Can inhibition of cyclin B1 destruction in G1 of mitosis be exploited to promote cancer cell death?

背景：CDK活性的振荡从G1期细胞周期开始驱动细胞周期进程。事实上，从G1的最早阶段开始，CDK活性在细胞决定进入细胞周期（通过启动DNA复制）或退出进入静止期中起着核心作用。重要的是，CDK仅在与细胞周期蛋白结合时才有活性。有许多细胞周期蛋白-CDK复合物，然而，只有细胞周期蛋白B1-CDK 1活性是必需的。至关重要的是，CDK活性水平受到高度调节，以正确的速度驱动进展，从而有足够的时间准确完成周期的每个阶段。为了促进这一点，细胞周期蛋白水平在健康细胞中被严格削弱。相反，细胞周期蛋白B1（B1）在许多人类癌症中过表达。此外，它的上调与不良预后、治疗抗性相关，并被认为是启动癌细胞与其微环境之间通信的信号。事实上，高表达的B1从G1开始就与CDK 1结合，导致肿瘤组织的侵袭性增殖。我们已经发现了一个新的基序在B1是至关重要的，它的调节，其中B1存在于卵母细胞减数分裂过量（参考）。在此之后，我们在有丝分裂中有令人兴奋的未发表数据，表明如果B1过表达，这个相同的基序对稳定B1水平G1很重要。值得注意的是，当我们抑制这种基序时，过表达B1的细胞就会死亡。 目标;1.目的：明确有丝分裂G1期过量B1表达调控的分子机制。2.在此之后，提出候选蛋白质，如果扰动，防止这种机制的过度B1的调节，迫使细胞dead.新奇和Timelty;我们的数据在有丝分裂是全新的，并提出了一个独特的机会，我们可以确定一个新的机制，我们可能会触发细胞死亡。值得注意的是，这种B1调节机制仅在过表达B1的细胞中是必需的，这意味着任何未来衍生的治疗方案都可以区分不稳定和健康的细胞。这是及时的，因为临床医生正在寻找保护健康细胞的有效治疗方法（限制一些替代治疗方法的经常使人衰弱的脱靶效应）。实验方法;学生将通过活的有丝分裂细胞中的Bio-ID分析，以及通过使用过表达B1的裂解细胞进行IP实验（有或无此关键基序），鉴定G1中新型细胞周期蛋白B1基序的结合伴侣（实验1）。然后，他们将在体内试验中确认候选蛋白，如BiFc，以确认邻近性（实验室1并与VisiTech合作）和体外（通过生成蛋白质以生物化学方式抑制相互作用;实验室2）。最后，我们将确定我们感兴趣的相互作用蛋白质中的突变，这些突变干扰了过量B1的调节，导致细胞死亡。

Background; Oscillations in CDK activity drive cell-cycle progression from the outset of the cell cycle in G1. Indeed, from the earliest stages of G1, CDK activity plays a central role in a cells 'decision' to either commit to cell-cycle entry (by initiating DNA replication) or exit into quiescence. Importantly, CDKs are only active when bound to cyclins. There are many cyclin-CDK complexes, however, only cyclin B1-CDK1 activity is essential. Critically, CDK activity levels are highly regulated to drive progression at the correct speed, allowing sufficient time to complete each stage of the cycle accurately. To facilitate this cyclin levels are strictly attenuated in healthy cells.In contrast, cyclin B1 (B1) is overexpressed in numerous human cancers. Further, its upregulation is associated with poor prognosis, treatment resistance, and suggested to act as a signal that initiates communications between cancer cells and their microenvironment. Indeed, highly expressed B1 binds to CDK1 from the outset in G1, contributing to aggressive proliferation in neoplastic tissues. We have revealed a novel motif in B1 that is critical for its regulation where B1 exists in excess in oocyte meiosis (ref). Following this, we have exciting unpublished data in mitosis demonstrating that this same motif is important to stabilise B1 levels G1 if B1 is overexpressed. Significantly, where we inhibit access to this motif, cells overexpressing B1 die. Objectives;1. To identify the molecular mechanism by which excessive levels of B1 are regulated in G1 of mitosis. 2. Following this, to propose candidate proteins which if perturbed, prevent this mechanism of excess B1 regulation, forcing cell death.Novelty and Timeliness; Our data in mitosis are entirely novel and present us with a unique opportunity, to identify a new mechanism by which we may trigger cell death. Significantly, this mechanism of B1 regulation is only essential in cells overexpressing B1, meaning that any future derived treatment programme could differentiate between unstable and healthy cells. This is timely as clinicians search for effective treatments that preserve healthy cells (limiting the often debilitating off target effects of some alternative treatments). Experimental Approach; The student will identify binding partners of the novel cyclin B1 motif in G1 by Bio-ID assay in live mitotic cells, and by IP experiments with lysed cells over expressing B1 with and without this critical motif (lab 1). They will then confirm candidate proteins in in-vivo assays such as BiFc to confirm proximity (lab 1 and in collaboration with VisiTech) and in-vitro (by generating proteins to characterise interactions biochemically; lab 2). Finally, we will identify mutations in our interacting protein(s) of interest that perturbs the regulation excess B1, resulting in cell death.