Progresses in the structure and biological function of Raf kinase inhibitory protein

rAf激酶抑制蛋白(rAf kInASE InHIbITOry PrOTEIn,rkIP)属于磷脂酰乙醇胺结合蛋白(PHOSPHATIdy-lETHAnOlAMInE bIndIng PrOTEIn,PEbP)家族,参与多种生理过程,可以交叉调控MAPk(MITOgEn-ACTIVATEdPrOTEIn kInASE)通路、gPCr(g-PrOTEIn-COuPlEd rECEPTOr)通路和nf-κb(nuClEAr fACTOr-κb)信号通路。细胞信号转导通路的异常会促进肿瘤的发生与发展。rkIP在肿瘤发生、发展中发挥着重要作用,影响肿瘤细胞的转移和治疗,是一种新的肿瘤诊断标志物。开展rkIP的结构生物学研究有助于理解rkIP发挥生理功能的结构基础,对揭示人类重大疾病(肿瘤)的分子机制以及研发用于细胞信号转导异常相关疾病的药物,都有着极其重要的科学意义。主要阐述了rkIP的结构特点、生物学功能及其与肿瘤疾病的关系。Raf kinase inhibitory protein(RKIP) is a member of the phosphatidylethanolamine-binding protein(PEBP) superfamily,which plays vital roles in many physiological processes and regulates MAPK,GPCR and NFκB signaling pathways.Abnormal signaling pathways will promote tumorigenesis and development.It has been reported that RKIP modulates tumors at several stages including tumorigenesis,development,metastasis and therapeutic response.By now RKIP has been considered as a new biomarker for cancer diagnosis.Therefore,it is highly significant to study the structure and function of RKIP,which helps us understand the structural basis and the molecular mechanism of RKIP-modulated signal pathways,and facilitate the drug development against diseases caused by abnormal signal transduction.This article reviewed the structural characteristics,biological functions of RKIP and its relationship with tumor.国家自然科学基金项目(30900233

H-1, C-13, N-15 backbone and side-chain resonance assignments of the human Raf-1 kinase inhibitor protein

Natural Science Foundation of China [30900233, 30730026]; Program of Shanghai Subject Chief Scientist [09XD1405100]Raf-1 kinase inhibitor protein (RKIP) plays a pivotal role in modulating multiple signaling networks. Here we report backbone and side chain resonance assignments of uniformly N-15, C-13 labeled human RKIP

Prion protein oligomer and its neurotoxicity

National Natural Science Foundation of China [31170717, 91129713, 30900233]The prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders. According to the protein only hypothesis, the key molecular event in the pathogenesis of prion disease is the conformational conversion of the host-derived cellular prion protein (PrPC) into a misfolded form (scrapie PrP, PrPSc). Increasing evidence has shown that the most infectious factor is the smaller subfibrillar oligomers formed by prion proteins. Both the prion oligomer and PrPSc are rich in -sheet structure and resistant to the proteolysis of proteinase K. The prion oligomer is soluble in physiologic environments whereas PrPSc is insoluble. Various prion oligomers are formed in different conditions. Prion oligomers exhibited more neurotoxicity both in vitro and in vivo than the fibrillar forms of PrPSc, implying that prion oligomers could be potential drug targets for attacking prion diseases. In this article, we describe recent experimental evidence regarding prion oligomers, with a special focus on prion oligomer formation and its neurotoxicity

Solution structure and backbone dynamics of human Raf-1 kinase inhibitor protein

Chinese National Science Foundation [30900233, 31170717, 91129713]; Natural Science Foundation of Fujian Province [2011J01246]Human Raf-1 kinase inhibitor protein (hRKIP) is a small multi-functional protein of 187 residues. It contains a conserved pocket, which binds a wide range of ligands from various small molecules to distinct proteins. To provide a structural basis for the ligand diversity of RKIP, we herein determined the solution structure of hRKIP, and analyzed its structural dynamics. In solution, hRKIP mainly comprises two anti-parallel beta sheets, two alpha helices and two 3(10) helices. NMR dynamic analysis reveals that the overall structure of hRKIP is rigid, but its C-terminal helix which is close to the ligand-binding site is mobile. In addition, residues around the ligand-binding pocket exhibit significant conformational exchange on the mu s-ms timescale. Conformational flexibility may allow the ligand-binding pocket and the C-terminal helix to adopt various conformations to interact with different substrates. This work may shed light on the underlying molecular mechanisms of how hRKIP recognizes and binds diverse substrate ligands. (C) 2013 Elsevier Inc. All rights reserved

H-1, C-13, N-15 backbone and side-chain resonance assignments of the human adenylate kinase 1 in apo form

Natural Science Foundation of Fujian Province [2011J01246]; Natural Science Foundation of China [30900233, 31170717, 91129713]; Program of Shanghai Subject Chief Scientist [09XD1405100]AK1 (Adenylate Kinase 1) plays crucial roles in processes such as cellular phosphotransfer networks, neuronal maturation and regeneration, gating of ABC transporter CFTR, tumor cell metabolism and myocardial energetic homeostasis. Here we report H-1, N-15 and C-13 backbone and side-chain resonance assignments of the human AK1 protein in apo form. This work lays the essential basis for the further structure determination of hAK1

Structural basis for RKIP binding with its substrate Raf1 kinase

Raf1 kinase inhibitor protein (RKIP) negatively regulates the Raf1/MEK/ERK pathway which is vital for cell growth and differentiation. It is also a biomarker in clinical cancer diagnosis. RKIP binds to the N-terminus of Raf1 kinase but little is known about the structural basis of RKIP binding with Raf1. Here, we demonstrate that the N-terminus of human Raf1 kinase (hRaf11-147aa) binds with human RKIP (hRKIP) at its ligand-binding pocket, loop "127-149", and the C-terminal helix by NMR experiments. D70, D72, E83, Y120, and Y181 were further verified as the key residues participating in the interaction of hRKIP and hRaf11-147aa. G143-R146 fragment was also critical for hRKIP binding with hRaf11-147aa, for its deletion decreased the binding affinity around 300 times, from 154 to 0.46 mM-1. Our results provide important structural clues for designing the lead compound that disrupts RKIP-Raf1 interaction. ? 2014 Springer Science+Business Media Dordrecht

H-1, C-13 and N-15 backbone and side-chain resonance assignments of reduced CcmG from Escherichia coli

Natural Science Foundation of Fujian Province [2011J01246]; Natural Science Foundation of China [30900233, 31170717, 91129713]; Program of Shanghai Subject Chief Scientist [09XD1405100]CcmG is a periplasmic, membrane-anchored protein widely distributed in a variety of species. In Escherichia coli, the CcmG protein always acts as a weak reductant in the electron transport chain during cytochrome c maturation (Ccm). Here we report H-1, N-15 and C-13 backbone and side-chain resonance assignments of the reduced CcmG protein (residues 19-185, renumbered as 1-167) from E. coli. This work lays the essential basis for the further structural and functional analysis of reduced CcmG

Fusion Expression of Human β-Defensin DEFB113 and Its Biological Activities

作者简介: 董靖( 1981-) ，男，汉族，陕西宝鸡人，中国药科大学博士研究生，研究方向: 微生物与生化药学。Tel: 021-50806600-3113;E-mail: ostrich81111@163. com。 通讯作者: 林东海，教授，博士生导师，Tel: 0592-2186078; E-mail: dhlin@ xmu. edu. cn。[中文文摘]DEFB113为人β-防御素家族新成员,其生物学功能尚不清楚。为了探索一种可以较低成本和较高产量表达活性DEFB113多肽的方法,将编码DEFB113成熟肽的序列克隆到载体pTWIN1上,并转化至E.coliBL21(DE3)中进行表达。结果表明融合蛋白主要以可溶形式存在;重组载体中融合标签的几丁质结合域(CBD)和内含肽(intein)分别使亲和层析纯化和融合标签自剪切一步完成。最终得到DEFB113多肽的产率为6～7 mg/LLB。经活性鉴定,该重组DEFB113多肽对于S.aureus,E.coli K12D31和C.albicans均表现出较强的抑制作用;另外DEFB113无明显的溶血活性,是一种较理想的多肽抗生素。该研究为进一步研究DEFB113的生理功能以及其它人β-防御素结构与功能奠定了基础。[英文文摘]DEFB113 is a recently identified human β-defensin,while its biological functions remain unclear.With the purpose of providing a low-cost and high-yielding approach to express DEFB113 for the determination of its bioactivities,the sequence encoding the mature peptide of DEFB113 was inserted into pTWIN1 and the recombinant transformed vector into E.coli BL21(DE3) for the recombinant expression.Most of the fusion proteins were expressed in a soluble form.Moreover,the chitin-binding domain(CBD) and the intein in the fusion tag facilitated the affinity chromatography and the auto-cleavage of the fusion tag,respectively.The resulting DEFB113 peptide was produced with a yield of 6.4 mg /L LB.The purified DEFB113 peptide exhibited potent antimicrobial activities against E.coli,S.aureus and C.albicans.In addition,the recombinant DEFB113 peptide caused little if any hemolysis towards human erythrocytes.In this respect,DEFB113 might exist as a promising peptide antibiotic.These attempts have laid the basis for further study on the physiological roles of DEFB113 as well as on the structure-function relationships of other DEFBs.上海市优秀学科带头人计划(A类)(No.09XD1405100