act as ‘inducers ‘ of the gene. 26 This control of gene expression acts relatively slowly as compared to feedback inhibition of metabolic reactions.

13. The constitutive cells was concluded to lack a repressor protein that is present in inducible bacteria and is gradually produced in the mated cells after its gene is introduced. This means that the repressorspecifically blocks gene expression; coding DNA is shut down when repressor protein binds to an upstream DNA repressor sequence. 27The repressor is released when its other site binds a low molecular weight inducer molecule. Specifically, expression of -galactosidase (and two adjacent genes) is inhibited when a lac repressor proteinbinds to its upstream DNA operator region. and mutant bacteria that cannot make repressor produce the enzyme constitutively. The lac repressor protein was isolated in 1966 by Walter Gilbert andBenno Muller-Hill.

14. Major developments from PaJaMa. i) Primarily, this experiment is the foundation of transcriptional control of gene expression byboth bacteria and eukaryotes. ii) Enzymes in synthetic pathways can be repressed by low molecular weight compounds, as well as thoseinvolved in catabolism; a metabolite can repress transcription of its biosynthetic pathway. Examples are the pathway of pyrimidine biosythesis by Richard Yates and Arthur Pardee, and for arginine by Luigi Gorini and Werner Maas (for a review see 30).iii) The broad biological roles of functional sites interacting with separate regulatory sites depends upon these concepts of repressor and regulatory DNA promoter sequences

15. Allostery. The two types of binding sites of proteins, one functional and the other regulatory, permit many types of biological reactions to be controlled by a molecule that has no structural similarity to the molecules acted upon. Jacques Monod combinedthree lines of research to create this allosteric concept,30 which he called “the second secret of life”: i) feedback inhibition with its catalytic and regulatory sites (see above), ii) a site for binding galactosidesto the lac repressor modifies another functional site thatbinds it to a DNA sequence, and iii) cooperative binding of oxygen to the four protein subunits of hemoglobin and which are modified by their interactions with CO2. For an historical review see ref. 36.

16. Mathematics of multi-subunit interaction. Allosteric activity depends on functional regulation by alternative structures of multiproteincomplexes. An early example is the Hill equation whichmathematically describes interactions of the four subunits of hemoglobin upon binding of O2. General allosteric equations have been described by two mathematical models, based upon alternativeactive and inactive conformations of subunits controlled by regulator binding. In one, the subunits conformations change in a concerted, all-or-none, manner. 38 In the other, each binding sequentially altersthe protein’s structure and changes the next binding affinity; technical methods, such as 3D protein structure determinations, are resolving this question of allosteric changes. 39

17. REGULATION OF MEMBRANE FUNCTIONS.Control by molecular location is seen at three levels, whose amounts and activities are regulated both genetically and environmentally First is extra-cellular vs. intra-cellular location. Many molecules generally must pass into a cell to metabolized. They cross the cell membrane via specific transport mechanisms that permit either passive entry or catalyze enzyme-like energy-dependent accumulation.Second are systems that move molecules between cytoplasm and organelles. For example, enzymes involved in DNA synthesis accumulate in the nucleus before S-phase. Third, enzymes are often assembled, onto protein scaffolds, into multi-protein complexes that perform cooperative functions.40 As an example of such interactions, compounds that specifically inhibit an isolated enzyme also inhibit others that are in the replitase complex.41 Individual mRNAs similarly have been localized in cells.42

18. Active transport of a molecule into the cell is a first step in many metabolic pathways. Kinetics of substrate uptake and enzymes are similar. It is therefore not surprising that trans-membrane transportis regulated and inhibited similarly to enzyme activity. Transport of galactosides across the membrane of E. coli is inducible;44 adjacent genes for -galactosidase and galactoside transport (permease) areco-induced by -galactosides, per the operon model proposed by Jacob and Monod.30 Molecules catalyzing transport were unknown in the 1950’s. One of the first transport-related molecules to be purifiedis a regulatory factor for sulfate transport.45 A transport system was demonstrated for uptake of sulfate ion into Salmonella typhimurium. Mutants that could not grow on sulfate were isolated by applying toxic chromate ion; they were defective in transport.

19. Cell surface membrane and transport are very important ineukaryote metabolism and regulation, e.g., the coupled transport of hydrogen ions across the mitochondrial membrane that produces ATP discovered by Peter Mitchell, or control of neuronal transmissionby regulated receptor-mediated transport of ions. Density-dependent contact inhibition of cell growth involves surface proteins such as cadherins, integrins, etc. that make connections to other cells and tothe extracellular matrix. Membranes of eukaryotic cells contain proteins with extra-cellular binding sites that are specific receptors for protein growth factors. These regulate these receptors’ intracellular tyrosine kinase activity, as shown by Joseph Schlessinger.47

20. MORE MECHANISMSEpigenetic controls can permit a cell to express only a subset of its genes, for example differently in liver than skin. Pioneering experiments by Werner Arber demonstrated DNA methylation protects bacterialDNA from hydrolysis of by restriction endonucleases, and by Ruth Sager who found that methylation is the basis of non-Mendelian inheritance of organelle genes in the eukaryotic alga Chlamydomonas.The effect of methylation then shifted from elimination of DNA to blocking gene expression in higher organisms. Methylation of DNA attracts enzymes that catalyze acetylation of histones and thereby changes of chromatin structure and activity. Mechanisms of histonemodifications and their effects on gene expression are under vigorous investigation.48

21. Information about the classes and functions of RNAs areincreasing dramatically. Mechanisms are newly discovered. that on the one hand regulate amounts and functions of mRNA, and on the other regulations by RNAs For an overview see ref. 49. Transcriptionalproduction of pre-mRNA is followed by its processing and splicing, which produces hundreds of mRNAs and then their corresponding proteins. 50 mRNA production is also controlled by complex reactions such as trans-splicing to their 5’ ends of short synthesis-regulatingleader RNAs in some organisms. 51 About 13,000 target relationships have been identified as complimentary seed sequences.52 Ribozymes catalyze molecular reactions. siRNAs can block translational activity,and importantly they activate specific mRNA degradation, 53 which takes place in cellularly localized P-bodies.

22. REGULATION OF CELL PROLIFERATIONResearch shifted from bacteria toward higher organisms in the 1960s, along with the rise of molecular biology. Techniques had progressed sufficiently to make in vitro culture of mammalian cells generally feasible. Functions in eukaryotic cells are controlled by interplay of genetic and environmental factors, and as with bacteriathese can regulate DNA and protein functions. Processes that involve an entire cell homeostasis take us into a new realm of regulation; these are at least an order of magnitude more complex than isgene expression or a metabolic pathway. The mechanisms that control gene expression and enzyme activity are applied in regulation of cellproliferation.

23. Cell cycle control. Regulation in eukaryotic cells is at a slower pace than controls in bacteria; completing the cycle can require a day or longer vs. an hour. The sequentially organized processes of cellproliferation are described as the cell cycle. Early research on the eukaryotic cycle is summarized,56 and has since often been reviewed.57 To produce two cells from one requires that all molecules, large and small, must be duplicated precisely. These syntheses takeplace at specific times, the most prominent example being duplication of DNA in mid-cycle S-phase, shown by Howard and Pelc in 1951.58 The cell cycle of bacteria had begun to be investigated by1960. Its duration as measured with synchronized E. coli depends oncarbon source, requiring over an hour on acetate and as short as 15 min on glucose.

24. Emergence from quiescence and transit through G1 is inhibited by density dependent physical-chemical interactions between adjacent cell surfaces. It is activated by proteins in serum, such as insulinderivedgrowth factor and epidermal growth factor. These bind toexternal receptors on the cell membrane, which activates intracellular auto-phosphorylation by the receptor’s tyrosine kinase. Alternatively,estrogen and androgen initiate proliferation of female and male sexrelated cells, respectively, and these relatively small molecules bind to receptors located to the nucleus. Both activations initiate kinasecascades that activate transcriptions. The cyclin proteins increase and then decrease in a specific sequence during the cycle, as discoveredby Tim Hunt and colleagues.62 They complex with and regulate several cyclin-dependent kinases (cdks), discovered by Paul Nurse.63

25. Other complicated mechanisms limit DNA replication to only once per cycle, and yet others control mitosis and daughter cell separation. Toward the end of G1 phase cyclin/cdk activities phosphorylate the retinoblastoma protein, causing its inactivation and release and activation of E2F-1, a transcription factor for many enzymes required for DNA synthesis. E2F-1 is also the (autocatalytic) factor for its own transcription, and therefore it increases dramatically at the G1/S boundary. But excessive E2F-1 is apoptotic, and so a feedback control must exist; perhaps inhibition by the end product dNTPs is responsible. Based upon this idea, a novel chemotherapeutic principle for action of agents that deplete dNTP pools has been suggested.64Feedback loops between plus and minus balancing controls are indicated, such that excess of one activates the opposite. A major question that determines detailed investigations of molecular mechanisms was at what point in the cell cycle growth is regulated.

26. Production and removal are balanced at every biological level. Specific multi-protein enzymatic machineries label and then degrade metabolites, RNAs, proteins, and cells. Such a major regulatory mechanism is proteolysis, the most dramatic alteration of a protein’s structure. It was discovered early to convert extracellular inactiveproteins (zymogens) to active enzymes; trypsinogen to trypsin is an example. But proteolysis usually eliminates intracellular activity,70 and the protein is in a steady state determined by balance with its synthesis. This is especially so for regulatory proteins includingcyclins that are produced transiently and are degraded when their roles are complete. Another prominent example is removal of proapoptotic P53, activated by its ubiquitination involving Mdm271 and then degraded by proteasomes.72 This control is a feedback loopbecause P53 induces Mdm2.

27. Cancer and mis-regulation. For regulatory mechanisms, ‘The pathological illuminates the normal’. Defective controls created by mutations and altering gene expressions are causal of cancer. Genetic material introduced by viruses can also cause cancer. These genetic level changes can produce either gain of an activity of an oncogene such as ras discovered by Ed Skolnik and shown by Robert Weinberg and Geoffery Cooper to be mutated in cancers. Mutational loss of a tumor suppressor gene such p53 or pRb was proposed by Ruth Sager.74 The latter are more frequent, and the more probable because the normal phenotype is dominant in fused normal plus cancer cells, as shown by Henry Harris and Boris Ephrussi.

28. Cancer cells require more oxygen, more energy, and more active metabolism than do normal cells, which are usually quiescent. A hallmark of cancer is deregulated cell proliferation, and changes of controls through the cell cycle are reported, particularly in G1 phase. Numerous changes of kinases and phosphorylations have been reported. Cyclin E is over expressed and modified in advanced cancers, and it provides a clinical marker.79 The tumor suppressing retinoblastoma protein is very frequently inactive or absent, and the E2F-1 protein that it negatively regulates is released to activate S phase transcriptions. The critical Restriction point control is relaxed or absent in cancers,80 the R protein can be more stable, a difference that provides a molecular basis for greater proliferative capacity.81

29. Of fundamental importance are the dynamic steady states between production and removal. These are active at all levels—molecular, cellular, and biological. Regulatory interactions create off-on switches between alternative pathways, negative feedback loops for limiting pathways, positive feedback loops that convert transient into sustainedsignals, feed-forward loops and successive activation pathways that amplify signals, as by MAP kinases.84 Systems biology—mathematicalcomputer models are being developed to grasp these interactions in large genetic and metabolic networks.85

30. References