肝细胞生成素在睾丸组织中的相互作用蛋白

肝细胞生成素（HPO）具有复杂的生理功能,在睾丸中的高表达提示其在生殖活动中的重要性,而不仅局限于肝再生.构建了酵母表达载体pGBKT7-HPO,采用酵母双杂交系统,以HPO为诱饵蛋白,从人睾丸cDNA文库中寻找能够与HPO相互作用的蛋白质.经过筛选、验证阳性克隆,并进行PCR、测序和序列比对,得到4种相互作用蛋白质：NADH脱氢酶1、钠/钾ATP酶β3亚基、磷脂酶C δ1以及附睾分泌蛋白.提示HPO可能参与了细胞的蛋白质合成,能量代谢等.通过对候选蛋白的研究,为探讨HPO对睾丸组织细胞功能的调节机制提供了重要的线索.     

肝细胞生成素家族成员的作用机制

肝细胞生成素通过激活MAPK和AP-1,以细胞因子的方式促进肝细胞的增殖.同时,该家族又具有巯基氧化还原酶活性,主要与线粒体参与的铁硫蛋白成熟和线粒体膜间隙蛋白的转运折叠有关.人们对肝细胞生成素家族成员功能的认识,已经超出了原来仅具有促细胞增殖功能的局限,发现其参与了更加广泛的生物过程.     

基于蛋白质跨外膜自转运系统的细菌细胞表面蛋白展示技术研究进展

革兰氏阴性菌Ⅴ型分泌系统是细菌病原蛋白分泌的主要途径之一,可分为Ⅴa-Ⅴe5个亚型,其中Ⅴa型(即经典的单体自转运蛋白)是细菌毒力和黏附因子向细胞外分泌的重要工具,其在内膜Sec易位子和外膜BAM蛋白复合体的协助下,通过2个连续的跨膜步骤介导蛋白质穿过阴性菌的内外膜.据信Va型是目前已知蛋白质跨膜转运时最简单的分泌途径...     

PCSK9降解低密度脂蛋白受体分子机制研究进展

血清低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)水平的升高是导致心血管疾病发生的主要危险因素。低密度脂蛋白受体(LDL receptor, LDLR)介导的低密度脂蛋白(low density lipoprotein, LDL)清除是决定循环中LDL-C水平的主要因素。LDL与细胞表面的LDLR结合后通过经典的网格蛋白小窝(clathrin-coated vesicles)内化进入细胞。在酸性核内体中,LDLR与LDL解离并循环回到细胞表面,释放的LDL将被运送到溶酶体中降解。前蛋白转化酶枯草溶菌素9 (proprotein convertase subtilisin kexin type 9, PCSK9)编码一种肝脏分泌型蛋白,其突变与LDL-C水平密切相关。前期研究已经证明,PCSK9直接与细胞表面的LDLR相互作用,二者一起通过网格蛋白小窝内化进入细胞。然而,在酸性核内体中,PCSK9和LDLR形成紧密的复合物,并进入溶酶体中进行降解,从而减少肝细胞表面LDLR的水平,降低肝脏对LDL-C的清除,该过程对于维持血浆中LDL在相对恒定的水平具有重要作用。因此,阻断PCSK9功能已成为治疗高胆固醇血症的新策略。本文综述了PCSK9的功能和机制研究的最新进展,并着重介绍了PCSK9抑制剂的研究进展,旨在为PCSK9-LDLR通路的研究和胆固醇代谢的调控提供参考。     

重组人肝细胞生成素的纯化及活性研究

人肝细胞生成素 ( human hepatopoietin,h HPO)是一种新型肝再生调控因子 .在大肠杆菌中表达的的重组 h HPO( rh HPO)是以包涵体的形式存在的 ,其表达量为菌体总蛋白的 2 0 % .包涵体经各种溶液洗涤后 ,用 8mol/L尿素裂解 ,裂解上清经凝胶过滤、复性和离子交换柱层析得到电泳纯的 rh HPO,经还原型 SDS- PAGE测定其分子量为 1 5k D.纯化 rh HPO的 N端氨基酸序列与其c DNA推导序列完全一致 ;纯化产物的氨基酸组成分析结果亦与 rh HPO氨基酸组成的理论值吻合 .生物学活性研究表明 ,rh HPO在体外具有刺激原代培养肝细胞增殖作用     

在笼型蛋白和脂筏介导的两种内吞途径中nephrin的磷酸化修饰动力学不同

研究肾小球裂隙膜的主要成分nephrin分子在细胞内的转运途径及不同转运途径对nephrin磷酸化的影响.分别应用笼型蛋白介导的内吞(clathrin-mediated endocytosis,CME)和脂筏介导的内吞(raft-mediated endocytosis,RME)标记物转铁蛋白和霍乱毒素B亚基对nephrin的内吞过程进行分析,并进一步应用两种内吞途径阻断物EPS15Δ和Dyn2aK44A,研究阻断nephrin的内吞途径对其磷酸化水平的影响.结果显示,nephrin通过笼型蛋白和脂筏介导的两种内吞途径以不同速率进行内吞;与Src酪氨酸激酶家族成员Fyn共表达时,细胞内nephrin酪氨酸磷酸化被增强,而在Src家族激酶抑制剂PP2的作用下,nephrin酪氨酸磷酸化被减弱,表明nephrin的磷酸化过程是Fyn依赖的;内吞20min时,笼型蛋白介导的内吞途径的特异性阻断物EPS15Δ降低了nephrin磷酸化水平、笼型蛋白和脂筏介导的内吞途径的通用抑制剂Dyn2aK44A则增加了nephrin的磷酸化水平,综上结果表明:单独阻断脂筏介导的内吞可引起nephrin的磷酸化水平增加,脂筏介导的内吞对nephrin磷酸化过程起下调作用.     

AP-1辅助激活因子JAB1的分子克隆及其与肝细胞生成素的相互作用

利用酵母双杂交方法,用肝细胞生成素(HPO)作为诱饵蛋白在人胎肝cDNA文库中筛选到能与HPO相互作用的蛋白：AP-1辅助激活因子JAB1.并用PCR方法从人胎肝cDNA文库中扩出JAB1全长cDNA,进行GST-JAB1原核融合蛋白表达与纯化.蛋白质结合实验表明,JAB1与人重组HPO以及COS7真核表达的HPO在体外有结合作用.     

节肢动物ABC转运蛋白及其介导的杀虫剂抗性

腺苷三磷酸结合盒转运蛋白（ATP-binding cassette transporter),简称ABC转运蛋白（ABC transporter）,是继细胞色素P450单加氧酶、羧酸酯酶、谷胱甘肽S-转移酶之后又一类参与解毒作用的重要蛋白家族,因其在杀虫剂解毒等方面起着非常重要的作用,近年来逐渐受到广泛关注。ABC转运蛋白是一大类跨膜蛋白,其核心结构通常由4个结构域组成,包括2个高度疏水的跨膜结构域（transmembrane domains , TMD）和2个核苷酸结合域（nucleotide binding domains, NBD）。根据序列相似性和保守结构域,可以把ABC转运蛋白家族分为8个亚家族,每个亚家族的成员数及功能不同。这类蛋白在各种生物体内均有分布,其主要功能包括转运物质、信号传导、细胞表面受体及参与细胞内DNA修复,转录及调节基因的表达过程等。此外,近年来的研究表明,ABC转运蛋白的突变或过表达不仅与节肢动物对化学农药的抗药性密切相关,而且在抗Bt毒素方面也起着非常重要的作用,对转Bt作物造成严重威胁。本文综述了节肢动物ABC转运蛋白的结构,ATP水解介导的作用机制,亚家族的分类、结构及生理功能,以及由ABC转运蛋白介导的抗药性研究进展,旨在深入了解ABC转运蛋白的研究现状及其在节肢动物抗药性方面的作用,为阐明节肢动物抗药性机制提供新的理论依据,对改进农业害虫的抗性监测和治理策略也具有一定的指导意义。     

ULK1蛋白激酶通过自噬及非自噬途径介导的生理、病理和疾病研究进展

ULK1 (unc-51 like autophagy activating kinase 1)是一种哺乳动物丝/苏氨酸激酶,其作为自噬起始复合物的关键分子可介导细胞发生经典自噬反应。经典自噬反应是细胞通过由一系列自噬相关蛋白质介导的自噬溶酶体途径,将废弃或受损的蛋白质、细胞器经过自噬体的包裹后与溶酶体结合,继而使蛋白质、细胞器在溶酶体内降解。因此, ULK1介导的经典自噬反应是细胞质量控制的重要组成部分。除了介导经典自噬反应以外,ULK1也发挥着独立于自噬反应之外的重要作用,比如:促进细胞凋亡、强化磷酸戊糖途径、调控固有免疫反应等。此外, ULK1在糖脂代谢、红细胞形成、内质网应激、肿瘤、神经系统疾病中也发挥着重要作用。鉴于ULK1的重要性,本综述围绕ULK1蛋白激酶参与的经典自噬反应和不依赖自噬的反应及其介导的生理、病理和疾病过程展开论述。     

ABC转运蛋白结构及在植物病原真菌中的功能研究进展

ABC(ATP-binding cassette)转运蛋白是最大的膜转运蛋白超家族之一,其主要功能是利用ATP水解产生的能量将底物进行逆浓度梯度运输.所有生物体都含有大量ABC蛋白.ABC蛋白位于细胞的不同空间,如细胞膜、液泡、线粒体和过氧化物酶体.通常,ABC转运蛋白由跨膜结构域(TMD)和核苷酸结合结构域(NBD)组成,分别与底物和ATP结合.NBD执行与ATP结合和水解,是ABC转运蛋白的动力引擎,TMD识别特异性配体.大多数ABC转运蛋白最初是通过研究生物体耐药性而被发现的,包括多效耐药(PDR)和多药耐药(MDR).本文对ABC转运蛋白的结构及作用机制,以及植物病原真菌中ABC转运蛋白功能的研究进展进行综述.     

Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes

Heat shock protein (HSP)70 can be released from tumor cells and stimulate a potent antitumor immune response. However, HSP70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. We have observed HSP70 release from intact human prostate carcinoma cell lines (PC-3 and LNCaP) by a mechanism independent of de novo HSP70 synthesis or cell death. This pathway is similar to one used by the leaderless protein IL-1beta. Our studies show that HSP70 release involves transit though an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of HSP70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of HSP70 into this secretory compartment appears to involve the ABC family transporter proteins and ABC transporter inhibitor glibenclamide antagonizes secretion. Although the cell signals involved in triggering stress induced HSP70 release though this lysosomal pathway are largely unknown, our experiments suggest a regulatory role for extracellular ATP. These mechanisms appear to be shared by IL-1beta secretion. Following release, we observed the binding of extracellular HSP70 to the cell surface of the prostate carcinoma cells. These findings suggest that secreted HSP70 can take part in paracrine or autocrine interactions with adjacent cell surfaces. Our experiments therefore suggest a mechanism for HSP70 secretion and binding to the surface of other cells that may be involved in recognition of the tumor cells by the immune system.     

The N terminus of the HasA protein and the SecB chaperone cooperate in the efficient targeting and secretion of HasA via the ATP-binding cassette transporter.

Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system. SecB also interacted specifically in vitro with the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.     

Alternative protein secretion: the Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast

To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor.     

A new pathway for protein export in Saccharomyces cerevisiae

Several physiologically important proteins lack a classical secretory signal sequence, yet they are secreted from cells. To investigate the secretion mechanism of such proteins, a representative mammalian protein that is exported by a nonclassical mechanism, galectin-1, has been expressed in yeast. Galectin-1 is exported across the yeast plasma membrane, and this export does not require the classical secretory pathway nor the yeast multidrug resistance-like protein Ste6p, the transporter for the peptide a factor. A screen for components of the export machinery has identified genes that are involved in nonclassical export. These findings demonstrate a new pathway for protein export that is distinct from the classical secretory pathway in yeast.     

Macrophage migration inhibitory factor directly interacts with hepatopoietin and regulates the proliferation of hepatoma cell

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in growth factor-dependent cell proliferation, cell cycle, angiogenesis, and tumorigenesis. Several studies have documented MIF expression in the sera following hepatic resection or in the course of liver cancer progression, but there is a paucity of information regarding the effect of MIF on hepatoma cells and relating mechanisms. In this paper, by [3H] thymidine incorporation, we found that exogenously added MIF could promote the proliferation of HepG2 in a dose-dependent manner. Hepatopoietin (HPO), as a liver-specific regeneration augmenter, could be induced by the expression of MIF in hepatoma cells. The activity of HPO promoter was increased, and its levels were enhanced after MIF was overexpressed in hepatoma cells. The similarities between HPO and MIF in structure and action led us to investigate their interaction and the inducing biological significance. Using yeast two-hybrid identification, we found that HPO interacted with MIF in yeast cells, and their binding ability was higher than that between HPO and JAB1 (Jun activation domain binding protein) or MIF and JAB1 in yeast cells. Their interaction was further verified by His pull-down assay in vitro and coimmunoprecipitation experiment in vivo. They were colocalized in the cytoplasm. Both HPO and MIF could bind to JAB1 and modulate the AP-1 pathway. When HPO and MIF were cotransfected into HepG2 cells, the binding activity of MIF to JAB1 was reduced, and the activity of AP-1 was improved. In contrast, MIF overexpressed in HepG2 was unable to interfere with the binding activity of HPO to JAB1, but its potentiation on AP-1 activity was reduced significantly. Taken together, these results indicate that MIF plays an important role in the proliferation of hepatoma cells, and the effect of MIF is in concert with HPO.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

Secretion of the Serratia marcescens HasA protein by an ABC transporter.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

The SecB chaperone is involved in the secretion of the Serratia marcescens HasA protein through an ABC transporter.

The secretion pathways of the heme-binding protein HasA from Serratia marcescens and of the metalloproteases A, B, C and G from Erwinia chrysanthemi have been reconstituted in Escherichia coli. They are secreted in a single step from the cytoplasm across both membranes of the Gram-negative envelope, after recognition of their specific C-terminal secretion signal by their cognate ABC transporter. We report strong evidence that both HasA and the metalloproteases bind the SecB chaperone involved in the export of several envelope proteins via the Sec pathway. We also show that the secretion of the HasA protein is strongly dependent upon SecB in the reconstituted system, whereas that of the proteases is not. HasA secretion in the original host is strongly inhibited by a protein known to interfere with E.coli SecB function. We propose that the proteins secreted by the ABC pathway may have to be unfolded for efficient secretion.     

The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide.

The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtDEC, prtEEC, and prtFEC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtFEC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF(EC) system.