Role of E3 ubiquitin ligases in insulin resistance

E3 ubiquitin ligases are a large family of proteins that catalyse the ubiquitination of many proteins for degradation by the 26S proteasome. E3 ubiquitin ligases play pivotal roles in the process of insulin resistance and diabetes. In this review, we summarize the currently available studies to analyse the potential role of E3 ubiquitin ligases in the development of insulin resistance. We propose two mechanisms by which E3 ubiquitin ligases can affect the process of insulin resistance. First, E3 ubiquitin ligases directly degrade the insulin receptor, insulin receptor substrate and other key insulin signalling molecules via the UPS. Second, E3 ubiquitin ligases indirectly regulate insulin signalling by regulating pro‐inflammatory mediators that are involved in the regulation of insulin signalling molecules, such as tumour necrosis factor‐α, interleukin (IL)‐6, IL‐4, IL‐13, IL‐1β, monocyte chemoattractant protein‐1 and hypoxia‐inducible factor 1α. Determining the mechanism by which E3 ubiquitin ligases affect the development of insulin resistance can identify a novel strategy to protect against insulin resistance and diabetes.     

Role of E3 ubiquitin ligases in gastric cancer

E3 ubiquitin ligases have an important role in carcinogenesis and include a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.So far,E3 ubiquitin ligases have been reported to have a role in a variety of biological processes including cell cycle regulation,cell proliferation,and apoptosis.Recently,several kinds of E3 ubiquitin ligases were demonstrated to be generally highly expressed in gastric cancer(GC) tissues and to contribute to carcinogenesis.In this review,we summarize thecurrent knowledge and information about the clinical significance of E3 ubiquitin ligases in GC.Bortezomib,a proteasome inhibitor,encouraged the evaluation of other components of the ubiquitin proteasome system for pharmaceutical intervention.The clinical value of novel treatment strategies targeting aberrant E3 ubiquitin ligases for GC are discussed in the review.     

Enhanced ubiquitination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases

Ubiquitin conjugation of proteins is critical for cell homeostasis and contributes to both cell survival and death. Here we studied ubiquitination of proteins in pressure overloaded (PO) myocardium in the context of cardiomyocyte survival. Analysis using a feline right ventricular pressure overload (RVPO) model revealed a robust and transient increase in ubiquitination of proteins present in the Triton X-100-insoluble fraction in 24 to 48 h PO myocardium, and confocal micrographs indicate this increase in ubiquitination occurs subsarcolemmaly near the intercalated disc area of cardiomyocytes. The ubiquitination was accompanied by changes in E3 ligases including Cbl, E6AP, Mdm2 and cIAP in the same period of PO, although atrophy-related E3 ligases, MuRF1 and MuRF3 were unaltered. Furthermore, Cbl displayed a substantial increase in both levels of expression and tyrosine phosphorylation in 48 h PO myocardium. Confocal studies revealed enrichment of Cbl at the intercalated discs of 48 h PO cardiomyocytes, as evidenced by its colocalization with N-cadherin. Although apoptosis was observed in 48 h PO myocardium by TUNEL staining, cardiomyocytes showing ubiquitin staining were not positive for TUNEL staining. Furthermore, 48 h PO resulted in the phosphorylation of inhibitor of nuclear factor kappa B (IkappaB), suggesting its ubiquitin-mediated degradation and the nuclear localization of NFkappaB for the expression of specific cell survival factors such as cIAPs. Together these data indicate that increased levels of E3 ligases that regulate cell homeostasis and promote cell survival could ubiquitinate multiple cytoskeletal protein targets and that these events that occur during the early phase of PO may contribute to both cardiomyocyte survival and hypertrophy.     

Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases

A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed “ER-associated degradation” (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed “Der3”) and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired the in vivo degradation of Doa10 substrates but had no apparent effect on the degradation of Hrd1 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the α2 helix of Ubc7 and Ub and that this transition state may be stabilized further by some E3 ligases, such as Hrd1, through additional interactions outside the RING domain.The ubiquitin (Ub) conjugation machinery employs three basic enzymatic activities, E1, E2, and E3, that work in concert to transfer Ub to client substrates and to form polyUb chains (1). Initially, an E1 Ub-activating enzyme forms a high-energy thioester bond with the C terminus of Ub, after which the Ub molecule is transferred to the active-site Cys of an E2 Ub-conjugating (Ubc) enzyme. The Ub-charged E2 binds to an E3 ligase and catalyzes the transfer of Ub to the ε-amino group of a Lys side chain within the substrate. Additional Ubs then can be ligated to the initial Ub molecule through sequential ubiquitylation cycles, ultimately forming a polyUb chain. Ub can be conjugated to itself via specific Lys residues, resulting in diverse types of chain linkages. Linkage through Lys48 is linked primarily to substrate degradation. Consequently, protein substrates carrying Lys48-linked polyUb chains bind to and are degraded by 26S proteasome.Although it is well established that E3 ligases activate Ub ligation by E2s via their RING domains, very little is actually known about the underlying regulatory mechanism. Several recent studies determined the structure of RING domain complexes with Ub-charged UbcH5 (2–4). In one of these studies, the structure in solution of Ub-charged UbcH5c together with the mouse E3 ligase E4B U-box domain revealed that Ub can adopt an array of “open” and “closed” conformations (2). The productive closed conformation promotes a nucleophilic attack on the Ub∼E2 thioester by an incoming Lys (acceptor) residue (2). A similar closed conformation was identified in the structures of UbcH5a and UbcH5b, together with their cognate RING domains (3, 4). Taken together, these structural studies suggest that RING domains can catalyze Ub transfer by stabilizing a transition state of a closed conformation of the E2-bound (donor) Ub (5).Among the fundamental intracellular functions of the Ub–proteasome system is maintenance of cellular protein quality control (PQC) by targeting a diverse array of transiently or permanently misfolded substrates for proteolysis. A central branch of PQC degradation takes place in the endoplasmic reticulum (ER) in a process termed “ER-associated degradation” (ERAD) (6). Despite the multitude of misfolded substrates, ERAD employs only a few E3–ligase complexes (7). In fact, the bakers'' yeast S. cerevisiae ERAD system employs only two Ub-ligation complexes, specified by their E3 ligase components, Hrd1 and Doa10 (8–12). Importantly, each of the two E3 ligase complexes recognizes a distinct set of substrates, with minor overlaps (13).Degradation by the yeast ERAD Ub-ligation system entails the combined activity of two E2 enzymes: Ubc6 and Ubc7 for the Doa10 pathway and Ubc1 and Ubc7 for the Hrd1 pathway (14, 15). The shared E2 enzyme, Ubc7, is a soluble cytosolic protein whose binding to either of the E3–ligase complexes at the ER membrane is mediated by the auxiliary ER membrane protein Cue1. Binding to Cue1 not only mediates the interaction with the E3–ligase complex but also protects Ubc7 from degradation and stimulates its Ub-transfer activity (16–20). Ubc7 is highly conserved in evolution, as evident from substantial sequence and structure similarities with its orthologs from other species (21). The human Ubc7 ortholog, Ube2g2 (21), functions together with several ER membrane-embedded E3 ligases, the best characterized of which is the tumor autocrine motility factor receptor, gp78 (22). Ubc7 and Ube2g2 are subjected to similar regulatory mechanisms: They bind to and are activated by the RING domains of their cognate E3s as well as by the E2-binding regions and CUE domains within Cue1 and gp78 (19, 20, 23–26). The evolutionarily conserved sequence, structure, and regulatory mechanisms of the Ubc7 E2s imply an essential physiological function.In this study we explored the role of helix α2 of Ubc7 in enzyme activation. Based on our in vivo and in vitro observations and on the available structural information, we propose a mechanism whereby activation of Ubc7, mediated by noncovalent interaction with Ub at helix α2, is differentially affected by the RING domains of its cognate E3 ligases Hrd1 and Doa10.     

E3 ubiquitin ligase GRAIL controls primary T cell activation and oral tolerance

T cell unresponsiveness or anergy is one of the mechanisms that maintain inactivity of self-reactive lymphocytes. E3 ubiquitin ligases are important mediators of the anergic state. The RING finger E3 ligase GRAIL is thought to selectively function in anergic T cells but its mechanism of action and its role in vivo are largely unknown. We show here that genetic deletion of Grail in mice leads not only to loss of an anergic phenotype in various models but also to hyperactivation of primary CD4⁺ T cells. Grail^−/− CD4⁺ T cells hyperproliferate in vitro to TCR stimulation alone or with concomitant anti-CD28 costimulation, with transient increased survival. In vitro differentiated T helper 1 cells show slight but significant hypersecretion of IFN-γ in Grail^−/− mice whereas Th2 and Th17 cytokine secretions are unchanged. Consistent with defective in vitro anergy, oral tolerance is abolished in vivo in OT-II TCR transgenic Grail^−/− mice fed with ovalbumin. In experimental allergic encephalitis, a model of organ-specific autoimmunity, oral tolerization with myelin basic protein was abrogated as well in Grail^−/− mice. On the protein level, Grail^−/− naïve T cells show no significant differences of total and phosphorylated levels of ZAP70, phospholipase Cγ1, and MAP kinases p38 and JNK but elevated baseline levels of MAP kinase ERK1/2. In summary, we define a role for GRAIL in primary T cell activation, survival, and differentiation. In addition, we formally prove an indispensable role for GRAIL in T cell anergy and oral tolerance—a promising, antigen-specific strategy to treat autoimmune diseases.     

Inhibitors of cullin-RING E3 ubiquitin ligase 4 with antitumor potential

Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4’s core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4’s substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds’ cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4–expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.

Cullin-RING (really intersting new gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest RING-type E3 family, consisting of ∼300 members, ∼50% of the E3s identified in humans (1, 2). CRLs target many critical regulators of cell division and signaling. Canonical CRLs are modular complexes, in which a cullin (CUL) subunit’s N-terminal domain assembles interchangeably with CUL-specific substrate receptors capable of binding a substrate. On the other hand, a CUL’s C-terminal domain (CTD) binds a RING finger protein, ROC1/RBX1 for CUL1 to 4 or ROC2 for CUL5, to form a core ligase complex. CRLs’ core ligase collaborates with E2 Ub-conjugating enzymes for transferring Ub(s) to the bound substrate or a Ub moiety on a growing Ub chain.A selective small-molecule modulator of CRLs’ function allows us to address mechanistic and phenotypic questions about its targets in biochemical, cell-based, and animal studies. To date, there is only one Food and Drug Administration (FDA)-approved E3 drug class that targets the substrate receptor cereblon (thalidomide/lenalidomide) (3). Current drug/probe discovery efforts against the Ub-proteasome system depend heavily on traditional methods that exploit the ability of small-molecule agents to disable an enzyme’s catalytic pocket. However, RING-type E3s are atypical enzymes and contribute to ubiquitination by mediating protein–protein interactions with substrate, E2, and Ub (1). High-resolution structural studies have shown that interactions involving E3’s RING domain, E2, and Ub are characterized by large, relatively flat interfaces (4). Such perceived “undruggable” features impose a significant barrier to structure-based ligand search using either virtual or fragment-based physical screening.To date, there are no reported small-molecule lead compounds targeting the catalytic activity of any CRL. To address this need, we have recently created a novel high-throughput screen (HTS) platform using the fluorescence (Förster) resonance energy transfer (FRET) K48 di-Ub assay (5). In this system, a FRET signal is generated as a result of covalent conjugation of two Ub molecules carrying a pair of matching fluorophores in a reaction that requires E1, E2 Cdc34, and an E3 CRL1 subcomplex (ROC1–CUL1 CTD). Fully functional Ub variants were created to allow only one nucleophilic attack that produces a single Ub–Ub isopeptide bond, thereby eliminating the complexity associated with polyubiquitin chain assembly to ensure a high degree of reproducibility for effective HTS. Each fluorophore is placed to either donor or receptor Ub at a specific site in a manner that satisfies optimal energy transfer. A pilot HTS identified a small-molecule compound, suramin (an antitrypanosomal drug), that can inhibit E3 CRL1 activity by disrupting its ability to recruit E2 Cdc34 (5). These observations have provided proof-of-principle evidence that an E2–E3 interface can be perturbed through small-molecule modulators. The current study describes a large-scale HTS and extensive follow-up hit-to-lead studies, which identified a class of small-molecule inhibitors against E3 CRLs.     

镉对小鼠附睾的损害及维生素E的拮抗作用

目的研究慢性染镉对小鼠附睾的损害及维生素E(VitaminE,VE)的保护作用。方法健康昆明种小鼠75只(体质量28～35g)随机分成3组:镉组(氯化镉2mg/kg,皮下注射,2次/周,共3个月)、VE组(染镉同时给VE10mg·kg-1·d-1,灌胃)和对照组(注射等量生理盐水)。通过HE染色、免疫组化细胞凋亡(Tunel法)染色和透射电镜技术观察小鼠附睾管上皮细胞细微结构的改变,对各组附睾管上皮细胞形态作立体定量分析,并对凋亡细胞进行计数。结果①镉组小鼠附睾上皮细胞明显受损,细胞出现变性、坏死。VE组仅少数小鼠附睾局部上皮细胞受到损伤。②镉组与对照组相比,小鼠附睾上皮细胞胞核的平均截面周长、平均截面积、平均表面积、平均体积和体积密度均显著缩小(P<0.05);VE组附睾上皮细胞胞核的上述参数值与对照组相比差异无统计学意义(P>0.05),而与镉组相比,相应形态参数值明显增大(P<0.05)。③凋亡细胞计数表明,镉组附睾上皮细胞凋亡比对照组明显增多(P<0.01);VE组细胞凋亡比镉组明显减少(P<0.01),而与对照组比较差异无统计学意义(P>0.05)。结论①慢性染镉可引起小鼠附睾管上皮细胞形态损害;②镉有促进小鼠附睾管上皮细胞凋亡的作用;③VE对镉引起的小鼠附睾管上皮细胞损害有保护作用;④VE对镉所致的小鼠附睾管上皮细胞凋亡有拮抗作用。     

Fluid shear stress and melatonin in combination activate anabolic proteins in MC3T3‐E1 osteoblast cells

In this study, we investigated whether fluid shear stress and melatonin in combination stimulate the anabolic proteins through the phosphorylation of extracellular signal‐regulated kinase (p‐ERK) in MC3T3‐E1 osteoblast cells. First, we researched why fluid shear stress and melatonin in combination influence cell survival. Fluid shear stress (1 hr) and melatonin (1 mm ) in combination reduced autophagic marker LC3‐II compared with fluid shear stress (1 hr) and/or melatonin (0.1 mm ). Under the same conditions for fluid shear stress, markers of cell survival signaling pathway p‐ERK, phosphorylation of serine‐threonine protein kinase (p‐Akt), phosphorylation of mammalian target of rapamycin (p‐mTOR), and p85‐S6K were investigated. p‐Akt, p‐mTOR (Ser 2481) expressions increased with the addition of 1 mm melatonin prior to 0.1 mm melatonin treatment. However, p‐S6K expression did not change significantly. Next, mitochondria activity including Bcl‐2, Bax, catalase, and Mn‐superoxide dismutase (Mn‐SOD) were studied. Expressions of Bcl‐2, Bax, and catalase proteins were low under fluid shear stress plus 1 mm melatonin compared with only fluid shear stress alone, whereas Mn‐SOD expression was high compared with conditions of no fluid shear stress. Finally, the anabolic proteins of bone, osteoprotegerin, type I collagen (collagen I), and bone sialoprotein II (BSP II) were checked. These proteins increased with combined fluid shear stress (1, 4 hr) and melatonin (0.1, 1 mm ). Together, these results suggest that fluid shear stress and melatonin in combination may increase the expression of anabolic proteins through the p‐ERK in MC3T3‐E1 osteoblast cells. Therefore, fluid shear stress in combination with melatonin may promote the anabolic response of osteoblasts.     

Metalloreductase Steap3 coordinates the regulation of iron homeostasis and inflammatory responses

Background

Iron and its homeostasis are intimately related to inflammatory responses, but the underlying molecular mechanisms are poorly understood. We investigated the role of Steap3 in regulating iron homeostasis in macrophages, and the effects of Steap3 depletion on host inflammatory responses.

Design and Methods

We analyzed bone marrow-derived macrophages and primary cultured hepatocytes from Steap3^-/- mouse models to investigate the roles of Steap3 in coordinately regulating iron homeostasis and inflammatory responses. First, we examined iron distribution and iron status in cells deficient in Steap3, as well as the requirement for the Steap3 gene during inflammatory responses. Secondly, we analyzed the regulation of Steap3 expression by inflammatory stimuli and thus, the influence of these stimuli on iron distribution and homeostasis.

Results

We found that Steap3 mRNA was expressed at high levels in macrophages and hepatocytes. Steap3 deficiency led to impaired iron homeostasis, causing abnormal iron distribution and a decreased availability of cytosolic iron in macrophages. Among STEAP family members, Steap3 mRNA was uniquely down-regulated in macrophages stimulated by lipopolysaccharides. To determine whether Steap3 regulated iron homeostasis during inflammatory stress, we treated Steap3^-/- mice with lipopolysaccharide, which produced greater iron accumulation in the vital tissues of these mice compared to in the tissues of wild-type controls. Furthermore, Steap3 depletion led to impaired induction of interferon-β, monocyte chemoattractant protein-5, and interferon induced protein-10 in macrophages via the TLR4-mediated signaling pathway.

Conclusions

Steap3 is important in regulating both iron homeostasis and TLR4-mediated inflammatory responses in macrophages. Steap3 deficiency causes abnormal iron status and homeostasis, which leads to impaired TLR4-mediated inflammatory responses in macrophages. Following inflammatory stimuli, Steap3 depletion causes dysregulated iron sequestration and distribution. Our results provide important insights into the function of Steap3 as a coordinate regulator of both iron homeostasis and innate immunity.     

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

PTEN is a tumor suppressor frequently mutated in cancer. Recent reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that regulates its stability and nuclear localization. We tested the physiological role of Nedd4-1 as a PTEN regulator by using cells and tissues derived from two independently generated strains of mice with their Nedd4-1 gene disrupted. PTEN stability and ubiquitination were indistinguishable between the wild-type and Nedd4-1-deficient cells, and an interaction between the two proteins could not be detected. Moreover, PTEN subcellular distribution, showing prominent cytoplasmic and nuclear staining, was independent of Nedd4-1 presence. Finally, activation of PKB/Akt, a major downstream target of cytoplasmic PTEN activity, and the ability of PTEN to transactivate the Rad51 promoter, a measure of its nuclear function, were unaffected by the loss of Nedd4-1. Taken together, our results fail to support a role for Nedd4-1 as the E3 ligase regulating PTEN stability and subcellular localization.     

E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells

The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90–client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition.Heat shock protein 90 (HSP90) is a molecular chaperone that facilitates the stabilization and activation of around 350 client proteins (see www.picard.ch/downloads/Hsp90interactors.pdf for an updated client protein list) (1). As well as being involved in a wide range of normal cellular processes, many HSP90 clients are oncogenic kinases that are hyperactivated by mutation or amplified/overexpressed in malignancies (2, 3). HSP90-mediated activation and stabilization of client proteins requires an ATP-driven chaperone cycle regulated by a number of cochaperones (4, 5). Pharmacologic inhibition of HSP90 disrupts this cycle and leads to the ubiquitin-mediated proteasomal degradation of client proteins (3, 6, 7).The proposed model is that clients are ubiquitinated and thus targeted to the proteasome by the action of one or more E3 ubiquitin ligases (8). Some evidence suggests that the U box-containing ligase CHIP is involved in the degradation of certain HSP90 clients (9, 10). However, the stability of protein kinase clients such as ERBB2 is not increased in CHIP^−/− cells treated with the first-in-class pharmacologic HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin [17-AAG, tanespimycin (11)] (10), suggesting that other E3 ubiquitin ligases are also involved.One study showed that the Cullin-RING ligase Cullin-5 (CUL5) is recruited to HSP90-containing complexes and is involved in the ubiquitination and degradation of the client ERBB2 following HSP90 inhibition (12). Cullin-RING ligases function as modular, multisubunit complexes that consist of a Cullin scaffold, a RING-H2 finger protein, a substrate-recognition subunit, and, in most cases, an adaptor that links the Cullin to the substrate recognition subunit (13, 14). In the case of CUL5, functional complexes consist of RBX2, Elongin-B, Elongin-C, and a SOCS-containing substrate receptor.Given the link between CUL5 and the HSP90 inhibitor-induced degradation of ERBB2 (12), we have investigated the role of Cullin-RING ligases with respect to HSP90’s protein kinase clients in human cancer cell lines. Our initial focused siRNA screen of 28 Cullin-RING ligase family members identified five genes, including CUL5, that were required for ERBB2 degradation following treatment with 17-AAG—which we use here as a representative HSP90 inhibitor and chemical tool to promote client protein turnover. We go on to show for the first time to our knowledge that RNAi silencing of CUL5 reduces the 17-AAG–induced degradation of four other structurally diverse protein kinase clients in addition to ERBB2. As well as reducing client ubiquitination, we were surprised to find that silencing CUL5 also delayed the much earlier loss of client activity upon HSP90 inhibition. This delayed loss of client activity correlated with the delay induced by CUL5 knockdown in cochaperone dissociation from inhibited HSP90–client protein complexes. A mutant CUL5 that cannot be neddylated impaired 17-AAG–induced client protein kinase degradation but did not affect loss of client activity or recruitment of clients and HSP90 to CUL5. Finally, silencing CUL5 decreased sensitivity to HSP90 inhibitors in cell lines encompassing four cancer types driven by five different oncogenic protein kinase clients of HSP90. We therefore conclude that CUL5 plays an important role in multiple aspects of the molecular and cellular response to HSP90 inhibition.     

Inhibition of angiogenesis and tumor growth by murine 7E3, the parent antibodyof c7E3 Fab (abciximab; ReoProTM)

Angiogenesis plays an essential role in the growth and dissemination of solid tumor cancers. The expression of endothelial cell integrin αvβ3 has been shown to increase during vascular proliferation associated with human tumors. Selective antagonists of αvβ3 can block angiogenesis and tumor growth by inducing programmed cell death in proliferating endothelial cells. Monoclonal antibody 7E3, an antagonist of the human, but not murine, integrins αvβ3 and αIIbβ3 (GPIIb/IIIa), inhibits platelet aggregation. It is the parent antibody of a mouse/human chimeric antibody fragment approved for adjunctive therapy of patients undergoing percutaneous coronary interventions to prevent ischemic complications (c7E3Fab; abciximab; ReoPro). To evaluate the potential of 7E3 to inhibit human angiogenesis and tumor growth independent of its antiplatelet effects, we established integrin αvβ3-negative human melanoma tumors in full-thickness human skin grafted onto SCID mice. The resulting tumors induce a human angiogenic response as assessed by the immunoreactivity of vascular cells with monoclonal antibodies specific for human CD31. Administration of 7E3 prevented or significantly inhibited the growth of tumors, and this effect correlated with a significant reduction in the number of blood vessels supplying the tumors. These results support the previous findings that blockade of integrin αvβ3 inhibits angiogenesis and tumor growth and indicates that dual inhibitors of αvβ3 and αIIbβ3 are effective in blocking tumor growth and angiogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

武汉地区基因Ⅳ型戊型肝炎病毒ORF3基因变异的检测和分析

目的检测并分析武汉地区基因Ⅳ型戊型肝炎（HEV）病毒开放读码框3（ORF3）基因变异情况。方法采用逆转录-巢式聚合酶链反应（RT-nPCR）扩增41株基因Ⅳ型HEVRNA两个基因片段（nt5020～nt5392和nt5347～nt5956;参考病毒株：EF570133）,并对PCR产物进行测序,然后使用ContigExpress将测序结果拼接（含有HEVORF3全序列）,使用MegAlign（DNAStar软件包）对ORF3进行比对,查看ORF3基因及其编码蛋白变异情况。结果41株基因Ⅳ型HEVRNA两个基因片段均扩增出来的毒株为18株,18株HEVORF3检测出47个位点存在核苷酸替换,其中21个替换发生在密码子第1、2位核苷酸,26个替换发生在密码子第3位核苷酸;其编码蛋白存在19个位点氨基酸变异。结论武汉地区基因Ⅳ型HEVORF3存在多个位点核苷酸替换,这些位点变异在急性戊型肝炎病情进展中有何意义,有待于进一步研究。     

Hepatitis E virus genotype 3 microbiological surveillance by the Spanish Reference Laboratory: geographic distribution and phylogenetic analysis of subtypes from 2009 to 2019

BackgroundHepatitis E virus genotype 3 (HEV-3) is widely distributed throughout Europe, with incidence of infections increasing in many countries. Belgium, Bulgaria, France, Germany, Italy, the Netherlands and the United Kingdom have reported the distribution of HEV-3 subtypes in cohorts of patients with hepatic disease.AimTo describe the distribution of the HEV-3 subtypes in Spain at national and autonomous community (AC) levels between 2009 and 2019. The study was also extended to Andorra.MethodsOf 5,197 samples received by the National Reference Laboratory during the study, 409 were HEV-RNA-positive. Among these, 294 (71.9%) were further typed based on an ORF2 sequence fragment, or, for a subset of 74, based on the full-coding genome sequence.ResultsHEV-3 was detected in 291 samples. The dominant subtype in Spain was HEV-3f (88.3%; 257/291), which occurred in all ACs, with no change in detection level over time. Within this subtype, three subclusters were characterised: HEV-3f-B, HEV-3f-A1 and HEV-3f-A2. The second most common HEV subtype was the recently described HEV-3m (7%; 21/291), with two subclusters identified: HEV-3m-A, which has been known since 2010, and HEV-3m-B, since 2014. The third most encountered subtype was HEV-3c (4.1%; 12/291), with a frequency not increasing over time, unlike observations in some European countries.ConclusionThe importance of the surveillance of HEV-3 subtype and subcluster circulation is yet to be assessed. This surveillance together with the comprehensive epidemiological characterisation of clinical cases, could support the identification of sources of transmission and the establishment of control measures nationally and internationally.     

维生素C、E 对柯萨奇B3病毒感染的心肌细胞的保护作用

目的探讨抗氧化剂维生素C、E在治疗病毒性心肌炎过程中的重要作用。方法体外培养乳鼠心肌细胞，分别在感染柯萨奇B3病毒前后加入一定浓度的维生素C、E及其合剂，待病毒对照组细胞病变达 时，以MTT法测定细胞存活率，应用全自动生化分析仪测定细胞上清液中的心肌酶活性。结果实验组的细胞存活率明显高于病毒对照组，心肌酶活性明显低于病毒对照组。结论维生素C、E对病毒感染的心肌细胞具有一定的保护作用，联合使用的效果明显优于单独使用。