The second guiding principle stems from the tremendous diversity of organisms that encode rhomboid enzymes. Since these include organisms that do not encode any known forms of cell-to-cell communication, sequence information implies that rhomboid proteins perform an ancient and fundamental role in cell biology. This function is not essential for cell survival, however, because several lineages are missing rhomboid
genes entirely, presumably by gene loss [21
]. Although defining the cellular functions of rhomboid proteases has proven a persistent challenge, focused investigations have succeeded in documenting the function of at least one rhomboid in nearly a dozen organisms (Table ). These functions are usually regulated by substrate trafficking, and fall into four broad categories (Figure ).
Figure 4 The cellular roles of rhomboid proteases fall into four categories. Top left: Rhomboid proteases initiate EGF signaling during Drosophila development. Rhomboid-1 is localized in the Golgi apparatus, and cleaves Spitz (green) after it is transported from (more ...)
First, rhomboid proteases initiate animal cell signaling by releasing growth factors from the membrane. This function emerged from detailed genetic study of Drosophila
development; rhomboid proteases are localized in the Golgi apparatus and act as the signal-generating component by cleaving Spitz to initiate the pathway in neighboring cells [16
]. Although a role in regulating EGF signaling is also seen in Caenorhabditis elegans
vulval development, CeROM-1 has a surprisingly minor role as a target of EGF signaling that sets up a paracrine loop to amplify and spread the signal [47
]. Even less is clear in mammals: recent investigations have localized rhomboid proteins to the secretory pathway and cell surface and begun to uncover increased rhomboid
expression in cancer cells with potential links to growth factor signaling [24
]. However, this is not limited to active rhomboid proteases; expression of the iRhom RHBDF1, which is localized in the endoplasmic reticulum in human epithelial cancer cells, increased secretion of the EGF ligand transforming growth factor-α [50
]. Accordingly, RHBDF1 silencing decreased pathway activation through EGF receptor (EGFR), ERK and AKT phosphorylation, and limited tumor growth in mice [51
]. The Drosophila
homolog, however, was recently found to have the opposite effect of decreasing EGFR signaling by promoting the ERAD-mediated degradation of EGF ligands [41
]. The basis of this remarkable discrepancy is currently unclear; knockout mouse studies are expected to provide clarity on the physiological roles of rhomboid proteins.
Recent studies have also placed the mitochondrial rhomboid protease at the nexus of key pathways that govern mitochondrial fusion, mitophagy and apoptosis. All mitochondrial rhomboid proteins are encoded in the nuclear genome, and imported into mitochondria. The main function of the mitochondrial rhomboid Pcp1 is to release the dynamin-like GTPase Mgm1 from the membrane [28
]. Because Mgm1 is essential for mitochondrial fusion and Mgm1 cleavage occurs only in healthy mitochondria, this limits fusion to occurring between healthy organelles [54
]. A similar function was described in Drosophila
], but genetic interactions soon revealed further complexity in metazoans; the mitochondrial rhomboid DmRho-7 also participates in the Parkin/PINK1 pathway that malfunctions in Parkinson's disease [55
]. It has recently become clear that the human mitochondrial rhomboid PARL cleaves PINK1 to suppress its ability to recruit the Parkin ubiquitin ligase onto mitochondria [56
]. Without PARL cleavage, PINK1 accumulates in mitochondria and fails to be recruited properly to damaged mitochondria. A PARL knockout mouse suffers tremendous atrophy several months after birth resulting from malformed mitochondria and elevated apoptosis, although without mitochondrial fusion defects [29
]. PARL has also been implicated in suppressing apoptosis in lymphocytes, potentially through a different substrate, High-temperature regulated A (HtrA, also called Omi) [59
]. Intriguingly, mutations in PARL have recently been found in Parkinson's disease patients [58
] and diabetes patients [60
], although the significance of these mutations for disease remains speculative.
The third category of rhomboid function was revealed in Providencia stuartii
, a Gram-negative bacterial pathogen. Genetic screens identified its rhomboid homolog, AarA, to be required for production of an unidentified signal for quorum sensing [61
]. Once the similarity to rhomboid was noted [63
], proteolytic activity of AarA was demonstrated against Spitz [20
], and AarA was found to partially rescue tissue development of Drosophila
mutant in rhomboid
]. Historically, the intriguing similarity of activating Drosophila
EGF signaling and producing an auto-inducer for bacterial quorum sensing, both by a rhomboid, received much attention [63
]. But the similarity proved to be superficial when the substrate was identified to be TatA, a component of the twin-arginine translocation machinery [66
]. As such, AarA removes a short amino-terminal extension, presumably to activate the machinery for signal secretion, rather than activating the signal itself. TatA from other bacteria, including E. coli
, lacks this short extension and is immediately active, and the AarA function is therefore an exception. Nevertheless, this is the only known function for a rhomboid protease in any prokaryote, and it dramatically highlights the apparent diversity of rhomboid function even within similar bacteria.
Finally, rhomboid proteases help to dismantle adhesive junctions in unicellular eukaryotic parasites. This is the only role that was discovered by searching for rhomboid targets using substrate specificity determinants [33
]. The adhesins of Plasmodium
are necessary for host-cell invasion, making them essential proteins for the survival of these obligate intracellular parasites [67
]. These parasites encode six or more rhomboid proteases, two of which in each organism are known to process these adhesins at the end of the invasion program [25
]. The precise need for this dismantling is not entirely clear, but has been thought to free the parasite from being tethered to the host plasma membrane. Recent knockdown experiments indicate that this processing is important for efficient invasion [71
], although the full extent is incompletely understood and may involve later functions during parasite replication within the host cell [72
]. Even the non-cell-invasive Entamoeba histolytica
encodes a highly active rhomboid protease, which is localized to the parasite surface but which relocalizes to phagosomes during feeding and the bud neck during immune evasion, perhaps to shed surface proteins, including lectins [73
]. The functions of other Plasmodium
rhomboid proteases not involved in invasion are not yet understood [75
], and many other parasites encode rhomboid enzymes whose functions have never been explored.
Perhaps the most powerful, yet subtle, guiding principle that can be deduced from the near ubiquity of rhomboid proteases is that they possess a biochemical property that is both very rare and highly useful: but what? Solving this riddle requires understanding the enzymatic features of rhomboid proteases, and remarkable progress has been made towards these goals (reviewed in [38
There is now proof beyond doubt that rhomboid enzymes are serine proteases. This includes reconstitution of proteolysis with pure proteins [17
], protease inhibitor profiling [16
], extensive analysis of residues essential for activity [16
], and structural visualization of catalytic residues and with a covalently bound inhibitor [34
]. Moreover, the initial paradox of how water is delivered to the membrane-immersed active site for hydrolysis was largely addressed by structural analyses [34
]: the active site lies submerged about 10 Å below the presumed membrane surface, but with an open cavity above the active site for water access (Figure ).
Structure-function analyses of rhomboid proteases have also revealed several unusual proteolytic properties that make them unlike most serine proteases. These differences are clear evidence of convergent evolution to a serine protease mechanism down an independent path. First, structural analysis indicates that nucleophilic catalysis is achieved by a histidine-serine catalytic pair, rather than the more common aspartate-histidine-serine catalytic triad [34
]. Catalytic dyads have been noted in a minority of exceptional serine proteases [79
]. The identity of the residues that stabilize the oxyanion transition state is uncertain, but this stabilization is most likely mediated by asparagine and/or histidine side-chains [36
] (Figure ). Use of an asparagine for oxyanion stabilization is uncommon but strikingly analogous to the mechanism of the conventional serine protease subtilisin [80
The third unusual catalytic property of rhomboid proteases relates to the direction in which substrates lie across the active site cleft relative to the catalytic residues. Although initially thought to be similar to nearly all other serine proteases [34
], identification of the substrate gate on the opposite side of GlpG relative to expectation mandated that substrates approach the catalytic residues from the so-called 'si' face [35
]. This stereochemical arrangement is very uncommon and had only been encountered in α/β-hydrolyses [82
]. Consistent with this stereochemistry are rhomboid's resistance to most canonical serine protease inhibitors and a weak but specific sensitivity to monocyclic β-lactams [16
]. It should be stressed that the definitive evidence for substrate orientation, identity of the oxyanion hole, and the nature of substrate stabilization await a co-structure with a peptide substrate.
Rhomboid proteases have been studied largely within the framework of an established serine protease precedent as a way to interpret rhomboid mechanism, which is instructive but does not help to understand how they are different. Although deciphering the specifics of the catalytic chemistry is essential for designing effective inhibitors, the key functional properties of rhomboid enzymes that are relevant to the cell are unlikely to be determined by its catalytic mechanism. These defining features most likely result from membrane-immersion of the enzyme, and more recent investigations have started to study rhomboid proteases as integral membrane proteins directly.
The greatest impact of membrane immersion is on how substrates and rhomboid proteases behave (as reviewed in [38
]). The closed ring of TM segments observed in the first crystal structure suggested that something must move to clear a path for lateral substrate entry [34
]. Only mutations that weaken TM5 packing with TM2 were found to enhance protease activity by up to ten-fold, thereby identifying the gate functionally [77
]. This dramatic enhancement also revealed that gate opening is the rate-limiting step for intramembrane proteolysis. Molecular dynamics simulations and structural analysis in a bicelle also suggest membrane thinning surrounding GlpG, but its mechanistic implications remain unclear [84
]. Investigating the role of the membrane in greater detail promises to reveal the defining features of the rhomboid proteolysis system.