Identification and characterization of phosphatases, master regulators of cellular stress signalling pathways

蛋白质的可逆磷酸化是一种翻译后修饰，控制着几乎所有细胞过程中涉及的一系列信号事件。几乎三分之一的细胞蛋白质被磷酸化，这突显了这个严格监管的信号网络的巨大重要性。为了控制这么多靶点的修饰，细胞编码了大约400个具有不同特异性的激酶。相比之下，蛋白质磷酸酶，即逆转蛋白质磷酸化的酶，长期以来被认为是非特异性酶，因为这些酶的编码基因数量相当少。此外，在体外实验中，纯化的催化实体对其底物表现出相当无差别的活性。然而，近年来的研究表明，许多磷酸酶是必需的寡聚酶，形成大量具有不同底物特异性的全酶。蛋白磷酸酶1(PP1)是真核细胞中主要的丝氨酸/苏氨酸去磷酸化蛋白。到目前为止，已确定了大约200个PP1调节亚基，这些亚基决定了它的特异性和亚细胞定位。尽管它们在控制细胞信号方面具有巨大的重要性，但人们对特定的PP1全酶的生物学知之甚少。在贝托洛蒂实验室逗留期间，我计划鉴定和鉴定新的磷酸酶全酶，在调节和终止细胞应激信号方面发挥重要作用。为此，我的目标是研究内质网(ER)和胞质应激条件下的PP1相互作用组，以确定参与这些途径的调节和信号终止的调节亚基。此外，我的目的是阐明与泛素结合酶UBE2J1去磷酸化有关的信号事件，UBE2J1是内质网蛋白质动态平衡的关键角色。UBE2J1在不同的压力下被磷酸化，这种修饰似乎是细胞生存所必需的。在第三个项目中，我将集中于一个特定的PP1调节亚基的特征，该亚单位可能参与胞内应激信号。总之，新的PP1全酶的鉴定和鉴定将加深我们对应激反应途径和相关疾病的理解。

The reversible phosphorylation of proteins is a post-translational modification controlling a wide range of signalling events involved in virtually all cellular processes. Almost one third of cellular proteins become phosphorylated, underlining the immense importance of this tightly regulated signalling network. To control the modification of that many targets, cells encode around 400 kinases with differing specificities. In contrast, protein phosphatases, which reverse the phosphorylation of proteins, were long thought to be unspecific enzymes due to the rather small number of genes encoding for these enzymes. Furthermore, the purified catalytic entities display a fairly non-discriminating activity towards their substrates in in vitro assays. However, research from recent years demonstrated that many phosphatases are obligatory oligomeric enzymes, forming a large number of holoenzymes with distinct substrate specificities. Protein phosphatase 1 (PP1) accounts for the majority of serine/threonine dephosphorylation events in eukaryotic cells. To date, approximately 200 regulatory subunits of PP1 are identified, which determine its specificity and subcellular localization. Despite their tremendous importance in controlling cellular signalling, little is known about the biology of specific PP1 holoenzymes. During my stay in the Bertolotti lab, I plan to identify and characterize new phosphatase holoenzymes, playing a major role in the regulation and termination of cellular stress signalling. For this purpose, I aim to investigate the PP1 interactome during endoplasmic reticulum (ER) and cytosolic stress conditions to identify regulatory subunits, which are involved in the regulation and signal termination of these pathways. Additionally, I aim to elucidate the signalling events involved in the dephosphorylation of the ubiquitin conjugating enzyme UBE2J1, which is a key player in protein homeostasis at the ER. UBE2J1 is phosphorylated upon different stresses and this modification appears to be essential for cellular survival. In a third project, I will focus on the characterization of a specific PP1 regulatory subunit potentially involved in cytosolic stress signalling. Altogether, the identification and characterization of new PP1 holoenzymes will deepen our understanding of stress response pathways and related diseases.