植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

Cloning and characterization of a gene encoding the human putative ubiquitin conjugating enzyme E2Z (UBE2Z)

Ubiquitination, the covalent attachment of the protein ubiquitin (Ub) to other cellular proteins, has been implicated in a number of important physiological processes. ubiquitin conjugating enzyme (E2) plays an important role in the ubiquitin system. Here we report the research about a human putative ubiquitin conjugating enzyme gene, UBE2Z. The length of UBE2Z cDNA is 3054 base pairs and contains an open reading frame encoding 246 amino acids. The UBE2Z gene was mapped to human chromosome 17q21.32 and consisted of 6 exons. RT-PCR showed that UBE2Z was widely expressed in human tissues, especially high in placenta, pancreas, spleen and testis. The UBE2Z-GFP fusion protein was located in both nucleus and cytoplasm of AD293 cells.     

Structural Basis for Non-Covalent Interaction Between Ubiquitin and the Ubiquitin Conjugating Enzyme Variant Human MMS2

Modification of proteins by post-translational covalent attachment of a single, or chain, of ubiquitin molecules serves as a signaling mechanism for a number of regulatory functions in eukaryotic cells. For example, proteins tagged with lysine-63 linked polyubiquitin chains are involved in error-free DNA repair. The catalysis of lysine-63 linked polyubiquitin chains involves the sequential activity of three enzymes (E1, E2, and E3) that ultimately transfer a ubiquitin thiolester intermediate to a protein target. The E2 responsible for catalysis of lysine-63 linked polyubiquitination is a protein heterodimer consisting of a canonical E2 known as Ubc13, and an E2-like protein, or ubiquitin conjugating enzyme variant (UEV), known as Mms2. We have determined the solution structure of the complex formed by human Mms2 and ubiquitin using high resolution, solution state nuclear magnetic resonance (NMR) spectroscopy. The structure of the Mms2–Ub complex provides important insights into the molecular basis underlying the catalysis of lysine-63 linked polyubiquitin chains.     

Characterization and function of an E2-17 kDa (UBE2D) in an invertebrate Haliotis diversicolor supertexta

Ubiquitin-conjugating enzymes (UBE2s or E2s) are characterized by the presence of a highly conserved ubiquitin-conjugating (UBC) domain, which predominantly determines the type of ubiquitin chains and directly controls the cellular fate of the substrate. In this study, an E2 homolog was identified and functionally characterized in abalone, which we named ab-UBE2D. The full-length cDNA consists of 1005 bp with an ORF encoding a protein of 147 amino acids. The deduced amino acid sequence shows ab-UBE2D shares conserved UBC domain with other E2 proteins and belongs to class I E2 enzyme family, which are further confirmed by phylogenetic tree analysis. Real-time PCR and western blot analyses showed that ab-UBE2D was ubiquitously expressed in abalone and the expression level of ab-UBE2d was significantly induced by LPS and Poly (I:C). Immunofluorescence microscopy staining demonstrated that native ab-UBE2D was mainly distributed in the cytoplast. Ubiquitination assay showed that ab-UBE2D had ubiquitin conjugating activity to form the enzyme-(Ub)n conjugates. Taken together, these results strongly suggest that ab-UBE2D is an E2 homolog and it may be involved in the immune response of abalone, Haliotis diversicolor supertexta.     

MOB2基因在真核细胞中的表达研究

通过RT-PCR从培养的HUVECs中扩增MOB2基因片段,将该片段克隆在真核表达载体pEGFP-C1中,转染NIH3T3细胞,经G418筛选,构建稳定表达细胞系,并用荧光显微镜和Western blot对其进行鉴定。经RT-PCR扩增出734 bp基因片段,经测序分析并与GenBank的DNA序列比对分析后,在NIH3T3细胞中表达。G418筛选后,细胞荧光信号较强,Western blot检测,细胞中的融合蛋白能够与抗MOB2的多抗结合。成功地扩增了HUVECs中的MOB2基因全长cDNA并进行真核表达与鉴定。     

Expression,purification and characterization of human ubiquitin-activating enzyme,UBE1

UBE1 plays an important role in the first step of ubiquitin–proteasome pathway to activate ubiquitin. Both the structure and biochemical property research of human UBE1 protein, and the activity analysis of those enzymes which are related with ubiquitination pathway, are based on high purity of UBE1 protein. To obtain human UBE1 protein, the full length of human UBE1 was expressed in E. coli and purified by Ni-NTA superflow sepharose and strep-tactin sepharose which based on UB–UBE1 high-energy thioester bonded intermediate complex. It was demonstrated that purified UBE1 could activate and conjugate UB to ubiquitin-conjugating enzyme E2s. The purified large amount of UBE1 could be used for in vitro studies of ubiquitin pathway and structural studies.     

Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems: comparison with the recombinant enzyme purified from Escherichia coli.

Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for biochemical and physical characterization. However, the effective use of heterologous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" protein. For human sulfotransferases, little attention has been paid to this despite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine-metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharomyces cerevisiae, COS-7, and V79 cells, by comparison of Km values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expression. This is the first report of heterologous expression of a cytosolic sulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell types, and the Km for dopamine under the conditions applied was approximately 1 microM in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE, electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with Mr approximately 68,000 as determined by light scattering and sedimentation analysis. Mass spectrometry revealed two species with experimentally determined masses of 34,272 and 34,348 which correspond to the native protein with either one or two 2-mercaptoethanol adducts. We conclude that the enzyme expressed in prokaryotic and eukaryotic heterologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations.     

Structural basis of generic versus specific E2–RING E3 interactions in protein ubiquitination

Protein ubiquitination is a fundamental regulatory component in eukaryotic cell biology, where a cascade of ubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymes assemble distinct ubiquitin signals on target proteins. E2s specify the type of ubiquitin signal generated, while E3s associate with the E2~Ub conjugate and select the substrate for ubiquitination. Thus, producing the right ubiquitin signal on the right target requires the right E2–E3 pair. The question of how over 600 E3s evolved to discriminate between 38 structurally related E2s has therefore been an area of intensive research, and with over 50 E2–E3 complex structures generated to date, the answer is beginning to emerge. The following review discusses the structural basis of generic E2–RING E3 interactions, contrasted with emerging themes that reveal how specificity can be achieved.     

Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2

Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.     

泛素链的形成机理

泛素化是真核细胞中重要的蛋白质翻译后修饰过程,通过靶向蛋白质降解或其他信号途径参与多种细胞功能.底物蛋白的多聚泛素化修饰是一个持续的过程,其中不仅涉及复杂泛素系统相关酶的参与,而且存在更为复杂的结构上相互作用与泛素链组装机理.不同的泛素链修饰决定了底物蛋白下游的不同命运,泛素结合酶E2在泛素链形成中的重要作用受到越来越...     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

A plant‐specific in vitro ubiquitination analysis system

Protein ubiquitination requires the concerted action of three enzymes: ubiquitin‐activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3). These ubiquitination enzymes belong to an abundant protein family that is encoded in all eukaryotic genomes. Describing their biochemical characteristics is an important part of their functional analysis. It has been recognized that various E2/E3 specificities exist, and that detection of E3 ubiquitination activity in vitro may depend on the recruitment of E2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes from Arabidopsis. It includes most varieties of Arabidopsis E2 proteins, which are tested with several RING‐finger type E3 ligases. This system permits determination of E3 activity in combination with most of the E2 sub‐groups that have been identified in the Arabidopsis genome. At the same time, E2/E3 specificities have also been explored. The components used in this system are all from plants, particularly Arabidopsis, making it very suitable for ubiquitination assays of plant proteins. Some E2 proteins that are not easily expressed in Escherichia coli were transiently expressed and purified from plants before use in ubiquitination assays. This system is also adaptable to proteins of species other than plants. In this system, we also analyzed two mutated forms of ubiquitin, K48R and K63R, to detect various types of ubiquitin conjugation.     

Expression,purification, and crystal structure of N-terminal domains of human ubiquitin-activating enzyme (E1)

Ubiquitin-activating enzyme (E1) is a key regulator in protein ubiquitination, which lies on the upstream of the ubiquitin-related pathways and determines the activation of the downstream enzyme cascade. Thus far, no structural information about the human ubiquitin-activating enzyme has been reported. We expressed and purified the N-terminal domains of human E1 and determined their crystal structures, which contain inactive adenylation domain (IAD) and the first catalytic cysteine half-domain (FCCH). This study presents the crystal structure of human E1 fragment for the first time. The main structure of both IAD and FCCH superimposed well with their corresponding domains in yeast Uba1, but their relative positions vary significantly. This work provides new structural insights in understanding the mechanisms of ubiquitin activation in humans.     

Solution structure of the ubiquitin-conjugating enzyme UbcH5B

The ubiquitination pathway is the main pathway for protein degradation in eukaryotic cells. The attachment of ubiquitin to a substrate protein is catalyzed by three types of enzymes, namely a ubiquitin activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Here, the structure of the human ubiquitin-conjugating enzyme (E2) UbcH5B has been solved by a combination of homology modeling, NMR relaxation data and automated NOE assignments. Comparison to E2 structures solved previously by X-ray crystallography or NMR shows in all cases the same compact fold, but differences are observed in the orientation of both N and C-terminal alpha-helices. The N-terminal helix that is involved in binding to ubiquitin ligases (E3) displays a different position, which could have consequences for precise E2-E3 recognition. In addition, multiple conformations of the side-chain of Asn77 are found in solution, which contrasts the single hydrogen-bonded conformation in the crystal structures of E2 enzymes. The possible implication of this conformational freedom of Asn77 for its catalytic function is discussed.     

E2-binding surface on Uba3 β-grasp domain undergoes a conformational transition

The covalent attachment of ubiquitin (Ub) and ubiquitin‐like (Ubl) proteins to various eukaryotic targets plays critical roles in regulating numerous cellular processes. E1‐activating enzymes are critical, because they catalyze activation of their cognate Ub/Ubl protein and are responsible for its transfer to the correct E2‐conjugating enzyme(s). The activating enzyme for neural‐precursor‐cell‐expressed developmentally downregulated 8 (NEDD8) is a heterodimer composed of APPBP1 and Uba3 subunits. The carboxyl terminal ubiquitin‐like β‐grasp domain of human Uba3 (Uba3‐βGD) has been suggested as a key E2‐binding site defining E2 specificity. In crystal structures of free E1 and the NEDD8‐E1 complex, the E2‐binding surface on the domain was missing from the electron density. However, when complexed with various E2s, this missing segment adopts a kinked α‐helix. Here, we demonstrate that Uba3‐βGD is an independently folded domain in solution and that residues involved in E2 binding are absent from the NMR spectrum, indicating that the E2‐binding surface on Uba3‐βGD interconverts between multiple conformations, analogous to a similar conformational transition observed in the E2‐binding surface of SUMO E1. These results suggest that access to multiple conformational substates is an important feature of the E1–E2 interaction. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.     

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin‐conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co‐crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.     

激光扫描共聚焦显微镜研究APOBEC3G蛋白的亚细胞定位

采用RT-PCR技术,从HIV的非允许性H9细胞中获得载脂蛋白B mRNA编辑酶催化多肽样蛋白3G(APOBEC3G)的全长cDNA。APOBEC3G cDNA全长1 155nt,编码384个氨基酸。将APOBEC3G克隆到真核表达载体pEGFP-C3上,转染CD4 HeLa细胞,激光扫描共聚焦显微镜下可观察到表达的GFP-APOBEC3G融合蛋白定位于细胞质。     

A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1

The effect of restrictive temperature on ubiquitin conjugation activity has been studied in cells of ts20, a temperature-sensitive cell cycle mutant of the Chinese hamster cell line E36. Ts20 is arrested in early G2 phase at nonpermissive temperature. Immunoblotting with antibodies to ubiquitin conjugates shows that conjugates disappear rapidly at restrictive temperatures in ts20 mutant but not in wild type E36 cells. The incorporation of 125I-ubiquitin into permeabilized ts20 cells is temperature-sensitive. Addition of extracts of another G2 phase mutant, FM3A ts85, with a temperature-sensitive ubiquitin activation enzyme (E1), to permeabilized ts20 cells at restrictive temperatures fails to complement their ubiquitin ligation activity. This indicates that the lesions in the two mutants are similar. Purified E1 from reticulocytes restores the conjugation activity of heat-inactivated permeabilized ts20 cells. Ubiquitin conjugation activity of cell-free extracts of ts20 cells was temperature-sensitive and could be restored by adding purified reticulocyte E1. Purified reticulocyte E2 or E3, on the other hand, did not restore the ubiquitin conjugation activity of heat-treated ts20 extracts. These results are consistent with the conclusion that ts20 has temperature-sensitive ubiquitin-activating enzyme (E1). The fact that two E1 mutants (ts20 and ts85) derived from different cell lines are arrested at the S/G2 boundary at restrictive temperatures strongly indicates that ubiquitin ligation is necessary for passage through this part of the cell cycle. The temperature thresholds of heat shock protein synthesis of ts20 and wild type E36 cells were identical. The implications of these findings with respect to a suggested role of ubiquitin in coupling between protein denaturation and the heat shock response are discussed.