Kinetic analysis of the conjugation of ubiquitin to picornavirus 3C proteases catalyzed by the mammalian ubiquitin-protein ligase E3alpha

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.     

The yeast Hex3.Slx8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation

Hex3 and Slx8 are Saccharomyces cerevisiae proteins with important functions in DNA damage control and maintenance of genomic stability. Both proteins have RING domains at their C termini. Such domains are common in ubiquitin and ubiquitin-like protein ligases (E3s), but little was known about the molecular functions of either protein. In this study we identified HEX3 as a high-copy suppressor of a temperature-sensitive small ubiquitin-related modifier (SUMO) protease mutant, ulp1ts, suggesting that it may affect cellular SUMO dynamics. Remarkably, even a complete deletion of ULP1 is strongly suppressed. Hex3 forms a heterodimer with Slx8. We found that the Hex3.Slx8 complex has a robust substrate-specific E3 ubiquitin ligase activity. In this E3 complex, Slx8 appears to bear the core ligase function, with Hex3 strongly enhancing its activity. Notably, SUMO attachment to a substrate stimulates its Hex3.Slx8-dependent ubiquitination, primarily through direct noncovalent interactions between SUMO and Hex3. Our data reveal a novel mechanism of substrate targeting in which sumoylation of a protein can help trigger its subsequent ubiquitination by recruiting a SUMO-binding ubiquitin ligase.     

Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis

Enterovirus 71 (EV71), a member of the Picornaviridae family, may cause serious clinical manifestations associated with the central nervous system. Enterovirus 3C protease is required for virus replication and can trigger host cell apoptosis via cleaving viral polyprotein precursor and cellular proteins, respectively. Although the role of the 3C protease in processing viral and cellular proteins has been established, very little is known about the modulation of EV71 3C function by host cellular factors. Here, we show that sumoylation promotes EV71 3C protein ubiquitination for degradation, correlating with a decrease of EV71 in virus replication and cell apoptosis. SUMO E2-conjugating enzyme Ubc9 was identified as an EV71 3C-interacting protein. Further studies revealed that EV71 3C can be SUMO (small ubiquitin-like modifier)-modified at residue Lys-52. Sumoylation down-regulated 3C protease activity in vitro and also 3C protein stability in cells, in agreement with data suggesting 3C K52R protein induced greater substrate cleavage and apoptosis in cells. More importantly, the recombinant EV71 3C K52R virus infection conferred more apoptotic phenotype and increased virus levels in culture cells, which also correlated with a mouse model showing increased levels of viral VP1 protein in intestine and neuron loss in the spinal cord with EV71 3C K52R recombinant viral infection. Finally, we show that EV71 3C amino acid residues 45-52 involved in Ubc9 interaction determined the extent of 3C sumoylation and protein stability. Our results uncover a previously undescribed cellular regulatory event against EV71 virus replication and host cell apoptosis by sumoylation at 3C protease.     

Localization and purification of two enzymes from Escherichia coli capable of hydrolyzing a signal peptide

The signal peptide generated during the maturation of prolipoprotein by the purified prolipoprotein signal peptidase can be isolated in substrate amounts (Dev, I. K., and Ray, P. H. (1984) J. Biol. Chem. 259, 11114-11120). This signal peptide is degraded predominantly from the carboxyl terminus by cell-free extracts of Escherichia coli. The signal peptide is degraded (at least 300-fold) more rapidly than other cellular proteins in E. coli. Greater than 90% of the signal peptide hydrolase activity is localized in the cytoplasm. Two enzymes from the cytoplasmic fraction responsible for the degradation of the signal peptide have been identified and purified to near homogeneity. The major activity is associated with a monomeric protein with a molecular weight of 68,000 (S.E. 3,400) as determined by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme appears to be similar to the oligopeptidase (Vimr, E. R., Green, L., and Miller, C. G. (1983) J. Bacteriol. 153, 1259-1265) that hydrolyzes N-acetyl tetra alanine. The second protein represents approximately 5% of the total cytoplasmic activity and has been shown to be a dimer with a monomer molecular weight of 81,000 (S.E. 5,300). This enzyme is similar to protease So (Chung, H. C., and Goldberg, A. L. (1983) J. Bacteriol. 154, 231-238).     

The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. In the frog, Xenopus laevis, the CDK inhibitor p27(Xic1) (Xic1) inhibits DNA synthesis by negatively regulating CDK2-cyclin E. Using the frog egg extract as a model system for the study of Xic1, studies have demonstrated that Xic1 protein levels are regulated by nuclear ubiquitination and proteolysis. To characterize the molecular mechanism that regulates Xic1 turnover, we have identified the minimal sequences of Xic1 that are necessary and sufficient for its nuclear ubiquitination and degradation. Using deletion mutagenesis, our studies indicated that the C-terminal 50 amino acids of Xic1 are critical for its proteolysis beyond a role in nuclear transport. Replacement of the Xic1 C terminus with the SV40 nuclear localization sequence resulted in the nuclear localization of Xic1 but not its ubiquitination or degradation. Our deletion studies also indicated that the CDK2-cyclin binding domain of Xic1 is important for its efficient retention in the nucleus. Further deletion analyses identified at least 3 lysine residues within the Xic1 C terminus that are targeted for specific ubiquitination. Importantly, our studies demonstrated that the Xic1 C-terminal 50 amino acids can serve as a nuclear degradation signal when fused to a stable heterologous nuclear protein. Moreover, a 30-amino-acid region within the C terminus of Xic1 can serve as a nuclear ubiquitination signal. To address the role of phosphorylation on Xic1 turnover, all the potential phosphorylation sites within the C-terminal 50 amino acids of Xic1 were mutated to alanine to prevent possible phosphorylation. This resulted in a Xic1 protein that was nevertheless degraded in a manner similar to wild-type Xic1, suggesting that phosphorylation of Xic1 is not critical for its nuclear ubiquitination or proteolysis.     

人泛素连接酶AREL1催化结构域的晶体结构

泛素化是一种重要的翻译后修饰,几乎调控着生命活动的所有方面.泛素连接酶是泛素化过程中唯一对底物蛋白质有特异性识别能力的一类酶,它们在泛素化过程中是不可或缺的,起到非常关键的作用.人抗凋亡E3泛素连接酶(AREL1)是HECT泛素连接酶家族成员之一,它能够泛素化促凋亡蛋白SMAC、HtrA2和ARTS,并通过蛋白酶体将它们降解,从而发挥抵抗细胞凋亡的作用.本文解析了3.2?分辨率的人AREL1蛋白催化结构域(AREL1HECT)的晶体结构,并将其与HECT家族中其他成员的结构进行了比对.尺寸排阻色谱和X射线小角散射的结果表明,AREL1HECT在溶液中是以多种聚集状态形式存在的,小角散射的3D模型进一步表明AREL1HECT在溶液中会发生二聚化.这些结果将为AREL1HECT与泛素复合物结构的解析及功能的分析提供坚实的结构基础,为揭示AREL1泛素化底物蛋白质的分子机制提供重要的依据.     

Signal peptidases and signal peptide hydrolases

Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two fromE. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the –3, –1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes inEscherichia coli that can degrade a signal peptidein vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptidein vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.     

Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response.

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen showed that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.     

Rubella virus cDNA. Sequence and expression of E1 envelope protein

A cDNA clone encoding the entire E1 envelope protein (410 amino acid residues) and a portion of the C-terminal end of the E2 envelope protein of the rubella virus has been isolated and characterized. DNA sequence analysis has revealed a region 20 nucleotides in length at the 3' end of the cloned cDNA which may be a replicase recognition site or a recognition site for encapsidation. The proteolytic cleavage site between the E1 and E2 proteins was localized based on the known amino-terminal sequence of the isolated E1 protein (Kalkkinen, N., Oker-Blom, C., and Pettersson, R. F. (1984) J. Gen. Virol. 65, 1549-1557) and the deduced amino acid sequence. The mature E1 protein is preceded by a set of 20 highly hydrophobic amino acid residues possessing characteristics of a signal peptide. This "signal peptide" is flanked on both sides by typical protease cleavage sites for trypsin-like enzyme and signal peptidase. The presence of a leader sequence in the E1 protein precursor may facilitate its translocation through the host cell membrane. The E1 protein of rubella virus shows no significant homology with alphavirus E1 envelope proteins. However, a stretch of 39 amino acids in the E1 protein of rubella virus (residues 262-300) was found to share a significant homology with the first 39 residues of bovine sperm histone. The position of 4 half-cystines and 8 arginines overlaps. The E1 protein of rubella virus has been successfully expressed in COS cells after transfecting them with rubella virus cDNA in simian virus 40-derived expression vector. This protein is antigenically similar to the one expressed by cells infected with rubella virus.     

Inducible expression of encephalomyocarditis virus 3C protease activity in stably transformed mouse cell lines.

An inducible expression vector system has been developed to facilitate the study of the effects of individual virus gene products on cell function. The vector utilizes the mouse metallothionein promoter carried on the bovine papillomavirus genome. Conditions which optimize the induced expression of open reading frames inserted downstream from the mouse metallothionein promoter have recently been described. In this communication we describe the use of this system to clone and express the encephalomyocarditis virus 3C protease in cultured mouse cells. Stably transformed cell lines could be induced to produce levels of 3C protease activity comparable to those observed during normal virus infection. In spite of this, no effects on cellular protein synthesis rate or membrane permeability were observed. It was also discovered that 3C protease as well as 3C protease-containing polyproteins are turned over. This was true not only in the induced cell clones, but also during the normal course of encephalomyocarditis virus infection, as well as in translation systems in vitro. This phenomenon was highly specific for this family of polypeptides, perhaps explaining their apparent lack of cytotoxic effects.     

Development of a high throughput scintillation proximity assay for hepatitis C virus NS3 protease that reduces the proportion of competitive inhibitors identified

A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC(50) values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening.     

Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.     

Rapid method for the preparation of encephalomyocarditis virus protease from rabbit reticulocyte lysates.

Fractionation of the reticulocyte lysate translation products of encephalomyocarditis virus RNA by ultracentrifugation showed that the viral proteins were distributed differentially in the supernatant and the ribosomal pellet fractions. The viral noncapsid proteins C and D, which contain the viral protease sequence, sedimented preferentially with the pellet fraction. Incubation of the resuspended pellet and subsequent centrifugation of the suspension resulted in cleavage of the protease from proteins C and D and separation of the enzyme from reticulocyte particulate proteins. Preparations thus obtained contained only three encephalomyocarditis virus proteins and were almost devoid of reticulocyte proteins.     

Modulation of protease activity to enhance the recovery of recombinant nucleocapsid protein of Nipah virus

The nucleocapsid (N) protein of Nipah virus (NiV) expressed in Escherichia coli (E. coli) is antigenic and immunogenic. A method to enhance the recovery of recombinant N protein of NiV produced in E. coli is described. A bioinformatics tool, PeptideCutter was used to identify potential protease and cleavage sites from the amino acid sequences deduced from the published DNA sequence of the N protein of NiV. The size of degraded protein was estimated by using the Western blot and PeptideCutter analyse. The identified proteases were serine proteases, hence, a range of serine protease inhibitors were tested to improve the recovery of the N protein. The relative amount of N protein of NiV was 2-fold higher with the addition of PMSF, compared to the control sample (without any protease inhibitor supplementation).     

In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation

Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.     

Identification of the polyprotein termination site on encephalomyocarditis viral RNA.

We show by sequence analysis of a 420-base-long region adjacent to the 3' polyadenylic acid of encephalomyocarditis viral RNA and by carboxy terminus analysis of protein E that the termination site of encephalomyocarditis virus polyprotein translation consists of two successive UAG codons located at positions 121 to 126 from the 3' polyadenylic acid.     

Site-specific mutations at a picornavirus VP3/VP1 cleavage site disrupt in vitro processing and assembly of capsid precursors.

Most proteolytic cleavages within the picornavirus polyproteins are carried out by viral protease 3C. For encephalomyocarditis virus, the protease 3C-catalyzed processing occurs between Gln-Gly or Gln-Ser amino acid pairs which are flanked by proline residues, but the sequence-specific constraints on recognition and cleavage by the enzyme are not completely understood. To examine alternative cleavage site sequences, we constructed a cDNA plasmid which expresses the viral L-P1-2A capsid precursor in vitro and introduced site-specific mutations into the Gln-Gly pair at the VP3/VP1 junction. The altered protein substrates were tested for cleavage activity in assays with protease 3C. The encephalomyocarditis virus 3C processed Gln-Ala as efficiently as its natural sites but did not cleave Gln-Val, Gln-Glu, Lys-Gly, Lys-Ala, Lys-Val, Lys-Glu, or Pro-Gly combinations. Displacement of the flanking proline residue by an engineered insertion slowed but did not prevent cleavage at this site. Also, a mutant defective in processing at the VP3/VP1 junction was unable to form 14S pentameric assembly intermediates in vitro.     

Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin

The secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.     

APC/C(Cdh1)-mediated degradation of the F-box protein NIPA is regulated by its association with Skp1

NIPA (Nuclear Interaction Partner of Alk kinase) is an F-box like protein that targets nuclear Cyclin B1 for degradation. Integrity and therefore activity of the SCF(NIPA) E3 ligase is regulated by cell-cycle-dependent phosphorylation of NIPA, restricting substrate ubiquitination to interphase. Here we show that phosphorylated NIPA is degraded in late mitosis in an APC/C(Cdh1)-dependent manner. Binding of the unphosphorylated form of NIPA to Skp1 interferes with binding to the APC/C-adaptor protein Cdh1 and therefore protects unphosphorylated NIPA from degradation in interphase. Our data thus define a novel mode of regulating APC/C-mediated ubiquitination.