Molecular mechanisms governing the ubiquitination signaling during KSHV cell entry and tumorigenesis

感染卡波西肉瘤疱疹病毒(KSHV)可增加患经典型卡波西病的风险 肉瘤(KS)、艾滋病相关KS(AIDS-KS)和淋巴增生性疾病，包括原发积液 HIV/AIDS中的淋巴瘤(PEL)和多中心Castleman病(MCD)。约30%-40%的艾滋病相关患者 在接受抗逆转录病毒药物治疗的HIV+患者中，癌症是由KSHV感染引起的，在许多人中 在这种情况下，它们是无法治愈的。新出现的研究表明，慢性炎症由 KSHV感染细胞中的转录因子，包括STAT3和NF-κB在KSHV的发育中起关键作用 在HIV(+)个体中与KSHV相关的恶性肿瘤。蛋白质泛素化，一种翻译后修饰， 在调节NF-κB和STAT3的激活、致病病毒进入靶细胞和 病毒感染细胞的存活。使用一种无偏见的Ubi-Scan蛋白质组学方法，我们已经确定了肿瘤 赖氨酸437(K437)上CADM1的特异性泛素化，这对STAT3和NF-3的慢性激活是必不可少的 κB与细胞增殖和存活的关系。CADM1-K63-UBI对于有效进入KSHV也至关重要 新感染KSHV后的靶细胞。通过基于siRNA文库的筛选，我们鉴定了E2泛素 结合酶UBC13和E3泛素连接酶Mib1，作为CADM1泛素化的必需因子。在……里面 此外，我们发现泛素化的CADM1是激活KSHV-1中的小GTP酶rac1所必需的。 感染细胞，进而对慢性STAT3和NF-κB的激活以及PEL细胞的增殖和 生死存亡。此外，使用基于siRNA文库的鸟核苷酸交换因子(GEF)的筛选，我们 还确定了全球环境基金TIAM1是激活rac1所必需的。因此，我们假设KSHV 从头感染诱导的UBC13和Mib1依赖的CADM1-K63泛素化导致激活 KSHV进入靶细胞所必需的TIAM1/RAC1、NF-κB和STAT3的潜伏期建立 和KSHV淋巴瘤细胞的存活和增殖。为了验证这一假设，我们提出了三个具体目标：1) CADM1-K63-Ubi及其相关分子在KSHV新生进入靶细胞中的作用机制 感染，2)CADM1-K63-Ubi及其相关分子在NF-κB和STAT3激活中的机制作用 KSHV新感染后，3)检测小分子抑制剂对CADM1-K63-Ubi的疗效 其相关分子采用PEL患者来源的异种移植(PDX)小鼠模型。从本网站获得的数据 该项目将揭示慢性STAT3和NF-κB激活以及KSHV有效进入靶点的机制 新感染KSHV后的细胞。这些发现将为KSHV的治疗干预铺平道路。 介导性高度侵袭性艾滋病相关恶性肿瘤，常见于美国各地，尤其是 南佛罗里达州是全国艾滋病毒发病率最高的地区之一。

Infection with Kaposi sarcoma herpesvirus (KSHV) has been shown to increase the risk of classic Kaposi's sarcoma (KS), AIDS-associated KS (AIDS-KS) and lymphoproliferative disorders, including primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD) in HIV/AIDS. About 30-40% of AIDS-related cancers in HIV+ individuals treated with antiretroviral drugs are caused due to KSHV infection, and in many circumstances they are incurable. Emerging studies have indicated that chronic inflammation mediated by transcription factors, including STAT3 and NF-κB, in KSHV-infected cells plays critical role in the development of KSHV-associated malignancies in HIV (+) individuals. Protein ubiquitination, a post-translational modification, plays key roles in the regulation of NF-κB and STAT3 activation, pathogenic virus entry into target cells, and survival of virus infected cells. Using an unbiased Ubi-Scan proteomics approach, we have identified tumor specific CADM1 ubiquitination at lysine 437 (K437), which is essential for chronic activation of STAT3 and NF- κB and the proliferation and survival of PEL cells. CADM1-K63-Ubi is also critical for efficient KSHV entry into target cells after KSHV de novo infection. Using siRNA library-based screening we identified the E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligase Mib1, as essential factors for CADM1 ubiquitination. In addition, we found that ubiquitinated CADM1 was required for the activation of the small GTPase Rac1 in KSHV- infected cells, which in turn was critical for chronic STAT3 and NF-κB activation and PEL cell proliferation and survival. Furthermore, using siRNA library-based screening of Guanine nucleotide exchange factors (GEFs), we have also determined that the GEF TIAM1 is required for Rac1 activation. Therefore, we hypothesize that KSHV de novo infection-induced rapid Ubc13 and Mib1-dependent CADM1-K63 ubiquitination leads to the activation of TIAM1/Rac1 and NF-κB and STAT3 essential for KSHV entry into target cells, KSHV latency establishment and KSHV lymphoma cells survival and proliferation. To test this hypothesis, we propose three specific Aims: 1) Mechanistic roles of CADM1-K63-Ubi and its associated molecules in KSHV entry into target cells after de novo infection, 2) Mechanistic roles of CADM1-K63-Ubi and its associated molecules in NF-κB and STAT3 activation after KSHV de novo infection, and 3) Examine the efficacy of small molecule inhibitors against CADM1-K63-Ubi and its associated molecules using PEL patient-derived xenograft (PDX) mouse model. Data obtained from this project will reveal the mechanisms of chronic STAT3 and NF-κB activation and efficient KSHV entry into target cells after KSHV de novo infection. These findings will pave the way for therapeutic intervention of KSHV- mediated highly aggressive AIDS associated malignancies, which are often seen throughout the US, particularly South Florida, which has one of the highest incidences of HIV in the country.