9.B.1 The Integral Membrane CAAX Protease (CAAX Protease) Family
Posttranslational lipidation modulates the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues, and in some cases, by additional proteolytic cuts. Pryor et al. (2013) determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. They hypothesized that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.
Isoprenoid groups are conjugated to proteins via cysteine residues of CaaX acceptor sequences in which the cysteine attachment site is followed by two aliphatic amino acid residues and one unspecified residue at the protein C-terminus. Isoprenylation is generally accompanied by two subsequent processing steps, proteolytic cleavage of the aaX residues and carboxymethylation of the newly exposed carbonyl group of the modified cysteine residue . Some isoprenylated proteins also undergo additional proteolytic processing, including an additional cleavage by the same protease that initially removes the aaX residues. At least two classes of enzymes are responsible for the cleavage of isoprenylated proteins and peptides. One of these is the ras-converting enzyme (Rce) family of Type II prenyl proteases, responsible for proteolytic processing of signal-transducing proteins including Ras and the Gγ subunits of heterotrimeric G protein complexes . The other is the Ste24p family of Type I prenyl proteases, first identified in yeast based on its role in maturation of the mating pheromone a-factor. Extensive characterization of the role of Ste24p in a-factor processing has been conducted in the yeast system. The proteolytic activity of Ste24p requires zinc, consistent with the fact that Ste24p contains the zinc metalloprotease signature motif HEXXH.
A human ortholog of Ste24p, ZMPSTE24 (Zinc MetalloProtease STE24), can complement the full function of yeast Ste24p. A substrate for ZMPSTE24 is prelamin A, the precursor to the nuclear intermediate filament protein lamin A. Lamins provide mechanical stability to the nuclear envelope, function as scaffolds for localization of other proteins and for cytoskeletal attachment, regulate chromatin, and are implicated in transcription and DNA repair and replication. Mutations in either ZMPSTE24 or the processing site of prelamin A are associated with a spectrum of premature-aging diseases referred to as progeria. The severity of different forms of progeria is reported to be correlated with extent of loss of ZMPSTE24 activity. Ste24p is localized to the endoplasmic reticulum membrane. Its proteolytic activity requires zinc, consistent with the fact that Ste24p contains the zinc metalloprotease signature motif HEXXH.
Ste24 enzymes, a family of eukaryotic integral membrane proteins, target prenylated substrates, but also some nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique 'alpha-barrel' structure consisting of seven transmembrane (TM) alpha-helices encircling a large intramembranous cavity (~14 000 A(3) ). The catalytic zinc, coordinated via a HExxH...E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been explored. Goblirsch et al. 2019 developed a model of Ste24 that provides a conceptual framework for this enzyme family. The model consists of an interfacial, zinc-containing 'ZMP Core' module surrounded by a 'ZMP Accessory' module, both capped by a TM helical 'alpha-barrel' module. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 conserved residues, apportioned unequally among the ZMP Core, ZMP Accessory, and alpha-barrel modules. This tripartite architecture representation of Ste24 provides a unified image of this enzyme family (Goblirsch et al. 2019). Yeast Ste24 has TC# 9.B.1.1.3 while human Ste24 has TC# 9.B.1.1.3. See these TC#s and Goblirsch and Wiener 2020 for more details.
The CAAX prenyl protease CAAX PP, ZMPSTE24 (FACE1, STE24). The 3-d structure (2.0 Å resolution) revealed a seven TMS α-helical barrel structure surrounding a large water-filled intramembrane chamber capped by a zinc metalloprotease domain with the catlytic site facing into the chamber. Mutations cause laminopathies (Quigley et al. 2013). Results showed: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C-terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations, and (5) water molecules associated with the surface of the internal cavity (Clark et al. 2017). A tripartite architecture of the human zinc metalloprotease Ste24 has been proposed (Goblirsch et al. 2019; see family description). ZMPSTE24 protects cells from SARS-CoV-2 spike-mediated pseudovirus infection and syncytia formation (Shilagardi et al. 2022).
CAAX PP of Homo sapiens (O75844)
The CAAX prenyl protease CAAX PP
CAAX PP of Arabidopsis thaliana (Q8RX88)
The CAAX integral membrane zinc prenyl metaloprotease, CAAX PP or Ste24p of 453 aas and 7 TMSs, found in every kingdom of eukaryotes. It's 3-D structure has been solved (Goblirsch and Wiener 2020). It is a factor responsible for processing the yeast mating a-factor pheromone. In animals, Ste24 processes prelamin, a component of the nuclear lamina; mutations in the human ortholog of Ste24 diminish its activity, giving rise to genetic diseases of accelerated aging (progerias). Additionally, lipodystrophy, acquired from the standard highly active antiretroviral therapy (HAART) used to treat AIDS patients, likely results from off-target interactions of HIV (aspartyl) protease inhibitor drugs with Ste24. Ste24 possesses a novel "α-barrel" structure, consisting of a ring of seven transmembrane α-helices enclosing a large (> 12,000 Å3) interior volume that contains the active-site and substrate-binding region; this "membrane-interior reaction chamber" is unprecedented in integral membrane protein structures. Additionally, the surface of the membrane-interior reaction chamber possesses a strikingly large negative electrostatic surface potential. Ste24p may be a key factor in several endoplasmic reticulum (ER) processes, including the unfolded protein response, a cellular stress response of the ER, and removal of misfolded proteins from the translocon. Ste24p is thus a "translocon unclogger". Goblirsch and Wiener 2020
have reviewed the sturcture and functions of Ste24. STE24p cleaves farnesylated prelamin A, the precursor of the nuclear scaffold protein lamin A (Wood et al. 2020).
CAAX PP of Sacharomyces cerevisiae (P47154)
Ste24 endopeptidase, CAAX PP of 407 aas and 7 TMSs. The crystal structure has been solved showing that the Ste24p core structure is a ring of seven TMSs enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is large enough to hold hundreds of water molecules, and is accessible to the external milieu by means of gaps between splayed transmembrane helices (Sanders and Hutchison 2018). Possibly cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection (Pryor et al. 2013). The active site is just under the interfacial lid of the barrel, with substrate entry and product exit through fenestrations located near the upper end of the barrel, just under the water-bilayer interface (Sanders and Hutchison 2018).
endopeptidase of Helicobacter pylori (J0MMU8)
Protease HtpX homologue of 295 aas
HtpX homologue of Leptospira interrogans
protease able to endoproteolytically degrade overproduced SecY and other membrane protein. Probably plays a role in the quality control of
integral membrane proteins (Arolas et al. 2014).
HtpX of E. coli
Peptidase M48 of 300 aas
Peptidase M48 Ste24p of Candidatus Saccharibacteria bacterium
Protease HtpX of 841 aas and 11 TMSs with an HtpX domain.
HtpX of Lokiarchaeum sp. GC14_75
M48 family metalloprotease of 405 aas and 4 TMSs in a 2 + 2 TMS arrangement.
Protease of Lachnoclostridium sp. An298
CAAX prenyl protease 2, RCE1 (Ashby, 1998). A member of the Abi Superfamily.
RCE1 of Saccharomyces cerevisiae (Q03530)
CAAX prenyl protease 2 of 304 aas
CAAX protease of Fusarum oxysporum
CAAX prenyl protease 2 of 277 aas
CAAX protease of Paramecium tetraurelia
CAAX prenyl protease 2
CAAX protease 2 of Trypanosoma cruzi
CAAX prenyl protease 2 of 311 aas
CAAX protease 2 of Arabidopsis thaliana
CAAX prenyl protease 2 of 237 aas
CAAX protease 2 of Entamoeba histolytica