Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain

Rhomboid intramembrane proteases initiate cell signaling during Drosophila development and Providencia bacterial growth by cleaving transmembrane ligand precursors. We have determined how specificity is achieved: Drosophila Rhomboid-1 is a site-specific protease that recognizes its substrate Spitz by a small region of the Spitz transmembrane domain (TMD). This substrate motif is necessary and sufficient for cleavage and is composed of residues known to disrupt helices. Rhomboids from diverse organisms including bacteria and vertebrates recognize the same substrate motif, suggesting that they use a universal targeting strategy. We used this information to search for other rhomboid substrates and identified a family of adhesion proteins from the human parasite Toxoplasma gondii, the TMDs of which were efficient substrates for rhomboid proteases. Intramembrane cleavage of these proteins is required for host cell invasion. These results provide an explanation of how rhomboid proteases achieve specificity, and allow some rhomboid substrates to be predicted from sequence information.     

An Arabidopsis Rhomboid homolog is an intramembrane protease in plants

Regulated intramembrane proteolysis (RIP) is a fundamental mechanism for controlling a wide range of cellular functions. The Drosophila protein Rhomboid-1 (Rho-1) is an intramembrane serine protease that cleaves epidermal growth factor receptor (EGFR) ligands to release active growth factors. Despite differences in the primary structure of Rhomboid proteins, the proteolytic activity and substrate specificity of these enzymes has been conserved in diverse organisms. Here, we show that an Arabidopsis Rhomboid protein AtRBL2 has proteolytic activity and substrate specificity. AtRBL2 cleaved the Drosophila ligands Spitz and Keren, but not similar proteins like TGFalpha, when expressed in mammalian cells, leading to the release of soluble ligands into the medium. These studies provide the first evidence that the determinants of RIP are present in plants.     

Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases.

The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFalpha-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but our analysis suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is, to our knowledge, the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and our results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread.     

The rhomboids: a near ubiquitous family of intramembrane serine proteases evolved via multiple horizontal gene transfers

Background  

The rhomboid family consists of polytopic membrane proteins, which show a level of evolutionary conservation that is unique among membrane proteins. The rhomboids are present in nearly all sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes. On the basis of experimental studies with the developmental regulator Rhomboid from Drosophila and the AarA protein from the bacterium Providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose signaling function is conserved in eukaryotes and prokaryotes.     

Rhomboid proteins: a role in keratinocyte proliferation and cancer

The Rhomboids represent a relatively recently discovered family of proteins, consisting in a variety of intramembrane serine proteases and their inactive homologues, the iRhoms. Rhomboids typically contain six or seven transmembrane domains (TMD) and have been classified into four subgroups: Secretase A and B, Presenilin-Associated-Rhomboid-Like (PARL) and iRhoms. Although the iRhoms, iRhom1 and iRhom2, have lost their protease activity during evolution, they retain key non-protease functions and have been implicated in the regulation of epidermal growth factor (EGF) signalling. EGF is moreover a substrate of RHBDL2, their active Rhomboid relative. Other substrates of RHBDL2 include members of the EphrinB family and thrombomodulin. RHBDL2 has also previously been demonstrated to be important in wound healing in cutaneous keratinocytes through the cleavage of thrombomodulin. Additional roles for these intriguing proteins seem likely to be revealed in the future. This review focuses on our current understanding of Rhomboids and, in particular, on RHBDL2 and iRhom2 and their roles in cellular processes and human disease.     

EGF receptor signalling: roles of star and rhomboid revealed.

Recent studies have clarified how the active form of the Drosophila EGF receptor ligand Spitz is produced: Star chaperones Spitz in the ER and mediates its transport to the Golgi, where the intramembrane serine protease Rhomboid cleaves the Spitz proprotein to initiate secretion.     

Rhomboid-like proteins in Apicomplexa: phylogeny and nomenclature

Rhomboids form a family of polytopic intramembrane serine proteases. In Toxoplasma gondii, an essential activity called microneme protein protease 1 (MPP1) cleaves secreted adhesive proteins within their transmembrane domains, at a site conserved in similar proteins of other Apicomplexa. Current evidence suggests that MPP1 is ubiquitous in the phylum and is encoded by a rhomboid gene. In this article, we present the current repertoire of rhomboid-like proteins in Apicomplexa using a nomenclature based on phylogenetic analyses.     

Rhomboid cleaves Star to regulate the levels of secreted Spitz

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S(2)R(+) cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.     

Regulated intramembrane proteolysis takes another twist.

Rhomboid, a seven-transmembrane domain protein, has been shown genetically to potentiate EGFR signaling via the TGFalpha-like ligand Spitz. Here we discuss recently published papers that identify Rhomboid as a novel serine protease, cleaving Spitz within its transmembrane domain.     

Versatility of EGF receptor ligand processing in insects

Processing of EGF-family ligands is an essential step in triggering the EGF receptor pathway, which fulfills a diverse set of roles during development and tissue maintenance. We describe a mechanism of ligand processing which is unique to insects, and possibly to other invertebrates. This mechanism relies on ligand precursor trafficking from the ER by a chaperone, Star (S), and precursor cleavage by Rhomboids, a family of intra-membrane protease. Remarkably, the ability of Rhomboids to cleave S as well, endows the pathway with additional diversity. Rhomboid isoforms which also reside in the ER inactivate the chaperone before any ligand was trafficked, thus significantly reducing the level of ligand that will eventually be processed and secreted. ER localization also serves as a critical feature in trafficking the entire ligand-processing machinery to axonal termini, as the ER extends throughout the axon. Finally, examination of diverse species of insects demonstrates the evolution of chaperone cleavability, indicating that the primordial processing machinery could support long-range signaling by the ligand. Altering the intracellular localization of critical components of a conserved signaling cassette therefore provides an evolutionary mechanism for modulation of signaling levels, and diversification of the biological settings where the pathway functions.     

Rhomboid proteases in plants - still in square one?

Rhomboids are ubiquitous intramembrane serine proteases the sequences of which are found in nearly all sequenced genomes, including those of plants. They were molecularly characterized in a number of organisms, and were found to play a role in a variety of biological functions including signaling, development, apoptosis, mitochondrial integrity, parasite invasion and more. Although rhomboid sequences are found in plants, very little is known about their function. Here, we present the current knowledge in the rhomboids field in general, and in plant rhomboids in particular. In addition, we discuss possible physiological roles of different plant rhomboids.     

The rhomboid protease family: a decade of progress on function and mechanism

Rhomboid proteases are the largest family of enzymes that hydrolyze peptide bonds within the cell membrane. Although discovered to be serine proteases only a decade ago, rhomboid proteases are already considered to be the best understood intramembrane proteases. The presence of rhomboid proteins in all domains of life emphasizes their importance but makes their evolutionary history difficult to chart with confidence. Phylogenetics nevertheless offers three guiding principles for interpreting rhomboid function. The near ubiquity of rhomboid proteases across evolution suggests broad, organizational roles that are not directly essential for cell survival. Functions have been deciphered in only about a dozen organisms and fall into four general categories: initiating cell signaling in animals, facilitating bacterial quorum sensing, regulating mitochondrial homeostasis, and dismantling adhesion complexes of parasitic protozoa. Although in no organism has the full complement of rhomboid function yet been elucidated, links to devastating human disease are emerging rapidly, including to Parkinson's disease, type II diabetes, cancer, and bacterial and malaria infection. Rhomboid proteases are unlike most proteolytic enzymes, because they are membrane-immersed; understanding how the membrane immersion affects their function remains a key challenge.     

Solution structure and dynamics of the N-terminal cytosolic domain of rhomboid intramembrane protease from Pseudomonas aeruginosa: insights into a functional role in intramembrane proteolysis

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.     

Diverse substrate recognition mechanisms for rhomboids; thrombomodulin is cleaved by Mammalian rhomboids

The rhomboids are a recently discovered family of intramembrane proteases that are conserved across evolution. Drosophila was the first organism in which they were characterized, where at least Rhomboids 1-3 activate EGF receptor signaling by releasing the active forms of EGF-like growth factors. Subsequent work has begun to shed light on the role of these proteases in bacteria and yeast, but nothing is known about the function of rhomboids in vertebrates beyond evidence that the subclass of mitochondrial rhomboids is conserved. Here, we report that the anticoagulant cell-surface protein thrombomodulin is the first mammalian protein to be a rhomboid substrate in a cell culture assay. The thrombomodulin transmembrane domain (TMD) is cleaved only by vertebrate RHBDL2-like rhomboids. Thrombomodulin TMD cleavage is directed not by sequences within the TMD, as is the case with Spitz but by its cytoplasmic domain, which, at least in some contexts, is necessary and sufficient to determine cleavage by RHBDL2. These data suggest that thrombomodulin could be a physiological substrate for rhomboid. Moreover, the discovery of a second mode of substrate recognition by rhomboids implies mechanistic diversity in this family of intramembrane proteases.     

Discrete models of autocrine cell communication in epithelial layers

Pattern formation in epithelial layers heavily relies on cell communication by secreted ligands. Whereas the experimentally observed signaling patterns can be visualized at single-cell resolution, a biophysical framework for their interpretation is currently lacking. To this end, we develop a family of discrete models of cell communication in epithelial layers. The models are based on the introduction of cell-to-cell coupling coefficients that characterize the spatial range of intercellular signaling by diffusing ligands. We derive the coupling coefficients as functions of geometric, cellular, and molecular parameters of the ligand transport problem. Using these coupling coefficients, we analyze a nonlinear model of positive feedback between ligand release and binding. In particular, we study criteria of existence of the patterns consisting of clusters of a few signaling cells, as well as the onset of signal propagation. We use our model to interpret recent experimental studies of the EGFR/Rhomboid/Spitz module in Drosophila development.     

Intramembrane Proteolysis of Mgm1 by the Mitochondrial Rhomboid Protease Is Highly Promiscuous Regarding the Sequence of the Cleaved Hydrophobic Segment

Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed ‘rhomboid cleavage region’ (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.     

A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands

Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. We have analysed four of these: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signalling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. Our data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands.     

Rhomboid function in the midline of the Drosophila CNS

Rhomboid (Rho), a cell surface, seven-transmembrane domain protein, participates in Spitz-dependent activation of the Drosophila EGF receptor (EGFR). By contrast to transient expression in other embryonic tissues, rho is expressed continuously in the embryonic and larval Midline Glia (MG) lineage and is required upstream of, or in parallel with, S, Spi, and EGFR to establish MG cell number. EGFR signaling is necessary for the expression of rho in the MG and sufficient to stimulate rho expression in additional MG progenitors. rho expression is required continuously from embryonic stage 9-17 to suppress apoptosis in the MG. Although rho misexpression can increase MG number through a non-cell autonomous mechanism, the pattern of normal rho expression suggests that it functions by enhancing autocrine or paracrine signaling among MG cells.     

家蚕EGFR配体的鉴定和验证

表皮生长因子受体(EGFR)是广泛存在于后生动物中的多功能受体,其配体的种类、活化方式、配体与EGFR之间的相互作用以及激活的信号通路在哺乳动物中研究得较为深入。而非脊椎动物中,EGFR配体在各物种间差异较大,目前缺乏对除果蝇以外的其他昆虫EGFR配体的认识。通过同源比对、结构域预测、m RNA翻译起始序列分析和系统进化树构建,在家蚕中鉴定到2个EGFR的配体,命名为Bm EGF-1和Bm EGF-2。Bm EGF-1与果蝇Spitz有较高的同源性和一致的Rhomboid识别序列,Bm EGF-2为Vein的同源分子。经原核表达和纯化获得了Bm EGF-1胞外区段,利用Sf9细胞分泌Bm EGFR胞外区段,并通过pull-down实验验证了两者之间存在相互作用。在Bm E细胞中表达Bm EGF-1后,通过Western blotting检测到ERK和p38 MAPK的磷酸化水平增强,说明其不仅能激活经典的ERK信号通路,还可能通过p38 MAPK信号通路参与其他生理过程,为进一步研究EGFR配体在家蚕中的生物学功能提供了参考。     

Rhomboid homologs in mycobacteria: insights from phylogeny and genomic analysis

Background  

Rhomboids are ubiquitous proteins with diverse functions in all life kingdoms, and are emerging as important factors in the biology of some pathogenic apicomplexa and Providencia stuartii. Although prokaryotic genomes contain one rhomboid, actinobacteria can have two or more copies whose sequences have not been analyzed for the presence putative rhomboid catalytic signatures. We report detailed phylogenetic and genomic analyses devoted to prokaryotic rhomboids of an important genus, Mycobacterium.