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Retroviral Subversion of ERAD and Intrinsic Immunity

ERAD 和内在免疫的逆转录病毒颠覆

基本信息

批准号：
8542800
负责人：
Jaquelin Page Dudley
金额：
$ 34.33万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-10 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8542800
关键词：
Affect Alzheimer&apos s Disease Amino Acids Antibodies Antibody Formation Area B-Cell Neoplasm B-Lymphocytes Bacteria Binding Biological Assay C-terminal Cations Cell Culture Techniques Cell Nucleus Cells Chromosomal translocation Cleaved cell Communicable Diseases Complex Cystic Fibrosis Cytidine Deaminase Cytidine Deaminase Inhibitor Cytosol DNA Double Strand Break Data Development Disease Dislocations Endoplasmic Reticulum Endoplasmic Reticulum Degradation Pathway Engineering Enzymes Failure Family Family member Fluorescence Genetic Transcription Genome Goals HIV Hormones Human Immune response Immunity Immunoglobulin Class Switching Immunoglobulin Somatic Hypermutation Immunoglobulin Switch Recombination Infection Knockout Mice Knowledge Length Libraries Malignant Neoplasms Mammary gland Mass Spectrum Analysis Mediating Membrane Messenger RNA Modeling Molecular Chaperones Monitor Mouse Mammary Tumor Virus Mus Mutagens Mutation N-terminal Nonsense Codon Nuclear Export Nuclear Import Oncogenic Oncogenic Viruses Paper Pathway interactions Peptide Signal Sequences Peptides Phenotype Plasmids Polyubiquitination Post-Translational Protein Processing Process Production Protein Analysis Proteins Proviruses RNA Regulatory Pathway Reporter Retroviridae Scheme Series Signal Transduction Superantigens Terminator Codon Testing Translations Ubiquitin Ubiquitin-Activating Enzymes Viral Viral Genome Viral Proteins Virus Virus Diseases Virus Replication Work activation-induced cytidine deaminase base cancer therapy effective therapy genetic regulatory protein human disease in vivo innovation leukemia malignant breast neoplasm multicatalytic endopeptidase complex mutant novel pathogen prevent protein expression protein folding protein misfolding signal peptidase trafficking transmission process viral RNA

项目摘要

DESCRIPTION (provided by applicant): Alzheimer's, cystic fibrosis, infectious diseases, and cancer all have a common feature: failure to efficiently destroy incorrectly folded or assembled proteins. Transmembrane and secreted proteins use a signal peptide (SP) to direct translation to the endoplasmic reticulum (ER), where an error-prone process of protein folding and modification occur. The ER-associated degradation (ERAD) process identifies misfolded proteins, leading to addition of ubiquitin chains using E1, E2, and E3 enzymes. Polyubiquitinated proteins then are extracted from the ER membrane, a process known as retrotranslocation or dislocation, and delivered to cytosolic proteasomes for degradation. Many pathogens, including viruses and bacteria, exploit ERAD to further their dissemination. Mouse mammary tumor virus (MMTV) is a complex retrovirus that uses ERAD for replication and induction of breast cancer and leukemia. MMTV produces a regulatory protein (Rem), which is directed to the ER by a long signal peptide (Rem-SP) containing all the functional motifs found in HIV Rev. Rem is cleaved by signal peptidase in the ER lumen to yield N-terminal Rem-SP and a unique C-terminus (RemCT). In an unprecedented pathway, Rem-SP is retrotranslocated to the cytosol, where it avoids proteasomal degradation prior to nuclear import. Rem-SP then binds viral RNA to allow efficient nuclear export and expression. Although retrotranslocation is believed to require polyubiquitination of target proteins, our preliminary data indicate that the E enzyme used for all known ERAD substrates is dispensable for Rem-SP retrotranslocation. Our hypothesis is that Rem-SP uses a previously unknown process that does not require polyubiquitination or the E1 enzyme Uba1 for retrotranslocation. In the first specific aim, two approaches will be used to identify cellular components involved in subversion of ERAD. C-terminal GFP tagging of Rem blocks retrotranslocation and will be used to purify a retrotranslocation intermediate and associated cellular proteins for analysis by mass spectrometry. An alternative approach will use a fluorescence-based reporter assay and a small-hairpin library to identify new cellular proteins involved in ERAD and Rem trafficking. In the second specific aim, exciting preliminary data have been presented showing that MMTV proviruses lacking RemCT expression have many G to A mutations compared to wild-type proviruses. Co-expression of Rem and activation-induced cytidine deaminase (AID), a known mutagen required for antibody somatic hypermutation and class switch recombination, results in AID degradation. We propose that MMTV is the first virus to antagonize AID to avoid both viral genome mutation and maturation of the MMTV-specific antibody response. This idea will be tested by monitoring RemCT localization and its ability to antagonize cellular cytidine deaminases both in cell culture and in knockout mice in vivo. Further understanding of ERAD and intrinsic immunity are essential to develop new treatments for cancer and pathogenic viral infections.

描述（由申请人提供）：阿尔茨海默病、囊性纤维化、传染病和癌症都具有共同的功能：不能有效地破坏错误折叠或组装的蛋白质。跨膜蛋白和分泌蛋白使用信号肽（SP）指导翻译至内质网（ER），在内质网中发生蛋白质折叠和修饰的易错过程。ER相关降解（ERAD）过程识别错误折叠的蛋白质，导致使用E1，E2和E3酶添加泛素链。然后，从ER膜提取多泛素化蛋白质，这一过程被称为逆转位或移位，并被递送到胞质蛋白酶体进行降解。许多病原体，包括病毒和细菌，利用ERAD来进一步传播。小鼠乳腺肿瘤病毒（MMTV）是一种复杂的逆转录病毒，它利用ERAD复制并诱导乳腺癌和白血病。MMTV产生调节蛋白（Rem），其通过含有HIV Rev中发现的所有功能基序的长信号肽（Rem-SP）导向ER。Rem在ER腔中被信号肽酶切割，产生N-末端Rem-SP和独特的C-末端（RemCT）。在一个前所未有的途径，Rem-SP是retrotranslocated细胞质，在那里它避免蛋白酶体降解之前，核输入。Rem-SP然后结合病毒RNA以允许有效的核输出和表达。虽然逆转录被认为需要靶蛋白的多泛素化，我们的初步数据表明，用于所有已知的ERAD底物的E酶是Rem-SP逆转录。我们的假设是，Rem-SP使用了一个以前未知的过程，不需要多聚泛素化或E1酶Uba 1的逆转位。在第一个具体的目标，两种方法将被用来确定参与颠覆ERAD的细胞成分。Rem的C-末端GFP标记阻断逆转录易位，并将用于纯化逆转录易位中间体和相关细胞蛋白，以通过质谱法进行分析。另一种方法将使用基于荧光的报告基因测定和小发夹文库来鉴定参与ERAD和Rem运输的新细胞蛋白。在第二个具体目标中，已经提出了令人兴奋的初步数据，显示与野生型前病毒相比，缺乏RemCT表达的MMTV前病毒具有许多G至A突变。Rem和激活诱导的胞苷脱氨酶（AID）（抗体体细胞超突变和类别转换重组所需的已知诱变剂）的共表达导致AID降解。我们认为MMTV是第一个拮抗AID的病毒，以避免病毒基因组突变和MMTV特异性抗体反应的成熟。将通过监测RemCT定位及其在细胞培养物和体内敲除小鼠中拮抗细胞胞苷脱氨酶的能力来测试这一想法。进一步了解ERAD和内在免疫对于开发癌症和致病性病毒感染的新疗法至关重要。