Mechanism of Chlamydia-induced centrosome amplification

描述（由申请人提供）：衣原体感染是美国最常报告的感染性疾病。沙眼衣原体是细菌性性传播感染的最常见原因，它与宫颈癌相关，既是一个独立的风险因素，也是人乳头瘤病毒的一个假定的辅助因素。然而，衣原体可能导致或促成癌症的机制尚不清楚。最近，C.沙眼衣原体已显示在受感染的细胞中诱导中心体异常，这是令人感兴趣的，因为中心体异常可导致基因组不稳定性，并且已在大多数人类癌症中检测到。我们有了一个新的发现，即中心体复制的关键调节因子HsSAS-6在C.沙眼感染此外，我们的研究提供了间接的证据表明，切割去除了在C-末端的HsSAS-6的降解基序，这是必要的调节HsSAS-6蛋白水平，使中心体复制只发生一次在细胞周期中。基于这些发现，我们的假设是，HsSAS- 6是由衣原体蛋白酶蛋白水解切割，产生一个截短的，但稳定的形式的蛋白质，导致中心体扩增正常的中心体复制失调。 该假设将通过2个特定目标进行检验： 在第一个目标中，将研究HsSAS-6裂解的后果，以了解衣原体感染如何导致中心体扩增。将检查在衣原体感染细胞中检测到的截短的HsSAS-6产物，以确定其是否是在细胞周期期间不像全长HsSAS-6那样发生降解的稳定形式。我们还将研究是否截短的HsSAS-6的表达是足以引起中心体扩增在没有衣原体感染。 在第二个目标中，将研究HsSAS-6的切割。HsSAS-6中的切割位点将通过蛋白质测序和质谱法确定。将建立可切割重组HsSAS-6以产生大量切割产物用于分析的体外切割测定。将测试一种充分表征的衣原体蛋白酶CPAF，以确定其是否是切割HsSAS-6的蛋白酶。CPAF是一个有希望的候选物，因为已知它在与HsSAS-6切割相同的时间过程中切割衣原体感染的细胞中的几种宿主蛋白。 这些研究的成功完成将有助于我们了解导致衣原体感染细胞中心体扩增的潜在机制，并可能为与衣原体相关的宫颈癌发病率增加提供病理生理学解释。沙眼感染公共卫生相关性：中心体通过其在细胞分裂和染色体分离中的作用影响基因组稳定性，在许多人类癌症中失调。此外，在感染细胞内细菌沙眼衣原体（Chlamydia trachomatis）期间，也检测到中心体异常，这与宫颈癌有关。由于我们已经发现，中心体数量的关键调节因子HsSAS-6在衣原体感染期间被切割，我们希望测试HsSAS-6在控制中心体数量中的功能失调是否将衣原体感染与基因组不稳定性和宫颈癌联系起来。本项目的目的是研究HsSAS-6切割对这种蛋白质的功能和调节的影响，并确定衣原体感染期间观察到的HsSAS-6切割产物是否足以诱导中心体异常。我们还旨在确定在感染这种流行病学上显著的细菌期间这种裂解事件的位点和分子要求。

DESCRIPTION (provided by applicant): Chlamydia infections are the most commonly reported infectious disease in the U.S. Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, and it has been associated with cervical cancer, both as an independent risk factor and as a putative co-factor for human papillomavirus. However, the mechanisms by which Chlamydia may cause or contribute to cancer are not understood. Recently, C. trachomatis has been shown to induce centrosome abnormalities in an infected cell, which is intriguing as centrosome abnormalities can lead to genomic instability and have been detected in the majority of human cancers. We have made a novel finding that a key regulator of centrosome duplication, HsSAS-6, is cleaved during a C. trachomatis infection. Furthermore, our studies provide indirect evidence that cleavage removes a degradation motif at the C-terminus of HsSAS-6 that is necessary for regulating HsSAS-6 protein levels so that centrosome duplication only occurs once in a cell cycle. Based on these findings, our hypothesis is that HsSAS- 6 is proteolytically cleaved by a chlamydial protease to produce a truncated but stabilized form of the protein that causes centrosome amplification by dysregulation of normal centrosome duplication. This hypothesis will be tested with 2 specific aims: In the first aim, the consequences of HsSAS-6 cleavage will be studied to understand how a chlamydial infection leads to centrosome amplification. The truncated HsSAS-6 product detected in Chlamydia-infected cells will be examined to determine if it is a stabilized form that does not undergo degradation during the cell cycle like full length HsSAS-6. We will also examine whether expression of truncated HsSAS-6 is sufficient to cause centrosome amplification in the absence of a Chlamydia infection. In the second aim, the cleavage of HsSAS-6 will be investigated. The cleavage site in HsSAS-6 will be determined by protein sequencing and mass spectrometry. An in vitro cleavage assay that can cleave recombinant HsSAS-6 to produce large amounts of the cleavage product for analysis will be established. A well-characterized chlamydial protease, CPAF, will be tested to determine if it is the protease that cleaves HsSAS-6. CPAF is a promising candidate because it is known to cleave several host proteins in a Chlamydia-infected cell over the same time course as cleavage of HsSAS-6. Successful completion of these studies will help us understand the underlying mechanisms that lead to centrosome amplification in Chlamydia-infected cells and may provide a pathophysiologic explanation for the increased incidence of cervical cancer associated with C. trachomatis infection. PUBLIC HEALTH RELEVANCE: The centrosome, which through its role in cell division and chromosome segregation has effects on genomic stability, is dysregulated in many human cancers. In addition, centrosome abnormalities have been detected during infections with the intracellular bacterium, Chlamydia trachomatis, which has been associated with cervical cancer. As we have found that the key regulator of centrosome number, HsSAS-6, is cleaved during a chlamydial infection, we wish to test whether the dysregulation of HsSAS-6 function in the control of centrosome number links Chlamydia infections to genomic instability and cervical cancer. It is the goal of this project to examine the effect of HsSAS-6 cleavage on the function and regulation of this protein and to determine if the HsSAS-6 cleavage products seen during a chlamydial infection are sufficient to induce centrosome abnormalities. We also aim to determine the site and molecular requirements for this cleavage event during an infection with this epidemiologically significant bacterium.