Solorio-Kirpichyan, K. | Search bioRxiv

bioRxiv2025

1-Deoxysphingolipids dysregulate membrane properties and cargo trafficking in the early secretory pathway

1-Deoxysphingolipids are non-canonical sphingolipids linked to several diseases, but their cellular effects are poorly understood. Here, we utilize lipid chemical biology approaches to investigate the role of 1-deoxysphingolipid metabolism on the properties and functions of secretory membranes. We first applied organelle-specific bioorthogonal labeling to visualize the subcellular distribution of metabolically tagged 1-deoxysphingolipids in RPE-1 cells, observing that they are retained in the endoplasmic reticulum (ER). We found that 1-deoxysphingolipids can be transported by the non-vesicular transporter CERT in vitro but are retained at ER exit sites (ERES) in cells, suggesting that they do not efficiently sort into vesicular carriers. Cells expressing disease-associated variants of serine palmitoyl-CoA transferase (SPT) accumulated long-chain 1-deoxysphingolipids, which reduced ER membrane fluidity and enlarged ERES. We observed that the rates of membrane protein release from the ER were altered in response to mutant SPT expression, in a manner that was dependent on the cargo affinity for ordered or disordered membranes. We propose that dysregulation of sphingolipid metabolism alters secretory membrane properties, which can then modulate protein trafficking.

cell biology↗

bioRxiv2024

An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimers disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4s kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Forster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4s enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl -ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4s active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4s catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

biochemistry↗