Cell-cycle Contol and the Stress Response (Ubiquitin and Proteasomes)

Excerpted from:

Keith D. Wilkinson, "Roles of Ubiquitinylation in Proteolysis and Cellular Regulation" Annual Reviews of Nutrition (1995) 15:161-169.

Used by permission of the publisher.

INTRODUCTION

Ubiquitin is a small protein, highly-conserved, and present universally in eukaryotic cells. It functions by being covalently attached to other proteins, resulting in the targeting of those proteins for specific cellular fates (1-8). The covalent linkage is an isopeptide bond between the C-terminus of glycine 76 of ubiquitin and the side-chain amino group of lysine on the target protein (9). We have argued that ubiquitin is a post-translational signal-sequence which is attached to a variety of cellular proteins and which can respond in context to the environment (10,11). The ultimate consequences of ubiquitinylation will depend on the protein to which it is attached, as well as the localization of the conjugate and complex enzymatic specificities. Only a few of those consequences are understood, and only protein degradation has been studied in any detail.

Ubiquitin-dependent proteolysis has been shown to respond to glucocorticoids during fasting (14), to TNF as may occur in cachexia (15,16), to metabolic acidosis (17), to interferon gamma elicited by viral infection (18,19), to feeding cycles (20), to heat-shock (21), and to the presence of damaged proteins (22). Many of these effects may be due to induction of the stress response (23). Short-lived proteins which are known to be substrates for the system include regulatory molecules such as cyclins (24), c-mos (25), p53 (26), c-myc (27), c-fos (27), c-jun (28), MAT[[alpha]]2 (29), and NF-[[kappa]]B (30). Thus, ubiquitin-dependent processes are important in a large number of basic regulatory and repair processes.

The overall process of ubiquitin-dependent metabolism can be thought of as involving four separate reactions: activation of ubiquitin requiring the expenditure of one ATP molecule; conjugation of ubiquitin to a variety of cellular proteins; proofreading of the conjugates to either regenerate the target protein by removal of ubiquitin or to commit the target protein to its fate by adding more ubiquitin molecules; and finally metabolism of the conjugates. In the case of proteolysis, this involves degradation of ubiquitinylated proteins by the proteasome (2) or the lysosome (3). Through these reactions, proteins are marked by ubiquitinylation and targeted for degradation in the cell. The energy contributed by ATP is utilized to exert specificity on the proteolysis step by specifically marking only certain proteins and delivering them to the proteolytic systems.

Ubiquitin

FORMATION OF POLYMERIC UBIQUITIN

Structure and chemistry of ubiquitin. Ubiquitin is a highly conserved 76 residue protein that is universally present in eukaryotic cells. We have previously described the structure of ubiquitin (31-33) and only a few points will be elaborated here. The three-dimensional structure of ubiquitin shows that the site of protein attachment, the C-terminus, protrudes from the globular body of the protein. Ubiquitin is a very stable protein which refolds rapidly. It is also stable to most proteases at neutral pH. These properties are probably advantageous for a protein which participates in the delivery of substrate proteins to proteases. Secondly, we found that ubiquitin undergoes a conformational change in a hydrophobic environment (11). As discussed below, this conformational change may be important in the "proofreading" of protein structure (11).

Structure of polymeric ubiquitin. The chemistry of ubiquitin is dominated by the formation and cleavage of peptide bonds at its carboxyl-terminus, gly-76. Ubiquitin is encoded by three classes of genes in all species studies (34). In yeast, UBI1, UBI2, and UBI3 genes encode two proteins, each consisting of a single copy of ubiquitin fused to a different zinc finger protein. These carboxyl extension proteins are proteolytically processed to yield ubiquitin and the zinc finger proteins, which are required for efficient ribosome biogenesis (35). These genes are expressed constitutively in rapidly growing cells and this arrangement may be a mechanism to coordinate rates of protein synthesis and protein degradation. The yeast UBI4 gene encodes a ubiquitin precursor consisting of head-to-tail repeats of the ubiquitin protein sequence. The number of repeats varies with the organism, but this transcript is strongly induced by stress and is regulated by a consensus heat shock promoter. The pro-protein synthesized from this gene must also be proteolytically processed to release monomeric ubiquitin. Finally, a number of "ubiquitin-like" fusion protein genes have been identified in mammallian and viral systems (6). The functions of these gene products are generally unknown, but these proteins may require proteolytic processing also.

In addition to these gene products, a second type of ubiquitin C-terminal derivative is formed when one or more ubiquitins are added to target proteins. The best characterized of these conjugates is formed by the attachment of the C-terminus of ubiquitin to the side-chain amino group of lys-48 of another ubiquitin (9). Long chains of ubiquitin are formed on target proteins and must be disassembled during or after proteolysis of the target protein. These polymeric ubiquitin molecules are linked by epsilon-amino amide bonds and are sufficient to target proteins for degradation (9). In some cases, other lysines on ubiquitin may be involved in poly-ubiquitinylation, although the significance and consequences of these alternative structures are not understood (Arthur Haas, private communication and Dan Finley, private communication). The crystal structure of K48 poly-ubiquitin has been solved for n=4 (36). Subunit contacts are largely polar and the molecule is shaped like a flattened tube with a hydrophobic stripe on one face of the molecule.

The nomenclature used here is as follows; Pro-ubiquitin is the polymeric ubiquitin product of the UBI4 gene in yeast or its homologue in other eukaryotes, while ubiquitin carboxyl extension proteins (UBCEP) are the zinc finger products of the UBI1, UBI2 and UBI3 genes in yeast, or their homologues in other eukaryotes (37). Ubiquitinylation refers to the formation of isopeptide bonds between the C-terminus of ubiquitin and [[epsilon]]-amino sidechains of proteins. Multi-ubiquitinylation refers to the addition of ubiquitin to several lysines on a protein. Poly-ubiquitinylation refers to the addition of several ubiquitins to a single lysine on a protein. Poly-ubiquitin chains are linked by isopeptide bonds between the C-terminus of ubiquitin and a lysine (usually K48) on another ubiquitin.

Enzymes of ubiquitin conjugation. The covalent attachment of ubiquitin targets proteins for specific cellular fates. Proteins can be mono- di- or poly-ubiquitinylated . In all cases, the bond is between an amino group of the target protein (usually the side chain of lysine) and the carboxyl-terminus of ubiquitin. The specificity of ubiquitinylation is not understood, but involves a complex series of reactions catalyzed by the first three enzymes of the system.

The first enzyme, the ubiquitin activating enzyme, catalyzes the formation of a thiol ester between the C-terminus of ubiquitin and a cysteine residue of the ubiquitin carrier protein(s). Much of what is known about the function and biological roles of ubiquitin-dependent processes has been discerned from the phenotypes of conditional lethal mutations in the ubiquitin activating enzyme (1,34). They cell cycle arrest in S and S/G2 upon shifting to the non-permissive temperature, fail to activate ubiquitin, show reduced degradation of abnormal and short-lived proteins, and fail to condense chromatin (38).

After ubiquitin is activated by thiol ester formation with E1, it undergoes a trans-thiol esterification with a cysteine residue on one of the E2 proteins. E2 genes have now been characterized in many organisms (34), including at least 10 separate genes in yeast. All share a central core of about 150 residues which are 35% identical and which contain the active site thiol. Class I enzymes consist of the core only and require E3 (ligase) to conjugate ubiquitin to target proteins. Class II enzymes have C-terminal extensions, which are often highly charged. These extensions are required for direct ubiquitinylation and are thought to be involved in substrate recognition (39,40). Class III enzymes have both C- and N-terminal extensions, and the N-terminal domains are also important in substrate selection (41,42). The small E2 proteins have been called ubiquitin carrier proteins because of the mechanistic similarity to fatty acid activation. Some of the larger E2's however, can conjugate ubiquitin directly to target proteins (43), or to ubiquitin (44) and have thus been referred to as ubiquitin conjugating enzymes.

In some cases, the binding of a target protein by a specific binding protein is followed by the association with a ubiquitin carrier protein and ubiquitinylation of the target protein. These binding proteins are termed E3 ligase(s), have affinity for substrate proteins, and must bind E2, at least transiently, to transfer the ubiquitin from this carrier protein to the target protein. Genetic and biochemical evidence (1,7) suggests that reticulocyte E3[[alpha]] (UBR1 in yeast) has a site which is specific for a basic residue at the N-terminus and a second site which is specific for a large hydrophobic residue at the N-terminus of the target protein. A second E3, E3[[beta]], shares many functional and physical properties with E3[[alpha]], but recognizes substrates with A, S, or T as the amino-terminal residue (45). Another yeast factor (E3-R) appears to cooperate with the RAD6 gene product (UBC2) in catalyzing the mono-ubiquitinylation of several proteins, which are otherwise not ubiquitinylated by this E2 (46). Finally, a recently cloned human protein, E6-AP, binds to the complex of papilloma virus E6 and p53 causing its ubiquitinylation and subsequent degradation (47).

PROPROTEIN PROCESSING AND DISASSEMBLY OF POLY-UBIQUITIN CHAINS.

The above discussion details the formation of ubiquitinylated proteins. Equally important is the proteolytic processing of the variety of polymeric forms of ubiquitin. There are a number of steps in ubiquitin metabolism which require proteolytic processing by cleavage of amide bonds at the C-terminus of ubiquitin. In general, these processing enzymes are thiol proteases with specific recognition sites for ubiquitin. The cleavage must occur at gly76 of ubiquitin in order to accurately process these polymers. Two types of enzymes with this specificity have been described: the ubiquitin carboxyl-terminal hydrolases, which are thiol proteases of approximately 25,000 daltons (48) and; ubiquitin-specific proteases, a group of thiol proteases between 100 and 150 kDa (49,50).

Proofreading of protein ubiquitinylation. In principle, these enzymes may stimulate or inhibit ubiquitin-dependent processes. Inhibition could result from the deconjugation of ubiquitin from either mono- or poly-ubiquitinylated protein substrates. These reactions would reverse the conjugation of ubiquitin and define a "futile cycle". We have postulated that this futile cycle is a proof-reading mechanism designed to assure that only damaged proteins become marked and degraded by this system (11). This hypothesis suggests that ubiquitin is conjugated to many proteins. If the protein is stable, proteases are present which will recognize the native conformation of ubiquitin and remove it. If however, the protein is damaged, unstable, or denatured, the exposed hydrophobicity could cause a conformational change of ubiquitin. It is this altered conformation which is recognized by the committed step in the pathway, poly-ubiquitinylation. Short-lived regulatory substrates may have evolved ubiquitinylation sites which trigger this conformational change, resulting in their poly-ubiquitinylation and subsequent degradation.

Proteolytic processing of poly-ubiquitin. Alternatively. the action of these processing enzymes could stimulate ubiquitin-dependent processes by increasing the steady state level of free ubiquitin and by the hydrolysis of poly-ubiquitin chains. Accumulation of the latter would be expected to inhibit proteolysis, since these chains bind tightly to subunit 5 of the 26S proteasome (51). Enzymes with this catalytic activity are also required for the processing of all known ubiquitin gene products and metabolites, including adventitiously formed adducts between ubiquitin and small cellular nucleophiles. Clearly it is important to understand which enzymes catalyze these reactions and to determine their distribution and regulation.

Ubiquitin carboxyl-terminal hydrolases. Ubiquitin carboxyl-terminal hydrolases (UCH) are a class of small cytoplasmic thiol proteases with specificity for cleavage of small esters and amides of the carboxyl-terminal glycine of ubiquitin. We have identified and characterized four UCH activities from bovine thymus. Using antibodies to one of these proteins, we cloned and sequenced a human cDNA for the major thymus isozyme, UCH-L3 (48). The UCH-L3 sequence shows considerable similarity to a yeast protein which exhibits the same catalytic activity; a newly identified Drosophila protein prominent in nurse cells (52) and human PGP 9.5 (UCH-L1), a neuronal-specific protein.

Isozyme L1 is strongly expressed in neuronal, neuroendocrine, and perhaps some fetal cells. Isozyme L3 is present mainly in hematopoietic cells. Many tissues and cells contain significant amounts of isozyme L2 which may be a "housekeeping" isozyme (53). The expression of UCH isozymes is also developmentally regulated. The appearance of UCH-L1 (PGP 9.5) immunoreactivity in developing mouse brain correlates with the arrival of the neuronal precursor cells at the neural plate and the elaboration of neural processes (I. N. M. Day and R. Thompson, private communication). It is induced in the gonads of fish undergoing the sexual transition from female to male (54), in several neural inclusion bodies (55), and in experimentally-induced axonal dystrophy (56). Its level is strongly down-regulated upon viral transformation of lung fibroblasts (57). Similarly, in Drosophila, the mRNA for uch-D is strongly expressed in the nurse cell, the ovary and the testis (52). The transcripts are also easily identified for the first few hours of development. By 4 to 6 hours of development, the levels of transcript drop markedly, consistent with the pattern usually seen for maternal transcripts. Thus, the expression of UCH isozymes is temporally and spatially regulated.

Ubiquitin-specific proteases. Another class of enzymes with this general activity (hydrolysis of esters and amides at the C-terminus of ubiquitin) has been characterized in yeast (49,50). Three genes were identified based on the ability to hydrolyze large ubiquitin-protein fusions, (UBP1, 809 amino acids; UBP2, 1264 amino acids; and UBP3, 912 amino acids). These proteins exhibit homology in limited regions around presumed catalytic residues. Figure 2 shows 14 UBP sequences based on homology around the active site cysteine and histidines (58). There are nine yeast sequences, two human, one mouse and one fruit fly. The sizes vary widely from 408 to over 2700 amino acids. Most of this length variation is at the N-terminal site of the cys box. Little is known about the pattern of expression of these isozymes.

PROTEOLYSIS OF UBIQUITINYLATED PROTEINS BY THE PROTEASOME

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