Theme 1 CONTROL OF ENZYME ACTIVITY

Transcription

1 Theme 1 CONTROL OF ENZYME ACTIVITY Included in this section: Enzyme Activity: Control Notes in study material as well as slides used in class. Please use the study material provide as well as Matthews and van Holde Biochemistry. Page number may differ (3 rd edition pp ; pp ) Why are controls necessary? Since there are pathways which operate in opposite directions it is clear that they cannot all operate at once. The main needs are 1. To avoid the potential problem of substrate cycles. 2. To link energy production to energy usage. 3. To respond to physiological changes the direction of flow after feeding will not be the same as that during fasting. Starvation will be different again. Control of enzyme activity essential to maintain homeostasis o Enzyme activities can be upregulated or downregulated depends on metabolic state or needs of a cell o Metabolic co-ordination necessary to prevent uncontrolled growth or catabolism prevent conflict of opposing metabolic pathways Glycogen synthesis and breakdown Glycolysis and gluconeogenesis Regulation of metabolic processes accomplished by controlling enzyme activity Uncontrolled cycling from one compound to the other would result in massive destruction of ATP and generation of heat. While bumble-bees actually do carry out this process to allow themselves to warm up enough to fly when the conditions are cold, it is not normally an advantage for such cycling to occur. 1

2 There is the potential for futile cycles at a number of other sites in metabolism these include glycogen synthesis and breakdown and fat synthesis and breakdown. Control cannot occur at steps that are common to the pathway in both directions but only at steps which are unique to a particular pathway. In practice there are advantages to the cell if it allows some cycling, therefore the cycles have been renamed substrate cycles rather than futile cycle. The advantage lies in the fact that some cycling results in more sensitive controls. How are enzyme activities controlled? There are two ways in which the rate of an enzyme catalysed reaction can be changed: 1. Change in the amount of an enzyme. 2. Change in the rate at which an enzyme operates. Control of enzyme levels - The concentrations of different enzymes vary widely in cellular extracts. Enzyme levels are controlled in large part by controlling the enzyme's rate of synthesis. Enzyme synthesis can often be induced or repressed by the presence or absence of certain metabolites. The rate of enzyme degradation can also be a factor in controlling enzyme levels. Metabolic control by varying the amounts of an enzyme The amount of an enzyme can be changed by alterations in either the rate of synthesis or the rate of destruction. Proteins in the body have a half-life which ranges from hours to days. Because of this half-life changes in amount take hours to days to have their effect. There are many examples of this time course of change in response to changing physiological conditions. These include An increase in lipoprotein lipase in lactating mammary glands. Changes in liver enzymes during the shift from the fed state to starvation. Increases in drug-metabolising enzymes following the intake of foreign compounds. The urea cycle, where the enzyme levels change in proportion to the nitrogen content of the diet, is a good example. In bacteria the induction of enzymes can be rapid, e.g. when E. coli is exposed to lactose the induction of -galactosidase occurs rapidly. 2

3 In general, however, changes in enzyme level in animals are relatively slow. Control of enzyme activity - The catalytic activity of an enzyme can be controlled in two ways: by reversible interaction with ligands and by covalent modification of the enzyme itself. Low molecular weight ligands can interact with enzymes and exert allosteric effects. Frequently, the first or most important step in a metabolic pathway is under allosteric control in this way, enabling a cell to turn on or turn off an entire pathway easily and efficiently. Covalent modifications include phosphorylations, ADPribosylation, and other, more complex alterations. Covalent modification often occurs as a result of action of regulatory cascades. Glycogen metabolism is regulated in this fashion. Enzymes that phosphorylate other enzymes are called protein kinases. Compartmentation - Eukaryotic cells contain many different organelles and enzymes are distributed unevenly throughout them. For example, RNA polymerases are found in the nucleus and the nucleolus, where DNA transcription occurs. Enzymes of the citric acid cycle, on the other hand, are found in the mitochondria (Figure 12.11). Enzymes of fatty acid synthesis are found in the cytoplasm, but enzymes for fatty acid degradation are found in mitochondria. Hormonal regulation - Cells must respond to changes in the environment and/or to signals from other cells. The process of transmitting this information from outside of the cell to inside the cell is called signal transduction (Figure 12.13). The extracellular messengers that carry the information include hormones, growth factors, neurotransmitters, and pheromones. Hormones are substances synthesized in specialized cells and carried via the blood to remote target cells. There the hormones interact with specific receptors, resulting in specific metabolic changes in the target cell. An example is the rapid generation of energy that results from secretion of adrenaline (epinephrine). The following two types of metabolic response to hormones are well understood: 1. Steroid hormones involve changes in gene expression. 2. Second messengers that control metabolic reactions are made in response to the binding of an extracellular substance (the first messenger). Common first messengers 3

4 include glucagon and insulin. Common second messengers include cyclic AMP (or camp) and phosphoinositides. Distributive control of metabolism - This concept recognizes that control of metabolic pathways is not simply a function of a single allosterically regulated enzyme in the pathway, but rather a function of all of the enzymes of a pathway. While enzymes catalyzing committed steps in pathways often play major roles in regulating the pathway, contributions can be made by all of the enzymes of the pathway Time and Control Medium term metabolic regulation (minutes) [S] and [P] Short term regulation (seconds) Allosteric Effectors Reversible Covalent modification Irreversible Proteolytic activation Stimulation and inhibition by control proteins Long term metabolic regulation (Hours/days) This Theme will not be discussed in depth in this module. Control of enzyme synthesis Transcriptional control Translational control Control of enzyme degradation Compartmentalization Metabolic control by regulation of the activities of enzymes in the cell This type of control can be very rapid. To explain why, the properties of enzymes must be discussed. Which enzymes in metabolic pathways are regulated? In a pathway of the type A B C D E 4

5 where E is the product of the pathway, it is typically found that E inhibits the A B conversion. This process is called feedback inhibition. This type of control makes good sense because no intermediates accumulate. In more complex pathways, such as fat metabolism or carbohydrate metabolism, the end product is often hard to identify but the basic principle is the same. Regulation of Enzyme Activity Enzymes function in assembly line-like fashion to catalyze the thousands of reactions occurring in cells each second. Coordinating and regulating enzymatic activities is essential for efficient functioning of cells. Several control mechanisms that do not involve covalent modification of the enzymes are possible: Substrate level control - In this control mechanism, high levels of the product of a reaction inhibit the ability of the small amounts of substrate present to react. An example is the first step in glycolysis, catalyzed by hexokinase. It is inhibited by the product of its action, glucose-6-phosphate. If glycolysis is blocked for any reason, glucose-6-phosphate accumulates. Feedback control - In this mechanism, the product of a series of reactions (like in an assembly line) inhibits the action of an earlier step in the process (usually the first step). Feedforward regulation occurs when a molecule in an assembly line reaction activates an enzyme ahead of it in the pathway. REMEMBER TO THINK ABOUT THIS SECTION IN THEME 2 Example 1. Hexokinase is allosterically inhibited by glucose-6-phosphate (G6P). That is, the enzyme for the first reaction of glycolysis is inhibited by the product of the first reaction. As a result, glucose and ATP (in reactions 1 and 3) are not committed to glycolysis unless necessary. Glucose (Glc) may be considered the central metabolite in almost all organisms. Energy and precursors for the biosynthesis of many cellular components are generated from Glc through the glycolytic pathway; on the other hand, Glc can also be stored for later use, as starch or glycogen. The first step in glycolysis is the phosphorylation of Glc; this keeps Glc into the cell and favors the diffusion of 5

6 extracellular Glc into the cell by reducing its intracellular concentration. The difference of cellular concentration between substrates and products makes the phosphorylation of Glc very favorable ( G= -33kJ/mol); as a result, this reaction constitutes an important site for regulation. Hexokinase (ATP: D-hexose 6-phospho-transferase, EC ) is the enzyme responsible for the catalysis of this reaction, exerting a key regulatory role in glycolysis. Although this enzyme has the ability to phosphorylate different hexoses, phosphorilation of Glc is, by far, its most important function. Four different isozymes of hexokinase have been identified in mammals -Type I, Type II, Type III, and Type IV. As the latter isozyme has a strong specificity for D-Glc, it is also known as glucokinase. The structure of Types I-III isozymes consists of two globular halves -the N-terminal and C-terminal regions- held together by a connecting helix and a few hydrogen bonds (100 kda). The structure of each half is similar to that of yeast hexokinase, which consists of two regions, i.e. the large and the small lobe; the glucose binding site is located in the bottom of the cleft between the two lobes. These three types of mammalian hexokinases appear to have evolved from an ancestral hexokinase of 50 kda, by gene duplication and fusion; consequently, they have extensive sequence repetition, both between them and between their N-terminal and C-terminal regions. A common feature of these isozymes, also present in the ancestral hexokinase, is the regulatory role of Glc phosphorylation through inhibition by the product (glucose-6- phosphate, Glc-6-P). However, each isozyme displays particular properties and differs from the others in its tissue specificity. For example, while Type II isozyme possesses catalytic activity in both halves, Type I and Type III isozymes have lost the catalytic activity of the N-terminal region. Hexokinase I exhibits a unique regulatory property: physiological levels of inorganic phosphate (Pi) can reverse the inhibition of Glc phosphorylation effected by Glc-6-P. This characteristic defines hexokinase I as an enzyme with a catabolic role, while the other hexokinases are considered to have mainly an anabolic role. This supports the fact that hexokinase I is particularly abundant in highly energy-demanding cells, as brain and red blood cells. exokinase.htm Use the links and pdf s provided to summarize this regulation and the impact from conformation changes 6

7 The nature of regulatory enzymes There are two basic ways in which the activity of enzymes can be changed 1. Allosteric regulation. 2. Covalent modification. Allosteric control of enzymes This type of control is particularly important in metabolic regulation. The usual result of the binding of an allosteric effector is a change in the affinity of the enzyme for its substrate(s). The effect can be positive, with an increase in affinity, or negative, with a decrease. Since enzymes generally are operating at around their K m this results in a change in the effective activity of the enzyme in the case of positive effectors and vice versa. In a few cases allosteric effectors change V max but this is unusual. The mechanism of allosteric control of enzymes Allosteric enzymes are made up of multiple protein subunits, or monomers. The subunits have catalytic activity but interact with each other, changing the shape of the velocity/substrate (V/S) plot. One result of the interaction of the subunits is that the V/S plot is sigmoidal rather than hyperbolic. Binding of a positive effector moves the plot to the left and a negative effector moves it to the right. Because the plot is sigmoidal the change in rate for a given change in substrate concentration is more marked, i.e. the sensitivity is increased. The sigmoid shape arises because the binding of one molecule of substrate increases the affinity of the enzyme for subsequent substrate molecules. This is known as positive homotropic cooperative substrate binding. Strictly this is not an allosteric effect since only one site is involved but it is often called allosteric. The extent of cooperative interactions can be estimated using a Hill plot, where log V o /V max V o is plotted against log [S]. The slope of the central region gives a slope of n, the Hill coefficient. Two mechanisms of cooperative binding have been suggested. In both the number of catalytic subunits in the high affinity state increases as the substrate concentration increases 7

8 The concerted model suggests that all the subunits in a given molecule of enzyme are in a high affinity or a low affinity state, with the balance lying to the low affinity side. Binding of a substrate molecule tips the equilibrium to the high affinity side. Allosteric modifiers alter the equilibrium between the low affinity (T or tense) and high affinity states (R or relaxed), with those that tip the equilibrium towards the T state being inhibitors and vice versa. The other model is the sequential model. This suggests that initially all the molecules are in a low affinity state. Binding of one substrate molecule changes a subunit from the T to the R state and eases the transition for an adjacent subunit. The next substrate that binds has an even more marked effect for a third subunit and so on. Also another type of control is found where a regulatory subunit is associated with the catalytic subunit, either activating or inhibiting it. The allosteric effector causes this subunit to dissociate. Allosteric enzymes - These enzymes are invariably multisubunit proteins, with multiple active sites. They exhibit cooperativity in substrate binding (homoallostery) and regulation of their activity by other, effector molecules (heteroallostery). Homoallostery - The effects of cooperative substrate binding on enzyme kinetics are shown in Figure

9 Figure 11.32: Effect of cooperative substrate binding on enzyme kinetics. Binding of one substrate favors binding of additional substrates. Cooperative binding favors reduction of KM for the binding of substrates after the initial one. Figure shows the effect of extreme homoallostery. At concentrations of S below a critical point, [S]c, the enzyme is almost inactive, but then changes activity rapidly with concentrations of S greater than [S]c. 9

10 Figure 11.33: Effect of extreme homoallostery. Heteroallostery - This type of allosteric control involves heteroallosteric effectors which may be either inhibitors or activators of binding. If an enzyme can exist in two conformational states, T and R, that differ dramatically in the strength with which substrate is bound or which differ significantly in the catalytic rate, then the kinetics of the enzyme can be controlled by any other substance that, in binding to the protein, shifts the T<=>R equilibrium. Allosteric inhibitors shift the equilibrium toward T and activators shift it toward the R state. Figure illustrates how heteroallosteric control of an enzyme affects the shape of a V-vs-[S] curve. Note that shifts toward the R state (activators) increase the velocity for a given substrate concentration, whereas shifts toward the T state have the opposite effect. 10

11 Figure 11.34: Heteroallosteric control of an enzyme. Reversibility of allosteric control Allosteric control is very rapid in both directions since the bonds involved are noncovalent. Allosteric control is a tremendously powerful metabolic concept The key point is that the allosteric effector need not have any structural relationship whatsoever to the enzyme regulated or even belong to the same area of metabolism. This property allows signals to be transmitted across pathways. Allosteric enzymes can respond to more than one regulator they are not confined to a single allosteric site. Therefore they can respond to signals from a number of different sources. This allows interaction between pathways, e.g. signals can be transmitted from the citric acid cycle to glycolysis, the level of ATP can affect glycolysis, etc. An enzyme does not respond to such signals in a yes/no way. Enzyme activity is a continuous variable. Cell functioning would not be possible without subtle control of this type. 11

12 Summary in ATCase PDF file. Control of enzyme activity by phosphorylation Although other forms of covalent modification occur phosphorylation is by far the most important. In this type of control protein kinases transfer a phosphate group from ATP to specific sites on proteins. This causes a conformational change and hence change in the properties of the protein. The highly charged nature of the phosphate group makes it very effective in causing conformational change. The most important phosphorylatable sites are serine and threonine, which have OH groups in their side chains. The two classes of controls intracellular mechanisms and those dependent on extracellular signals Allosterically regulated enzymes give a very rapid response but those regulated by phosphorylation require the processes of phosphorylation and dephosphorylation to be regulated. This regulation is mainly by extracellular agents, such as hormones. Allosteric controls coordinate the flow of molecules within the cell. A second group of controls allows cells to respond to external controls such as hormones. This allows coordination at the whole organism level. Phosphorylation is mainly involved in responses to external signals. 12

13 Covalent Modification Is a Means of Regulating Enzyme Activity The covalent attachment of another molecule can modify the activity of enzymes and many other proteins. In these instances, a donor molecule provides a functional moiety that modifies the properties of the enzyme. Most modifications are reversible. Phosphorylation and dephosphorylation are the most common but not the only means of covalent modification. Histones proteins that assist in the packaging of DNA into chromosomes as well as in gene regulation are rapidly acetylated and deacetylated in vivo. More heavily acetylated histones are associated with genes that are being actively transcribed. The acetyltransferase and deacetylase enzymes are themselves regulated by phosphorylation, showing that the covalent modification of histones may be controlled by the covalent modification of the modifying enzymes. Modification is not readily reversible in some cases. Some proteins in signaltransduction pathways, such as Ras and Src (a protein tyrosine kinase), are localized to the cytoplasmic face of the plasma membrane by the irreversible attachment of a lipid group. Fixed in this location, the proteins are better able to receive and transmit information that is being passed along their signaling pathways. The attachment of ubiquitin, a protein comprising 72 amino acids, is a signal that a protein is to be destroyed, the ultimate means of regulation. Cyclin, an important protein in cell-cycle regulation, must be ubiquitinated and destroyed before a cell can enter anaphase and proceed through the cell cycle. Virtually all the metabolic processes that we will examine are regulated in part by covalent modification. Indeed, the allosteric properties of many enzymes are modified by covalent modification. The following table lists some of the common covalent modifications. 13

14 Table. Common covalent modifications of protein activity Modification Donor molecule Example of modified protein Protein function Phosphorylation ATP Glycogen phosphorylase Glucose homeostasis; energy transduction Acetylation Acetyl CoA Histones DNA packing; transcription Myristoylation Myristoyl CoA Src Signal transduction ADPribosylation NAD RNA polymerase Transcription Farnesylation Farnesyl pyrophosphate Ras Signal transduction - g-carboxylation HCO 3 Thrombin Blood clotting Sulfation 3 -Phosphoadenosine-5 - phosphosulfate Fibrinogen Blood-clot formation Ubiquitination Ubiquitin Cyclin Control of cell cycle Phosphorylation Is a Highly Effective Means of Regulating the Activities of Target Proteins The activities of many enzymes, membrane channels, and other target proteins are regulated by phosphorylation, the most prevalent reversible covalent modification. Indeed, we will see this regulatory mechanism in virtually every metabolic process in eukaryotic cells. The enzymes catalyzing phosphorylation reactions are called protein kinases, which constitute one of the largest protein families known, with more than 100 homologous enzymes in yeast and more than 550 in human beings. This multiplicity of enzymes allows regulation to be fine-tuned according to a specific tissue, time, or substrate. The terminal (g) phosphoryl group of ATP is transferred to specific serine and threonine residues by one class of protein kinases and to specific tyrosine residues by another. 14

15 The acceptors in protein phosphorylation reactions are located inside cells, where the phosphoryl-group donor ATP is abundant. Proteins that are entirely extracellular are not regulated by reversible phosphorylation. Protein phosphatases reverse the effects of kinases by catalyzing the hydrolytic removal of phosphoryl groups attached to proteins. The unmodified hydroxyl-containing side chain is regenerated and orthophosphate (P i ) is produced. It is important to note that phosphorylation and dephosphorylation are not the reverse of one another; each is essentially irreversible under physiological conditions. Furthermore, both reactions take place at negligible rates in the absence of enzymes. Thus, phosphorylation of a protein substrate will take place only through the action of a specific protein kinase and at the expense of ATP cleavage, and dephosphorylation will result only through the action of a phosphatase. The rate of cycling between the phosphorylated and the dephosphorylated states depends on the relative activities of kinases and phosphatases. Note that the net outcome of the two reactions is the hydrolysis of ATP to ADP and P i, which has a DG of -12 kcal mol -1 (-50 kj mol -1 ) under cellular conditions. This highly favorable free-energy change ensures that target proteins cycle unidirectionally between unphosphorylated and phosphorylated forms. 15

16 Phosphorylation is a highly effective means of controlling the activity of proteins for structural, thermodynamic, kinetic, and regulatory reasons: 1. A phosphoryl group adds two negative charges to a modified protein. Electrostatic interactions in the unmodified protein can be disrupted and new electrostatic interactions can be formed. Such structural changes can markedly alter substrate binding and catalytic activity. 2. A phosphate group can form three or more hydrogen bonds. The tetrahedral geometry of the phosphoryl group makes these hydrogen bonds highly directional, allowing for specific interactions with hydrogen-bond donors. 3. The free energy of phosphorylation is large. Of the -12 kcal mol -1 (-50 kj mol -1 ) provided by ATP, about half is consumed in making phosphorylation irreversible; the other half is conserved in the phosphorylated protein. Recall that a free-energy change of 1.36 kcal mol -1 (5.69 kj mol -1 ) corresponds to a factor of 10 in an equilibrium constant. Hence, phosphorylation can change the conformational equilibrium between different functional states by a large factor, of the order of ATP is the cellular energy currency. The use of this compound as a phosphoryl group donor links the energy status of the cell to the regulation of metabolism. 4. Phosphorylation and dephosphorylation can take place in less than a second or over a span of hours. The kinetics can be adjusted to meet the timing needs of a physiological process. 5. Phosphorylation often evokes highly amplified effects. A single activated kinase can phosphorylate hundreds of target proteins in a short interval. Further amplification can take place because the target proteins may be enzymes, each of which can then transform a large number of substrate molecules. 16

17 Protein kinases vary in their degree of specificity. Dedicated protein kinases phosphorylate a single protein or several closely related ones. Multifunctional protein kinases modify many different targets; they have a wide reach and can coordinate diverse processes. Comparisons of amino acid sequences of many phosphorylation sites show that a multifunctional kinase recognizes related sequences. For example, the consensus sequence recognized by protein kinase A is Arg-Arg-X-Ser-Z or Arg-Arg-X-Thr-Z, in which X is a small residue, Z is a large hydrophobic one, and Ser or Thr is the site of phosphorylation. It should be noted that this sequence is not absolutely required. Lysine, for example, can substitute for one of the arginine residues but with some loss of affinity. Short synthetic peptides containing a consensus motif are nearly always phosphorylated by serine- threonine protein kinases. Thus, the primary determinant of specificity is the amino acid sequence surrounding the serine or threonine phosphorylation site. However, distant residues can contribute to specificity. For instance, changes in protein conformation may alter the accessibility of a possible phosphorylation site. Proteolytic Activation Another example of covalent enzyme activation is proteolytic cleavage, found in the pancreatic proteases (such as trypsin, chymotrypsin, elastase, and carboxypeptidase). These enzymes are synthesized in the pancreas in an inactive form because if they were active in the pancreas, they would digest the pancreatic tissue. Rather, they are made as slightly longer, catalytically inactive molecules called zymogens (trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase, respectively). The zymogens must be cleaved proteolytically in the intestine to yield the active enzymes (Figure 11.39). If a small amount of protease becomes active in the pancreas, it can have painful or fatal consequences (i.e., acute pancreatitis). The pancreas protects itself from active protease action by synthesis of a protein called the secretory pancreatic trypsin inhibitor. The binding between trypsin and its inhibitor is one of the strongest noncovalent interactions known in biochemistry. The intestinal tissue is protected somewhat from damage by proteases by its glycosylated surface. 17

18 Figure 11.39: Zymogen activation by proteolytic cleavage. The first step is activation of trypsin in the duodenum. A hexapeptide is removed from the N-terminal end of trypsinogen by enteropeptidase,a protease secreted by duodenal cells. This yields active trypsin, which then activates the other zymogens by specific proteolytic cleavages. Trypsin will also activate other trypsinogen molecules in an autocatalytic process. Activation of a few trypsinogens ultimately leads to activation of many trypsins. Activation of chymotrypsinogen is shown in Figure First, trypsin cleaves the bond between arginine 15 and isoleucine 16. Notice that the N-terminal peptide remains attached to the rest of the molecule due to the disulfide bond between residues 1 and 122. The enzyme is activated by the cleavage due to changes in the conformation of the molecule. These include: 18

19 Figure 11.40: Activation of chymotrypsinogen. Enzyme Degradation: The Ubiquitin System General Information Ubiquitin (Ub) is a small protein that is composed of 76 amino acids. This protein is found only in eukaryotic organisms and is not found in either eubacteria or archaebacteria. Among eukaryotes, ubiquitin is highly conserved, meaning that the amino acid sequence does not differ much when very different organisms are compared. For example, there are only 3 differences in the sequence when Ub from yeast is compared to human Ub. This strong sequence conservation suggests that the vast majority of amino acids that make up Ub are essential as apparently any mutations that have occurred over evolutionary history have been removed by natural selection. Ub is a heat-stable protein that folds up into a compact globular structure. It is found throughout the cell (thus, giving rise to its name) and can exist either in free form or as part of a complex with other proteins. In the latter case, Ub is attached (conjugated) to proteins through a covalent bond between the glycine at the C-terminal end of Ub and the side chains of lysine on the proteins. Single Ub molecules can be conjugated to 19

20 the lysine of these proteins, or more commonly, Ub-chains can be attached. Conjugation is a process that depends on the hydrolysis of ATP. Ub is involved in many cell processes. For example, Ub is conjugated to the protein cyclin during the G1 phase of mitosis and thus plays an important role in regulating the cell cycle. Ub conjugation is also involved in DNA repair, embryogenesis, the regulation of transcription, and apoptosis (programmed cell death). Ub is encoded by a family of genes whose translation products are fusion proteins. The Ub genes typically exist in two states (see the figure below): A. The ubiquitin gene (red) can be fused to a ribosomal protein gene (blue) giving rise to a translation product that is a Ub-ribosomal fusion protein; B. Ub genes can exist as a linear repeat, meaning the translation product is comprised of a linear chain of Ub-molecules fused together (a polyubiquitin molecule). After the fusion proteins are synthesized, another protein called Ub-C-term hydrolase cleaves the fusion proteins at the C-terminal end of Ub. This either liberates an individual Ub and ribosomal protein (as shown in the figure) or liberates a set of Ub monomers from the polyubiquitin (not shown). 2012: Write as short assay on how ubiquitination lead to protein degradation. 20