蛋白酶体β5亚单位过表达对氧化环境下人晶状体上皮细胞的影响

【目的】探讨蛋白酶体筇亚单位（PSMB5）基因过表达对氧化条件下晶状体上皮细胞的影响。【方法】构建重组质粒pcDNA3．1-PSMB5并将其转染人人晶状体上皮细胞株SRAO1／04中，形成稳定表达，同时设pcDNA3．1空载体为对照组。RT—PCR法及Western blot法分别检测PSMB5基因及蛋白的表达情况。H2O2分别作用PSMB5转染细胞及空载体转染细胞，MTT法检测细胞增殖能力的变化。【结果】转染后用G418筛选3周后，获得PSMB5转染及空载体转染的G418抗性的细胞克隆。转染PSMB5基因的细胞PSMB5 mRNA表达强度明显增高．而空载体转染细胞与未转染细胞无显著差异：转染PSMB5基因的细胞的PSMB5蛋白表达也显著高于空载体转染细胞。低浓度H2O2作用后，转染PSMB5基因的细胞增殖能力高于空载体转染细胞。【结论】低浓度氧化环境下蛋白酶体历亚单位PSMB5过表达能对人晶状体上皮细胞具有保护作用。     

UBE2S drives elongation of K11-linked ubiquitin chains by the Anaphase-Promoting Complex

The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.     

DangER: protein ovERload. Targeting protein degradation to treat myeloma

Myeloma is a malignancy of the antibody-producing plasma cells and, as such, these cells synthesize large quantities of unfolded or misfolded immunoglobulin. The build-up of excess protein triggers a number of downstream signal transduction cascades, including endoplasmic reticulum stress and autophagy. As a result, myeloma cells are uniquely reliant on these and other protein handling pathways for their survival. Strategies aimed at targeting this vulnerability have proved successful with the proteasome inhibitor, bortezomib, already licensed for clinical use. In addition to the proteasome, various other points within the protein handling pathways are also the subject of drug discovery projects, with some already progressing into clinical trials. These include compounds directed against heat shock proteins, the unfolded protein response and pathways both upstream and downstream of the proteasome. More recently, the role of autophagy has been recognized in myeloma. In this review, we discuss the various pathways used by myeloma cells for survival, with particular emphasis on the emerging role and conundrum of autophagy, as well as highlighting pre-clinical research on novel inhibitors targeting protein handling pathways.     

Poxvirus ankyrin repeat proteins are a unique class of F-box proteins that associate with cellular SCF1 ubiquitin ligase complexes

F-box proteins direct the degradation of an extensive range of proteins via the ubiquitin-proteasome system. Members of this large family of proteins are typically bipartite. They recruit specific substrates through a substrate-binding domain and, via the F-box, link these to core components of a major class of ubiquitin ligases (SCF1). F-box proteins thus determine the specificity of SCF1-mediated ubiquitination. F-box-like motifs were recently detected in poxvirus ankyrin repeat (ANK) proteins but clear compositional differences to typical F-box proteins raise questions regarding the classification and function of the motif. Here we show that all five ANK proteins of a representative poxvirus, Orf virus, interact in vivo with core components of the SCF1 ubiquitin ligase complex. Interaction is dependent on the poxviral F-box-like motif and the adaptor subunit of the complex (SKP1). The viral protein does not block enzymatic activity of the complex. These observations identify the poxviral motif as a functional F-box. They also identify a new class of F-box that in contrast to cellular counterparts is truncated, has an extreme C-terminal location and is paired with an ANK protein-binding domain. ANK proteins constitute the largest family of poxviral proteins but their function and the significance of their abundance have remained an enigma. We propose that poxviruses use these unique ANK/F-box proteins to dictate target specificity to SCF1 ubiquitin ligases and thereby exploit the cell's ubiquitin-proteasome machinery.     

Proteasome inhibition in hematologic malignancies

Hematologic malignancies, including multiple myeloma (MM), will account for more than 100,000 new cases of cancer and over 57,000 deaths in the United States in 2003. Treatment of MM is a serious challenge, because despite a variety of available therapies, median survival is short. A new therapeutic area focuses on inhibiting the activity of the proteasome, a 26S protease complex involved in cell cycle regulation, cell adhesion, inflammation, and protein turnover. The novel proteasome inhibitor, bortezomib (Velcade®), was recently approved for use in patients with refractory and relapsed MM and to date is the only proteasome inhibitor to have entered clinical trials. Bortezomib has demonstrated activity with manageable toxicity in a variety of hematologic malignancies in addition to MM, including leukemia and non‐Hodgkin's lymphoma. This article reviews clinical information on bortezomib in hematologic malignancies both as monotherapy and in combination with dexamethasone. Preliminary reports of bortezomib in combination with Doxil (pegylated liposomal doxorubicin), melphalan, and thalidomide are discussed, and current trials are described. Available data suggest that bortezomib will be useful in the treatment of a variety of hematologic malignancies.     

Pentosidine accumulation in human oocytes and their correlation to age-related apoptosis

Age-related atresia of ovarian follicles is characterized by apoptosis of the constituent cells. Recent studies have indicated that dysfunction of the proteasome and endoplasmic reticulum and subsequent apoptosis in the presence of oxidative stress have relevance to aging. The aim of this study was to assess the involvement of these processes in age-related follicular atresia. Formalin-fixed, paraffin-embedded sections of ovaries obtained at surgery from 74 women (age: 21-54 y) were examined with the terminal deoxynucleotidyl transferase-mediated, dUTP-biotin nick-end labeling (TUNEL) method and an immunohistochemical technique. Primary antibodies used in immunohistochemistry were against pentosidine, ubiquitin and caspase 12. Histological localization of these substances in oocytes was observed by light microscopy, and labeling indices of these cells were evaluated by regression analysis. Positive signals for pentosidine, ubiquitin, caspase 12, and TUNEL were detectable in oocytes of the primordial, primary and their atretic follicles. Regression analysis revealed an age-related increase in the labeling indices for pentosidine, ubiquitin, caspase 12, and TUNEL. These results suggest that pentosidine accumulation in human oocytes is related to apoptosis and increases with age. Further studies will be necessary to clarify the involvement of pentosidine accumulation, proteasome inhibition, and endoplasmic reticulum stress in age-related apoptosis of oocytes in human ovaries.     

泛素-蛋白酶体系统与呼吸系统疾病

蛋白是细胞功能的主要执行者,蛋白的质量和数量与细胞功能和疾病有着密切的联系。蛋白的降解系统控制着蛋白的数量,泛素一蛋白酶体系统是细胞内蛋白的主要降解系统,负责细胞内大部分蛋白的降解。对于泛素一蛋白酶体与心血管、肿瘤和神经系统的关系目前研究的比较多,一些新的I艋床药物也应运到临床为患者带来福音。而该系统与呼吸系统疾病关系的研究并不多,了解泛素一蛋白酶体系统在呼吸系统疾病中的作用对于呼吸系统疾病的防治有重要的意义。     

Transcription from the gene encoding the herpesvirus entry receptor nectin-1 (HveC) in nervous tissue of adult mouse

Hepatitis C virus core protein, in addition to being a component of the viral capsid, has a number of regulatory functions. Here we showed two bodies of evidence indicating that a fraction of the core protein species is a substrate of the ubiquitin (Ub)-proteasome pathway of targeted proteolysis. First, the core protein processing the C-terminal hydrophobic region is metabolically unstable, and incubation with a proteasome inhibitor led to a significant accumulation of the protein. Second, an in vivo ubiquitylation assay indicates conjugation of multi-Ub chain to the unstable core protein. In contrast, a stable form of core protein, p21, is also able to be ubiquitylated, but it links to a single or only a few Ub moiety. Therefore, processing event(s) at the C-terminal hydrophobic domain of HCV core protein may affect the ubiquitylation pathway, particularly the efficiency of the multi-Ub chain assembly, resulting in stable, matured core proteins.