Inhibition of ubiquitin-dependent proteolysis by des-Gly-Gly-ubiquitin: implications for the mechanism of polyubiquitin synthesis

Cleavage of the two carboxyl-terminal glycine residues from native ubiquitin yields the proteolysis-incompetent derivative des-Gly-Gly-ubiquitin. We report here that this derivative inhibits the ATP-dependent degradation of casein and is multi-ubiquitinated but not degraded by reticulocyte lysates. Inhibition of proteolysis diminished with increasing concentration of native ubiquitin, but was not reduced by increased casein concentration. Cleavage of the last four residues from ubiquitin yielded a derivative that was a weaker inhibitor of proteolysis and a poorer substrate for ubiquitination. These results suggest that the conjugation of ubiquitin to ubiquitin during polyubiquitin synthesis involves a specific conjugation system that recognizes ubiquitin and some of its derivatives, but not general proteolysis substrates, as ubiquitin acceptors.     

The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor.

Programmed proteolysis of proteins such as mitotic cyclins and Cut2/Pds1p requires a 9-residue conserved motif known as the destruction box (D-box). Strong expression of protein fragments containing destruction boxes, such as the first 70 residues of Cdc13 (N70), inhibits the growth of Schizosaccharomyces pombe at metaphase. This inhibition can be overcome either by removal of all lysine residues from N70 using site-directed mutagenesis (K0-N70) or by raising the concentration of intracellular ubiquitin. Consistent with the idea that competition for ubiquitin accounts for some of its inhibitory effects, wild-type N70 not only stabilized D-box proteins, but also Rum1 and Cdc18, which are degraded by a different pathway. The K0-N70 construct was neither polyubiquitinated nor degraded in vitro, but it blocked the growth of strains of yeast in which anaphase-promoting complex/cyclosome (APC/C) function was compromised by mutation, and specifically inhibited proteolysis of APC/C substrates in vivo. Both K0-N70 and 20-residue D-box peptides blocked polyubiquitination of other D-box-containing substrates in a cell-free ubiquitination assay system. These data suggest the existence of a D-box receptor protein that recognizes D-boxes prior to ubiquitination.     

A specific inhibitor of the ubiquitin activating enzyme: synthesis and characterization of adenosyl-phospho-ubiquitinol, a nonhydrolyzable ubiquitin adenylate analogue

A nonhydrolyzable analogue of ubiquitin adenylate has been synthesized for use as a specific inhibitor of the ubiquitination of proteins. Ubiquitin adenylate is a tightly bound intermediate formed by the ubiquitin activating enzyme. The inhibitor adenosyl-phospho-ubiquitinol (APU) is the phosphodiester of adenosine and the C-terminal alcohol derived from ubiquitin. APU is isosteric with the normal reaction intermediate, the mixed anhydride of ubiquitin and AMP, but results from the replacement of the carbonyl oxygen of Gly76 with a methylene group. This stable analogue would be expected to bind to both ubiquitin and adenosine subsites and result in a tightly bound competitive inhibitor of ubiquitin activation. APU inhibits the ATP-PPi exchange reaction catalyzed by the purified ubiquitin activating enzyme in a manner competitive with ATP (Ki = 50 nM) and noncompetitive with ubiquitin (Ki = 35 nM). AMP has no effect on the inhibition, confirming that the inhibitor binds to the free form of the enzyme and not the thiol ester form. This inhibition constant is 10-fold lower than the dissociation constants for each substrate and 30-1000-fold lower than the respective Km values for ubiquitin and ATP. APU also effectively inhibits conjugation of ubiquitin to endogenous proteins catalyzed by reticulocyte fraction II with an apparent Ki of 0.75 microM. This weaker inhibition is consistent with the fact that activation of ubiquitin is not rate limiting in the conjugation reactions catalyzed by fraction II. APU is similarly effective as an inhibitor of the ubiquitin-dependent proteolysis of beta-lactoglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions

We have established a Saccharomyces cerevisiae genetic system that expresses the fusion protein ubiquitin-metallothionein. We have evaluated the effects of amino-terminal ubiquitination of metallothionein on the stability and function of metallothionein. The fusion protein of wild type ubiquitin and metallothionein was rapidly processed in vivo to release free ubiquitin and metallothionein. Site-directed mutants of ubiquitin-metallothionein expressed in yeast were used to study the specificity of the (alpha-NH2-ubiquitin) protein endopeptidases. The data suggest that amino-terminal ubiquitination is not a signal for the proteolysis of yeast metallothionein in yeast. We have also discovered that expression of selected ubiquitin mutants blocked the growth of yeast. The data suggest that in addition to its function as a proteolytic signal, ubiquitination of proteins plays multiple roles in the cell.     

Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.     

MDA-9/syntenin interacts with ubiquitin via a novel ubiquitin-binding motif

Ubiquitination appears to be involved in proteasome-dependent proteolysis and in the membrane trafficking system including endocytosis and exocytosis. In this study, we identified MDA-9/syntenin as a novel ubiquitin-binding protein by a yeast two-hybrid system using modified ubiquitin in which lysine 48 is substituted by arginine. It has been reported that MDA-9/syntenin is a membrane-associated protein and regulates a cellular process involving endocytosis and intracellular transport. We found that MDA-9/syntenin binds to ubiquitin by a non-covalent bond and is ubiquitinated covalently. MDA-9/syntenin has no ubiquitin-binding motifs that have so far been reported, suggesting that MDA-9/syntenin physically interacts with ubiquitin via a novel binding motif. MDA-9/syntenin is stable in the cell, suggesting that ubiquitin binding of MDA-9/syntenin or ubiquitination of MDA-9/syntenin is not related to proteolysis. Furthermore, we showed that overexpression of wild-type MDA-9/syntenin enhances formation of filopodia, whereas MDA-9/syntenin lacking the PDZ domain inhibits the formation of filopodia, suggesting that MDA-9/syntenin plays an important role via interaction with ubiquitin in the regulation of cancer metastasis and invasion.     

Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.     

TIP120A associates with unneddylated cullin 1 and regulates its neddylation

The cullin-containing E3 ubiquitin ligases play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. We recently identified TIP120A as a cullin-interacting protein and found that TIP120A functions as a negative regulator of a ubiquitin ligase by interfering with the binding of Skp1 and an F box protein to CUL1. Here we show that TIP120A binds to the unneddylated CUL1 but not the neddylated one. The association of TIP120A with CUL1 requires both the N-terminal stalk and the C-terminal globular domain of CUL1. TIP120A efficiently inhibits neddylation of CUL1 but does not affect substrate-independent ubiquitination by CUL1/Rbx1, implying that it blocks the access of Nedd8 to the conjugation site but does not interfere with the interaction of the ubiquitin-conjugating enzyme with Rbx1. Our data suggest that the association/dissociation of TIP120A coupled to neddylation/deneddylation of CUL1 may play an important role in assembly and disassembly of Skp1-Cdc53/cullin-F box ubiquitin ligases.     

Centrosomal microtubule nucleation activity is inhibited by BRCA1-dependent ubiquitination

In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of gamma-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of gamma-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of gamma-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.     

Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly‐ubiquitinated by the proteasome‐associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat‐shock or arsenite treatment, when poly‐ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin‐conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.     

The N-terminal Regulatory Domain of Cyclin A Contains Redundant Ubiquitination Targeting Sequences and Acceptor Sites

Cyclin A is destroyed during mitosis by the ubiquitin-proteasome system. Like cyclin B, a destruction box (D-box) motif is required for the destruction of cyclin A. However, Cyclin A degradation is more complicated than cyclin B because cyclin A’s D-box motif is more extensive and proteolysis involves complex signaling in some organisms. In this study, we found that in addition to the D-box, the region between residues 123-157 also contributed to the ubiquitination and degradation of human cyclin A. Indeed, removal of the bulk of the N-terminal regulatory domain was needed to completely stabilize cyclin A and eliminate ubiquitination. A putative second RxxL motif around residue 138 played only a minor role in cyclin A degradation. To distinguish between sequences recognized by the ubiquitination machinery and the ubiquitin acceptor sites per se, we utilized a novel approach involving in vitro cleavage of cyclin A after ubiquitination. We found that several lysine residues proximal to the D-box (Lys37, Lys54, and Lys68) were ubiquitin acceptor sites. Cyclin A lacking the three lysine residues was degraded slower than the wild-type protein. Although these lysines were normally used, ubiquitination could shift to other cryptic sites when the preferred sites were unavailable, suggesting the exact positions of the ubiquitin chains also contributed to degradation. Together, these data revealed that ubiquitination does not occur randomly on cyclin A and open up questions on the precise function of the D-box.     

Identification and mechanistic studies of a novel ubiquitin E1 inhibitor

Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.     

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Delivery of ubiquitinated substrates to protein-unfolding machines

Recent work has shown that ubiquitination leads to recognition of target proteins by diverse ubiquitin receptors. One family of receptors delivers the ubiquitinated proteins to the proteasome resulting in ATP-dependent substrate unfolding and proteolysis. A related family of ubiquitin-binding proteins seems to recruit ubiquitinated proteins to Cdc48, an ATPase ring complex that can also unfold proteins. Some targets seem to dock at Cdc48 before the proteasome does, in an ordered pathway. The intimate interplay between the proteasome and Cdc48, mediated in part by loosely associated ubiquitin receptors, has important functions in cellular regulation.     

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.     

The ubiquitin ligase SCF(Grr1) is required for Gal2p degradation in the yeast Saccharomyces cerevisiae

F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCF(Grr1), including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCF(Grr1)-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1Delta mutant cells imply that Gal2p is not the primary target of SCF(Grr1) ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.     

A small molecule ubiquitination inhibitor blocks NF-kappa B-dependent cytokine expression in cells and rats

A small molecule inhibitor of NF-kappaB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) alpha-induced IkappaB(alpha) degradation in MM6 cells but not the degradation of beta-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFalpha, interleukin-1beta, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC(50) values below 1 microm. Ro106-9920 also blocked TNFalpha production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require betaTRCP but associates with IkappaB(alpha) and will ubiquitinate IkappaB(alpha) S32E,S36E (IkappaB(alpha)(ee)) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IkappaB(alpha)(ee) ubiquitination with an IC(50) value of 2.3 +/- 0.09 microm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF(betaTRCP), the putative E3 for IkappaB(alpha) ubiquitination. Ro106-9920 was observed to be selective for IkappaB(alpha)(ee) ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFalpha-induced IkappaB(alpha) degradation and NF-kappaB activation.     

E3 ubiquitin-protein ligase activity of Parkin is dependent on cooperative interaction of RING finger (TRIAD) elements

The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinson's disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity.