人朊蛋白相关蛋白Shadoo的表达、纯化及抗体的制备

目的克隆、表达和纯化人朊蛋白相关Shadoo（SHO）蛋白并制备抗Shadoo的多克隆抗体。方法提取仓鼠各组织的mRNA，分析SHO基因在仓鼠各组织中转录水平的差异;提取人DNA，PCR方法获得人SHO基因，克隆至融合表达载体，在大肠埃希菌中表达人SHO蛋白并纯化;以纯化蛋白为抗原免疫家兔制备抗血清，用Western blot方法分别检测与重组及内源性SHO蛋白的免疫反应性。结果SHO基因在仓鼠各组织中的转录水平差异很大，脑组织转录水平高。在大肠埃希菌中表达了分子质量单位为37 ku的人SHO融合蛋白，制备的SHO蛋白抗体可与重组的SHO蛋白反应，可识别内源性的SHO。结论成功表达纯化人SHO融合蛋白并制备了抗SHO抗体，为研究SHO和正常朊蛋白之间的关系打下初步基础。     

New insights into cellular prion protein (PrP) functions: The “ying and yang” of a relevant protein

The conversion of cellular prion protein (PrP^c), a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrP^sc) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrP^c is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. Recent studies have confirmed its participation in basic physiological processes such as cell proliferation and the regulation of cellular homeostasis. Other studies indicate that PrP^c interacts with several molecules to activate signaling cascades with a high number of cellular effects. To determine PrP^c functions, transgenic mouse models have been generated in the last decade. In particular, mice lacking specific domains of the PrP^c protein have revealed the contribution of these domains to neurodegenerative processes. A dual role of PrP^c has been shown, since most authors report protective roles for this protein while others describe pro-apoptotic functions. In this review, we summarize new findings on PrP^c functions, especially those related to neural degeneration and cell signaling.     

Family reunion--the ZIP/prion gene family

Prion diseases are fatal neurodegenerative diseases of humans and animals which, in addition to sporadic and familial modes of manifestation, can be acquired via an infectious route of propagation. In disease, the prion protein (PrP(C)) undergoes a structural transition to its disease-causing form (PrP(Sc)) with profoundly different physicochemical properties. Surprisingly, despite intense interest in the prion protein, its function in the context of other cellular activities has largely remained elusive. We recently employed quantitative mass spectrometry to characterize the interactome of the prion protein in a murine neuroblastoma cell line (N2a), an established cell model for prion replication. Extensive bioinformatic analyses subsequently established an evolutionary link between the prion gene family and the family of ZIP (Zrt-, Irt-like protein) metal ion transporters. More specifically, sequence alignments, structural threading data and multiple additional pieces of evidence placed a ZIP5/ZIP6/ZIP10-like ancestor gene at the root of the PrP gene family. In this review we examine the biology of prion proteins and ZIP transporters from the viewpoint of a shared phylogenetic origin. We summarize and compare available data that shed light on genetics, function, expression, signaling, post-translational modifications and metal binding preferences of PrP and ZIP family members. Finally, we explore data indicative of retropositional origins of the prion gene founder and discuss a possible function for the prion-like (PL) domain within ZIP transporters. While throughout the article emphasis is placed on ZIP proteins, the intent is to highlight connections between PrP and ZIP transporters and uncover promising directions for future research.     

朊蛋白相关蛋白Shadoo与朊病毒病

朊病毒病,即传染性海绵状脑病(transmissible spongiform encephalopathies,TSEs),是一类传染性、致死性神经退行性疾病.在朊病毒病的病理过程中,细胞正常朊蛋白PrPC转化为异常构象的PrPSc是至关重要的,但是PrPC的正常生理功能仍不清楚.国外学者利用比较基因组学发现了一个新的朊蛋白相关蛋白-Shadoo(Sho).Sho与PrPC在氨基酸序列和细胞定位的相似性及主要在脑组织表达,使它成为一个非常值得研究的PrP相关蛋白.对Sho可能存在的与PrPC重叠的功能甚至直接相互作用的研究工作,将对今后揭示PrPC正常生理功能以及揭示Prion病发病机制具有重要现实意义.     

Overexpression of Shadoo protein in transgenic mice does not impact the pathogenesis of scrapie

Shadoo is a glycoprotein expressed in the adult brain that is an interacting protein of prion protein; however, its function remains to be determined. To elucidate its role in prion pathogenesis, we generated transgenic mice overexpressing wild-type (wt) Shadoo driven by the murine PrP promoter. Expression of the murine Sprn transgene significantly increased brain Shadoo protein levels in all three mouse lines generated. Following infection with mouse-adapted scrapie strain 22L, all transgenic mice tested exhibited characteristics of scrapie disease. Importantly, there was no correlation between the expression level or incubation time of Shadoo with disease phenotype. We therefore conclude that Shadoo has little or no influence on the outcome of transmissible spongiform encephalopathy (TSE) disease in transgenic mice.