Characterization of Mortalin's interaction with HIV-1 Nef and role in Nef vesicle

Mortalin 与 HIV-1 Nef 相互作用的表征以及在 Nef 囊泡中的作用

• 批准号: 8071385
• 负责人: MARTIN NEVILLE SHELTON
• 金额: $ 3.89万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-27 至 2012-01-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8071385
• 关键词: Accounting; Acquired Immunodeficiency Syndrome; Affect; Antibodies; Antiviral Agents; Apoptosis; Apoptotic; Binding; Binding Sites; Biological Assay; CD4 Positive T Lymphocytes; CXCR4 Receptors; Cell Lineage; Cell Survival; Cell physiology; Cells; Cessation of life; Data; Development; Engineering; Family member; Fluorescence; Functional disorder; Generations; HIV; HIV-1; Health; Heat-Shock Proteins 70; Human; Infection; Injection of therapeutic agent; Jurkat Cells; Knowledge; Legal patent; Length; Life; Literature; Manuscripts; Maps; MicroRNAs; Modification; Mus; N-terminal; Pathogenesis; Pathway interactions; Patients; Peptides; Plasma; Play; Prevention; Procedures; Process; Production; Protein p53; Proteins; Recombinants; Research; Rest; Role; SIV; T-Cell Depletion; T-Lymphocyte; Testing; Transfection; Tubulin; Tumor Suppressor Proteins; United States; Vaccines; Vesicle; Viral Proteins; Work; base; cell killing; expression vector; extracellular; functional disability; in vivo; inhibitor/antagonist; mortalin; nef Protein; novel therapeutics; prevent; protein transport; research study; success; therapeutic target; trafficking; virus infection mechanism

DESCRIPTION (provided by applicant): There is evidence that secreted HIV-1 Nef plays an important role in affecting T cell function and in the survival of bystander uninfected cells in vivo. However, the mechanism(s) by which Nef is secreted has not been elucidated. We hypothesize that cellular proteins which interact with Nef regulate its secretion. Intracellularly, Nef is known to interact with the endosomal trafficking pathway, and has been shown to upregulate the exosomal machinery of the host cell. Furthermore, our group has mapped highly conserved Nef motifs in the N-terminal regions of both HIV and SIV Nef that are critical for secretion, and have engineered a peptide based on one of these Nef secretion motifs, the Secretion Modification Region (SMR). This SMRwt peptide, which contains only the SMR motif and which inhibits Nef vesicle secretion, was used to co-immunoprecipitate four cellular binding partners. One of these host cell proteins was identified as heat shock protein 70 family member, mortalin. Though much of the literature on mortalin centers on its role in blocking apoptosis through its interaction with the tumor suppressor protein p53, there is evidence that it is also involved in exosomal trafficking. As Nef also contains a p53-binding site in its N-terminal region, it is possible that the full-length Nef protein does not bind mortalin directly, but rather interacts with it via their mutual interactions with p53. We hypothesize that Nef and mortalin directly interact allowing Nef to catalyze the intracellular secretary process and release itself in exosomes-like vesicles. This is currently being tested through the following experiments, (i) FLAG-tagged mortalin is being used to co-immunoprecipitate wild type Nef-GFP from Jurkat cells to verify that, in addition to binding the SMRwt peptide, mortalin interacts with the full-length Nef protein; and, (ii) a binding assay with bacterial recombinant wild type Nef-GFP and purified FLAG-tagged mortalin is being used to determine if Nef interacts directly with mortalin, or indirectly via an unidentified host cell protein. Since the SMRwt peptide has been shown to inhibit Nef secretion, presumably by out-competing it for a cellular binding partner, we hypothesize that this inhibition is a result of disrupting mortalin's interaction with Nef. If true, mortalin is necessary for Nef secretion, and disrupting this interaction by other means will also inhibit Nef's secretion in exosome-like vesicles. To test this, the effect on Nef secretion will be assessed following (i) knockdown of the expression of mortalin using microRNA (miRNA); and, (ii) blocking of mortalin's interaction with Nef using antibody inhibition. The results of these experiments will generate fundamental knowledge of the cellular component(s) of Nef secretion. Given the proven difficulty in targeting Nef directly, this knowledge will open new therapeutic avenues to prevent AIDS pathogenesis through the newly identified host cell trafficking proteins. PUBLIC HEALTH RELEVANCE: The development of an HIV/AIDS vaccine has proven to be more difficult than anticipated, and the production of new antiviral drugs, many of them targeting viral proteins, has provided the greatest success in reducing AIDS deaths. This research will confirm the identification of a new set of cellular trafficking proteins involved in progression to AIDS, which could be potential therapeutic targets.

描述（由申请人提供）：有证据表明，分泌的HIV-1 NEF在影响T细胞功能和体内未感染细胞的存活中起着重要作用。但是，尚未阐明分泌NEF的机制。我们假设与NEF相互作用的细胞蛋白调节其分泌。众所周知，NEF在细胞内与内体运输途径相互作用，并已证明可以上调宿主细胞的外泌体机械。此外，我们的小组已经在HIV和SIV NEF的N末端区域绘制了高度保守的NEF基序，这对于分泌至关重要，并基于这些NEF分泌基序中的一个（分泌修改区域（SMR））设计了肽。该SMRWT肽仅包含SMR基序并抑制NEF囊泡分泌，用于共免疫沉淀，以供四个细胞结合伴侣。这些宿主细胞蛋白之一被鉴定为热休克蛋白70家族成员莫尔塔林。尽管有关洛尔替林的许多文献都集中在其与肿瘤抑制蛋白p53相互作用通过相互作用来阻断细胞凋亡中的作用，但有证据表明它也参与外泌体贩运。由于NEF在其N末端区域还包含一个p53结合位点，因此全长NEF蛋白可能不会直接结合凡尔蛋白，而是通过与p53的相互作用与之相互作用。我们假设NEF和Mortalin直接相互作用，从而使NEF催化细胞内秘书过程并在外泌体样囊泡中释放。目前正在通过以下实验对此进行测试，（i）标记为标记的mortalin可用于从Jurkat细胞中共免疫沉淀野生型Nef-GFP，以验证，除了结合SMRWT肽外，Mortalin与全长NEF NEF NEF蛋白相互作用； （ii）使用细菌重组野生型NEF-GFP和纯化的FLAG标记的Mortalin的结合测定法被用于确定NEF是否直接与Mortalin相互作用，或通过未识别的宿主细胞蛋白间接相互作用。由于SMRWT肽已被证明可以抑制NEF分泌，这可能是通过将其用于细胞结合伴侣的能力，因此我们假设这种抑制是破坏Mortalin与NEF相互作用的结果。如果是真的，凡尔林对于NEF分泌是必需的，并且通过其他方式破坏这种相互作用也将抑制NEF在外泌体样囊泡中的分泌。为了测试这一点，将在使用microRNA（miRNA）敲除玛alin蛋白的表达后评估对NEF分泌的影响； （ii）使用抗体抑制作用阻断莫达林与NEF的相互作用。这些实验的结果将产生对NEF分泌的细胞成分的基本知识。鉴于可证明的难以直接靶向NEF，因此该知识将开放新的治疗途径，以防止通过新确定的宿主细胞运输蛋白来防止艾滋病发病机理。 公共卫生相关性：事实证明，艾滋病毒/艾滋病疫苗的开发比预期的要困难得多，而新抗病毒药的生产（其中许多针对病毒蛋白的靶向）在减少艾滋病死亡方面取得了最大的成功。这项研究将确定鉴定出一组参与艾滋病进展的细胞运输蛋白，这可能是潜在的治疗靶标。