Mechanism of Chlamydia-induced centrosome amplification

描述(申请人提供)：衣原体感染是美国最常见的传染病。沙眼衣原体是细菌性传播感染的最常见原因，它与宫颈癌有关，既是一个独立的风险因素，也是人类乳头瘤病毒的假定辅助因素。然而，衣原体可能导致或促进癌症的机制尚不清楚。最近，沙眼衣原体被证明可以在感染细胞中诱导中心体异常，这很有趣，因为中心体异常可以导致基因组不稳定，并且已经在大多数人类癌症中检测到。我们有一个新的发现，中心体复制的关键调控因子HsSAS-6在沙眼衣原体感染期间被切割。此外，我们的研究提供了间接证据，表明切割去除了HsSAS-6 C末端的一个降解基序，该基序对于调节HsSAS-6蛋白水平是必要的，从而使中心体复制在细胞周期中只发生一次。基于这些发现，我们的假设是HsSAS-6被衣原体蛋白水解酶切割，产生一种截短但稳定的蛋白质形式，通过对正常中心体复制的失调而导致中心体放大。 这一假设将通过两个具体目标进行检验： 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。将对在衣原体感染细胞中检测到的截短的HsSAS-6产物进行检查，以确定它是否是一种稳定的形式，在细胞周期中不会像全长HsSAS-6那样经历降解。我们还将检查在没有衣原体感染的情况下，截短的HsSAS-6的表达是否足以引起中心体扩增。 在第二个目的中，将研究HsSAS-6的切割。HsSAS-6的裂解位点将通过蛋白质测序和质谱分析来确定。将建立一种能够切割重组HsSAS-6以产生大量切割产物用于分析的体外切割试验。一种特性良好的衣原体蛋白水解酶CPAF将进行测试，以确定它是否是裂解HsSAS-6的蛋白酶。CPAF是一个很有希望的候选者，因为已知它在衣原体感染细胞中切割几个宿主蛋白的过程与切割HsSAS-6的时间过程相同。 这些研究的成功完成将有助于我们了解导致沙眼衣原体感染细胞中心体扩增的潜在机制，并可能为沙眼衣原体感染相关的宫颈癌发病率增加提供病理生理学解释。与公共卫生相关：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的稳定性，在许多人类癌症中处于失调状态。此外，在感染细胞内细菌沙眼衣原体的过程中也检测到中心体异常，沙眼衣原体与宫颈癌有关。由于我们已经发现中心体数量的关键调控因子HsSAS-6在衣原体感染过程中被切割，我们希望测试HsSAS-6在控制中心体数量方面的功能失调是否与衣原体感染和基因组不稳定和宫颈癌有关。本项目的目标是研究HsSAS-6裂解对该蛋白的功能和调节的影响，并确定衣原体感染期间看到的HsSAS-6裂解产物是否足以引起中心体异常。我们的目标也是确定在感染这种具有流行病学意义的细菌期间，这种分裂事件的位置和分子要求。

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.