rhomboids: 7 years of a new protease family
TRANSCRIPT
Seminars in Cell & Developmental Biology 20 (2009) 231–239
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Seminars in Cell & Developmental Biology
journa l homepage: www.e lsev ier .com/ locate /semcdb
Review
Rhomboids: 7 years of a new protease family
Matthew FreemanMRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
a r t i c l e i n f o
Article history:Available online 17 October 2008
Keywords:Rhomboids
a b s t r a c t
Drosophila Rhomboid-1 was discovered to be the first known intramembrane serine protease about 7years ago. The study of the rhomboid-like family has since blossomed, and the purpose of this review isto take stock of where the field is, and how it may progress in the next few years. Three major themes arethe increasing understanding of the biological roles of rhomboids, the detailed information we now have
Intramembrane proteaseEGF receptorParasiteMD
about their function and mechanism, and the promising leads they offer as medical targets.© 2008 Elsevier Ltd. All rights reserved.
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itochondriaisease mechanism
ontents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2312. New proteases from old genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2323. Milestones since the initial discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2324. What have we learnt about the biological roles of rhomboids?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
4.1. Rhomboid in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2334.2. Mitochondrial rhomboids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2334.3. Rhomboids in protozoan parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
5. What have we learnt about the enzymology and structure of rhomboids? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2346. Rhomboids—the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
6.1. Mammalian rhomboids—the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2356.2. Parasite rhomboids—the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2356.3. Mitochondrial rhomboids—the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2356.4. Bacterial rhomboids—the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2356.5. Plant rhomboids—the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
7. Systematic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2367.1. Substrate identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2367.2. Rhomboid inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
8. Rhomboid regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
9. Structural biology of rhomboids—the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23610. iRhoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23711. Conclusions and medical prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations: ADAM, a disintegrin and metalloprotease protein; EGFR, epider-al growth factor receptor; ER, endoplasmic reticulum; TGF�, transforming growth
actor alpha; TMD, transmembrane domain.E-mail address: [email protected].
1
iocs
084-9521/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2008.10.006
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
. Introduction
Rhomboids were identified about 7 years ago as the firstntramembrane serine proteases [1]. Since then, they have devel-ped from being the newest members of the mysterious andontroversial intramembrane proteases, to being the best under-tood of these now established enzymes. Nevertheless, our
2 velopmental Biology 20 (2009) 231–239
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Fig. 1. Schematic view of a rhomboid—an intramembrane serine protease. The cat-alytic serine is shown in red and the histidine in green. The transmembrane helixcontaining the catalytic serine is shorter than the rest, allowing access of water tothe active site (see Fig. 4).
Fig. 2. Trafficking controls EGFR activation in Drosophila. The TGF�-like ligand,Spitz, is confined to the endoplasmic reticulum (ER) until it encounters Star (red),wwt
absaitctp
3
2understanding both the biological roles of rhomboids, and the
32 M. Freeman / Seminars in Cell & De
nderstanding of rhomboids is still rudimentary in many aspects.fter 7 years we can take stock, and my goal here is to summarise
he progress that has been made in that time, to highlight thosereas where recent progress has been most rapid, and to lookorward to what may be the main developments over the nextew years. Several recent reviews [2–5] have discussed rhomboidesearch in more detail than I will here, and I shall refer to them asppropriate. Rhomboids appear to be conserved in all eukaryotesnd most prokaryotes, and we already know many biological func-ions, so there are almost unlimited potential research directions.n additional aim of this review is therefore to propose what I con-ider to be reasonable priorities for the immediate future—what arehe main questions that there is a prospect of being able to answer?
. New proteases from old genetics
The mechanistic and functional study of proteases is a large andature field; it has medical and biological significance and is the
ocus of much research in academia and industry. New proteasesre usually instantly recognisable and classifiable by their simi-arity to known proteases. The discovery of the intramembraneroteases was different. Over the last 10 years, each of the fournown classes have emerged from unexpected areas of biology [6].homboids were discovered from a systematic analysis of epider-al growth factor receptor (EGFR) signalling in Drosophila. The fly
GFR mediates multiple important intercellular signalling eventsuring development [7], and Drosophila genetics in a number of
abs was used to identify the main components of this importantignalling pathway. By the late 1990s, a number of approachesad highlighted the rhomboid gene (named after the abnormallyhaped heads of mutant larvae [8]) as the primary activator ofGFR signalling [9–15]. Moreover, the use of genetic mosaics, whereutant cells can be juxtaposed with wild-type cells in a develop-
ng tissue, proved that Rhomboid (now Rhomboid-1) was neededn the signal emitting cell rather than the signal receiving cell16].
Despite these conclusions about the function of Rhomboid-in EGFR signalling, its molecular mechanism was not obvious.
he primary EGFR activating ligand in Drosophila is called Spitz,nd is homologous to mammalian TGF�. As with TGF�, Spitzs synthesised as a transmembrane protein and the extracellularomain must be released from the membrane to become an active
igand [17,18]. The genetic evidence suggested that Rhomboid-might trigger this proteolytic release of Spitz [16], but since
he Rhomboid-1 protein was not a recognisable protease, thereas no molecular underpinning for this proposal. Of course, oncehomboid-1 was found to be a novel protease – the first intramem-rane serine protease (Fig. 1) – the existing Drosophila geneticvidence allowed rapid progress in understanding the details ofow rhomboids control EGFR signalling in flies [1,19–21].
An important feature of the mechanism that had not been pre-icted by genetics was the importance of controlled intracellularrafficking of the protein machinery that triggers the release ofctive Spitz [19–21]. Similar genetic approaches that led to thedentification of Rhomboid-1, pointed to the importance in EGFRignalling of another mysterious membrane protein, called Star12,14,15,22,23]. It too acts in the signal sending cell and provideso clues about its function from its sequence. Expression of Spitz,homboid-1 and Star in cell culture, later supported by in vivoata, showed that Star provides the second key to Spitz activa-
ion (Fig. 2) [19–21]. Unexpectedly, Spitz turns out to be stuck inhe endoplasmic reticulum upon synthesis. It can only exit intohe later secretory pathway in the presence of Star, which has atill poorly understood trafficking function. Rhomboid-1 is con-ned to post-ER compartments, so is unable to cleave Spitz in theeieas
hich traffics it to the Golgi apparatus. Rhomboid (dark blue) is located in the Golgi,here it cleaves Spitz. Rhomboid may also cleave Star, limiting its ability to recycle
o the ER [78]. Once cleaved, the active Spitz ligand is secreted.
bsence of Star. Once released by Rhomboid-1 from its transmem-rane tether, soluble (and therefore active) Spitz ligand is free to beecreted from the cell. This mechanism therefore limits the cleav-ge of Spitz by Rhomboid-1 by segregating enzyme and substratento different membrane compartments, and then using regulatedrafficking to control their meeting. This strategy of exploiting theellular machinery of membrane protein trafficking to regulatehe cleavage of rhomboid substrates is a novel way of regulatingroteolysis.
. Milestones since the initial discovery
This first wave of rhomboid research was complete by around001. Since then, progress has been rapid on many fronts, in
nzymology of how they work. In this section, I will outline themportant milestones over the last 7 years. Fig. 3 outlines the differ-nt classes of protein in the eukaryotic rhomboid-like family [24],nd Table 1 summarises the biological contexts where potentialubstrates of a particular rhomboid have been identified.
M. Freeman / Seminars in Cell & Developmental Biology 20 (2009) 231–239 233
Fig. 3. The eukaryotic rhomboid-like family. The members indicated in blue are the active rhomboids.
Table 1Rhomboids and their natural substrates (confirmed and proposed).
Species Rhomboid Substrate Evidencea References
Human RHBDL2 Thrombomodulin Cell culture [63]Human RHBDL2 EphrinB3 Cell culture [62]Mouse RHBDL2 Thrombomodulin Cell culture [63]Mouse PARL OPA1 Cell culture, genetics [36]Mouse PARL Omi/HtrA2 In vivo, genetics, cell culture [43]Drosophila Rhomboid-1,-2,-3 Spitz Cell culture, in vitro, in vivo, genetics [1,19–21,79]Drosophila Rhomboid-1,-2,-3 Gurken Cell culture, in vitro, genetics [52,79–82]Drosophila Rhomboid-1,-2,-3 Keren Cell culture, in vitro, genetics [79,83–85]Drosophila Rhomboid-1,-3 Star Cell culture, genetics [78]Drosophila Rhomboid-7 Omi In vivo, genetics [44]Drosophila Rhomboid-7 Pink1 In vivo, genetics [44]C. elegans ROM-1 LIN-3L Genetics [64]S. cerevisae Rbd1/Pcp1 Mgm1 In vivo, genetics [31,32]S. cerevisae Rbd1/Pcp1 Ccp1 In vivo, genetics [30]T. gondiib TgROM1-5 TgMIC2, TgMIC6, TgMic12, TgAMA1 Cell culture [45,86–90]P. falciparumb PfROM1, PfROM4,
PfROM7, PfROM8PfAMA1; PfEBA-175 and other EBLadhesins; RBL adhesins; TRAPadhesins; PfCTRP; PfMAEBL
Cell culture, genetics (in case of PfROM4and EBA-175 [91])
[89,91,92]
E. histolytica EhROM1 Lectin EHI 044650 Cell culture [50]P. stuartii AarA TatA Cell culture, in vitro, in vivo, genetics [25,26,29]
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a Cell culture: cleavage confirmed in cell culture assay. In vitro: cleavage confironfirmed in natural cells. Genetics: genetic evidence to support physiological sign
b Due to the complexity and number of rhomboids and possible substrates, these
. What have we learnt about the biological roles ofhomboids?
.1. Rhomboid in bacteria
After the discovery that rhomboids regulate EGFR signalling inrosophila, and that they are conserved across evolution, their
unction(s) beyond growth factor signalling became an obviousentre of attention. A specific case was the role of bacterial rhom-oids, which might provide some insight into their primordialunctions. Furthermore, the bacterium Providencia stuartii, was thenly organism beyond Drosophila, where genetic analysis had high-ighted a biological role for a rhomboid-like protein [25,26]. Theraditional view of bacteria as free living cells has been amended byhe discovery of the extent to which they communicate with eachther by diffusible signals, in a process generically termed quorumensing. Quorum sensing signals control many aspects of bacterialehaviour, gene expression and fate decisions [27], but there aretill major gaps in our understanding of the molecular mechanismsf signalling.
The laboratory of Philip Rather had identified the Providenciarotein AarA as required for the generation of a quorum sensingignal [25]. AarA is a rhomboid, implying that in this bacterium,s in Drosophila, a rhomboid participated in the production of anntercellular signal. In a spectacular example of common enzyme
unction across evolution, it was also shown that ProvidenciaarA could trigger EGFR signalling when expressed in flies; thatrosophila Rhomboid-1 could rescue the Providencia phenotypeaused by AarA loss; and that the fly and bacterial enzymes sharedubstrate specificity [26,28]. Recent work has now uncovered thewopio
in cell free reaction with detergent solubilisation. In vivo: physiological cleavagee of cleavage (NB does not prove direct protease/substrate relationship).tial rhomboid/substrate pairs have not been separated.
olecular function of AarA, and its participation in quorum sens-ng turns out to be less direct than the generation of active Spitz inrosophila [29]. The relevant substrate turns out not to be a sig-alling molecule itself, but instead the membrane protein TatA,he main subunit of the twin arginine translocase, a membraneransporter of fully folded proteins. Until the Providencia quorumensing signal is identified – presumably accelerated by the recentelease of the genome sequence – the relationship between TatAranslocation and quorum sensing remains elusive, although it isikely that either the signal itself or something required for its pro-uction relies on TatA.
.2. Mitochondrial rhomboids
The apparently analagous functions of the two first rhomboidunctions to be studied – intercellular signalling in Drosophiland Providencia – led to a question of whether signalling woulde a common thread between all rhomboids. This was ruled outhen a rhomboid in the yeast Saccharomyces cerevisiae was found
o reside and function in mitochondria [30–32]. This yeast rhom-oid, called Rbd1 or Pcp1, is located in the inner membrane of theitochondria, and two physiological substrates have been identi-
ed, the most important of which is Mgm1, a dynamin-like GTPaseequired for mitochondrial membrane fusion [31,32]. Mitochon-ria are dynamic organelles and their degree of fragmentation,
hich can vary between cell types, is highly regulated by a balancef fusion and fission. Mgm1 is synthesised as a transmembranerotein but to be active, a proportion must be released into the
ntermembrane space as a soluble enzyme; this release is carriedut by Rbd1/Pcp1 [31–33]. Drosophila mitochondrial rhomboids
2 velopm
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34 M. Freeman / Seminars in Cell & De
lso regulate mitochondrial fusion [34], although it has not beenstablished whether this is dependent on the Drosophila homo-ogue of Mgm1, Opa.
The role of the mammalian mitochondrial rhomboid, presenilinssociated rhomboid-like [35] (PARL, although the implied connec-ion with presenilin is an artefact), is proving difficult to untangle.
ouse knockouts of PARL show a mitochondrial phenotype butnstead of affecting membrane fusion, the primary defects appearo be in cristae junctional sealing, in turn regulating cytochrome celease and apoptosis [36]. There is some evidence to suggest thathis PARL function is mediated by OPA1 (the mammalian ortho-ogue of Mgm1 [37]), but the weight of evidence currently suggestshat OPA1 processing depends largely on PARL-independent mech-nisms, and that OPA1 is not a major substrate of PARL in mammals36,38–42]. Consistent with this idea, two other PARL substratesave recently been identified. In mammals, the membrane teth-red protease Omi/HtrA2 is cleaved by PARL, releasing it into thentermembrane space [43]. Similarly in Drosophila, Omi and Pinkinase, both associated with mitochondrial defects in Parkinson’sisease, are PARL substrates [44]. Importantly, the fly work also pro-ides genetic support for a functional relationship between PARL,mi and Pink, as well as Parkin, the Parkinson’s disease-relatedrotein. The exact function of mitochondrial rhomboids in animalells is therefore still rather unclear but there is strong evidenceo implicate them in membrane dynamics and related apoptoticegulation.
.3. Rhomboids in protozoan parasites
A potential role in parasites has been one of the most stud-ed areas of rhomboid research in the last 5 years. The possibilityhat they may be involved in the invasion of host cells by apicom-lexan parasites (Plasmodium, Toxoplasma, Cryptosporidium, etc.)as first suggested by the discovery that a group of cell surface
dhesins in Toxoplasma were rhomboid substrates [45]. But theiscovery of a protein that can be cleaved by a rhomboid is very dif-erent from proving a physiologically important relationship, andhis has been harder to confirm, largely because the genetics of thepicomplexa is difficult. Nevertheless, the growing number of caseshere adhesin like proteins have been shown to be good substrates
f parasite rhomboids, combined with some functional hints, sup-orts the general idea [reviewed in 2,5]. Although there are manyariations on the theme, adhesins are parasite cell surface proteinshat engage with host receptors in the early stages of invasion.fter actin-driven ingression to the host cell, the adhesins muste cleaved for the invasion to be complete [46]. The identity of theroteases that perform this important cleavage has been the focusf much attention and there are clearly several different types ofnzymes involved [47,48], with the rhomboids making a good caseo be included on the list.
There are now a significant number of adhesins and rhomboids,oth in Toxoplasma and Plasmodium (the malaria parasite), forhich this kind of relationship has been proposed or demonstrated
reviewed in 2, 5]. The story is not simple and it is likely that somef the rhomboids are involved in other parasite functions, just asome of the adhesins are cleaved by other proteases. For exam-le, loss of the Toxoplasma ROM1 leads to growth defects but no
nvasion phenotype [49]. Nevertheless, the biochemical and celliological evidence is generally persuasive and this is an area wheredvances are likely to occur in the near future. As in other exam-
les of understanding rhomboid function, the ability to establishhe consequence of loss of a particular rhomboid – primarily byenetic techniques – is the key to proving a causal link.For largely historical reasons, the primary focus of parasiteesearch with rhomboids has been on the apicomplexa, but a recent
eifos
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eport has also identified a rhomboid and substrate in the extra-ellular parasite Entamoeba histolytica [50], which causes amoebicysentery, and is a major human health burden. In this case theubstrate is a lectin associated with immune evasion, suggestingn interesting and different possible role for a rhomboid in par-site pathogenesis. Again, however, functional evidence will beeeded to promote this from the status of intriguing possibilityo something more like a validated medical target. But the valuef these kinds of studies remains high: they provide clear, focusednd testable hypotheses.
. What have we learnt about the enzymology andtructure of rhomboids?
The initial work defining rhomboid function in Drosophila EGFRignalling depended on a combination of genetics and cell biol-gy. Because intramembrane proteolysis was such an unexpectedechanism, there was a strong desire to understand rhomboid
nzymology. A milestone was the development of a cell-freenzyme assay system. This was a technical challenge, since thehomboid active site is embedded within the lipid bilayer and bothnzyme and substrate are insoluble, but it was achieved almostimultaneously by three groups [51–53]. The details varied but wereonceptually similar: detergent solubilised rhomboid expressed inacteria was mixed with a potential substrate TMD, and cleavageonitored. These studies revealed important elements of rhom-
oid enzymology, showing for example, that rhomboids did noteed cofactors, used an atypical serine catalytic dyad, and coulde influenced by lipid composition [51–53]. They were less use-ul for meaningful kinetic studies, since the cleavage takes placen detergent micelles, and are not simple first order reactions—theartitioning of substrate and enzyme into micelles contributing toverall rates.
The development of assays for the activity of purified rhomboidslso provided a foundation for crystallography studies. The publi-ation, by several groups, of the structures of the rhomboid GlpGrom E. coli and H. influenzae was a major mechanistic achievement54–57] (Fig. 4). Not only did these structures reveal much aboutow rhomboids work but, as the first structures of any intramem-rane proteases, they represent major landmarks [reviewed in,4,58,59]. They do, however, also leave many unanswered ques-ions. They explain how the active site is accessible to water androve that it is, as postulated, in the plane of the membrane; buthey do not provide more than hints about how substrates accesshe active site. They do establish that the active site resembles alassical serine protease; but they do not reveal the precise mech-nism of cleavage. Finally, they do suggest how transmembraneomains can be substrates; but they do not indicate what are theeterminants of specificity. Many of these questions will be betternswered once the structure of a complex of enzyme and substrateas been solved, but more generally, these questions illustrate thebvious need for functional studies, genetic and biochemical, toomplement structural analysis.
. Rhomboids—the future
In the remainder of this review, I look forward and try to pre-ict where the next advances will occur. Given the almost limitlessumber of rhomboids that exist in such a diverse range of species,
ach with their own repertoire of substrates, this is a goal thats doomed to fail: there is no way of predicting all the rhomboidunctions that will emerge from unexpected areas—indeed, this isne of the exciting features of the field. But this universe of pos-ibilities also imposes a discipline on those of us hoping to make
M. Freeman / Seminars in Cell & Developmental Biology 20 (2009) 231–239 235
F ordina
paatba
6
ukekbsEesmsAttbitpviraprmibmtwk
mdtsg
ocf
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rptiofdtisw–ribatph
6
rtadcaisimpp
ig. 4. Crystal structure of GlpG, an E. coli rhomboid. Colour scheme as in Fig. 1. Cobove.
rogress. We need to focus on the ‘big questions’, whether theyre fundamental or applied; structural, biochemical or biological;nd whether they are driven by the rhomboids themselves or byhe biology in which they participate. So even with expectation ofeing significantly wrong, it is perhaps still reasonable to speculatebout what the areas of focus will be, and should be.
.1. Mammalian rhomboids—the future
Notwithstanding the value of genetic model organisms forncovering mechanistic insight, a very obvious gap in our currentnowledge of rhomboid function is their function in mammals,specially humans. Apart from the mitochondrial rhomboids, wenow nothing about the role of any of the mammalian rhom-oids. One obvious question is whether they participate in EGFRignalling in mammals, as they do in Drosophila. Since aberrantGFR signalling is strongly associated with cancer and other dis-ases [60], this question has been near the top of the priority listince their role in flies was discovered. Like in Drosophila, mostammalian EGFR ligands are membrane tethered upon synthe-
is; but they are known to be processed by metalloproteases of theDAM family, such as TACE, so there is no obvious gap that needs
o be filled by a rhomboid [61]. Moreover, there have been reportshat specific EGFR ligands cannot be cleaved by specific rhom-oids [62,63]. On the other hand, the genetic evidence for ADAM
nvolvement does not rule out additional secretase enzymes, andhe conservation between Drosophila and mammalian signallingathways tends to be very high (moreover, a rhomboid also acti-ates EGFR in C. elegans [64]). There has also been one report thatndirectly hints at a possible relationship between a mammalianhomboid-like protein (albeit an inactive protease) and EGFR lig-nds [65]. That particular story does not provide a compellinghysiological link and, overall, the question of whether mammalianhomboids participate in EGFR signalling remains open. Two otherammalian substrates with no relationship to the EGFR have been
dentified—thrombomodulin and EphB3 [62,63]. These have noteen physiologically validated and therefore must be treated as noore than hypothesis generators, needing to be tested by func-
ional studies. All these questions, about EGFR ligands and others,ill presumably be addressed in the future by studies of mouse
nockouts.Our relative lack of knowledge of what rhomboids do in mam-
als illustrates a general important point that has emerged in theiscussion above, and will recur below: it is not sufficient to showhat a protein can be cleaved by a rhomboid; instead it is neces-ary to show that this cleavage is physiologically significant. Theold standard for this is genetics, where the consequence of loss-
6
P
ates from [55]. Left panel, side view; right panel, magnification of active site from
f-function of a rhomboid can be assessed, but even where thisannot be easily achieved, it remains crucial to recognise the needor functional data.
.2. Parasite rhomboids—the future
As outlined above, a particularly active area of rhomboidesearch has been their possible function in host cell invasion byarasites [reviewed in 2,5,48]. The possibility that they might beargets for intervention in the causative agents of some of the mostmportant human diseases is of course of great significance. With-ut wishing to labour the point, this is another context whereunctional studies are desperately needed to back up the currentata on potential substrates. With such a strong incentive, and withhe development of Toxoplasma as a genetically tractable organ-sm (as well as some genetics being possible in Plasmodium), it isafe to predict that progress will be rapid. Key questions includehether blocking rhomboid function can prevent parasite invasionor indeed other aspects of the life cycle; whether the parasite
homboids are druggable – not just whether the enzyme can benhibited but also whether inhibitors with drug-like properties cane found and whether they can access the site of enzyme action;nd whether specificity and redundancy issues might confound aherapeutic strategy. Of course, these questions will arise for anyotential medical use of rhomboid inhibitors, and the potentialurdles may well be overcome.
.3. Mitochondrial rhomboids—the future
The study of rhomboids in mitochondria has been anotherapidly moving field in the last few years [reviewed in 66–68]. Inhis case, the genetic approach has been taken, and it is a caution-ry tale about not assuming that genetics is a panacea for revealingetailed molecular understanding. As described above, the mito-hondrial roles of rhomboids are controversial and, although therere themes that run throughout all the work, notably their functionn the morphology and dynamics of mitochondrial membranes, thepecific conclusions remain disputed. As with parasite rhomboids,t seems likely that significant future progress will be driven by
edical potential. The clinical significance of mitochondrial mor-hology, the intrinsic apoptotic response, and the Pink/Omi/Parkinathway is obvious.
.4. Bacterial rhomboids—the future
The discovery that rhomboids are needed for quorum sensing inrovidencia [26] is exciting and provides a molecular explanation
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36 M. Freeman / Seminars in Cell & De
or some fascinating genetics, but it does not explain the function ofost bacterial rhomboids. Providencia is one of only a small num-
er of bacteria in which TatA has a rhomboid-cleaved N-terminalxtension [29]. In most species, TatA exists in a form that is equiv-lent to the truncated and active form in Providencia. This leads tohe following key questions: (1) In those minority of bacteria withrovidencia-like extended TatA genes, do rhomboids always cleavend activate the translocase? (2) What is the logic of this activationtep? (3) In the majority of bacteria with the short form of TatA,hat do rhomboids do? They have been highly conserved but weave no insight yet into their functions beyond the probably specialase of Providencia.
.5. Plant rhomboids—the future
Plant genomes hold the current record for the number ofhomboids: 13 in Arabidopsis and 12 in rice [24]. They exist inhloroplasts as well as mitochondria but, as in animal cells, most areredicted to reside in the secretory pathway [24,69]. Nothing is yetnown about their function so we cannot guess why the family hasecome expanded. Since Arabidopsis is a powerful genetic model70], and most genes have already been mutated, it is a good bet thatt will not be long before rhomboid phenotypes start to be reportednd the biology of intramembrane proteolysis in plants starts to benderstood. It has recently been reported that an Arabidopsis plas-id rhomboid may cleave a translocon component, Tic40, althoughhe biological meaning of this result is not yet clear [71].
. Systematic approaches
.1. Substrate identification
It would greatly accelerate our knowledge and understanding ofhomboids and the biological themes in which they participate, ifhere were a systematic approach to discovering their function. Thiss likely to be an area of progress in the near future. The questions straightforward: if the gene of interest is a protease, its functions likely to be defined by its substrate. Identifying substrates haseen a long-term bottleneck for all protease research but increas-
ngly sophisticated techniques are being developed [reviewed in2]. Three approaches that have great promise for systematic iden-ification of rhomboid substrates are bioinformatics, proteomicsnd biochemical techniques (see Table 1 for summary of currentlynown and proposed rhomboid substrates). The first depends onefining the determinants of rhomboid substrate specificity: whatakes one TMD a substrate and another uncleavable? The main
eterminant seems to be instability of the substrate transmem-rane alpha-helix [45,53,73]. This provides a basis for bioinformaticearching for potential substrates, although it has not been possi-le to make strong predictions with this rather imprecise structuralroperty. Nevertheless, this approach can identify likely candidates45,50,62,63], and will be refined if more specific determinants areound. The second promising approach relies on the recent explo-ion of proteomic techniques for identifying protease substratesreviewed in 72], and while these have not yet been applied tohomboids, there is every reason to believe they can be successful.one are yet able to provide all substrates with high confidence but,s with the bioinformatic approaches, if they provide candidates,his will already be of substantial value. The third approach is more
raditional and relies on biochemical techniques to detect bind-ng between enzyme and substrate. The enzyme may be wild-typer mutated to prevent cleavage; and the binding may be enhancedy, for example, chemical cross-linking. Although such an approachan work [44], it is also true that not all substrates will bind enzymesaamms
ental Biology 20 (2009) 231–239
ith sufficient affinity, and that membrane proteins are particularlyrone to binding artefacts.
.2. Rhomboid inhibitors
Another systematic approach to probing rhomboid function,ould be to use specific chemical inhibitors. These would be
xtremely valuable fundamental tools as well as potentially provid-ng leads for drug development, if and when validated therapeuticpportunities arise. To date, however, no specific inhibitors haveeen reported. This is probably more a consequence of the rela-ive youth of the field rather than of any particular difficulty withnhibiting rhomboids. Enzymes are good targets for inhibitors, andlthough located in the membrane, the rhomboid active site ispen to the aqueous environment [reviewed in 3, 4, 58]. Neverthe-ess, inhibitor development is a major undertaking, requiring eitherirected approaches based on designing molecules with appropri-te substrate-like features, or the development of high-throughputssays that can be used for screening large libraries of randomompounds, or possibly structurally based fragment screening. Inractice, however, it is probably safe to assume that first generationhomboid inhibitors will become available in the not-too-distantuture.
. Rhomboid regulation
Surprisingly little is yet understood about rhomboid regula-ion and this is another area ripe for progress. What has becomencreasingly clear is that many if not all rhomboids share a novelegulatory strategy that takes advantage of the membrane locationf both them and their substrates: the segregation of substrate fromnzyme [reviewed in 5]. This exploits the precise and highly reg-lated machinery of intracellular membrane protein trafficking toreatly limit the number of potential substrates to which a rhom-oid molecule has physical access. It is a regulatory logic that isnly fully available to intramembrane proteases (where enzymend substrate are both transmembrane proteins) and it appears toe a profound and extensive control mechanism for rhomboids. Itay partially explain why the activity of rhomboids appears less
egulated than other proteases, which have access to a much greaterniverse of possible substrates.
Despite this control by membrane trafficking, it is likely thathere are other ways in which rhomboids are regulated. For exam-le, it is notable that the cytoplasmic domains of rhomboids areften large, are not conserved between different family members,nd can contain recognisable motifs [74,75]. These domains, whicht least in some contexts are not essential for basic proteolyticctivity [19], may be a rich source of regulatory potential.
. Structural biology of rhomboids—the future
The recent solution of the crystal structures of bacterial rhom-oids has been of great value in understanding these enzymesreviewed in 3,4,76]. It has transformed our knowledge of theirnzymology from a position of inference and educated guess, intoclear picture of some of the basic mechanistic features. The
wo major goals in the structural biology of rhomboids are nowo solve the structure of eukaryotic rhomboids – many of which
nd, perhaps even more importantly, to solve the structure ofn enzyme/substrate complex. This latter will suggest answers toany of the remaining questions about the enzymatic mechanism,ost of which centre on how the substrate gets access to the active
ite, and what the exact mechanism of catalysis is.
M. Freeman / Seminars in Cell & Developmental Biology 20 (2009) 231–239 237
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ig. 5. Schematic view of an iRhom (left panel). The characteristic features are the mi-terminus. The right panel, reiterates Fig. 1 and serves as a comparison between iR
0. iRhoms
One final area to highlight in this brief survey of what the futuref rhomboid research may hold is the mysterious class of inactivehomboid-like proteins called the iRhoms (Fig. 5; see also Fig. 3).hese are highly conserved in all animal species, implying selectiveressure and therefore function, but are clearly not active proteases24]. One possibility is that they may have a regulatory function,ut nothing is known about them biochemically or genetically, sony current prediction is very speculative. One, human iRhom1 (orHBDF1), has been reported to interact with EGFR ligands and toarticipate in EGFR signalling in Drosophila when expressed inies [65]. This study was not able to assign a physiological roleo iRhom1, and its apparent ability to promote signalling in fliesespite a lack of enzymatic activity is also perplexing. It has alsoecently been reported that iRhom1 knockdown can affect growthf tumour cells [77]. These reports do little to clarify iRhom mech-nism but they add to the pressure to understand them better.
1. Conclusions and medical prospects
I have outlined the progress that has been made over the lasteven years of rhomboid research and suggested some predictionsbout future progress. The predictions have been categorised underistinct headings but this is misleading. In fact, research into rhom-oids is highly integrated: biological advances suggest mechanisticxplanations, structural insight explains biochemical phenomena,nd fundamental biology implies medical potential. In practice,herefore, these different territories will almost certainly advancen unison. And of course, the hallmark of science is its ability tourprise, so it is certain that some of the above predictions will berong, and even more certain that unexpected advances will beade.Finally, a thread that has run throughout this review is the poten-
ial for rhomboids to have medical significance. A priori, it would bextraordinary if a large family of proteases, conserved across evolu-ion and catalysing such a powerful control mechanism as release ofrotein domains from membranes, did not have medical relevance.
ndeed, there are already several intriguing indications to supporthis [reviewed in 2,5], ranging from cancer to parasites, and from
acterial infection to metabolic disorders. But it is important totress that none of these potential uses are yet fully validated, andlso to remember how long and tortuous is the path from targeto drug. Nevertheless, rhomboids seem like a good longterm bet asedical targets, and it may well be that by the time it comes to
[
atalytic residues, the large loop between TMD1 and 2, and the extended cytoplasmicand active rhomboids.
urvey the next seven years of progress, their clinical importances well established.
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