Chapter 2 - Regulation of protein activities
If DNA is the cell’s library and RNA is the messenger, proteins are the workers
(RNA does some work, but proportionally much less than proteins). They build the cell
by constructing (or being part of) macromolecular structures; they move the cell, or move
things about within the cell; they manage the metabolism of the cell; they manage the
way the cell interior communicates with the extracellular fluid; they accomplish the
replication of DNA and division of the cell.
Thus, the regulation of cellular function is primarily brought about by regulating
protein function. For many kinds of protein, this is measured as an ‘activity’, where
activity refers to the amount of a specific function that occurs per unit time. If, for
example, we consider a protein that functions as an enzyme, its activity is the rate at
which it converts substrate into product. If the protein is a membrane transport protein,
then its activity is the rate at which it moves a molecule from one side of the membrane
to the other. The maintenance of intracellular homeostasis in the face of changing
conditions therefore can be brought about by altering the activities of various proteins.
There are two general ways in which protein activities are altered: (1) transcriptional
regulation; (2) post-translational regulation. Translational regulation is also possible, but
is probably less significant overall.
Post-translational regulation of protein activities
Post-translational regulation of protein activity can be a very rapid form of regulation.
It occurs in proteins that have already been synthesized, and can be brought about via a
variety of strategies:
(1) Reversible Protein Phosphorylation
Perhaps the most common mechanism of post-translational regulation of intracellular
proteins is reversible phosphorylation. A phosphate is transferred from ATP to a
specific amino acid(s) on the target protein. This can be sufficient to alter the basic
function of the protein.
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The discovery of the first protein to be regulated by this mechanism was made in
the 1950s by two Eds, Edmond Fischer and Edwin Krebs, with the enzyme glycogen
phosphorylase. Glycogen phosphorylase is abundant in skeletal muscle, where it
catalyzes the breakdown of glycogen to individual subunits of glucose (that are then
further oxidized to fuel contraction). Fischer and Krebs demonstrated that glycogen
phosphorylase could be converted from an inactive form to an active form simply by
phosphorylation.
They further showed that another enzyme, glycogen phosphorylase kinase,
catalyzed this reaction. Of course, having been activated, it would be nice if a
mechanism existed to inactivate glycogen phosphorylase when its activity was not
needed. This is accomplished by a phosphatase (phosphoprotein phosphatase), which
dephosphorylates glycogen phosphorylase, thus returning it to its inactive conformation.
Subsequent to these initial findings, it was discovered that this was just the tip of
the iceberg. It turns out that an enormous number of proteins are regulated by reversible
phosphorylation; indeed this is an almost ubiquitous strategy, involved in regulating most
cellular processes. It has been estimated that 1% of the human genome encoded by
protein kinases and phosphatases, that regulate other proteins by reversible
phosphorylation. This is not to say it is the only strategy; rather, there are multiple,
overlaid regulatory pathways. Reversible phosphorylation is a very rapid event, allowing
immediate transitions in protein activities. Of course, this would be essential in the rest
to work transition of skeletal muscle.
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(2) Other chemical modifications of proteins
Particularly with the advent of mass spectrometric analysis of proteins, it has become
possible to identify a host of other chemical modifications that occur in specific proteins.
For example, some proteins are regulated by the intracellular red-ox environment. An
example of this is the red-ox sensitive transcription factor Ref-1 (APE1) that also
catalyzes an important step in DNA repair. Other proteins are reversibly acetylated, as in
the DNA-packaging histones that are deacetylated by sirtuins in a process that appears to
have major implications for regulating DNA transcription and thus cellular function.
Another important example is hypoxia inducible factor -1α (HIF-1α), which is
hydroxylated by prolyl hydroxylase (see chapter 4), leading to its rapid degradation.
(3) Cleavage of specific domains
Many proteins’ activities are regulated by removal of an inhibitory domain. The most
famous examples of this form of regulation are probably the digestive enzymes, which
must be synthesized and transported to the site(s) of their action without digesting the
body along the way. For example, gastric pepsinogen contains a 44 amino acid domain
that renders the enzyme completely inactive until it reaches the highly acidic gastric
lumen. The low pH alters the conformation of pepsinogen, allowing an autocatalytic
cleavage of the inactivating domain and thus formation of active pepsin. Of course, this
form of regulation is not reversible.
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(4) Movement between subcellular compartments
Many proteins have specific functions that are necessarily confined to a certain
cellular compartment. Alternatively, they may have different functions at different subcellular locations. In this instance, simple translocation from one location to another will
be sufficient to initiate an activity. Ref-1 (APE1) provides an example of this: redox
modification (oxidation) of Ref-1 causes it to move from the cytosol to the nucleus,
where it fulfills its dual roles in transcriptional regulation and DNA repair. Another
example of this is the sequestration of proteins within intracellular vesicles. Following an
appropriate signal, these protein-containing vesicles can be fused with a membrane, thus
immediately altering the amount of the protein at the functional site. Examples of this
include the aquaporins of the renal epithelium, which regulate water movement, and
insulin-stimulated glucose transporters of various tissues.
(5) Reversible association/dissocation
Proteins that function as multimers (containing multiple subunits) may be inactive in
monomeric form. For example the mitochondrial superoxide dismutase MnSOD is a
functional homotetramer, i.e. four identical protein subunits associate to form the active
enzyme. Hemoglobin is also a homotetramer. Any modification that results in
dissociation of the multimeric structure may have the effect of inhibiting activity.
(6) Modification of immediate environment
Integral membrane proteins have domains that function in a radically different
environment than soluble proteins, i.e. the lipid environment within a phospholipid
bilayer. Direct interactions between these membrane proteins and their surrounding
phospholipids may alter their activities. For example, the mitochondrial inner membrane
protein β-hydroxybutyrate dehydrogenase has a requirement for phosphatidylcholine.
Other mitochondrial inner membrane proteins maintain important interactions with the
unique phospholipid cardiolipin. Even the composition of fatty acyl chains can
appreciably alter the activities of membrane spanning proteins.
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Regulation by varying protein amount
All cellular components turn over, i.e. they are continually being synthesized as
they are also being degraded (particularly when damaged). As a result, the amount of a
protein present in the cell at any time, actually represents the steady-state balance that
exists between synthesis and degradation. Alterations in either process can therefore alter
the amount of any individual cellular protein in the cell. First we will consider regulation
of protein synthesis from their respective genes.
Transcriptional regulation of gene expression
A common form of cellular adaptation to changing conditions involves changing
the number of a specific protein that is present in the cell by increasing the rate of
synthesis. For example, human skeletal muscle that has been trained by habitual longdistance running will adapt by maintaining more mitochondria, and all of the proteins that
comprise mitochondria, in each muscle cell. Every step between transcription of a gene
and translation of mRNA is a potential regulatory site, as outlined in the figure below.
However, probably the most important mechanism involves the direct stimulation or
repression of gene transcription, and this will be the focus of the remaining discussion.
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Most cells, at any given moment, are expressing only 10-20% of their genome.
This is because, despite all cells within an individual having the same genes (there are
some exceptions to this general rule), different cell types have vastly different functions
and one type may simply have no need to transcribe a particular gene. Gene transcription
involves a number of steps, to expose and unwind DNA, transcribe it to RNA, process to
mRNA, export it from the nucleus and translate it to protein. Gene transcription requires
assembly of a ‘transcriptional complex’ on the unwound DNA that contains RNA
polymerase II, as well as multiple factors involved in recognizing transcriptional start
sites and maintaining interactions with transcription factors and transcriptional modifiers.
The transcriptional complex assembles at the gene promoter, as specific nucleotide
sequence recognized by the various proteins. In many genes, the promoter has a
sequence that corresponds roughly to 5’-GNGTATA(A/T)A(A/T)-3’ (where N can be
any nucleotide) and is called a ‘TATA box’. The assembly of the transcriptional
complex on the TATA box is illustrated in the figure below from Boron and Boulpaep.
Note that this represents the ‘basal transcriptional machinery’ to which various accessory
factors may bind. Assembly of the transcriptional complex involves the sequential
binding of proteins to specific DNA sequences, such as a TATA box (see below).
Typically, transcription factor IID (TFIID) recognizes the TATA box and is the first to
bind it., followed by TFIIA and TFIIB, RNA polymerase II, and so on. Some of the
general transcription factors are actually protein kinases (see above) that phosphorylate
and thus modify the activity of, RNA polymerase II.
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The basal transcriptional machinery alone is not capable of efficient transcription,
and requires additional transcription factors to stimulate its activity. The extent and
diversity of such transcription factors is an ongoing area of discovery in biology. The
figure below shows one example of how specific transcription factors may interact with
DNA and the basal transcriptional machinery.
Transcription factors recognize specific DNA sequences as well as other proteins
that are part of the transcriptional complex. These specific DNA sequences, or regulatory
elements, are often located upstream (5’) to the protein-coding region of a gene. Many
hormones exert their actions primarily by coordinating interactions between the
transcriptional complex and these regulatory elements. The latter may be either positive
(enhancers) or negative (silencers) regulatory elements. The transcription factors that
bind enhancers are termed ‘activators’ and those that bind silencers are termed
‘repressors’.
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Many hormones exert their intracellular effects by binding receptors and
becoming transcription factors that regulate transcriptional activity selectively via their
interactions with specific regulatory elements. Thyroid hormone (thyroxine) provides a
good example of this. Thyroxine stimulates the expression of a broad suite genes whose
products are enzymes, transporters and other proteins crucial for energy metabolism.
To increase ATP production, it is necessary to simultaneously increase the
expression of many genes, in a coordinated way. This is possible because the genes for
all of these proteins that must be regulated by thyroxine contain a regulatory element that
is recognized by the thyroxine-receptor complex. This ‘thyroxine response element’, or
TRE has one of the following consensus sequences:
5' AGGTCA 3' or 5’ TAAGGTCA 3’
These sequences, typically occurring in strings (sometimes palindromic) upstream of
thyroxine regulated genes, confer regulatory control over the rate of their transcription.
A similar design exists for other soluble hormone receptors.
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Intracellular signal transduction pathways
Taking a step back from the actual mechanisms by which intracellular activities
can be modified, we can examine what causes these mechanisms to be turned on or off.
In many cases, the signal arrives at the cell as a hormone, and the response begins when
the hormone binds a specific receptor.
Intracellular and nuclear receptors
Receptors for membrane-phospholipid soluble hormones, like the thyroxine
receptor, may be localized to the cytosol or nucleus. These receptors, once activated by
their hormone ligand, translocated to the nucleus where they function as transcription
factors (as outlined above). In addition to thyroxine hormone receptors, a wide variety of
hormone receptors, including those for many steroid-based hormones, share the same
basic modular structure. Specific domains of these receptors are responsible for
responsible for hormone binding, nuclear localization, or DNA binding. In the figure
below, the numbers inside these domains indicates the amino acid sequence identity with
the prototypical glucocorticoid receptor.
G-protein coupled receptors
Many receptors are localized to the cell membrane, where they are may span the
membrane and be in communication with both extracellular and intracellular
compartments. The fluid nature of the cell membrane provides lateral mobility to these
receptors, which can therefore interact with other membrane proteins. Many such
mobile, membrane-bound receptors interact with G-proteins. G-proteins (GTP-binding
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proteins) are peripheral proteins that are anchored in the cell membrane but have an
active site within the cytosol. G-proteins are heterotrimers, composed of an α, β and γ
subunit. There is substantial diversity in these individual subunits, with 16 different α, 5
β and 11 γ isoforms present in mammalian tissues. Thus, via various different
combinations, it is possible to generate hundreds of different types of G-proteins.
G-proteins exist in either an inactive or an active state. The inactive G-protein is
bound to GDP and the active G-protein is bound to GTP. Activation, via binding of
GTP, is mediated by specific membrane receptors following the binding of their ligands.
Once activated, G-proteins dissociate into an α and a βγ subunit, which both move within
the plane of the membrane and come in contact with various effector molecules that
transduce the message received by the receptor into a specific cellular response. The
exact nature of the response depends upon which G-proteins have been activated and
which down-stream effector molecules they stimulate. For example, Gα5 stimulates
adenylyl cyclase and Gα1 inhibits it. The G-protein subunits interact with adenylyl
cyclase, phosphodiesterase, phospholipases, ion channels and other proteins. Thus, the
same hormone may elicit different responses in different cells by interacting with
different downstream effectors. G-proteins are capable of eliciting a huge range of
intracellular responses.
A number of serious human diseases involve dysfunction of G-proteins and a
concomitant loss of homeostatic regulation One important example is the Cholera toxin.
Vibrio cholerae is a bacterium that infects the gastrointestinal system. It enters intestinal
epithelial cells, where it catalyzes the ADP-ribosylation the α-subunit of G-proteins. The
result of this change is that this subunit is unable to convert GTP to GDP, thus trapping it
permanently in the active conformation and overstimulating various downstream
effectors. Importantly, in the intestinal epithelium adenylyl cyclase becomes
overstimulated, causing a huge movement of Cl- ions across the membrane and a
concomitant loss of water from cells resulting in diarrhea.
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The ultimate effect of G-protein coupled receptor activation is the alteration of
some second messenger within the cell. Second messengers include cyclic AMP
(cAMP), inositol triphosphate, diacylglycerol, calcium, and arachadonic acid metabolites.
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Thus, for example, a G-protein may stimulate adenylyl cyclase to catalyze the conversion
of ATP to cAMP within the cell. cAMP, in turn may stimulate a protein kinase and thus
the phosphorylation of many different proteins in the cell.
cAMP dependent protein kinase A is composed of two catalytic and two
regulatory subunits. Binding of cAMP by the regulatory subunits results in their
dissociation from the catalytic subunits, which then become active and catalyze the
phosphorylation of specific amino acids on downstream proteins. When the intracellular
[cAMP] decreases again, the PKA complex reassembles and the catalytic subunits are
inhibited. In addition to PKA, cAMP is capable of interacting with a number of other
proteins within the cell to generate various other effects including, for example,
transcriptional changes.
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Many hormones exert their functions via this pathway: receptor → G-protein →
adenlyl cyclase → PKA. For example, water reabsorption in the mammalian kidney is
regulated by aquaporin (AQP), a protein that acts as a water channel. The hormone
arginine vasopressin (antidiuretic hormone) stimulates water reabsorption in the kidney
by eliciting a transfer of AQP from intracellular vesicles to the cell membrane using this
signal transduction pathway. Similarly, the epinephrine-induced activation of glycogen
breakdown to glucose in skeletal muscle is regulated by this same signal transduction
pathway.
In addition to adenylyl cyclase, G-proteins interact with phospholipases.
Phospholipases catalyze the cleavage of phospholipids into phosphate headgroups and
diacylglycerol (DAG) tails. For example, the relatively unabundant phospholipids
phosphatidylinositol is converted to inositol triphosphate (IP3) and DAG by the G-protein
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mediated activation of phospholipases. IP3 and DAG in turn activate various intracellular
targets, including the release of Ca2+ from intracellular stores.
Calcium, itself an intracellular second messenger, binds to numerous molecules to
coordinate a variety of responses, depending upon which Ca2+-sensitive molecules are
present within the cell. Many cells contain the protein calmodulin which, when bound to
four molecules of Ca2+ acts as a protein kinase to phosphorylate downstream protein
targets. For example, the contraction of smooth muscle cells is stimulated by this signal
transduction pathway.
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Receptors that are catalytic
A number of receptors in the cell membrane are also enzymes. These receptors
are membrane-spanning, with extracellular and intracellular domains. When the
extracellular domain is bound by the respective hormone, a conformational change occurs
in the intracellular domain, altering its catalytic activity. In many cases this catalytic
domain functions as either a kinase or a phosphatase, phosphorylating or
dephosphorylating, respectively, specific cellular proteins. As outlined above, such
changes in phosphorylation state significantly modify the activity of cellular proteins. An
example of this is the insulin receptor which, when bound by insulin, phosphorylates
tyrosines on target proteins, bringing about a wide range of responses that are cell type
specific. In some tissues, it stimulates recruitment of glucose transporters (GLUT 4) to
the cell membrane. Other catalytic receptors reversibly phosphorylate threonines or
serines.
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Another class of catalytic receptors are the guanylyl cyclases. The catalytic
domain of these receptors converts GTP to cGMP which, in turn, regulates cGMPdependent protein kinases, thus regulating a range of target proteins. The receptor for the
hormone atrial natriuretic peptide (ANP), which is released by cells in the heart in
response to excessive stretch, is an example of this type of membrane bound catalytic
receptor. Some receptor guanylyl cyclases are soluble. For example, the receptor for
nitric oxide (NO), a short-lived gas that functions as a signalling molecule in the
circulatory and nervous systems, is a soluble guanylyl cyclase. In vascular smooth
muscle cells of the circulatory system, stimulation of the soluble receptor guanylyl
cyclase with NO may increase the cytosolic concentration of cGMP by 50-fold, thus
stimulating and inhibiting a variety of cGMP-sensitive proteins. In smooth muscle cells
this elicits relaxation; this signalling mechanism is the molecular basis of the male
erection.
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Regulated protein degradation
Proteins are in a continual state of dynamic flux: existing proteins are damaged
and must be removed from the cell because they become dysfunctional, or even
dangerous to cell viability. Thus, the half-life of any individual protein can be
determined. Most protein degradation is highly regulated and the major pathway by
which this occurs is via ubiquitinylation and proteasomal degradation. Ubiquitin is a
small (76 amino acid) protein that is covalently attached to proteins that are targeted for
destruction. The process is catalyzed by a group of enzymes named E1, E2 and E3.
Protein ubiquitinylation involves the sequential addition of individual ubiquitin subunits
so that they may form quite long chains. The proteasome recognizes and degrades
proteins thus labelled into small peptides and individual amino acids that can then be
recycled by serving as substrates for proteins that are being newly synthesized.
Ubiquitin-mediated degradation is a key regulatory mechanism for two important
proteins that play roles in cancer: p53 and HIF-1 (see Chapter 4). The figure below
shows some general functions of the ubiquitin-proteasome system.
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