Elucidation of cystatin B associated signaling pathways to reduce HIV reservoirs

阐明胱抑素 B 相关信号通路以减少 HIV 储存

• 批准号: 8401015
• 负责人: Linda E Rivera
• 金额: $ 5.71万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-10 至 2015-01-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401015
• 关键词: Affect; Antiviral Agents; Antiviral Response; Biological Assay; Blood - brain barrier anatomy; Brain; CD8-Positive T-Lymphocytes; Clinical; Cystatins; Cysteine Proteinase Inhibitors; Data; Development; Disease; Genes; Goals; HIV; HIV Infections; HIV-1; In Situ; Infection; Interferon Regulatory Factor 1; Interferons; Knowledge; Laboratories; Lead; Ligation; Long Terminal Repeats; Luciferases; Mediating; Microglia; Modification; Mononuclear; Natural Immunity; Neuraxis; Neurocognitive; Pathway interactions; Phagocytes; Pharmacological Treatment; Phosphorylation; Prevention; Proteins; Proteomics; Regulation; Reporting; Research; Role; STAT1 gene; Serine; Signal Pathway; Signaling Protein; Site; Testing; Tissues; Tyrosine; Tyrosine Phosphorylation; Viral; Virus; Work; antiretroviral therapy; base; design; genetic regulatory protein; immune activation; innovation; macrophage; migration; monocyte; neurotoxicity; novel; novel therapeutic intervention; prevent; response; stefin

DESCRIPTION (provided by applicant): Monocytes, macrophages, and microglia are mononuclear phagocytes important in innate immunity, but also key reservoirs of HIV in the central nervous system. These reservoirs represent a challenge to HIV-1 eradication since they remain producing virus in tissue despite the presence of antiretroviral therapy. Cystatin B expression is positively correlated with HIV replication and decreased levels STAT-1 phosphorylation (STAT-1PY) in monocyte-derived macrophages (MDM). However, the players and the mechanism by which this occurs are unknown. This work is expected to elucidate the mechanism by which cystatin B contributes to HIV-1 replication by regulation of STAT-1PY in macrophage reservoirs. The objective of in this particular application is to define the relationships and signaling pathways between cystatin B, STAT-1PY, interferon (IFN), and HIV persistence in MDM. To attain the objective of this proposal, we will test hypothesis that cystatin B promotes HIV persistence by interacting with the STAT-1PY and additional proteins, including those related to IFN signaling pathway. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratories. The rationale of the proposed research is that understanding the role of cystatin B in HIV replication will permit the modulation of this protein or its interacting proteins and decrease HIV within macrophage reservoirs. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Define the cystatin B/STAT-1 signaling pathways and proteins associated to STAT-1 phosphorylation and HIV replication in macrophages; and 2) Identify the role of Cystatin B in the JAK/STAT-1 pathway during HIV infection and IFN-2 activation. Under the first aim we will use In situ Proximity Ligation Assay (PLA) (Olink Biosciences) to confirm, localize and quantify this protein interaction in uninfected and HIV infected MDM and determine if cystatin B interacts directly or indirectly with STAT-1. We will also identify the specific protein pathways associated to cystatin that are subject to modification by phosphorylation using spectral counting based quantification. Under the second aim, we will determine the effect of cystatin B in LTR mediated HIV replication and IFN induced antiviral response using the luciferase assay and correlate our findings with what happens in the MDM. This approach is innovative because, the role of cystatin B as a regulatory protein for in HIV replication has not been demonstrated using proteomics approaches and the interacting partners have not been found. The proposed research is significant because it will reveal novel mechanisms of HIV persistence that could be targeted for new therapeutic approaches directed to eliminate HIV in macrophage reservoirs.

描述（由申请人提供）：单核细胞、巨噬细胞和小胶质细胞是先天免疫中重要的单核吞噬细胞，也是中枢神经系统中 HIV 的关键储存库。这些储存库对根除 HIV-1 构成了挑战，因为尽管存在抗逆转录病毒治疗，它们仍然在组织中产生病毒。胱抑素 B 表达与 HIV 复制和单核细胞源性巨噬细胞 (MDM) 中 STAT-1 磷酸化 (STAT-1PY) 水平降低呈正相关。然而，参与者和发生这种情况的机制尚不清楚。这项工作有望阐明胱抑素 B 通过调节巨噬细胞储库中的 STAT-1PY 促进 HIV-1 复制的机制。此特定应用的目的是确定 MDM 中胱抑素 B、STAT-1PY、干扰素 (IFN) 和 HIV 持续存在之间的关系和信号通路。为了实现该提案的目标，我们将测试胱抑素 B 通过与 STAT-1PY 和其他蛋白质（包括与 IFN 信号通路相关的蛋白质）相互作用来促进 HIV 持续存在的假设。该假设是根据申请人实验室产生的初步数据制定的。这项研究的基本原理是，了解胱抑素 B 在 HIV 复制中的作用将允许调节该蛋白或其相互作用蛋白，并减少巨噬细胞储存库内的 HIV。在强有力的初步数据的指导下，这一假设将通过追求两个具体目标进行检验：1）定义胱抑素B/STAT-1信号通路以及与巨噬细胞中STAT-1磷酸化和HIV复制相关的蛋白质； 2) 确定 Cystatin B 在 HIV 感染和 IFN-2 激活过程中 JAK/STAT-1 通路中的作用。在第一个目标下，我们将使用原位邻近连接测定 (PLA) (Olink Biosciences) 来确认、定位和量化未感染和 HIV 感染的 MDM 中的这种蛋白质相互作用，并确定胱抑素 B 是否直接或间接与 STAT-1 相互作用。我们还将使用基于光谱计数的定量方法来确定与半胱氨酸蛋白酶抑制剂相关的特定蛋白质途径，这些蛋白质途径会受到磷酸化修饰。在第二个目标下，我们将使用荧光素酶测定确定胱抑素 B 在 LTR 介导的 HIV 复制和 IFN 诱导的抗病毒反应中的作用，并将我们的发现与 MDM 中发生的情况相关联。这种方法具有创新性，因为尚未使用蛋白质组学方法证明半胱氨酸蛋白酶抑制剂 B 作为 HIV 复制调节蛋白的作用，并且尚未发现相互作用的伙伴。拟议的研究意义重大，因为它将揭示艾滋病毒持续存在的新机制，可以作为消除巨噬细胞储存库中艾滋病毒的新治疗方法的目标。