The role of lysosomes and subcellular distribution of lysosomal enzymes in early acute pancreatitis

急性胰腺炎的特点是消化蛋白酶（酶原）的过早和细胞内激活。胰蛋白酶原被溶酶体蛋白酶组织蛋白酶B （CTSB）激活，因为这两种酶距离很近。据推测，共定位是通过溶酶体和分泌囊泡的融合介导的，从而使胰蛋白酶原在细胞内活化。我们之前的工作表明，其他组织蛋白酶，即CTSD和CTSC，也参与急性胰腺炎。我们可以进一步证明组织蛋白酶相互作用。在初步的研究中，我们可以表明，在腺泡细胞中溶酶体的选择性渗透导致细胞内CTSB活性的降低，但不会导致胰蛋白酶原活性的降低。该项目的目的是确定胰腺炎发病时蛋白酶的过早激活是否涉及溶酶体作为细胞器，溶酶体酶在分泌室中的错误分选，或溶酶体与分泌囊泡的融合。因此，溶酶体渗透对急性胰腺炎的影响将通过体内和体外实验进行研究。此外，将在敲除CLN8的小鼠中研究组织蛋白酶的亚细胞分布和急性胰腺炎的严重程度，CLN8负责溶酶体酶的细胞内运输。第三，溶酶体与包括分泌囊泡在内的亚细胞区室的相互作用将在小鼠模型中进行研究，该模型具有条件胰特异性敲除Rab7（一种GTPase，参与溶酶体与其他细胞器的融合）。

Acute pancreatitis is characterized by a premature and intracellular activation of digestive proteases (zymogens). Trypsinogen is activated by the lysosomal protease cathepsin B (CTSB), given that both enzymes are in close proximity. It is hypothesized that colocalization is mediated by fusion of lysosomes and secretory vesicles, enabling intracellular activation of trypsinogen.Our previous work has shown that other cathepsins, namely CTSD and CTSC, are also involved in acute pancreatitis. We could further demonstrate that cathepsins interact with each other. In preliminary studies we could show that a selective permeabilization of lysosomes in acinar cells leads to a reduction of intracellular CTSB activity, but not of trypsinogen activation.The aim of this project is to determine whether premature protease activation at the onset of pancreatitis involves lysosomes as organelles, missorting of lysosomal enzymes into the secretory compartment, or the fusion of lysosomes with secretory vesicles. Therefore effects of lysosomal permeabilization on acute pancreatitis will be examined by ex vivo and in vivo experiments. In addition, the subcellular distribution of cathepsins and severity of acute pancreatitis will be investigated in mice with knockout for CLN8, which is responsible for intracellular transport of lysosomal enzymes. Third, the interaction of lysosomes with subcellular compartments including secretory vesicles will be investigated in a mouse model with a conditional pancreas-specific knockout for Rab7, a GTPase, being involved in fusion of lysosomes with other organelles.