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9.B.104 The Rhomboid Protease Family

Structures of the prokaryotic homologue of rhomboid proteases reveal a core of six transmembrane helices, with the active-site residues residing in a hydrophilic cavity in the center of the membrane. The native environment of rhomboid protease is a lipid bilayer. Akiyama and Maegawa 2007 characterized sequence features in LY2 that allow efficient cleavage by the E. coli GlpG rhomboid protease (TC# 9/B/104.1.1) and identified two elements, a hydrophilic region encompassing the cleavage site and helix-destabilizing residues in the downstream hydrophobic region. GlpG prefers residues with a small side chain and a negative charge at the P1 and P1' sites, respectively.

Crystals of GlpG in a lipid environment were obtained at 1.7 Å resolution (Vinothkumar, 2011). The structure revealed well-ordered and partly ordered lipid molecules forming an annulus around the protein. Lipid molecules adapt to the surface features of the protein and arrange such that they match the hydrophobic thickness of GlpG. Wang et al. 2006 describe the 2.1 Å resolution crystal structure of the GlpG core domain. Residues previously shown to be involved in catalysis, including a Ser-His dyad and several water molecules, are found in the protein interior at a depth below the membrane surface. This active site is accessible by substrate through a large 'V-shaped' opening that faces laterally towards the lipid, but is blocked by a half-submerged loop structure. Thus, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. A gating mechanism for GlpG that involved movement of TMS 5 controls substrate access to its hydrophilic active site (Wang et al. 2006). It has been proposed that the channels through the rhomboid protease homologues, the derlins (see TC#s 3.A.16 and 3.A.26), some of which have lost their protease activities, serve as protein channels that transport ER lumen proteins into the cytoplasm for protease-mediated degradation (Neal et al. 2018), but see also Wu and Rapoport 2018 for another possibility involving Hrd1 as the channel.

The primary function of rhomboids is to cleave integral membrane proteins to release signalling molecules. These signals, when disrupted, can contribute to various diseases. The Ser-His catalytic dyad is buried within the membrane. The substrate entry gate is composed of helix 5 and loop 5 (Lazareno-Saez et al., 2011). Lazareno-Saez et al. (2011) compared the open and closed conformations of GlpG. Possibly loop 4 acts as an anchor for the substrate gate. Membrane immersion bestows rhomboid proteases with the ability to identify substrates primarily based on reading their intrinsic transmembrane dynamics (Moin and Urban 2012).

The ER-associated degradation (ERAD) pathway serves as an important cellular safeguard by directing incorrectly folded and unassembled proteins from the ER of eukaryotes to the proteasome. Fleig et al. (2012) showed that the evolutionarily conserved rhomboid family protein RHBDL4 is a ubiquitin-dependent ER-resident intramembrane protease that is upregulated upon ER stress. RHBDL4 cleaves single-spanning and polytopic membrane proteins with unstable transmembrane helices, leading to their degradation by the canonical ERAD machinery. RHBDL4 specifically binds the AAA -ATPase p97, suggesting that proteolytic processing and dislocation into the cytosol are functionally linked. The phylogenetic relationship between rhomboids and the ERAD factor derlin suggests that substrates for intramembrane proteolysis and protein dislocation are recruited by a shared mechanism.

The BAG6 complex is an upstream loading factor for tail-anchored membrane proteins entering the TRC40-dependent pathway for posttranslational delivery to the endoplasmic reticulum.  BAG6 also enhances proteasomal degradation of mislocalized proteins by selectively promoting their ubiquitination. BAG6-dependent ubiquitination of mislocalized proteins is reversible, and the glutamine-rich tetratricopeptide repeat-containing protein α, (SGTA) antagonizes this process. Promoting the deubiquitination of mislocalized proteins that are already covalently modified reverses the actions of BAG6, inhibiting its capacity to promote substrate-specific degradation (Leznicki and High 2012). 

From proteases that cleave peptide bonds in the plane of the membrane, rhomboids have evolved into a heterogeneous superfamily with a wide range of different mechanistic properties (Bergbold and Lemberg 2013). In mammals, 14 family members have been annotated based on a shared conserved membrane-integral rhomboid core domain.  Homologues include intramembrane serine proteases and diverse proteolytically inactive proteins. While the function of rhomboid proteases is the proteolytic release of membrane-tethered factors, rhomboid pseudoproteases, including iRhoms and derlins, interact with their clients without cleaving them. It has become evident that specific recognition of membrane protein substrates by the rhomboid fold reflects a spectrum of cellular functions ranging from growth factor activation and trafficking control to membrane protein degradation (Bergbold and Lemberg 2013).

With their catalytic rate limited by diffusion, rhomboids fold to distort surrounding lipids, overcome the viscosity limit of the membrane, and accelerate its search for substrates. Thus, evolution can boost the diffusion of enzymes in the crowded and viscous environment of the membrane. Some rhomboid proteins that lost their catalytic residues still play important roles in membrane biology. Derlins, for example, facilitate ER associated degradation of damaged proteins to safeguard the health of a cell.Possibly derlins disrupt local lipid interactions to help the Hrd1 channel (TC# 3.A.16) translocate damaged proteins across the ER membrane (Kreutzberger et al. 2019).

References associated with 9.B.104 family:

Akiyama, Y. and S. Maegawa. (2007). Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease. Mol. Microbiol. 64: 1028-1037. 17501925
Bergbold, N. and M.K. Lemberg. (2013). Emerging role of rhomboid family proteins in mammalian biology and disease. Biochim. Biophys. Acta. 1828: 2840-2848. 23562403
Fleig, L., N. Bergbold, P. Sahasrabudhe, B. Geiger, L. Kaltak, and M.K. Lemberg. (2012). Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of membrane proteins. Mol. Cell 47: 558-569. 22795130
Kreutzberger, A.J.B., M. Ji, J. Aaron, L. Mihaljević, and S. Urban. (2019). Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion. Science 363:. 30705155
Lazareno-Saez, C., C.L. Brooks, and M.J. Lemieux. (2011). Structural comparison of substrate entry gate for rhomboid intramembrane peptidases. Biochem. Cell Biol. 89: 216-223. 21455272
Lemieux, M.J., S.J. Fischer, M.M. Cherney, K.S. Bateman, and M.N. James. (2007). The crystal structure of the rhomboid peptidase from Haemophilus influenzae provides insight into intramembrane proteolysis. Proc. Natl. Acad. Sci. USA 104: 750-754. 17210913
Leznicki, P. and S. High. (2012). SGTA antagonizes BAG6-mediated protein triage. Proc. Natl. Acad. Sci. USA 109: 19214-19219. 23129660
Liu, G., S.E. Beaton, A.G. Grieve, R. Evans, M. Rogers, K. Strisovsky, F.A. Armstrong, M. Freeman, R.M. Exley, and C.M. Tang. (2020). Bacterial rhomboid proteases mediate quality control of orphan membrane proteins. EMBO. J. 39: e102922. 32337752
Meissner C., Lorenz H., Hehn B. and Lemberg MK. (201). Intramembrane protease PARL defines a negative regulator of PINK1- and PARK2/Parkin-dependent mitophagy. Autophagy. 11(9):1484-98. 26101826
Moin, S.M. and S. Urban. (2012). Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics. Elife 1: e00173. 23150798
Nakagawa, T., A. Guichard, C.P. Castro, Y. Xiao, M. Rizen, H.Z. Zhang, D. Hu, A. Bang, J. Helms, E. Bier, and R. Derynck. (2005). Characterization of a human rhomboid homolog, p100hRho/RHBDF1, which interacts with TGF-alpha family ligands. Dev Dyn 233: 1315-1331. 15965977
Neal, S., P.A. Jaeger, S.H. Duttke, C. Benner, C. K Glass, T. Ideker, and R.Y. Hampton. (2018). The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Membrane Proteins. Mol. Cell 69: 306-320.e4. 29351849
Sanders, C.R. and J.M. Hutchison. (2018). Membrane properties that shape the evolution of membrane enzymes. Curr. Opin. Struct. Biol. 51: 80-91. [Epub: Ahead of Print] 29597094
Vinothkumar, K.R. (2011). Structure of rhomboid protease in a lipid environment. J. Mol. Biol. 407: 232-247. 21256137
Wang, Y., Y. Zhang, and Y. Ha. (2006). Crystal structure of a rhomboid family intramembrane protease. Nature 444: 179-180. 17051161
Wu, X. and T.A. Rapoport. (2018). Mechanistic insights into ER-associated protein degradation. Curr. Opin. Cell Biol. 53: 22-28. [Epub: Ahead of Print] 29719269
Wu, Z., N. Yan, L. Feng, A. Oberstein, H. Yan, R.P. Baker, L. Gu, P.D. Jeffrey, S. Urban, and Y. Shi. (2006). Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry. Nat Struct Mol Biol 13: 1084-1091. 17099694