Project Description Project Description

Dariusz Pytel

Mapping the functional interplay between PERK and sphingolipids.  

The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) activates a cell adaptive program called the unfolded protein response (UPR). UPR engagement is essential for translation attenuation, transactivation of ER chaperone gene promoters, and activation of protein degradation pathways, all of which contribute to stress resolution. My long-term goal is to elucidate the contribution of the UPR to tumor progression and more specifically to define the contribution of the UPR to the maintenance of lipid homeostasis and signaling. Protein misfolding in the ER triggers the activation of three transmembrane protein kinases, Ire1a (alpha) , Ire1b (beta) and the PKR-like ER kinase (PERK) and the transmembrane transcription factor ATF6. Together these three proteins transduce signals from the stressed ER resulting in increased ER chaperone expression while simultaneously down-regulating cellular protein synthesis. Because PERK functions as a key regulator of cell survival following micro-environmental stresses commonly encountered by tumors, (hypoxia, nutrient limitation, increased protein biosynthetic demand and alterations in membrane lipid composition) PERK inhibition has significant potential as an anti-cancer therapy. In addition to functioning as a canonical protein kinase, we recently demonstrated that PERK possesses intrinsic lipid kinase activity wherein PERK directly phosphorylates diacylglycerol to generate phosphatidic acid (PA). Through PA generation, PERK can regulate Akt, mTOR, and MAPK pathways. This novel lipid kinase activity suggests that PERK plays an extensive role in tumorigenesis and insulin signaling and can provide a new strategy for therapeutically targeting the PI3K/mTOR pathway. In addition to lipid kinase activity, recent studies have identified connections between sphingolipids (SPLs) and ER stress signaling, suggesting an intricate network of PERK and lipid regulatory interactions. Stemming from these observations, I performed preliminary studies assessing the ability of a small subset of structurally varied SPLs, to regulate PERK. Interestingly, I have identified a set of long-chain synthetic ceramide (Cer) analogs, D-erythro-C16-analog (LCL-345) and D-erythro-C18-analog (LCL-461), that strongly inhibit PERK catalytic activity in vitro. Given the pro-apoptotic activity of certain SPLs, we propose that SPL-dependent regulation of PERK will determine the ability of PERK to regulate cell fate. The specific goal of this proposal is to determine the structure-activity relationships (SAR) between PERK and SPLs in in vitro and in vivo models. My central hypothesis is that PERK is regulated by specific SPLs, and through this interaction, they can control pro-survival PERK signaling. To address this hypothesis, the following two aims are proposed: 1) Determine the structural characteristics of sphingolipids and PERK that control regulatory interactions in vitro, 2) Determine the impact of sphingolipids/ceramides on PERK in vivo.

 

 

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