The physiological roles and regulation of DPP9 - an intracellular prolyl dipeptidase

这项提议的重点是DPP9，一种细胞内的氨基二肽酶，它具有从第二位(或丙氨酸)残基(NH2-Xaa-Pro/Ala)的蛋白质的N-末端切割二肽的独特能力。以前，我们发现DPP9定位于细胞核和胞浆，在那里它是含有Pro的多肽的切割限速，并被SUMO1以变构的方式激活。其他人的研究表明，DPP9在细胞迁移、细胞凋亡、下垂、癌症和新生儿存活中发挥作用。其潜在的分子机制还知之甚少。为了更深入地了解DPP9的功能，我们搜索了与DPP9相互作用的蛋白质，认为这些蛋白质包括DPP9底物和调节因子。已鉴定的蛋白质之一是丝状素A(Flna)，这是一种使肌动蛋白细丝交叉连接的蛋白质。在这一资助期间，我们显示了FLNA将DPP9招募到Syk，这是B细胞信号转导中的一种中心激酶。这导致了DPP9对Syk的N-末端处理，证明Syk是一种新的DPP9底物。DPP9切割产生一个新的Syk N-末端，Ser位于1位。脉冲追逐结合诱变研究表明，该Ser对Syk的稳定性有负面影响。一直以来，DPP9沉默或抑制稳定了Syk，从而调节了Syk介导的信号，将DPP9定义为Syk的一种新的负调控因子。此外，我们的结果表明，DPP9是N末端规则通路的上游组成部分，是一条基于蛋白质第一个N末端残基的调节降解途径。除了肌动蛋白外，Flna还与多种蛋白质相互作用，包括丝氨酸/苏氨酸激酶、PKCs和参与DNA损伤修复的蛋白质。我们现在报道，DPP9处理PKCA和PKCG的N末端。初步工作表明，DPP9与PKCs的相互作用除了活性部位外，还包括第二结合部位(外位体)。在下一个资助期，我们的目标是在了解DPP9与其底物的相互作用以及外显子是否有助于底物专一性方面迈出决定性的一步。我们将研究DPP9对PKCs的切割是否由Flna介导，以及这些切割事件的分子和细胞结果是什么。我们将研究的第二个蛋白质是BRCA2，它是双链DNA断裂(DSB)修复的中心成分。我们发现DPP9在细胞内与BRCA2相互作用，并证明它能裂解与BRCA2 N末端相对应的多肽。细胞暴露于丝裂霉素C，导致DSB的形成，导致DPP9-BRCA2相互作用的数量增加，提示DPP9在调节DSB修复中发挥作用。我们计划进一步详细分析DPP9在体外对BRCA2的切割，DPP9处理BRCA2的细胞结果，以及DPP9可能参与调节DSB的修复。我们将特别关注N-端规则通路，因为DPP9裂解会暴露BRCA2 N-端的不稳定残基。

The focus of this proposal is DPP9, an intra-cellular amino-dipeptidase, which has the unique ability to cleave off dipeptides from the N-termini of proteins having a Pro (or Ala) residue in second position (NH2-Xaa-Pro/Ala). Previously, we showed that DPP9 localizes to the nucleus and to the cytosol, where it is rate limiting for cleavage of Pro-containing peptides, and is activated in an allosteric manner by SUMO1. Works from others show a role for DPP9 in cell migration, apoptosis, pyroptosis, cancer and neonatal survival. The underlying molecular mechanisms are poorly understood. For more insight into DPP9 functions, we searched for DPP9-interacting proteins, arguing that these include DPP9 substrates and regulators. One of the identified proteins was Filamin A (FLNA), a protein that cross-links actin filaments. In this funding period we showed that FLNA recruits DPP9 to Syk, a central kinase in B-cell signaling. This resulted in N-terminal processing of Syk by DPP9, demonstrating Syk as a novel DPP9 substrate. Cleavage by DPP9 produced a neo Syk N-terminus with a Ser in position 1. Pulse-chases combined with mutagenesis studies revealed that this Ser negatively influences Syk stability. Consistently, DPP9 silencing or inhibition stabilized Syk, thereby modulating Syk-mediated signaling, defining DPP9 as a novel negative regulator of Syk. Furthermore, our results show that DPP9 is an up-stream component of the N-end rule pathway, a route for regulated degradation of proteins based on their first N-terminal residue.Apart from actin, FLNA interacts with multiple proteins, including the Ser/Thr Kinases PKCs and proteins involved in DNA damage repair. We now report that DPP9 processes the N-terminus of PKCa and PKCg. Preliminary work suggests that the interaction of DPP9 with PKCs involves a second binding site (exosite) in addition to the active site. In the next funding period we aim to take a decisive step towards understanding the interactions of DPP9 with its substrates, and whether an exosite contributes to substrate specificity. We will investigate whether cleavage of PKCs by DPP9 is mediated by FLNA, and what the molecular and cellular outcomes of these cleavage events are. A second protein we will investigate is BRCA2, a central component in the repair of double strand DNA breaks (DSB). We show that DPP9 interacts with BRCA2 in cells, and demonstrate that it cleaves a peptide corresponding to BRCA2 N-terminus. Exposure of cells to Mitomycin C, which causes the formation of DSB, leads to an increase in the number of DPP9 - BRCA2 interactions, suggesting a role for DPP9 in regulating DSB repair. We plan to further analyze in detail the cleavage of BRCA2 by DPP9 in vitro, the cellular outcomes of BRCA2 processing by DPP9, and the possible involvement of DPP9 in regulating the repair of DSB. A special focus will be given to the N-end rule pathway, since DPP9 cleavage exposes a destabilizing residue in BRCA2 N-terminus.