most biochemists were very busy successfully creating thi

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 1940s. 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
cells 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
enzymes 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 1940s 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 enzymes
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 cells
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 proteins 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 inducible44 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 1950s. 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
receptors tyrosine kinase. Alternatively,estroge
n 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 proteins
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 levelsmolecular, 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
biologymathematicalcomputer models are being
developed to grasp these interactions in large
genetic and metabolic networks.85
30
References