Biology subjects

Strisovsky, K.

Publications and source records attributed to Strisovsky, K..

10 recordsLinked to original sources

bioRxiv2026

Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes.

Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical for keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.

cell biology↗

bioRxiv2026

Rhomboid protease Rhbdl2 regulates macrophage recruitment and wound regeneration in zebrafish

Tissue regeneration requires tight control of immune cell behavior, yet the mechanisms that restrain immune-driven regenerative responses remain poorly defined. Here, we identify the rhomboid intramembrane serine protease Rhbdl2 as a critical regulator of regeneration in zebrafish. We generated rhbdl2 mutants by CRISPR Cas9 and found that it does not affect normal development, but triggers enhanced regenerative growth following injury, accompanied by increased macrophage accumulation at the wound site, which is accompanied by increased early apoptosis and proliferation. Proteomic analyses reveal increased Rac2 protein levels in rhbdl2 mutants, indicating dysregulated immune signaling. Functionally, Rac2 morpholino oligonucleotides mediated knockdown in rhbdl2 mutant larvae suppresses the elevated macrophage recruitment and enhanced tissue regenerative phenotype. Together, these findings uncover Rhbdl2 as an immune checkpoint that constrains macrophage driven enhanced regeneration, with vast implications for inflammatory disease, fibrosis, and tumor immune interactions.

immunology↗

bioRxiv2025

A critical role for the ER Membrane Complex (EMC) in lipid droplet homeostasis

The ER Membrane complex (EMC) facilitates insertion of multiple classes of integral membrane protein into the lipid bilayer of the endoplasmic reticulum (ER). Recent years have seen significant progress in understanding the structural aspects of EMC function, but the client protein/s enforcing its evolutionary conservation throughout eukaryotic kingdoms remain to be determined. Given reported role/s for the EMC in lipid homeostasis, we deleted the essential EMC3 subunit in the liver and adipose tissues of mice and examined the impact of EMC loss in a range of metabolic models. Intriguingly, absence of EMC3 in either the liver or adipose tissues results in defective storage of triglycerides and perturbations in lipid droplet (LD) homeostasis. EMC-deficient mouse adipose tissues were defective in their ability to mediate fat storage in response to a high fat diet and were unable to support non-shivering thermogenesis. Proteomic analyses of EMC3-deficient tissues found reduced levels of FITM2, a key regulator of LD biogenesis and ER homeostasis, identifying it as a novel EMC client. Strikingly, deletion of the EMC3 homolog dPob in the Drosophila fat body, which shares overlapping functions with mammalian liver/adipose tissues, revealed similar defects in LD homeostasis. Together, our results indicate that the EMC plays a key evolutionarily conserved role in biogenesis of machinery maintaining triglyceride storage in metazoans.

biochemistry↗

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↗

bioRxiv2024

Engineering cardiolipin binding to an artificial membrane protein

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

biochemistry↗

bioRxiv2022

Semaphorin 4B is an ADAM17-cleaved inhibitor of adipocyte thermogenesis

ObjectiveThe metalloprotease ADAM17 (also called TACE) plays fundamental roles in homeostasis by shedding key signaling molecules from the cell surface. Although its importance for the immune system and epithelial tissues is well-documented, little is known about the role of ADAM17 in metabolic homeostasis. The purpose of this study was to determine the impact of ADAM17 expression, specifically in adipose tissues, on metabolic homeostasis. MethodsWe used histopathology, molecular, proteomic, transcriptomic, in vivo integrative physiological and ex vivo biochemical approaches to determine the impact of adipose tissue-specific deletion of ADAM17 upon adipocyte and whole organism metabolic physiology. ResultsADAM17adipoq-cre{Delta}/{Delta} mice exhibited a hypermetabolic phenotype characterized by elevated energy consumption and increased levels of adipocyte thermogenic gene expression. On a high fat diet, these mice were more thermogenic, while exhibiting elevated expression levels of genes associated with lipid oxidation and lipolysis. This hypermetabolic phenotype protected mutant mice from obesogenic challenge, limiting weight gain, hepatosteatosis and insulin resistance. Activation of beta-adrenoceptors by the neurotransmitter norepinephrine, a key regulator of adipocyte physiology, triggered the shedding of ADAM17 substrates, and regulated ADAM17 expression at the mRNA and protein levels, hence identifying a functional connection between thermogenic licensing and the regulation of ADAM17. Proteomic studies identified Semaphorin 4B (SEMA4B), as a novel ADAM17-shed adipokine, whose expression is regulated by physiological thermogenic cues that acts to dampen thermogenic responses in adipocytes. Transcriptomic data showed that cleaved SEMA4B acts in an autocrine manner in brown adipocytes to dampen the expression of genes involved in thermogenesis, adipogenesis, lipid uptake, storage and catabolism. ConclusionOur findings identify a novel ADAM17-dependent axis, regulated by beta-adrenoceptors and mediated by the ADAM17-cleaved form of SEMA4B, that may act to limit uncontrolled energy depletion during thermogenesis.

physiology↗

bioRxiv2021

EMC is required for biogenesis and membrane insertion of Xport-A, an essential chaperone of rhodopsin-1 and the TRP channel

Insertion of hydrophobic transmembrane domains (TMDs) into the endoplasmic reticulum (ER) lipid bilayer is an essential step during eukaryotic membrane protein biogenesis. The ER membrane complex (EMC) functions as an insertase for TMDs of low hydrophobicity and is required for the biogenesis of a subset of tail-anchored (TA) and polytopic membrane proteins, including rhodopsin-1 (Rh1) and the TRP channel. To better understand the physiological implications of membrane protein biogenesis dependent on the EMC, we performed a bioinformatic analysis to predict TA proteins present in the Drosophila proteome. From 254 predicted TA proteins, subsequent genetic screening in Drosophila larval eye discs led to the identification of 2 proteins that require EMC for their biogenesis: farinelli (fan) and Xport-A. Fan is required for sperm individualization and male fertility in Drosophila and we now show that EMC is also required for these important biological processes. Interestingly, Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC loss of function mutations is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and increasing the hydrophobicity of Xport-A TMD rendered its membrane insertion to become EMC-independent. Moreover, these EMC-independent Xport-A mutants rescued Rh1 and TRP biogenesis in EMC mutants. Our data establish that EMC can impact the biogenesis of polytopic membrane proteins indirectly, by controlling the biogenesis and membrane insertion of an essential protein co-factor.

cell biology↗

bioRxiv2020

Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease, RHBDL2. We show that RHBDL2 prevents stochastic signalling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved, disease-linked proline residue is responsible for RHBDL2 recognising only the active conformation of Orai1, and cleavage by RHBDL2 is required to sharpen switch-like signalling triggered by store-operated calcium entry. Loss of RHBDL2 control of Orai1 causes severe dysregulation of CRAC channel effectors including transcription factor activation, inflammatory cytokine expression and T cell activation. We propose that this seek-and-destroy function may represent an ancient activity of rhomboid proteases in degrading unwanted signalling proteins.

biochemistry↗

bioRxiv2018

Dimerization of rhomboid protease RHBDL2 in lipid membranes addressed by FRET with MC simulations

Many membrane proteins are thought to function as oligomers, but measuring membrane protein dimerization in native lipid membranes is particularly challenging. Forster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are non-invasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. The effects inherent to such two-dimensional systems, such as excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and FCS we introduce methods to measure surface protein density and to estimate kappa squared, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined 2D environments. We then use FRET and FCS to analyze the dimerization of human rhomboid protease RHBDL2 in its native lipid membranes. While previous reports have proposed that rhomboid proteases dimerize and this allosterically activates them, we find no evidence for stable oligomers of RHBDL2 in lipid membranes of human cells. This indicates that the rhomboid transmembrane core may be intrinsically monomeric. Finally, our findings will find use in the application of FRET and FCS for the analysis of oligomerization of transmembrane proteins in lipid membranes.

biophysics↗

bioRxiv2018

iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires ADAM17/TACE, which cleaves TNF from its transmembrane tether, releasing it for signalling. The trafficking of ADAM17/TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. Here we report a novel protein, that we name iTAP (iRhom tail-associated protein) that binds to iRhoms, enhancing the stability of iRhoms and TACE, preventing their degradation in lysosomes. iTAP-null primary human macrophages, or tissues from iTAP KO mice, are dramatically depleted in the levels of iRhom2 and active TACE, and are, consequently, profoundly impaired in TNF production. Our work illustrates iTAP as a physiological rheostat controlling TNF signalling and a novel target for the control of inflammation.

biochemistry↗