Purification and characterization of arginyl-tRNA-protein transferase from rabbit reticulocytes. Its involvement in post-translational modification and degradation of acidic NH2 termini substrates of the ubiquitin pathway

Conjugation of ubiquitin to certain proteins can trigger their degradation. A major question concerns the structural features of a protein which make it susceptible to ubiquitin ligation. Recent studies have shown that the selection of proteins for degradation occurs most probably on a binding site of the ubiquitin-protein ligase (E3). It was shown that a free alpha-NH2 group is one important feature of the protein structure recognized by the ubiquitin-ligating enzyme. Proteins with basic or bulky hydrophobic residues in the NH2-terminal position are recognized by the ligase, marked by ubiquitin, and degraded. This is not true, however, for proteins with an acidic residue in this position. We have previously shown that a tRNA-dependent post-translational conjugation of arginine to acidic NH2 termini of proteins is essential for their degradation via the ubiquitin pathway, and we speculated that this modification is required for their recognition by the ligase. In the present study we have partially purified from rabbit reticulocytes the modifying enzyme, arginyl-tRNA-protein transferase, and characterized it. We have separated the enzyme from other known components of the ubiquitin system and shown that it is specifically required for degradation of proteins with either an aspartate or glutamate residue in their NH2-terminal position. We have shown that the action of the transferase is required for conjugation of ubiquitin to the substrate and most probably for its recognition by the ligase. The enzyme in its native form has a molecular mass of about 360 kDa. It appears to be a complex between several molecules of arginyl-tRNA synthetase and arginyl-tRNA-protein transferase.     

Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent

Reticulocyte lysate contains all the components of the ubiquitin-dependent proteolytic system. Several proteins are degraded in reticulocyte lysate in a ubiquitin-dependent manner. However, none of the proteins studied has a short intracellular half-life. We have investigated the degradation of ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells. ODC is efficiently degraded in reticulocyte lysate depleted of the ubiquitin activating enzyme, E1, in fraction II of reticulocyte lysate completely lacking ubiquitin, and in fraction II depleted of the entire complex of enzymes responsible for the ligation of ubiquitin to target proteins. The degradation of ODC is ATP dependent. Therefore, our results demonstrate that in addition to the ubiquitin-dependent proteolytic pathway, reticulocyte lysate contains at least one additional ATP-dependent proteolytic pathway. In vitro synthesized ODC served as a substrate in the present degradation study. Its successful utilization establishes a general strategy for investigating the degradation of short-lived proteins (for which a corresponding cDNA is available), that constitute a very small fraction of cellular proteins and for which purification is difficult or impossible. In contrast to ODC synthesized in vitro, that isolated from cells was not degraded by the reticulocyte lysate degradation system, suggesting that post-translational modifications may be involved in regulating ODC degradation.     

Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem f central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free α-NH₂ group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteo-lytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptile to ubiquitin-mediated degradation.     

Ubiquitin and its kin: how close are the family ties?

Modification of proteins by the covalent attachment of ubiquitin is known to target them for degradation by proteasomes. Several proteins have been discovered recently that are related to ubiquitin or function similarly. Some of these proteins act as modifiers; others bear ubiquitin-like domains embedded in their polypeptide chain but do not form conjugates with cellular proteins. Ubiquitin-like proteins mediate an impressive range of cellular functions, including cell-cycle progression, DNA repair and apoptosis. Recent discoveries endorse the view that, in many cases, the function of the relatives of ubiquitin is linked to the ubiquitin pathway.     

Mechanisms and regulation of the degradation of cyclin B

The degradation of the cyclin B subunit of protein kinase Cdk1/cyclin B is required for inactivation of the kinase and exit from mitosis. Cyclin B is degraded by the ubiquitin pathway, a system involved in most selective protein degradation in eukaryotic cells. In this pathway, proteins are targeted for degradation by ligation to ubiquitin, a process carried out by the sequential action of three enzymes: the ubiquitin-activating enzyme E1, a ubiquitin-carrier protein E2 and a ubiquitin-protein ligase E3. In the system responsible for cyclin B degradation, the E3-like function is carried out by a large complex called cyclosome or anaphase-promoting complex (APC). In the early embryonic cell cycles, the cyclosome is inactive in the interphase, but becomes active at the end of mitosis. Activation requires phosphorylation of the cyclosome/APC by protein kinase Cdk1/cyclin B. The lag kinetics of cyclosome activation may be explained by Suc1-assisted multiple phosphorylations of partly phosphorylated complex. The presence of a Fizzy/Cdc20-like protein is necessary for maximal activity of the mitotic form of cyclosome/APC in cyclin-ubiquitin ligation.     

Ubiquitin and intracellular protein degradation.

In eukaryotes, the ubiquitin-dependent protoelytic pathway is one of the major routes by which intracellular proteins are selectively destroyed. Recent work has shown that conjugation of ubiquitin to substrate proteins is mediated by a remarkably diverse array of enzymes. Proteolytic targeting may also be regulated at steps between ubiquitination of the substrate and its degradation to peptides by the multisubunit 26S protease. The complexity of the ubiquitin system suggests a central role for protein turnover in eukaryotic cell regulation.     

The protein substrate binding site of the ubiquitin-protein ligase system

In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.     

类泛素蛋白--SUMO

SUMO(small ubiquitin-related modifier)是泛素(ubiquitin)类蛋白家族的重要成员之一。尽管SUMO的生化反应途径与泛素相似,但不像泛素那样诱导底物蛋白降解。SUMO化能够使蛋白质更加稳定,进而调节许多关键的细胞活动。现从分类、结构、生化途径和生物学功能等方面介绍SUMO及SUMO化过程。     

SUMO与乳腺癌

小泛素修饰物（Small ubiquitin-like modifier,SUMO）是结构上与泛素类似的一种修饰蛋白,能与一些特定的靶蛋白共价连接。与泛素介导蛋白质的降解不同,SUMO化修饰调控主要对靶蛋白的功能,如在蛋白质的稳定性、细胞定位、信号转导、基因转录调控等方面均发挥着重要的作用。最近的研究表明：SUMO与乳腺癌的发生发展密切相关,它是通过SUMO化修饰参与并影响雌激素受体信号通路来实现的,本文将就此做一综述。     

Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines

Summary. Protein degradation mediated by the ubiquitin/proteasome system is the major route for the degradation of cellular proteins. In this pathway the ubiquitination of the target proteins is manifested via the concerted action of several enzymes. The ubiquinated proteins are then recognized and degraded by the 26S proteasome. There are few reports of proteins degraded by the 26S protesome without ubiquitination, with ornithine decarboxylase being the most notable representative of this group. Interestingly, while the degradation of ODC is independent of ubiquitination, the degradation of other enzymes of the polyamine biosynthesis pathway is ubiquitin dependent. The present review describes the degradation of enzymes and regulators of the polyamine biosynthesis pathway.     

Ubiquitin-protein ligases in muscle wasting

Muscle wasting occurs when rates of protein degradation outstrip rates of protein synthesis. Accelerated rates of protein degradation develop in atrophying muscle largely through activation of the ubiquitin-proteasome pathway. The complexity of the ubiquitination process, however, has hampered our understanding of how this pathway is activated in atrophying muscles and which enzymes of the ubiquitin conjugation system are responsible. Recent studies demonstrate that two ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MuRF1 are critical in the development of muscle atrophy. Other experiments implicate E2(14k) and E3alpha, of the N-end rule pathway, as important players in the process. It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as to develop inhibitors of, these E3s.     

The role of ubiquitin linkages on α-synuclein induced-toxicity in a Drosophila model of Parkinson's disease

Parkinson's disease (PD) is a common movement disorder marked by the loss of dopaminergic (DA) neurons in the brain stem and the presence of intraneuronal inclusions designated as Lewy bodies (LB). The cause of neurodegeneration in PD is not clear, but it has been suggested that protein misfolding and aggregation contribute significantly to the development of the disease. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Recent studies suggested that different types of ubiquitin linkages can modulate these two pathways in the process of protein degradation. In this study, we found that co-expression of ubiquitin can rescue neurons from α-syn-induced neurotoxicity in a Drosophila model of PD. This neuroprotection is dependent on the formation of lysine 48 polyubiquitin linkage which is known to target protein degradation via the proteasome. Consistent with our results that we observed in vivo , we found that ubiquitin co-expression in the cell can facilitate cellular protein degradation by the proteasome in a lysine 48 polyubiquitin-dependent manner. Taken together, these results suggest that facilitation of proteasomal protein degradation can be a potential therapeutic approach for PD.     

Interplay between autophagy and proteasome during protein turnover

Protein homeostasis is epitomized by an equilibrium between protein biosynthesis and degradation: the ‘life and death’ of proteins. Approximately one-third of newly synthesized proteins are degraded. As such, protein turnover is required to maintain cellular integrity and survival. Autophagy and the ubiquitin–proteasome system (UPS) are the two principal degradation pathways in eukaryotes. Both pathways orchestrate many cellular processes during development and upon environmental stimuli. Ubiquitination of degradation targets is used as a ‘death’ signal by both processes. Recent findings revealed a direct functional link between both pathways. Here, we summarize key findings in the field of protein homeostasis, with an emphasis on the newly revealed crosstalk between both degradation machineries and how it is decided which pathway facilitates target degradation.     

N-terminal ubiquitination: more protein substrates join in

The ubiquitin-proteasome system (UPS) is involved in selective targeting of innumerable cellular proteins through a complex pathway that plays important roles in a broad array of processes. An important step in the proteolytic cascade is specific recognition of the substrate by one of many ubiquitin ligases, E3s, which is followed by generation of the polyubiquitin degradation signal. For most substrates, it is believed that the first ubiquitin moiety is conjugated, through its C-terminal Gly76 residue, to an sigma-NH2 group of an internal Lys residue. Recent findings indicate that, for several proteins, the first ubiquitin moiety is fused linearly to the alpha-NH2 group of the N-terminal residue. An important biological question relates to the evolutionary requirement for an alternative mode of ubiquitination.     

Ubiquitin sorts proteins into the intralumenal degradative compartment of the late-endosome/vacuole

Many studies have demonstrated a role for ubiquitin (Ub) in the down-regulation of cell surface proteins. In yeast, down-regulation is marked by the internalization of proteins, followed by their delivery to the lumen of the vacuole where both the cytosolic and lumenal domains are degraded. It is generally believed that the regulatory step of this process is internalization from the plasma membrane and that protein delivery to the lysosome or vacuole is by default. By separating the process of internalization from degradation, we demonstrate that incorporation of proteins into intralumenal vesicles represents a distinct sorting step along the endocytic pathway that is controlled by recognition of ubiquitin. We show that attachment of a single ubiquitin can serve as a specific sorting signal for the degradative pathway by redirecting recycling Golgi proteins and resident vacuolar proteins into intralumenal vesicles of the yeast vacuole. This pathway is independent of PtdIns(3,5) P2 and does not rely on the specific composition of transmembrane domain segments. These data provide a physiological basis for how ubiquitination of cell surface proteins guides their degradation and removal from the recycling pathway.     

p53 regulation by ubiquitin

The ubiquitination pathway is a highly dynamic and coordinated process that regulates degradation as well as numerous processes of proteins within a cell. The p53 tumor suppressor and several factors in the pathway are regulated by ubiquitin as well as ubiquitin-like proteins. These modifications are critical for the function of p53 and control both the degradation of the protein as well as localization and activity. Importantly, more recent studies have identified deubiquitination enzymes that can specifically remove ubiquitin moieties from p53 or other factors in the pathway, and the reversible nature of this process adds yet another layer of regulatory control of p53. This review highlights the recent advances in our knowledge of ubiquitin and the p53 pathway.     

Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1

Protein quality control and subsequent elimination of terminally misfolded proteins occurs via the ubiquitin-proteasome system. Tagging of misfolded proteins with ubiquitin for degradation depends on a cascade of reactions involving an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). While ubiquitin ligases responsible for targeting misfolded secretory proteins to proteasomal degradation (ERAD) have been uncovered, no such E3 enzymes have been found for elimination of misfolded cytoplasmic proteins in yeast. Here we report on the discovery of Ubr1, the E3 ligase of the N-end rule pathway, to be responsible for targeting misfolded cytosoplasmic protein to proteasomal degradation.     

Transferring substrates to the 26S proteasome

Ubiquitin-dependent protein degradation is not only involved in the recycling of amino acids from damaged or misfolded proteins but also represents an essential and deftly controlled mechanism for modulating the levels of key regulatory proteins. Chains of ubiquitin conjugated to a substrate protein specifically target it for degradation by the 26S proteasome, a huge multi-subunit protein complex found in all eukaryotic cells. Recent reports have clarified some of the molecular mechanisms involved in the transfer of ubiquitinated substrates from the ubiquitination machinery to the proteasome. This novel substrate transportation step in the ubiquitin-proteasome pathway seems to occur either directly or indirectly via certain substrate-recruiting proteins and appears to involve chaperones.