BIOLOGICAL OVERVIEW

To understand the role and significance of rhomboid (rho), is to grasp development in a microcosm. rhomboid is involved at the very earliest stage in morphogenesis, prior to fertilization, when the dorsoventral axis in the oocyte is determined. Rho protein is detected on the apical surface of dorsal-anterior follicle cells during stage 9, and in egg chambers during stage 10. rho expression correlates with the function of gurken and the Epidermal growth factor receptor. Interference with rho function results in ventralization of the embryo. Thus rho is required maternally in conjunction with gurken and Egf-r for the induction of dorso-ventral polarity. In this case Rhomboid seems to function in cells receiving the Gurken signal, that is in cells bearing the Egf-r (Ruohola-Baker, 1993).

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 remained obscure, but this 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 the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and these results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread (Urban, 2001).

Although Rhomboid-1 does not contain any obvious sequence homology domains, it has the characteristics of a serine protease. (1) Four of its six essential residues parallel the residues required for a serine protease catalytic triad charge-relay system (S217, H281, and N169) and an oxyanion stabilization site (consisting of a glycine two residues away from the active serine, and the serine itself; G215 and S217). These are the two active site determinants of serine proteases, and these four essential residues account for all of the amino acids known to participate directly in the serine protease catalytic mechanism. (2) These residues are absolutely conserved in all Rhomboids, and their mutation to even very similar residues (i.e., G215A, S217T, and S217C) abolishes Rhomboid-1 activity. These are hallmarks of active site residues. (3) The location of the essential residues is highly suggestive of a serine protease active site; both G215 and S217 occur in the conserved GASGG motif, which is remarkably similar to the conserved GDSGG motif surrounding the active serine of 200 different serine proteases. Furthermore, the essential residues N169 and H281 occur at the same height in their transmembrane domains (TMDs) as the GASGG motif, consistent with the proposal that they associate with S217 to generate a catalytic triad. Finally, Spitz processing is directly inhibited by the specific serine protease inhibitors DCI and TPCK, and Rhomboid-1 itself becomes limiting in their presence, suggesting that Rhomboid-1 is their direct target and thus the serine protease responsible for Spitz cleavage (Urban, 2001).

The proposed Rhomboid-1 catalytic triad is unusual, as it contains an asparagine rather than the more common aspartate. The central importance of this aspartate, however, is uncertain, since serine proteases with catalytic dyads of only serine and histidine have been identified, and even in those enzymes with catalytic triads, the aspartate is 100-fold less sensitive to mutation than the serine or histidine. In the case of Rhomboid-1, although N169 is essential in the assay presented in this study, there is evidence that in other contexts, its mutation leaves residual Rhomboid-1 activity. Further support for the possibility that N169 replaces an aspartate in a catalytic triad comes from the mechanism of some cysteine proteases, whose catalytic mechanisms are identical to serine proteases: they use catalytic triads with an asparagine to orient the histidine. Overall, the idea is favored that N169 does form part of the catalytic triad, but without a structural analysis of the active site, the possibility remains that it instead could be involved in oxyanion stabilization. In summary, although several mechanistic questions remain, these results strongly suggest that Rhomboid-1 is a serine protease that catalyses proteolysis in a membrane bilayer. Since no intramembrane serine protease is listed in either the MEROPS protease or EC enzyme databases, Rhomboid-1 and RHBDL2 appear to be the first examples of this kind of enzyme (Urban, 2001).

It is not clear how Rhomboid-1 functions within the lipid bilayer. Proteases catalyze hydrolysis of the peptide bond and thus require water to be accessible to their active sites. Although the Rhomboid-1 active site is situated within the membrane bilayer, helical packing among the Rhomboid-1 TMDs could provide an aqueous environment surrounding its active site. Consistent with this idea, there is a conserved helical repeat of charged and/or polar residues in TMD II that contributes the putative catalytic triad residue N169; this could form an aqueous environment around the active site. This polar face is also likely to mediate associations with other TMDs. TMD VI, which contributes the putative catalytic triad residue H281, is also predicted to contribute to TMD interactions: it contains two tandem GxxxG motifs known to mediate strong associations between transmembrane helices of the same orientation. The only helices in Rhomboid-1 of the same orientation as TMD VI are TMDs II and IV, which contribute the active site residues N169 and S217, respectively. Thus, TMDs II, IV, and VI might associate to generate the putative catalytic triad while the polar face of TMD II could provide the local aqueous environment required for catalysis (Urban, 2001).

Proteases in general cannot cleave folded proteins, and most TMDs adopt a helical conformation, with the amino acid side chains facing outward, sterically hindering access to the peptide backbone. The putative aqueous cavity of the Rhomboid-1 active site could force the hydrophobic Spitz TMD to change conformation, allowing cleavage. Alternatively, it has been proposed that intramembrane proteases function by partially unfolding their helical substrates, extending them into the cytosol and thus simultaneously unwinding the helix and providing an aqueous environment for proteolysis to occur. The essential residues W151 and R152 in the large lumenal loop of Rhomboid-1 could be involved in substrate unwinding from the lumenal side. Note that the helical packing and substrate unwinding models are not necessarily mutually exclusive, and various aspects of each model may prove to be important for intramembrane proteolysis by Rhomboid-1. Ultimately, direct biochemical analysis of purified Rhomboid-1 protein activity will be required to answer many of these questions, but this has not yet been achieved for any intramembrane protease, despite intensive study. However, the observations that Rhomboid-1 does not require endoproteolytic activation or other Drosophila cofactors suggests that these important goals may be achievable (Urban, 2001).

Although many other membrane-bound growth factors are activated by proteolytic release, Spitz is unusual, because it is cleaved within its TMD. Regulated intramembrane proteolysis (RIP) has recently emerged as a novel mechanism for controlling several important signaling pathways, including Notch receptor activation and cholesterol biosynthesis, by the release of cytoplasmic transcription factor domains from membrane-anchored proteins. As with other known RIP proteases, Rhomboid-1 is a polytopic membrane protein that is a member of a large protein family with homologs in many species. However, previously described RIP proteases have been limited to either aspartyl or metalloproteases, while Rhomboid is a serine protease. Beyond this mechanistic distinction, there are two major differences between the pathways involved in current examples of RIP and Spitz cleavage by Rhomboid-1 (Urban, 2001).

(1) Previously characterized examples of RIP result in the cytoplasmic release of either membrane-tethered transcription factors or proteins that are required for the activation of transcription factors. Conversely, Spitz cleavage releases a growth factor into the lumen of the Golgi apparatus, which is then secreted as an active signal for the EGF receptor in neighboring cells. (2) There is also a clear distinction between the mechanisms regulating intramembrane cleavage. All other known RIP proteases are widely expressed and have broad substrate specificity. Intramembrane cleavage is regulated by a prior cleavage that removes the bulk of the lumenal or extracellular portions of the target protein. Only after this cleavage takes place can the intramembrane proteases recognize and cleave their substrates. Conversely, Rhomboid-1 activity is regulated primarily by its transcription; Rhomboid-1 expression is tightly regulated and precisely prefigures EGF receptor signaling during Drosophila development. Furthermore, Rhomboid-1 is site-specific in its cleavage ability, since it specifically cleaves Spitz but not similar proteins such as TGFalpha. The human Rhomboids, too, show specificity, since RHBDL2 but not RHBDL cleaves Spitz (Urban, 2001).

Since Drosophila Rhomboid-1 is the prototype of a family consisting of over 75 proteins, both in prokaryotes and eukaryotes, understanding its mode of action has implications beyond Drosophila signaling and development. Furthermore, conservation of a proteolytic function and biochemical mechanism in a human Rhomboid, coupled with the absolute conservation of the putative catalytic residues, suggests that all Rhomboids are intramembrane serine proteases. Although their physiological roles remain unclear, it is notable that most, but not all, organisms have Rhomboids. This is consistent with a role in important but not essential processes, for example, intercellular signaling. Intriguingly, recent analysis supports this notion. Only one Rhomboid member outside Drosophila has been studied. In the human pathogenic bacterium Providencia stuartii, the Rhomboid-like AarA protein is involved in promoting the release of an unknown factor that regulates virulence in response to cell population size (Rather, 1999; Gallio, 2000). Gram-negative bacteria like Providencia use peptide-based factors as quorum sensing signals, and in some cases these are proteolytically released from precursors. The observations made for the AarA protein represent the first example of gram negative bacterium using peptide-mediated signaling. Peptide signaliing has, until now, been reported only for gram-positives, whereas gram-negatives commonly use acylated homoserine-lactones as mediators of cell communications (Gallio, personal communication to the editor of The Interactive Fly, 2002). Thus, although the current evidence is very limited, it is notable that even in a bacterium, Rhomboid-dependent proteolysis may be involved in signal production during cell communication. Proteases control many aspects of cell regulation; they also have substantial clinical significance. Defining the substrates of other Rhomboids should thus reveal their physiological and perhaps pathological roles in humans and other species (Urban, 2001).

The intimate link between Rhomboid and the EGF-receptor (Torpedo) is demonstrated by two phenomena. Rho seems to be required for amplification of the Egf-r signal, and excessive Rho results in the downregulation of EGF-R mRNA. Ectopic rho expression results in a rapid disappearance of EGF-R mRNA, suggesting that there is a fairly direct link between the two (Sturtevant, 1993). Spatially restricted processing of Spitz may be responsible for Efg-r graded activation. The Rhomboid and Star proteins have been suggested, on the basis of genetic interactions, to act as modulators of Egf-r signaling. No alteration in Egf-r autophosphorylation or the pattern of MAP kinase activation by secreted Spitz is observed when the Rho and Star proteins are coexpressed with Egf-r in S2 cells. In embryos mutant for rho or Star the ventralizing effect of secreted Spitz is epistatic, suggesting that Rho and Star may normally facilitate processing of the Spitz precursor (Schweitzer, 1995). Thus, a model is favored in which Rho protein is required for the activation of an Egf-R ligand, the Spitz transmembrane protein, by processing it into a functional form (Okabe, 1997).

Rhomboid appears to be an essential element in the development of the extremely delicate and fascinating vein structure of the insect wing. The segment polarity genes (engrailed, hedgehog, patched, cubitus interruptus, fused and decapentaplegic) are involved in the process of wing formation even earlier than rhomboid. These genes give positional information that will define the geometry of vein placement. They set the stage. The genes araucan and caupolican code for two divergent homeodomain proteins that are involved in establishing the prepattern for rhomboid, thus interacting with its position-specific enhancers to establish transcription at the sites of future veins (Gomez-Skarmeta, 1996).

The next level in the developmental hierarchy involves the initiation of vein formation. Staining for Rhomboid protein reveals a pattern that coincides with the future sites of wing veins. Mutations in some vein promotion genes result in lack of individual veins, while other mutants may lack portions of several veins. Vein promotion genes such as drifter (aka ventral veinless), hairless, Notch, hairy and extramachrochaete, are involved in the conversion of boundaries determined by the segmentation genes and other genes like araucan and caupolican, into patterns of rhomboid expression. Many of these vein promotion genes are also required for neurogenesis.

Other successive steps occur: vein extention, dorsal-ventral vein induction, and suppression of intervein differentiation. Each of these necessary steps requires different sets of genes. Intervein differentiation for example, is regulated by integrins, an important set of cell adhesion genes. The significance of rhomboid's involvement in venation is related to Egf-r signaling. It is the pattern of rhomboid expression that regulates the strength of the Egf-r signal. In turn, torpedo regulates downstream genes that do the actual vein building. Thus a hierarchical pattern of gene activity reaches from the segmentation genes expressed in the embryo and larvae, through rhomboid all the way to the adult structure of the wing (Sturdevant, 1995).

The chordotonal (Ch) organ, an internal stretch receptor located in the subepidermal layer, is one of the major sensory organs in the peripheral nervous system of Drosophila. Clues as to Rhomboid's function are provided in an analysis of the role of Rhomboid in the determination of Ch organ precursor cells (COPs). The rhomboid gene and the activity of the Drosophila Epidermal growth factor receptor (Egf-R) signaling pathway are necessary to specifically induce three of the eight COPs in an embryonic abdominal hemisegment. The cell-lineage analysis of COPs indicates that each of the eight COPs originate from an individual undifferentiated ectodermal cell. The eight COPs in each abdominal hemisegment seem to be determined by a two-phase induction: first, five COPs are determined by the action of the proneural gene atonal and neurogenic genes. Subsequently, these five COPs start to express the rho gene, and rho activates the Efg-R-signaling pathway in neighboring cells and induces argos expression. Three of these argos-expressing cells differentiate into the three remaining COPs and they prevent neighboring cells from becoming extra COPs. In the five atonal dependent COPs, Egf-R signaling activity is required, but this signaling does not seem to involve the cell autonomous activity of Rho. In rho null mutants five chordotonal organs remain intact. However, rho expression is required to activate Egf-R in adjacent cells, and these three adjacent cells express the neuronal marker asense. Argos functions from the second wave of cells as a lateral inhibitor, restricting the number of recruited cells to the original three. As the rho-expressing first wave of COPs is adjacent to the three argos and asense expressing double postive COPs, Argos may function to prevent the continuance of Egf-R-signal activation in additional neighboring cells. A model is favored in which Rho protein is required for the activation of an Egf-R ligand, the Spitz transmembrane protein, by processing it into the functional soluble form. An alternative model, invalid at least in Ch organ determination but still valid for follicle cell determination in oogenesis, suggests that Rho protein is expressed in cells that require the activation of the Egf-R signaling pathway, and that Rho protein interacts with Egf-R protein directly or indirectly to amplify Egf-R signaling (Okabe, 1997).

cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster

This study characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE many additional leg enhancers were identified, suggesting that this logic is broadly used by many genes during Drosophila limb development (Newcomb, 2018).

The EGFR signaling pathway is widely used in animal development, and is frequently a target in human disease and developmental abnormalities. Yet despite its importance in animal biology, many questions remain about how this pathway functions. Among these questions is whether secreted ligands that activate this pathway can induce distinct cell fates in a concentration-dependent manner. This study tests this idea by specifically eliminating a single source of EGFR ligands from the center of the Drosophila leg imaginal disc, which fate maps to the distal-most region of the adult leg. One plausible scenario is that this single source of secreted EGFR ligands, which is referred to as the EOC, activates distinct gene expression responses at different distances from this source. Alternatively, eliminating ligands secreted from the EOC might only affect gene expression locally, close to or within the EOC. Taken together, the current data are most consistent with the second scenario. This conclusion is largely supported by the observations that CRM deletions that eliminate vn and rho expression from the EOC have mild developmental consequences, both in the L3 leg imaginal discs and adult legs. These phenotypes are significantly weaker than those generated when the entire EGFR pathway is compromised using a temperature sensitive allele of the EGFR receptor. The difference between these two phenotypes is most likely explained by removing only a single source of EGFR ligands in the enhancer deletion experiments versus affecting EGFR signaling throughout the leg disc in the Egfr^tsla experiments. This explanation is further supported by the observation that there are indeed additional CRMs, some of which were defined in this study, that drive EGFR ligand production in more medial ring-like patterns during the L3 stage (Newcomb, 2018).

One possible caveat to these conclusions is that there are a total of seven rho-like protease genes in the Drosophila genome that could, in principle, play a role in distal leg development. This study focused on rho and roughoid ru, based on previous results showing that triple rho ru vn clones generate severe leg truncations that phenocopy strong Egfr^tsla truncations. In addition, it is noted that if other rho family proteases were active in the EOC, leg truncations and patterning defects would not be expected in the leg discs of the rho^rhoE-Df vn^vnE-Df double mutant, because those proteases should be able to produce active Spi. These observations suggest that the remaining five rho-like protease genes play a minor (or no) role in leg development. However, this conclusion will ultimately benefit from further genetic and expression analysis of these additional rho-like genes (Newcomb, 2018).

An additional previous observation that contrasts with the suggestion that EOC activity has only a limited role in specifying distal leg fates is the partial rescue of the PD axis when only a small number of distal leg cells were wild type in legs containing large rho ru vn clones. However, it is noted that even in these 'rescued' legs, medial defects in PD patterning were apparent. It is also noteworthy that in these earlier experiments, only adult legs were examined. When the same experiment was repeated, but L3 discs were analyzed, it was found that rho ru vn clones generated phenotypes that were very similar to those produced by the double vnE rhoE enhancer deletions. Taken together, these observations suggest that timing must be considered in the interpretation of these experiments. When assayed at the late L3 stage, both enhancer deletion and rho ru vn clone experiments argue that EOC activity is limited to specifying only the most distal fates, marked by the expression of al and C15. Starting in mid L3, and perhaps continuing into pupal development, there are additional sources of EGFR ligands that, when compromised, can affect adult leg morphology. Nevertheless, at least at the L3 stage, these data suggest that EGFR ligands produced from the EOC have a limited and local role in specifying distal leg fates (Newcomb, 2018).

Integration of inputs from signaling pathways and organ selector genes at CRMs in order to execute distinct developmental programs is a recurrent theme during animal development. This study identified two leg EGFR ligand CRMs that integrate the inputs from the Wg and Dpp signaling pathways and the leg selector genes Dll and/or Sp1 in a manner that is very similar to a previously characterized leg enhancer DllLT. In addition, when the same regulatory logic was applied to the whole genome, a battery of leg enhancer elements was identified. Interestingly, each of these enhancers drives expression in a specific manner with slightly different timing despite the fact that many of the inputs are shared. It is conceivable that the different expression patterns directed by these enhancers are in part a consequence of additional inputs and/or the difference in the TF binding site grammar. In support of this idea, vnE and rhoE differ in the number of binding sites for many inputs and vnE requires Sp1 while rhoE does not. Both of these differences may contribute to the earlier onset of vnE expression compared to rhoE. The remaining enhancer elements identified in this study direct a plethora of PD-biased leg expression patterns -- ranging from ubiquitous, to central and 'ring' patterns (see Genome-wide analysis of combinatorial inputs of Dll, Sp1, Wg, and Dpp in leg discs), which likely integrate inputs in addition to the ones described here. Future studies of these CRMs would help reveal the complex network of regulation that orchestrates leg development in the fruit fly. Such detailed understanding of the cis-regulatory architecture of fly leg development would likely give insights into organogenesis and evolution in other animals as well (Newcomb, 2018).

The EGFR signaling pathway has tremendous oncogenic potential and understanding the various mechanisms regulating its activation is not only interesting from the point of view of animal development but also has important practical implications. While the core components of the EGFR pathway have been thoroughly studied because of their potent tumorigenic capability in humans, little is known about the transcriptional regulation of EGFR ligands that bind the receptor and activate the pathway. The reiterative use of EGFR signaling in many developmental processes implies that different cis-regulatory elements are likely utilized by each EGFR ligand in different organs and tissues in order to correctly read the diverse cues in any specific developmental context. It is conceivable that genomic variation in EGFR pathway CRMs might lead to a predisposition to different types of EGFR-dependent tumors in humans, since such CRMs may respond to potent growth-promoting signaling pathways, such as Wnt and BMP (Newcomb, 2018).

This study has characterized in detail two Drosophila EGFR CRMs, vnE and rhoE, and showed how they integrate the cues from two transcription factors, Dll and Sp1, and two signaling pathways, Wg and Dpp, in order to execute a leg patterning developmental program. Analogous EGFR CRMs are likely to exist in mammals, especially because complex interactions between BMP, Wnt, Shh, multiple Dlx paralogs and other factors, are implicated in the induction of FGF signaling in mammalian limb development. Consistent with this idea, specific single nucleotide polymorphisms (SNPs) in humans in non-coding loci of genes encoding EGFR ligands have been shown to be associated with different types of cancer. Such loci may be enhancer elements analogous to vnE and rhoE. It is also noted that the regulatory logic uncovered in this study is likely to be relevant to many CRMs and genes that share spatial and temporal expression programs. Exploiting this regulatory logic in other systems might streamline the identification of enhancer elements that will aid in the discovery of mechanisms that are relevant to EGFR-related human disease and developmental birth defects (Newcomb, 2018).

GENE STRUCTURE

cDNA clone length - 2.5 kb

Bases in 5' UTR -321

Exons - three

Bases in 3' UTR - 1485 and 1493 at the two polyA sites detected

PROTEIN STRUCTURE

Amino Acids - 355

Structural Domains

There is an EKEKE sequence motif in what is predicted to be the amino-terminal cytoplasmic portion of the protein, a PEST sequence associated with proteins of short half life, and seven putative transmembrane regions. There are 14 copies of an ATTA motif in the 3'UTR. It is a motif that confers rapid mRNA turnover. No homology to G-protein coupled receptors has been found (Bier, 1990).

Six sequences from the Berkeley Drosophila Genome Project database were identified that exhibit high similarity to rhomboid. These include rhomboid-2 (CG12083), rhomboid-3 (CG1214) and rhomboid-4. Both rhomboid-2 and rhomboid-3 are cytologically located very close to the rhomboid-1 (rhomboid) gene on the third chromosome, whereas rhomboid-4 (CG1697) has been mapped to position 10C on the X chromosome by polytene chromosome in situ hybridization. Full length cDNAs were isolated for each of the new genes and their sequences were compared. The most highly conserved region spans the seven transmembrane domains; the hydrophilic amino terminus is strikingly divergent. This pattern of similarity is very like that between Drosophila rhomboid-1 and its recently identified mammalian homologs (Pascall, 1998), and suggests that the transmembrane domains provide a core function for Rhomboid-like proteins. A phylogenetic tree derived from these sequences indicates that rhomboid-3 is most closely related to rhomboid-1, followed by rhomboid-2; rhomboid-4 is the least related. The amino-terminal region of Rhomboid-4 contains two tandemly arranged EF-hand motifs that are putative calcium-binding domains. There are three further rhomboid-like genes predicted (rhomboid-5, rhomboid-6, and rhomboid-7). Rhomboid-5 (CG5364) is located at 31C; Rhomboid-6 (CG17212) at 33C, and Rhomboid-7 (CG8972) at 48E. The most conserved region encompasses the transmembrane domains, while diverging in the hydrophilic amino termini. This striking conservation of rhomboid-like genes suggests that the primordial function of these proteins is a fundamental cellular process. The restriction of Drosophila Rhomboid-1 and Rhomboid-3 function to Egfr signaling presumably represents a specialization of this original function (Wasserman, 2000).

EVOLUTIONARY HOMOLOGS

The Drosophila regulatory protein Rhomboid has been demonstrated genetically to facilitate signalling within the Spitz/epidermal growth factor receptor/mitogen-activated protein kinase pathway. Using a PCR-based strategy, a human cDNA has been cloned that encodes a protein that has high sequence similarity to Rhomboid. The encoded protein, termed rhomboid-related protein (RRP), is predicted to contain seven transmembrane domains. Northern analysis indicates that RRP mRNA is expressed at highest levels in brain and kidney (Pascall, 1998).

In Drosophila, the seven-pass transmembrane protein Rhomboid (Rho) is a crucial positive modulator of EGF signaling playing a substantial role in patterning of the ventral neuroectoderm and fate specification of neuroblasts. The cloning and expression pattern of Ventrhoid (Vrho), the novel evolutionarily conserved vertebrate cDNA related to fruit fly rho, is described. Most importantly, like rho in Drosophila, Vrho is also expressed in a spatially restricted manner. Vrho expression is most prominent along the developing ventral neural tube, and is also detectable in the ventral forebrain, prospective diencephalon, otic vesicles, mandibular arches, cranial sensory placodes, last formed pair of somites and hindgut in midgestational mouse embryos (Jaszai, 2002).

Rhomboid-1 is a serine protease that cleaves the membrane domain of the Drosophila EGF-family protein, Spitz, to release a soluble growth factor. Several vertebrate rhomboid-like proteins have been identified, although their substrates and functions remain unknown. The human rhomboid, RHBDL2, cleaves the membrane domain of Drosophila Spitz when the proteins are co-expressed in mammalian cells. However, the membrane domains of several mammalian EGF-family proteins were not cleaved by RHBDL2, suggesting that the endogenous targets of the human protease are not EGF-related factors. The amino acid sequence at the luminal face of the membrane domain of a substrate protein determines whether it is cleaved by RHBDL2. Based on this finding, B-type ephrins are predicted as potential RHBDL2 substrates. One of these, ephrinB3, was cleaved so efficiently by the protease that little ephrinB3 was detected on the surface of cells co-expressing RHBDL2. These results raise the possibility that RHBDL2-mediated proteolytic processing may regulate intercellular interactions between ephrinB3 and eph receptors (Pascall, 2004).

The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes. Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA. Mgm1 and its human ortholog OPA1, associated with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation. Mgm1 and its orthologs exist in two forms of different lengths. To obtain new insights into their biogenesis and function, these isoforms have been characterized. The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1). The large isoform is an integral inner membrane protein facing the intermembrane space. Furthermore, the conversion of l-Mgm1 into s-Mgm1 is dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphology. Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1. Expression of s-Mgm1 can partially complement the Deltapcp1 phenotype. Expression of both isoforms but not of either isoform alone was able to partially complement the Deltamgm1 phenotype. Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphology and maintenance of mitochondrial DNA (Herlan, 2003).

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. 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 (Lohi, 2004).

The role of thrombomodulin in the protein C anticoagulation pathway is well established. It is expressed on endothelial cells that line the blood vessels where it forms a complex with the clotting factor thrombin, inhibiting thrombin's interaction with fibrinogen. At the same time, the thrombin-thrombomodulin complex activates protein C, which proteolyses the activated coagulation factors Va and VIIIa. These two activities give thrombomodulin an important anticoagulant role. Beyond this, the biology of thrombomodulin is less well understood although it has been implicated in many processes including inflammation, adhesion, tumorigenesis, and embryonic development (Lohi, 2004 and references therein).

A circulating form of thrombomodulin, shed from the cell surface, is normally present in plasma and other fluids, implying that it is cleaved under physiological conditions. But it is not known whether soluble thrombomodulin has a function or whether it is merely a marker of endothelial cell damage. Circulating products representing a variety of cleavage sites can be found in plasma. Most correspond to proteolysis in the region between the membrane and the EGF repeats, but some are large enough potentially to correspond to intramembrane cleavage. Little is known about the proteases responsible for thrombomodulin shedding, although neutrophil-derived enzymes including elastase, proteinase-3, and cathepsin G have been implicated. The discovery that thrombomodulin is efficiently and specifically cleaved by RHBDL2, coupled with the observations reported here that most TMDs are not rhomboid substrates, suggests that this cleavage may be physiologically significant. If so, this would be the first vertebrate rhomboid substrate to be discovered and would represent a new biological function for the rhomboid family of proteases, which are conserved throughout evolution. Beyond the obvious significance of a potential role for RHBDL2 in thrombomodulin release, this work has implications for studying rhomboids. One of the most efficient methods for probing rhomboid function is to identify substrates: these provide insight into the cellular processes that rhomboids mediate. The knowledge that a second type of substrate recognition mechanism can be used by some rhomboids might influence strategies for finding rhomboid substrates. Finally, the discovery of a second mode of substrate recognition by rhomboids implies mechanistic diversity in this family of intramembrane proteases. Note, however, that the two recognition mechanisms uncovered are not mutually exclusive: as well as cleaving thrombomodulin, human, mouse, and zebrafish, RHBDL2s can also cleave Spitz and do so by recognizing the standard Spitz TMD motifs, implying that these are enzymes with dual specificities (Lohi, 2004).

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. This study reports 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. Endogenous RHBDL2 activity was identifed in several tumour cell lines (Adrain, 2011: PubMed).

A subset of membrane-altering agents and gamma-secretase modulators provoke nonsubstrate cleavage by rhomboid proteases

Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, this study found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects (Urban, 2014; PubMed).

Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. This study reports that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function (Cortesio, 2015).

date revised: 11 June 2022 

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