Biological function and molecular mechanisms of intramembrane proteoylsis by Signal Peptide Peptidase Like-3 (SPPL3)

信号肽肽酶样3 （SPPL3）是SPP/SPPL家族的一种天冬氨酸膜内蛋白酶。在该项目的第一个资助期内，我们确定了SPPL3作为蛋白质糖基化的调节因子。通过切割高尔基体中的聚糖修饰酶，SPPL3降低了它们的催化活性，因此，SPPL3的过表达导致许多细胞蛋白的低糖基化，而SPPL3的表达减少则导致高糖基化。因此，调整SPPL3的表达水平可以使细胞快速改变分泌途径中蛋白质的聚糖模式。因此，我们在第二个资助期询问了SPPL3的表达是如何调控的。我们发现SPPL2c的表达，另一个只在精子中表达的成员，切割SNARE蛋白，从而改变分泌途径中的蛋白质运输。SPPL3的表达以及聚糖修饰酶的表达被改变，因为它们保留在内质网中。然而，这仅在睾丸中具有生理相关性，而SPPL3在所有其他组织中的表达调节方式不同。有趣的是，胰岛素抑制SPPL3的表达，我们有证据表明mTORC通路参与了这一调控。葡萄糖饥饿强烈增加SPPL3的表达，正如预期的那样，导致细胞糖蛋白的低糖基化。值得注意的是，葡萄糖饥饿条件下SPPL3的稳定似乎与mTORC信号传导无关。基于此，我们现在将确定葡萄糖饥饿时调节SPPL3表达的精确信号事件，并解读胰岛素诱导的mTORC激活与SPPL3表达下调之间的联系。此外，我们将阐明其他营养物质对SPPL3表达的影响，并确定潜在的信号级联。最后，我们将尝试将细胞培养模型系统中SPPL3调控机制的发现与我们在SPPL3 -/-小鼠中观察到的表型联系起来。为此，我们已经开始建立条件SPPL3 -/-小鼠，这将使我们能够单独分析观察到的表型。该项目的研究结果将最终提供SPPL3调控的详细图谱，并有助于全面了解其生理功能。这可能为开发治疗肥胖症或2型糖尿病等疾病的新策略提供基础。

Signal Peptide Peptidase-like 3 (SPPL3) is an aspartyl intramembrane protease of the SPP/SPPL family. Within the first funding period of this project, we identified SPPL3 as a regulator of protein glycosylation. By cleavage of glycan-modifying enzymes in the Golgi, SPPL3 reduces their catalytic activity and, therefore, overexpression of SPPL3 results in hypoglycosylation of many cellular proteins, while reduced SPPL3 expression induces hyperglycosylation. Thus, adapting the expression level of SPPL3 enables cells to rapidly change the glycan pattern of proteins within the secretory pathway. Consequently, we asked in the second funding period how expression of SPPL3 is regulated. We found that expression of SPPL2c, another member that is exclusively expressed in spermatids, cleaves SNARE proteins and, thus, alters protein transport in the secretory pathway. By that SPPL3 expression as well as expression of glycan modifying enzymes is altered since they are retained in the endoplasmic reticulum. However, this is only of physiological relevance in testis, while regulation of SPPL3 expression in all other tissues occurs differently. Interestingly, insulin suppresses SPPL3 expression and we have evidence that the mTORC pathway is involved in this regulation. Glucose starvation strongly increases SPPL3 expression and, as expected, results in hypoglycosylation of cellular glycoproteins. Of note, stabilization of SPPL3 under conditions of glucose starvation seems to be independent of mTORC signaling. Based on this, we will now identify the precise signaling events that regulate SPPL3 expression upon glucose starvation and decipher the link between insulin induced mTORC activation and downregulation of SPPL3 expression. Additionally, we will elucidate the impact of other nutrients on SPPL3 expression and identify the underlying signaling cascades. Finally, we will try to link the findings on the mechanisms of SPPL3 regulation from cell culture model systems to the phenotypes we observe in SPPL3 -/- mice. To this end, we have already started to establish conditional SPPL3 -/- mice that will allow us to analyze the observed phenotypes individually.The results of this project will finally provide a detailed picture of SPPL3 regulation and help to fully understand its physiological function. This may provide the basis to develop new strategies for treatment of for instance obesity or Diabetes type 2.