SHARPIN的研究进展

人类线性泛素链相关蛋白SHARPIN在哺乳动物体内广泛存在,主要分布于大脑、心脏和睾丸等器官细胞的胞质内,可通过线性泛素化调节介导NF-kB信号通路激活等途径对机体产生多种生理效应,特别是在炎症和免疫系统的发展中可能起着重要的作用。另外,SHARPIN与肿瘤发生、发展、转归的关系更成为近年来研究的热点,目前发现在多种肿瘤中SHARPIN表达均升高,但机制尚未明确。本文就SHARPIN的分子结构、与机体多种疾病发生发展的相关机制展开综述,并对SHARPIN未来的研究方向进行展望。     

Protein stability regulators screening assay (Pro-SRSA):protein degradation meets the CRISPR-Cas9 librar y

The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled“A genome-scale CRISPR–Cas9 screening method for protein stability reveals novel regulators of Cdc25A,”recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR–Cas9) library with a dual-lfuorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our ifndings, we are conifdent that this effcient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.     

The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation

Acetaminophen (APAP) overdose is a major cause of liver injury. Neural precursor cell expressed developmentally downregulated 4–1 (NEDD4-1) is an E3 ubiquitin ligase that has been implicated in the pathogenesis of numerous liver diseases; however, its role in APAP-induced liver injury (AILI) is unclear. Thus, this study aimed to investigate the role of NEDD4-1 in the pathogenesis of AILI. We found that NEDD4-1 was dramatically downregulated in response to APAP treatment in mouse livers and isolated mouse hepatocytes. Hepatocyte-specific NEDD4-1 knockout exacerbated APAP-induced mitochondrial damage and the resultant hepatocyte necrosis and liver injury, while hepatocyte-specific NEDD4-1 overexpression mitigated these pathological events both in vivo and in vitro. Additionally, hepatocyte NEDD4-1 deficiency led to marked accumulation of voltage-dependent anion channel 1 (VDAC1) and increased VDAC1 oligomerization. Furthermore, VDAC1 knockdown alleviated AILI and weakened the exacerbation of AILI caused by hepatocyte NEDD4-1 deficiency. Mechanistically, NEDD4-1 was found to interact with the PPTY motif of VDAC1 through its WW domain and regulate K48-linked ubiquitination and degradation of VDAC1. Our present study indicates that NEDD4-1 is a suppressor of AILI and functions by regulating the degradation of VDAC1.     

A phase I pharmacodynamic trial of bortezomib in combination with doxorubicin in patients with advanced cancer

Purpose  This phase I trial sought to define the toxicity, maximally tolerated dose (MTD) and pharmacodynamics of a combination of bortezomib and doxorubicin in patients with advanced malignancies. Patients and methods  Twenty-six patients were treated with bortezomib intravenously on days 1, 4, 8 and 11, with doxorubicin also administered intravenously on days 1 and 8, both in a 21-day cycle. Dosing ranged from 1.0 mg/m² of bortezomib with 15 mg/m² of doxorubicin to 1.5 mg/m² of bortezomib with 20 mg/m² of doxorubicin. Pharmacodynamic studies performed included assessment of levels of 20S proteasome activity and ubiquitin-protein conjugates. Results  The combination of bortezomib and doxorubicin was generally well tolerated. There were two dose limiting toxicities (DLT) at dose cohort 3 (1.3 mg/m² bortezomib, 20 mg/m² doxorubicin) and 2 DLT at dose cohort 3a (1.5 mg/m² bortezomib, 15 mg/m² doxorubicin). DLT seen included neutropenia, thrombocytopenia, and neuropathy. In addition, one patient developed grade 3 central nervous system toxicity in cycle 2 (not a DLT). One patient with hormone refractory prostate cancer had a partial response. Proteasome inhibition in whole blood was demonstrated and an increase in ubiquitin-protein conjugates was observed in peripheral blood mononuclear cells of most patients. Conclusions  Bortezomib and doxorubicin can be administered safely. The recommended phase II dose for this 21-day cycle is bortezomib 1.3 mg/m² intravenously on days 1, 4, 8 and 11, and doxorubicin 20 mg/m² intravenously on days 1 and 8. This combination may be of special interest in multiple myeloma, given the activity of both drugs in that disease. Supported by grant: U01 CA062491 “Early Clinical Trials of Anti-Cancer Agents With Phase I Emphasis, NCI” and M01 RR03186 “General Clinical Research Center Program of The National Center for Research Resources, NIH”.     

黑色素瘤抗原泛素连接酶的研究进展

黑色素瘤抗原（melanoma-associated antigen gene, MAGE）被视为一种肿瘤标志物和免疫治疗的热门对象。研究发现MAGE联合E3-RING泛素连接酶形成MAGE-RING连接酶复合体（MAGE-RING ligases, MRLs）,成为泛素化的调控因子,从而调控连接酶的活性、底物的特异性和亚细胞定位。该文就MRLs的结构、活化机制和转录功能作一综述。     

Alteration of alpha-spectrin ubiquitination after hemorrhagic shock

Background

RBC deformability after trauma and hemorrhagic shock (T/HS) leads to the microcirculatory dysfunction and clinical manifestations of organ failure. However, the cellular mechanism of this phenomenon remains unknown. Spectrins are important for the shape and physical properties of erythrocytes, such as deformability and resistance to mechanical stress. Previous studies have shown that erythrocyte α-spectrin is ubiquitinated. Studies of sickled cells and aged erythrocytes, 2 conditions known to have decreased RBC deformability, have shown decreased α-spectrin ubiquitination, which may contribute to the inability of these cells to change shape. It was hypothesized that decreased α-spectrin ubiquitination could participate in the mechanism(s) whereby T/HS erythrocytes become less deformable.

Methods

The level of α-spectrin ubiquitination in erythrocytes isolated from T/HS rats was determined and compared with erythrocytes from control sham-shocked (T/SS) animals. After T/SS (n = 4) or T/HS (n = 7), α- and β-spectrin subunits were isolated using a low ionic-strength buffer at 37°C for 30 minutes. The relative amount of ubiquitinated α-spectrin was evaluated by Western blotting using a monoclonal antibody to ubiquitin.

Results

The relative level of α-spectrin ubiquitination (normalized to total α-spectrin in the same preparation) was found to be significantly decreased after T/HS (.319 ± .03) compared with T/SS control erythrocytes (.485 ± .06, P < .05). To evaluate the content and relative amounts of the other membrane proteins, the profiles of T/HS and T/SS erythrocytes were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This did not reveal any significant quantitative differences between T/SS and T/HS spectrin or other membrane proteins.

Conclusions

The finding of decreased α-spectrin ubiquitination after T/HS suggests that this mechanism could contribute to increased rigidity of the erythrocyte membrane.     

卵巢相关F-box蛋白的功能及研究进展

F-box蛋白广泛存在于真核生物和原核生物中,可以通过泛素-蛋白酶体途径参与调控细胞周期、细胞凋亡、细胞信号转导和肿瘤发生等多种生物学过程。近年来研究提示卵巢中存在多种F-box蛋白的表达,F-box蛋白功能紊乱可导致卵巢生殖和内分泌功能发生障碍及卵巢疾病,出现卵泡发育不良、卵巢早衰（POF）及卵巢癌等病理反应。随着对细胞内泛素化作用研究的不断深入,越来越多F-box蛋白通过调控细胞周期和信号传导通路等途径影响卵巢功能的作用机制逐渐被揭示,这也为相关卵巢疾病的临床诊治提供了理论基础和新思路。现就与卵巢相关的F-box蛋白的功能及研究进展进行综述。     

泛素化调控与中药抗炎

炎症性疾病是一类极为常见且危害极大的疾病,以中药抗炎来减轻或治疗炎症性疾病在临床上已得到广泛应用,但其分子机制研究尚处于起步阶段。泛素是一类极为保守的小分子功能蛋白,其通过对关键信号分子蛋白的泛素化修饰过程,而对炎症等生理和病理活动发挥重要调控作用。从泛素/去泛素角度来探究中药对炎症信号途径的调控作用,将为中药抗炎机制研究提供崭新思路。