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Matthew Freeman - One of the best experts on this subject based on the ideXlab platform.
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spatial proteomics reveal that the protein phosphatase ptp1b interacts with and may modify tyrosine phosphorylation of the Rhomboid Protease rhbdl4
Journal of Biological Chemistry, 2019Co-Authors: Kyojiro N Ikeda, Matthew FreemanAbstract:: Rhomboid-like proteins are evolutionarily conserved, ubiquitous polytopic membrane proteins, including the canonical Rhomboid intramembrane serine Proteases and also others that have lost Protease activity during evolution. We still have much to learn about their cellular roles, and evidence suggests that some may have more than one function. For example, RHBDL4 (Rhomboid-like protein 4) is an endoplasmic reticulum (ER)-resident Protease that forms a ternary complex with ubiquitinated substrates and p97/VCP (valosin-containing protein), a major driver of ER-associated degradation (ERAD). RHBDL4 is required for ERAD of some substrates, such as the pre-T-cell receptor α chain (pTα) and has also been shown to cleave amyloid precursor protein to trigger its secretion. In another case, RHBDL4 enables the release of full-length transforming growth factor α in exosomes. Using the proximity proteomic method BioID, here we screened for proteins that interact with or are in close proximity to RHBDL4. Bioinformatics analyses revealed that BioID hits of RHBDL4 overlap with factors related to protein stress at the ER, including proteins that interact with p97/VCP. PTP1B (protein-tyrosine phosphatase nonreceptor type 1, also called PTPN1) was also identified as a potential proximity factor and interactor of RHBDL4. Analysis of RHBDL4 peptides highlighted the presence of tyrosine phosphorylation at the cytoplasmic RHBDL4 C terminus. Site-directed mutagenesis targeting these tyrosine residues revealed that their phosphorylation modifies binding of RHBDL4 to p97/VCP and Lys63-linked ubiquitinated proteins. Our work lays a critical foundation for future mechanistic studies of the roles of RHBDL4 in ERAD and other important cellular pathways.
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Quantitative proteomics screen identifies a substrate repertoire of Rhomboid Protease RHBDL2 in human cells and implicates it in epithelial homeostasis
Scientific Reports, 2017Co-Authors: Nicholas Johnson, Jana Březinová, Elaine Stephens, Emma Burbridge, Matthew Freeman, Colin Adrain, Kvido StrisovskyAbstract:Rhomboids are intramembrane serine Proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of Rhomboid Protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface Protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloProteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.
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Rhomboid intramembrane Protease rhbdl4 triggers er export and non canonical secretion of membrane anchored tgfα
Scientific Reports, 2016Co-Authors: Lina Wunderle, Kvido Strisovsky, Matthew Freeman, Colin Adrain, Julia D Knopf, Nathalie Kuhnle, Aymeric Morle, Beate Hehn, Marius K LembergAbstract:Rhomboid intramembrane Proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian Rhomboid Protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this Rhomboid Protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.
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structure of Rhomboid Protease in complex with β lactam inhibitors defines the s2 cavity
Structure, 2013Co-Authors: Kutti R Vinothkumar, O A Pierrat, Jonathan M Large, Matthew FreemanAbstract:Rhomboids are evolutionarily conserved serine Proteases that cleave transmembrane proteins within the membrane. The increasing number of known Rhomboid functions in prokaryotes and eukaryotes makes them attractive drug targets. Here, we describe structures of the Escherichia coli Rhomboid GlpG in complex with β-lactam inhibitors. The inhibitors form a single bond to the catalytic serine and the carbonyl oxygen of the inhibitor faces away from the oxyanion hole. The hydrophobic N-substituent of β-lactam inhibitors points into a cavity within the enzyme, providing a structural explanation for the specificity of β-lactams on Rhomboid Proteases. This same cavity probably represents the S2′ substrate binding site of GlpG. We suggest that the structural changes in β-lactam inhibitor binding reflect the state of the enzyme at an initial stage of substrate binding to the active site. The structural insights from these enzyme-inhibitor complexes provide a starting point for structure-based design for Rhomboid inhibitors.
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mammalian egf receptor activation by the Rhomboid Protease rhbdl2
EMBO Reports, 2011Co-Authors: Colin Adrain, Kvido Strisovsky, Marius K Lemberg, Markus Zettl, Landian Hu, Matthew FreemanAbstract:The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, Rhomboid intramembrane Proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloProteases. We report here that EGF is an efficient substrate of the mammalian Rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines.
Kvido Strisovsky - One of the best experts on this subject based on the ideXlab platform.
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discovery and validation of 2 styryl substituted benzoxazin 4 ones as a novel scaffold for Rhomboid Protease inhibitors
Bioorganic & Medicinal Chemistry Letters, 2018Co-Authors: Parul Goel, Kvido Strisovsky, Thorsten Jumpertz, Anežka Ticha, Isabella Ogorek, David C Mikles, Martin Hubalek, Claus U Pietrzik, Boris Schmidt, Sascha WeggenAbstract:Abstract Rhomboids are intramembrane serine Proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and Rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific Rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli Rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel Rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine Protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel Rhomboid inhibitor scaffold with ample opportunity for optimization.
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dimerization of Rhomboid Protease rhbdl2 in lipid membranes addressed by fret with mc simulations
bioRxiv, 2018Co-Authors: Jan Skerle, Jana Humpolickova, Petra Rampirova, Edita Polachova, Ljubina Adamkova, Anna Suchankova, David Jakubec, Kvido StrisovskyAbstract: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.
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Quantitative proteomics screen identifies a substrate repertoire of Rhomboid Protease RHBDL2 in human cells and implicates it in epithelial homeostasis
Scientific Reports, 2017Co-Authors: Nicholas Johnson, Jana Březinová, Elaine Stephens, Emma Burbridge, Matthew Freeman, Colin Adrain, Kvido StrisovskyAbstract:Rhomboids are intramembrane serine Proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of Rhomboid Protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface Protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloProteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.
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Activity Assays for Rhomboid Proteases.
Methods in Enzymology, 2016Co-Authors: Elena Arutyunova, Kvido Strisovsky, M.j. LemieuxAbstract:Abstract Rhomboids are ubiquitous intramembrane serine Proteases that are involved in various signaling pathways. This fascinating class of Proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli Rhomboid GlpG with various inhibitors revealed the catalytic mechanism for Rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay Rhomboid Protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study Rhomboid Protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of K M and V max to measure catalytic parameters, using detergent solubilized Rhomboids with TatA, the only known substrate for bacterial Rhomboids, and the model substrate fluorescently labeled casein.
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Rhomboid Protease inhibitors emerging tools and future therapeutics
Seminars in Cell & Developmental Biology, 2016Co-Authors: Kvido StrisovskyAbstract:Abstract Rhomboid-family intramembrane serine Proteases are evolutionarily widespread. Their functions in different organisms are gradually being uncovered and already suggest medical relevance for infectious diseases and cancer. In contrast to these advances, selective inhibitors that could serve as efficient tools for investigation of physiological functions of Rhomboids, validation of their disease relevance or as templates for drug development are lacking. In this review I extract what is known about Rhomboid Protease mechanism and specificity, examine the currently used inhibitors, their mechanism of action and limitations, and conclude by proposing routes for future development of Rhomboid Protease inhibitors.
Sinisa Urban - One of the best experts on this subject based on the ideXlab platform.
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Ten catalytic snapshots of Rhomboid intramembrane proteolysis from gate opening to peptide release
Nature Structural & Molecular Biology, 2019Co-Authors: Rosanna P Baker, Ming Ji, Sinisa UrbanAbstract:Time-resolved crystallography captures Escherichia coli Rhomboid Protease GlpG in different steps of catalysis, revealing how substrate reaches the active site and reaction intermediates. Protein cleavage inside the cell membrane triggers various pathophysiological signaling pathways, but the mechanism of catalysis is poorly understood. We solved ten structures of the Escherichia coli Rhomboid Protease in a bicelle membrane undergoing time-resolved steps that encompass the entire proteolytic reaction on a transmembrane substrate and an aldehyde inhibitor. Extensive gate opening accompanied substrate, but not inhibitor, binding, revealing that substrates and inhibitors take different paths to the active site. Catalysis unexpectedly commenced with, and was guided through subsequent catalytic steps by, motions of an extracellular loop, with local contributions from active site residues. We even captured the elusive tetrahedral intermediate that is uncleaved but covalently attached to the catalytic serine, about which the substrate was forced to bend dramatically. This unexpectedly stable intermediate indicates Rhomboid catalysis uses an unprecedented reaction coordinate that may involve mechanically stressing the peptide bond, and could be selectively targeted by inhibitors.
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a golgi Rhomboid Protease rbd2 recruits cdc48 to cleave yeast srebp
The EMBO Journal, 2016Co-Authors: Jiwon Hwang, Diedre Ribbens, Sumana Raychaudhuri, Leah Cairns, He Gu, Adam Frost, Sinisa Urban, Peter J EspenshadeAbstract:Hypoxic growth of fungi requires sterol regulatory element‐binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane‐anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a Rhomboid family Protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi‐localized Protease that cleaves the transmembrane segment of the TatA Rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA‐ATPase Cdc48 through a C‐terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48‐binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation. ![][1] The sterol‐responsive transcriptional regulator SREBP is proteolytically activated by sequential action of the Dsc ubiquitin ligase and Rbd2 Rhomboid Protease in the Golgi, representing a novel control point in the fission yeast SREBP pathway. [1]: /embed/graphic-1.gif
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a trichomonas vaginalis Rhomboid Protease and its substrate modulate parasite attachment and cytolysis of host cells
PLOS Pathogens, 2015Co-Authors: Angelica M Riestra, Sinisa Urban, Shiv K Gandhi, Michael J Sweredoski, Annie Moradian, Sonja Hess, Patricia J JohnsonAbstract:Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis Rhomboid Proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine Proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model Rhomboid Protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.
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cytosolic extensions directly regulate a Rhomboid Protease by modulating substrate gating
Nature, 2015Co-Authors: Rosanna P Baker, Sinisa UrbanAbstract:Calcium potently stimulates proteolysis by endogenous Rhomboid-4, an intramembrane Protease that contains a cytoplasmic calcium-binding EF-hand domain. The active sites of intramembrane Proteases are deep in the middle of the lipid bilayer. This means that the substrate, which is a transmembrane domain of an integral membrane protein, needs to overcome energetic/entropic barriers to leave the hydrophobic environment of the cellular membrane and enter the active site of the Protease. In this manuscript, Rosanna P. Baker and Sinisa Urban show that addition of calcium leads to the activation of Rhomboid-4, an intramembrane Protease that contains an EF-hand domain helix-loop-helix structural domain, a feature found in in a large family of calcium-binding proteins. They determined that the relevant calcium-binding site is formed by a pair of cytoplasmic loops and showed that calcium increases the catalytic rate of this Protease by triggering the opening of the lateral substrate gate within the membrane. Intramembrane Proteases catalyse the signal-generating step of various cell signalling pathways, and continue to be implicated in diseases ranging from malaria infection to Parkinsonian neurodegeneration1,2,3. Despite playing such decisive roles, it remains unclear whether or how these membrane-immersed enzymes might be regulated directly. To address this limitation, here we focus on intramembrane Proteases containing domains known to exert regulatory functions in other contexts, and characterize a Rhomboid Protease that harbours calcium-binding EF-hands. We find calcium potently stimulates proteolysis by endogenous Rhomboid-4 in Drosophila cells, and, remarkably, when Rhomboid-4 is purified and reconstituted in liposomes. Interestingly, deleting the amino-terminal EF-hands activates proteolysis prematurely, while residues in cytoplasmic loops connecting distal transmembrane segments mediate calcium stimulation. Rhomboid regulation is not orchestrated by either dimerization or substrate interactions. Instead, calcium increases catalytic rate by promoting substrate gating. Substrates with cleavage sites outside the membrane can be cleaved but lose the capacity to be regulated. These observations indicate substrate gating is not an essential step in catalysis, but instead evolved as a mechanism for regulating proteolysis inside the membrane. Moreover, these insights provide new approaches for studying Rhomboid functions by investigating upstream inputs that trigger proteolysis.
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Membrane immersion allows Rhomboid Proteases to achieve specificity by reading transmembrane segment dynamics
eLife, 2012Co-Authors: Syed M. Moin, Sinisa UrbanAbstract:Proteases are enzymes that break the peptide bonds that hold proteins together, and have a central role in many physiological processes, including digestion, blood clotting and programmed cell death. An important characteristic of Proteases is that they are highly selective, only cutting proteins that contain well-defined sequences of amino acids in accessible regions. Proteases that are soluble in water have been studied for over a century and are now well understood, as are Proteases that need to be tethered to the membrane of a cell to work properly. In 1997 researchers discovered a Protease that was immersed in the cell membrane, and it soon became clear that these intramembrane Proteases were widespread and involved in a wide range of processes in cells. Examples of intramembrane Proteases include γ-secretase, which is implicated in Alzheimer's disease, and various site-2 Proteases that regulate pathogenic circuits in bacteria. There are many similarities between soluble and intramembrane Proteases. However, given that intramembrane Proteases evolved within the hydrophobic environment of the membrane, whereas soluble Proteases evolved in an aqueous environment, there should there should also be significant differences between them. The best understood intramembrane Proteases in terms of their biochemistry are probably the Rhomboid Proteases. However, most studies of their function have been performed in detergent systems rather than in real membranes. Moin and Urban now report that the main strategy used by Rhomboid Proteases to identity the proteins that they selectively cut is completely different from that used by soluble Proteases. Through a combination of biochemical and spectroscopic methods, they have discovered that Rhomboid Proteases identify the proteins they act on mainly by detecting changes in dynamic behavior: only those proteins that lose a stable helical structure when they exit the lipid phase to interact with the Rhomboid Protease will be cut by the Rhomboid Protease. Soluble Proteases, on the other hand, achieve specificity by looking for proteins with a particular sequence of amino acids. The novel strategy used by Rhomboid Proteases allows them to patrol the membrane for unstable helices and selectively cut them. This discovery provides the first explanation of why these complicated enzymes evolved to have active sites immersed within the cell membrane.
Marius K Lemberg - One of the best experts on this subject based on the ideXlab platform.
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Probing the Activity of Eukaryotic Rhomboid Proteases In Vitro.
Methods in Enzymology, 2016Co-Authors: Baptiste Cordier, Marius K LembergAbstract:Abstract Proteolysis within the membrane is a recent concept in biology. Rhomboid intramembrane serine Proteases are conserved in evolution and serve as key switches in diverse cellular pathways ranging from signaling to protein degradation. Since deregulation of intramembrane proteolysis can lead to severe diseases including neurodegenerative disorders, dissecting their enzymatic function and specificity becomes crucial. As membrane proteins, their solubilization, and purification are technically challenging. As a start point for a comprehensive in vitro characterization of eukaryotic Rhomboid Proteases, we depict in this chapter a robust workflow to find the best conditions to obtain pure and active enzymes from a bacterial expression system. To monitor the integrity of their active site and visualize substrate cleavage, various established activity assays including activity-based labeling and gel-based cleavage assays are described. These methods are illustrated by use of the Escherichia coli Rhomboid Protease GlpG and human RHBDL2 as an example.
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Inactive Rhomboid proteins: New mechanisms with implications in health and disease
Seminars in Cell & Developmental Biology, 2016Co-Authors: Marius K Lemberg, Colin AdrainAbstract:Abstract Rhomboids, Proteases containing an unusual membrane-integral serine Protease active site, were first identified in Drosophila, where they fulfill an essential role in epidermal growth factor receptor signaling, by cleaving membrane-tethered growth factor precursors. It has recently become apparent that eukaryotic genomes harbor conserved catalytically inactive Rhomboid Protease homologs, including derlins and iRhoms. Here we highlight how loss of proteolytic activity was followed in evolution by impressive functional diversification, enabling these pseudoProteases to fulfill crucial roles within the secretory pathway, including protein degradation, trafficking regulation, and inflammatory signaling. We distil the current understanding of the roles of Rhomboid pseudoProteases in development and disease. Finally, we address mechanistically how versatile features of proteolytically active Rhomboids have been elaborated to serve the sophisticated functions of their pseudoProtease cousins. By comparing functional and structural clues, we highlight common principles shared by the Rhomboid superfamily, and make mechanistic predictions.
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Rhomboid intramembrane Protease rhbdl4 triggers er export and non canonical secretion of membrane anchored tgfα
Scientific Reports, 2016Co-Authors: Lina Wunderle, Kvido Strisovsky, Matthew Freeman, Colin Adrain, Julia D Knopf, Nathalie Kuhnle, Aymeric Morle, Beate Hehn, Marius K LembergAbstract:Rhomboid intramembrane Proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian Rhomboid Protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this Rhomboid Protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.
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ubiquitin dependent intramembrane Rhomboid Protease promotes erad of membrane proteins
Molecular Cell, 2012Co-Authors: Lina Fleig, Nina Bergbold, Priyanka Sahasrabudhe, Beate Geiger, Lejla Kaltak, Marius K LembergAbstract:The ER-associated degradation (ERAD) pathway serves as an important cellular safeguard by directing incorrectly folded and unassembled proteins from the ER to the proteasome. Still, however, little is known about the components mediating ERAD of membrane proteins. Here we show that the evolutionary conserved Rhomboid family protein RHBDL4 is a ubiquitin-dependent ER-resident intramembrane Protease that is upregulated upon ER stress. RHBDL4 cleaves single-spanning and polytopic membrane proteins with unstable transmembrane helices, leading to their degradation by the canonical ERAD machinery. RHBDL4 specifically binds the AAA+-ATPase p97, suggesting that proteolytic processing and dislocation into the cytosol are functionally linked. The phylogenetic relationship between Rhomboids and the ERAD factor derlin suggests that substrates for intramembrane proteolysis and protein dislocation are recruited by a shared mechanism.
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mammalian egf receptor activation by the Rhomboid Protease rhbdl2
EMBO Reports, 2011Co-Authors: Colin Adrain, Kvido Strisovsky, Marius K Lemberg, Markus Zettl, Landian Hu, Matthew FreemanAbstract:The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, Rhomboid intramembrane Proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloProteases. We report here that EGF is an efficient substrate of the mammalian Rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines.
N. Bishara - One of the best experts on this subject based on the ideXlab platform.
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The mitochondrial Rhomboid Protease PSARL is a new candidate gene for type 2 diabetes
Diabetologia, 2005Co-Authors: K. Walder, L. Kerr-bayles, A. Civitarese, J. Jowett, J. Curran, K. Elliott, J. Trevaskis, N. Bishara, P. Zimmet, L. MandarinoAbstract:Aims/hypothesis This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes. Methods We screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions. Results We identified a mitochondrial intramembrane Protease, known as presenilins-associated Rhomboid-like protein ( PSARL ) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus , and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic–euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, accounting for 5% of the variation in plasma insulin in elderly subjects. Conclusions/interpretation Variation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.
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the mitochondrial Rhomboid Protease psarl is a new candidate gene for type 2 diabetes
Diabetologia, 2005Co-Authors: K. Walder, A. Civitarese, J. Jowett, J. Trevaskis, Lyndal Kerrbayles, Joanne E Curran, Katherine S Elliott, N. BisharaAbstract:Aims/hypothesis This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes.