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期细胞周期的开始驱动细胞周期的进展。事实上，从G1的最早阶段开始，CDK活性就在细胞“决定”要么进入细胞周期(通过启动DNA复制)，要么退出静止状态中发挥核心作用。重要的是，CDK只有在与细胞周期蛋白结合时才是活性的。细胞周期蛋白-CDK复合体很多，但只有细胞周期蛋白B1-CDK1活性是必需的。关键的是，CDK活性水平受到高度调控，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，在健康细胞中细胞周期蛋白水平被严格减弱。相比之下，细胞周期蛋白B1(B1)在许多人类癌症中过度表达。此外，它的表达上调与不良的预后、治疗耐药有关，并被建议作为启动癌细胞与其微环境之间通讯的信号。事实上，在G1中，高表达的B1从一开始就与CDK1结合，促进了肿瘤组织的侵袭性增殖。我们已经揭示了B1中的一个新的基序，该基序对其调控至关重要，其中B1在卵母细胞减数分裂(REF)中过度存在。随后，我们在有丝分裂方面有了令人兴奋的未发表的数据，表明如果B1过度表达，这个相同的基序对于稳定B1水平G1是重要的。值得注意的是，当我们抑制这个基序的访问时，过度表达B1的细胞就会死亡。目的：1.确定在有丝分裂的G1期调节过高水平B1的分子机制。2.随后，提出候选蛋白质，如果受到干扰，可以防止这种过度调节B1的机制，迫使细胞死亡。新颖性和及时性；我们在有丝分裂中的数据是全新的，为我们提供了一个独特的机会，以确定我们可能触发细胞死亡的新机制。值得注意的是，这种B1调节机制只有在过度表达B1的细胞中才是必不可少的，这意味着未来的任何派生治疗方案都可以区分不稳定细胞和健康细胞。这是及时的，因为临床医生正在寻找有效的治疗方法来保存健康细胞(限制一些替代治疗往往使人虚弱的靶向效应)。实验方法：学生将通过在活的有丝分裂细胞中的Bio-ID分析，以及通过裂解细胞在含有和不含有这一关键基序的情况下过度表达B1的IP实验，来确定新的细胞周期蛋白B1基序在G1中的结合伙伴(实验1)。然后，他们将在BIFC等体内测试中确认候选蛋白质，以确认接近(实验室1并与VisiTech合作)和体外(通过产生蛋白质来描述相互作用的生化特征；实验室2)。最后，我们将确定我们感兴趣的相互作用蛋白(S)中的突变，该突变扰乱了过量的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 Following this, we have exciting unpublished data in mitosis demonstrating that this same motif is important to stabilise B1 levels G1 if B1 is overexpressed.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.