Rhomboid protease

Rhomboids are intramembrane serine proteases. The other types of intramembrane protease are aspartyl- and metallo-proteases, respectively. The presenilins and signal peptide peptidase-like family, which are intramembrane aspartyl proteases, cleave substrates that include the Notch receptor and the amyloid precursor protein, which is implicated in Alzheimer's disease. The site-2 protease family, which are intramembrane metalloproteases, regulate among other things cholesterol biosynthesis and stress responses in bacteria. The different intramembrane protease families are evolutionarily and mechanistically unrelated, but there are clear common functional themes that link them. Rhomboids are perhaps the best characterised class. == History ==

Rhomboids were first named after a mutation in the fruit fly Drosophila, discovered in a famous genetic screen that led to a Nobel Prize for Christiane Nüsslein-Volhard and Eric Wieschaus. In that screen they found a number of mutants with similar phenotypes: 'pointy' embryonic head skeletons. genetic analysis later proved that this group of genes were members of the epidermal growth factor (EGF) receptor signalling pathway, The molecular function of rhomboid took a bit longer to unravel but a combination of genetics and molecular techniques led to the discovery that Drosophila rhomboid and other members of the family were the first known intramembrane serine proteases. == Function ==

Rhomboids were first discovered as proteases that regulate EGF receptor signalling in Drosophila. By releasing the extracellular domain of the growth factor Spitz, from its transmembrane precursor, rhomboid triggers signalling. • Later, Drosophilas' Rhomboid-1 was shown to regulate sleep, through a new function of an already-discovered mechanism. the cleavage of the anticoagulant protein thrombomodulin and wound healing. • All eukaryotes have a mitochondrial rhomboid. In yeast this has been shown to control mitochondrial function and morphology by regulating membrane fusion via the cleavage of a dynamin-like GTPase called Mgm1p, the orthologue of human OPA1. In Drosophila, the mitochondrial rhomboid (Rhomboid-7) Drosophila Opa1 and Rhomboid-7 appear to have the same relationship as in yeast. is implicated in cell death and there is increasing evidence of an important role in Parkinson's disease; • Apicomplexan parasites (including Plasmodium, the agent that causes malaria, and Toxoplasma) rhomboids are used to reposition between attachment to a target cell and entry, and most microneme-produced adhesins are released from the microneme by rhomboids. Rhomboids have also been implicated in the pathogenicity of other parasites. In Toxoplasma specifically, some serpins inhibit rhomboids. • Rhomboids control EGF receptor signaling in Caenorhabditis elegans as in Drosophila. == Structure ==

Rhomboids were the first intramembrane proteases for which a high resolution crystal structure was solved. These structures confirmed predictions that rhomboids have a core of six transmembrane domains, and that the catalytic site depends on a serine and histidine catalytic dyad. The structures also explained how a proteolytic reaction, which requires water molecules, can occur in the hydrophobic environment of a lipid bilayer: one of the central mysteries of intramembrane proteases. The active site of rhomboid protease is in a hydrophilic indentation, in principle accessible to water from the bulk solution. The active site of rhomboid protease is protected laterally from the lipid bilayer by its six constituent transmembrane helices, suggesting that substrate access to rhomboid active site is regulated. One area of uncertainty has been the route of substrate access. Substrates were initially proposed to enter between transmembrane segments (TMSs) 1 and 3, This notion is also supported by the fact that mutations in TMS 5 have only a marginal effect on the thermodynamic stability of rhomboid, unlike other regions of the molecule. Very recently, the first ever co-crystal structure of an intramembrane protease confirms and extends this substrate access model and provides implications for the mechanism of other rhomboid-superfamily proteins. E. coli's GlpG is unusual for its low enzyme/substrate binding affinity. and propose that the surface of TMSs 2 and 5 rather serves as an "intramembrane exosite" mediating the recognition of substrate TMS. The rhomboid ortholog in D. suzukii is Dsuz\DS10_00004507. == Enzymatic specificity ==

Rhomboids do not cleave all transmembrane domains. In fact, they are highly specific, with a limited number of substrates. Most natural Rhomboid substrates known so far are type 1 single transmembrane domain proteins, with their amino termini in the luminal/extracellular compartment. However, recent studies suggested that type 2 membrane protein (i.e. with opposite topology: the amino terminus is cytoplasmic), or even multipass membrane proteins could act as rhomboid substrates. The specificity of rhomboids underlies their ability to control functions in a wide range of biological processes and, in turn, understanding what makes a particular transmembrane domain into a rhomboid substrate can shed light on rhomboid function in different contexts. Initial work indicated that rhomboids recognise instability of the transmembrane alpha-helix at the site of cleavage as the main substrate determinant. More recently, it has been found that rhomboid substrates are defined by two separable elements: the transmembrane domain and a primary sequence motif in or immediately adjacent to it. The principles of substrate TMS recognition by rhomboid remain poorly understood, but numerous lines of evidence indicate that rhomboids (and perhaps also other intramembrane proteases) somehow recognise the structural flexibility or dynamics of transmembrane domain of their substrates. Full appreciation of the biophysical and structural principles involved will require structural characterisation of the complex of rhomboid with the full transmembrane substrate. As a first step towards this goal, a recent co-crystal structure of the enzyme in complex with a substrate-derived peptide containing mechanism-based inhibitor explains the observed recognition motif sequence preferences in rhomboid substrates structurally, and provides a significant advance in the current understanding of rhomboid specificity and mechanism of rhomboid-family proteins. == Medical significance ==

The diversity of biological functions already known to depend on rhomboids is reflected in evidence that rhomboids play a role in a variety of diseases including cancer, parasite infection, == The rhomboid-like family ==

Rhomboid proteases appear to be conserved in all eukaryotes and the vast majority of prokaryotes. Bioinformatic analysis highlights that some members of the rhomboid family lack the amino acid residues essential for proteolysis, implying that they cannot cleave substrates. These 'pseudoproteases' include a subfamily that have been named the iRhoms (also known as RHBDF1 and RHBDF2). iRhoms can promote the ER associated degradation (ERAD) of EGF receptor ligands in Drosophila, thus providing a mechanism for regulating EGF receptor activity in the brain. This implies that the fundamental cellular quality control mechanism is exploited by multicellular organisms to regulate signalling between cells. In mice, iRhoms are key trafficking chaperones required for the ER export of ADAM17/TACE and its maturation. iRhoms are thus required for the TNF-alpha and EGF receptor signalling, making them medically highly attractive. Phylogenetic analysis indicates that rhomboids are in fact members of a larger rhomboid-like superfamily or clan, which includes the derlin proteins, also involved in ERAD. Kinetoplastids have an unusually small rhomboid family repertoire, in Trypanosoma brucei XP 001561764 and XP 001561544, and in T. cruzi XP 805971, XP 802860, and XP 821055. Various rhomboid family proteins are vital to Toxoplasma gondii virulence and motility, including TgMIC2, TgMIC6, various AMA1 variants including TgAMA1, TgROM1, TgROM4, and TgROM5. Trypanosome mitochondria have TimRhom I and TimRhom II (two rhomboid family members with proteolytic function deactivated) in their presequence translocases. The difficulty in finding greater similarity either to eukaryotic or bacterial relatives may mean these came as part of the original mitochondrial progenitor. Rhomboid-relatives may be membrane transport proteins in the ERAD and SELMA systems. == iRhoms ==

iRhoms are rhomboid-like proteins, but are not proteases. As with rhomboids they were first discovered in Drosophilae. To the contrary of rhomboids, however, iRhoms inhibit EGFr signaling. Knockout mice for iRhom2 have severe immune compromise. == References ==