Defective hepatic response to interferon and activation of suppressor of cytokine signaling 3 in chronic hepatitis C

BACKGROUND & AIMS: Approximately half of hepatitis C virus (HCV)-infected patients do not respond to current interferon (IFN)-alpha combination therapy. To understand IFN-alpha resistance in vivo, we examined the dynamic responses to both type I and type II IFNs, human IFN (hIFN)-alpha, -gamma, and consensus IFN, in the chimpanzee model. METHODS: Naive and HCV-infected chimpanzees were treated with 3 forms of hIFNs in vivo. Quantitative real-time polymerase chain reaction was performed to evaluate the expression of IFN-stimulated genes (ISGs) in both peripheral blood mononuclear cells and liver to compare the responses to hIFN between naive and infected chimpanzees. The hepatic expression of IFN signaling components and inhibitory regulators including suppressor of cytokine signaling 3 (SOCS3) were assessed. SOCS3 expression was also evaluated in the liver of HCV-infected patients undergoing IFN treatment. RESULTS: The in vivo responses to all 3 hIFNs were much lower in the HCV-infected chimpanzees than those in the naive chimpanzees. This defect was particularly evident in the liver because induction of hepatic ISGs was barely detectable in the infected animals. Following IFN administration, the expression of SOCS3 was significantly up-regulated, possibly through induction of interleukin-6, in the liver of HCV-infected chimpanzees. HCV-infected humans also showed a differential pattern of hepatic SOCS3 expression in response to IFN that is associated with treatment response. CONCLUSIONS: Our data indicate a predominantly defective hepatic response to IFN in HCV-infected chimpanzees, which is probably mediated through the activation of SOCS3 and may explain the nonresponse of many HCV patients to IFN-based therapy.     

从癫痫模型大鼠海马神经元中克隆caspase 3的新底物PIASI

目的 从癫痫模型大鼠海马凋亡神经元中克隆caspase-3的新底物。方法 制作癫痫模型大鼠海马组织cDNA文库；PCR获得caspase-3的P12和P17两亚基，然后分别定向插入pBridge质粒，构建三杂交诱饵栽体；进行酵母三杂交筛库。结果 建立成功癫痫模型大鼠海马组织cDNA文库，构建了大鼠caspase-3基因的酵母三杂交诱饵栽体，并通过了caspase-3与eIF2n之间的相互作用验证，筛库获得caspase-3的新底物PIASI。结论 用酵母三杂交系统寻找caspase-3下游底物具有可行性，从癫痫模型大鼠海马的cDNA文库中筛库获得caspase-3的新底物PIASl，为进一步研究caspase-3在癫痫发作引起的海马损伤中的作用奠定了基础。     

Alterations of ubiquitin related proteins in the pathology and development of schizophrenia: Evidence from human and animal studies

Gene expression analyses in post-mortem schizophrenia brains suggest that a number of ubiquitin proteasome system (UPS) genes are associated with schizophrenia; however the status of UPS proteins in the schizophrenia brain is largely unknown. Ubiquitin related proteins are inherently involved in memory, neuronal survival and morphology, which are processes implicated in neurodevelopmental disorders such as schizophrenia. We examined levels of five UPS proteins (Protein Inhibitor of Activated STAT2 [PIAS2], F-Box and Leucine rich repeat protein 21 [FBXL21], Mouse Double Minute 2 homolog [MDM2], Ubiquitin Carboxyl-Terminal Hydrolase-L1 [UCHL1] and Ubiquitin Conjugating Enzyme E2D1 [UBE2D1]) involved in these neuronal processes, within the dorsolateral prefrontal cortex of post-mortem schizophrenia subjects and matched controls (n = 30/group), in addition to across neurodevelopmental time-points (juvenile, adolescent and adult stages of life), utilizing a well-established neurodevelopmental phencyclidine (PCP) animal model of schizophrenia. We observed significant reductions in PIAS2, FBXL21 and MDM2 in schizophrenia subjects compared to controls (p-values ranging from 0.002 to 0.004). In our developmental PCP model, MDM2 protein was significantly reduced in adult PCP-treated rats compared to controls (p = 0.034). Additionally, FBXL21 (p = 0.022) and UCHL1 (p = 0.022) were significantly decreased, whilst UBE2D1 was increased (p = 0.022), in juvenile phencyclidine-treated rats compared to controls. This is the first study reporting alterations of UPS proteins in post-mortem human schizophrenia subjects and in a neurodevelopmental model of schizophrenia. The findings from this study provide strong support for a role of these UPS proteins in the pathology and development of schizophrenia.     

PIAS3绿色荧光蛋白表达载体构建及其对胶质瘤U251细胞增殖和凋亡的影响

目的：构建PIAS3绿色荧光蛋白真核表达载体,探讨外源性PIAS3对胶质瘤细胞U251周期和凋亡的影响。方法：利用RT-PCR扩增人全长PIAS3编码序列,并将其克隆到带有绿色荧光蛋白的真核表达载体pEGFP-N1中,得到重组质粒pEGFP-N1-PIAS3。应用脂质体法转染体外培养的人胶质瘤U251细胞株。荧光显微镜观察蛋白表达,用RT-PCR和Westernblot检测PIAS3的表达,Annexin V2FITC和PI双染流式细胞术检测细胞凋亡,流式细胞术检测细胞增殖周期变化。结果：转染PIAS3基因的细胞株外源目的基因和蛋白表达增强,瞬时转染48 h后,光镜下可见部分细胞变圆,部分细胞脱落死亡;对照组和未转染组无明显变化。流式细胞仪分析显示,pEGFP-N1-PIAS3转染组凋亡细胞增多,细胞凋亡率显著高于未转染组和pEGFP-N1转染组,P=0.0073;U251细胞转染pEGFP-N1-PIAS3后细胞周期阻滞于S期,S期细胞比例增高,P=0.025,G2期细胞比例降低。结论：外源性PIAS3使U251阻滞在S期,诱导胶质瘤细胞凋亡。     

Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein

Sumoylation of the papillomavirus (PV) origin binding helicase E1 protein is critical for its function. Consequently, factors modulating the sumoylation of E1 could ultimately alter the outcome of a papillomavirus infection. We investigated the role played by phosphorylation and two known SUMO E3 ligases, RanBP2 and PIAS proteins, on the sumoylation of E1. E1 sumoylation was unaffected by phosphorylation as both wild-type and pseudo-phosphorylation mutants of BPV E1 exhibited similar sumoylation profiles. RanBP2 bound to BPV E1, but not to HPV11 E1, and lacked sumoylation enhancing activity for either E1. In contrast, proteins of the PIAS family (except PIASy) bound to both BPV and HPV11 E1 and stimulated their sumoylation. The structural integrity of the RING finger domain of the PIAS proteins was required for their E3 SUMO ligase activity on PV E1 sumoylation but was dispensable for their PV E1 binding activity. Miz1, the PIAS protein exerting the strongest E1 sumoylation enhancing activity, favored SUMO1 versus SUMO2 as the modifier and was shown to be transcribed in a keratinocyte cell line. This study indicates PIAS proteins as possible modulators of PV E1 sumoylation during papillomavirus infections.