Kvido Strisovsky

Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates.

Summary Members of the widespread rhomboid family of intramembrane proteases cleave transmembrane domain (TMD) proteins to regulate processes as diverse as EGF receptor signaling, mitochondrial dynamics, and invasion by apicomplexan parasites. However, lack of information about their substrates means that the biological role of most rhomboids remains obscure. Knowledge of how rhomboids recognize their substrates would illuminate their mechanism and might also allow substrate prediction. Previous work has suggested that rhomboid substrates are specified by helical instability in their TMD. Here we demonstrate that rhomboids instead primarily recognize a specific sequence surrounding the cleavage site. This recognition motif is necessary for substrate cleavage, it determines the cleavage site, and it is more strictly required than TM helix-destabilizing residues. Our work demonstrates that intramembrane proteases can be sequence specific and that genome-wide substrate prediction based on their recognition motifs is feasible.

Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase.

The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and is required for the production of an unknown quorum-sensing molecule. In a screen to identify rhomboid-encoding genes from Proteus mirabilis, tatA was identified as a multicopy suppressor and restored extracellular signal production as well as complementing all other phenotypes of a Prov. stuartii aarA mutant. TatA is a component of the twin-arginine translocase (Tat) protein secretion pathway and likely forms a secretion pore. By contrast, the native tatA gene of Prov. stuartii in multicopy did not suppress an aarA mutation. We find that TatA in Prov. stuartii has a short N-terminal extension that was atypical of TatA proteins from most other bacteria. This extension was proteolytically removed by AarA both in vivo and in vitro. A Prov. stuartii TatA protein missing the first 7 aa restored the ability to rescue the aarA-dependent phenotypes. To verify that loss of the Tat system was responsible for the various phenotypes exhibited by an aarA mutant, a tatC-null allele was constructed. The tatC mutant exhibited the same phenotypes as an aarA mutant and was epistatic to aarA. These data provide a molecular explanation for the requirement of AarA in quorum-sensing and uncover a function for the Tat protein export system in the production of secreted signaling molecules. Finally, TatA represents a validated natural substrate for a prokaryotic rhomboid protease.

Rhomboid Family Pseudoproteases Use the ER Quality Control Machinery to Regulate Intercellular Signaling

Summary Intramembrane proteolysis governs many cellular control processes, but little is known about how intramembrane proteases are regulated. iRhoms are a conserved subfamily of proteins related to rhomboid intramembrane serine proteases that lack key catalytic residues. We have used a combination of genetics and cell biology to determine that these “pseudoproteases” inhibit rhomboid-dependent signaling by the epidermal growth factor receptor pathway in Drosophila, thereby regulating sleep. iRhoms prevent the cleavage of potential rhomboid substrates by promoting their destabilization by endoplasmic reticulum (ER)-associated degradation; this mechanism has been conserved in mammalian cells. The exploitation of the intrinsic quality control machinery of the ER represents a new mode of regulation of intercellular signaling. Inactive cognates of enzymes are common, but their functions are mostly unclear; our data indicate that pseudoenzymes can readily evolve into regulatory proteins, suggesting that this may be a significant evolutionary mechanism.

The structural basis for catalysis and substrate specificity of a rhomboid protease

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism‐based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S1‐ and S2′‐binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor‐bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.

Mammalian EGF receptor activation by the rhomboid protease RHBDL2

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.

Monocyclic β-lactams are selective, mechanism-based inhibitors of rhomboid intramembrane proteases.

Rhomboids are relatively recently discovered intramembrane serine proteases that are conserved throughout evolution. They have a wide range of biological functions, and there is also much speculation about their potential medical relevance. Although rhomboids are weakly inhibited by some broad-spectrum serine protease inhibitors, no potent and specific inhibitors have been identified for these enzymes, which are mechanistically distinct from and evolutionarily unrelated to the classical soluble serine proteases. Here we report a new biochemical assay for rhomboid function based on the use of quenched fluorescent substrate peptides. We have developed this assay into a high-throughput format and have undertaken an inhibitor and activator screen of approximately 58,000 small molecules. This has led to the identification of a new class of rhomboid inhibitors, a series of monocyclic β-lactams, which are more potent than any previous inhibitor. They show selectivity, both for rhomboids over the soluble serine protease chymotrypsin and also, importantly, between different rhomboids; they can inhibit mammalian as well as bacterial rhomboids; and they are effective both in vitro and in vivo. These compounds represent important templates for further inhibitor development, which could have an impact both on biological understanding of rhomboid function and potential future drug development.

Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures.

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl‐chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl‐CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate‐like manner, and their co‐crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid‐like protein superfamily may have similar substrate or client‐protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.

Structural and mechanistic principles of intramembrane proteolysis – lessons from rhomboids

Intramembrane proteases cleave membrane proteins in their transmembrane helices to regulate a wide range of biological processes. They catalyse hydrolytic reactions within the hydrophobic environment of lipid membranes where water is normally excluded. How? Do the different classes of intramembrane proteases share any mechanistic principles? In this review these questions will be discussed in view of the crystal structures of prokaryotic members of the three known catalytic types of intramembrane proteases published over the past 7 years. Rhomboids, the intramembrane serine proteases that are the best understood family, will be the initial area of focus, and the principles that have arisen from a number of structural and biochemical studies will be considered. The site‐2 metalloprotease and GXGD‐type aspartyl protease structures will then be discussed, with parallels drawn and differences highlighted between these enzymes and the rhomboids. Despite the significant advances achieved so far, to obtain a detailed understanding of the mechanism of any intramembrane protease, high‐resolution structural information on the substrate–enzyme complex is required. This remains a major challenge for the field.

Why cells need intramembrane proteases - a mechanistic perspective.

Intramembrane proteases (IMPRs) cleave transmembrane proteins inside the lipid bilayer. They regulate a growing number of biological processes, and our knowledge about the evolutionary and functional niches these unusual enzymes have filled is slowly unravelling. Although structures of representative members of four IMPR families have been solved, the mechanism of substrate recognition and cleavage is still poorly understood. Here I offer a view on substrate recognition by IMPRs from the angle of their biological functions. Zooming in on rhomboid proteases I delineate the emerging principles and areas of contention, and argue that by studying the mechanisms, specificity and natural substrate repertoires of IMPRs we can understand the properties for which they have been selected in evolution.

Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα.

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.