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

本提案的重点是DPP9，一种细胞内氨基二肽酶，具有从具有Pro（或Ala）残基的蛋白质的n端剪切二肽的独特能力（NH2-Xaa-Pro/Ala）。在此之前，我们发现DPP9定位于细胞核和细胞质，在那里它限制了含pro肽的切割速率，并被SUMO1以变抗方式激活。其他人的研究表明DPP9在细胞迁移、凋亡、焦亡、癌症和新生儿存活中发挥作用。其潜在的分子机制尚不清楚。为了更深入地了解DPP9的功能，我们搜索了DPP9相互作用蛋白，认为这些蛋白包括DPP9底物和调节因子。其中一种鉴定的蛋白质是丝蛋白A (FLNA)，一种交联肌动蛋白丝的蛋白质。在这个资助期，我们发现FLNA将DPP9招募到Syk上，Syk是b细胞信号传导的一种中心激酶。这导致Syk的n端被DPP9加工，证明Syk是一种新的DPP9底物。DPP9的裂解产生了一个新的Syk n端，Ser位于1位。脉冲追逐结合诱变研究表明，这种Ser对Syk的稳定性有负面影响。一致地，DPP9的沉默或抑制稳定了Syk，从而调节了Syk介导的信号传导，将DPP9定义为Syk的一种新的负调控因子。此外，我们的研究结果表明，DPP9是n端规则途径的上游组成部分，n端规则途径是基于蛋白质的第一个n端残基调节蛋白质降解的途径。除肌动蛋白外，FLNA还与多种蛋白相互作用，包括丝氨酸/苏氨酸激酶PKCs和参与DNA损伤修复的蛋白。我们现在报道DPP9处理PKCa和PKCg的n端。初步研究表明，DPP9与PKCs的相互作用除了活性位点外，还涉及第二个结合位点（外源位点）。在下一个资助期，我们的目标是迈出决定性的一步，了解DPP9与其底物的相互作用，以及外源位点是否有助于底物特异性。我们将研究DPP9切割PKCs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。我们将研究的第二种蛋白质是BRCA2，它是修复双链DNA断裂（DSB）的核心成分。我们证明DPP9与细胞中的BRCA2相互作用，并证明它切割了一个与BRCA2 n端相对应的肽。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，这表明DPP9在调节DSB修复中起作用。我们计划进一步详细分析DPP9在体外切割BRCA2， DPP9加工BRCA2的细胞结果，以及DPP9可能参与调节DSB修复。我们将特别关注n端规则通路，因为DPP9的切割暴露了BRCA2 n端不稳定的残基。

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.