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Site-2 Protease Substrate Specificity and Coupling in Trans by a PDZ

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Site-2 Protease Substrate Specificity and Coupling in Trans by a PDZ Site-2 protease substrate specificity and coupling in trans by a PDZ-substrate adapter protein Jessica S. Schneidera,b, Shilpa P. Reddya, Hock Y. Ea, Henry W. Evansc, and Michael S. Glickmana,b,c,1 aImmunology Program, Sloan-Kettering Institute, New York, NY 10021; bProgram in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065; and cSummer Undergraduate Research Program, Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, New York NY 10065 Edited by Michael S. Wolfe, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, and accepted by the Editorial Board October 14, 2013 (received for review April 1, 2013) Site-2 proteases (S2Ps) are intramembrane metalloproteases that Methanocaldococcus jannaschii S2P will cleave the Caeno- cleave transmembrane substrates in all domains of life. Many S2Ps, rhabditis elegans transmembrane protein Ced-9 (18). These results including human S2P and Mycobacterium tuberculosis Rip1, have strongly suggest that the substrate specificity of S2Ps is not de- multiple substrates in vivo, which are often transcriptional regu- termined by the primary sequence of the cleaved transmembrane lators. However, S2Ps will also cleave transmembrane sequences segment and that additional determinants of protease specificity of nonsubstrate proteins, suggesting additional specificity deter- exist in vivo (19). minants. Many S2Ps also contain a PDZ domain, the function of Another poorly understood feature of S2Ps is the mechanism which is poorly understood. Here, we identify an M. tuberculosis by which S1P and S2P cleavage events are coupled. Many S2Ps, protein, PDZ-interacting protease regulator 1 (Ppr1), which bridges including E. coli RseP, Salmonella enterica RseP, Human S2P, between the Rip1 PDZ domain and anti-sigma factor M (Anti-SigM), and M. tuberculosis Rip1, contain PDZ domains. Substantial a Rip1 substrate, but not Anti-SigK or Anti-SigL, also Rip1 substrates. evidence indicates that these PDZ domains integrate S1 and site-2 In vivo analyses of Ppr1 function indicate that it prevents nonspe- cleavage by preventing the S2P from cleaving before the S1P. In cific activation of the Rip1 pathway while coupling Rip1 cleavage E. coli, RseP lacking its PDZ domain can complement the es- of Anti-SigM, but not Anti-SigL, to site-1 proteolysis. Our results sential function of RseP but decouples RseP activity from S1 support a model of S2P substrate specificity in which a substrate- – proteolysis (20 23). However, the mechanism by which the PDZ MICROBIOLOGY specific adapter protein tethers the S2P to its substrate while hold- domain modulates S2P activity is not known. Despite the usual ing the protease inactive through its PDZ domain. function of PDZ domains in protein–protein interactions, and particularly in nucleating signaling complexes (24), no S2P PDZ intramembrane proteolysis | signal transduction | protein interactors have been identified. Tuberculosis | Sigma factor In M. tuberculosis, we have previously identified the Rip1 S2P as a major virulence determinant with three anti-sigma factor egulated intramembrane proteolysis (RIP) is a widely dis- substrates (3, 4). Here, we identify a Rip1-PDZ interacting pro- Rtributed mechanism of signal transduction across membranes tein, PDZ-interacting protease regulator (Ppr)1, which holds Rip1 in which specialized intramembrane cleaving proteases (iCLIPs) inactive while tethering Rip1 to one of its substrates, RsmA. After cleave the transmembrane segments of substrate proteins (1, 2). S1 cleavage, Ppr1 specifically facilitates Rip1 cleavage of RsmA The site-2 (S2) proteases (S2Ps) are one class of iCLIP that is but not other Rip1 substrates. We propose a model of S2P substrate widely distributed from bacteria to human cells and participate specificity and coupling in which the S2P PDZ domain nucleates in such diverse pathways as lipid biosynthesis (1, 3, 4), sporula- distinct signaling complexes through substrate-specific adapter tion (5), membrane stress (6, 7), alginate production (8), and polar proteins that both hold the S2P inactive and tether it to its substrate. morphogenesis (9). In bacteria, PDZ domain containing S2Ps often control the release of extracytoplasmic function sigma Results factors from the membrane through proteolysis of their cognate Membrane Topology of Rip1 and Its Three Anti-Sigma Factor Substrates. membrane-bound anti-sigma factors (10, 11). S2Ps are so named Many S2Ps, including E. coli RseP, are predicted to have four because the intramembrane cleavage event follows cleavage by transmembrane (TM) domains with a PDZ domain between a site-1 (S1) protease (S1P). The S1P first cleaves the periplasmic thesecondandthirdTMs(Fig.1A and ref. 25), although the domain of the anti-sigma factor in response to an extracellular reported structure of M. jannaschii S2P has six TM segments signal. Only after S1 cleavage does the S2P cleave near the cy- toplasmic side of the transmembrane domain of the anti-sigma Significance factor, liberating the anti-sigma/sigma factor complex from the membrane into the cytoplasm where the sigma factor can asso- fi Site-2 proteases are ubiquitously distributed, and yet their ciate with RNA polymerase. A speci c extracytoplasmic stimulus regulation and substrate recognition remain poorly understood. can thus be coupled to a transcriptional response through a co- Here, we describe a model for site-2 protease regulation in which ordinated proteolytic cascade. regulated intramembrane protease 1 (Rip1), a site-2 protease One of the major unanswered questions in S2P-mediated RIP Mycobacterium tuberculosis fi crucial for virulence in ,istethered is how these proteases achieve speci city in signaling. In many to one of its substrates by an adapter protein, PDZ-interacting cases, S2Ps have multiple substrates, including human S2P, protease regulator (Ppr) 1. which cleaves SREBPs, ATF6, and CREB-H (12, 13); Bacillus subtilis RasP, which cleaves both RsiW as well as FtsL (14, Author contributions: J.S.S. and M.S.G. designed research; J.S.S., S.P.R., H.Y.E, and H.W.E. per- 15); and Mycobacterium tuberculosis regulated intramembrane formed research; J.S.S., S.P.R., and M.S.G. analyzed data; and J.S.S. and M.S.G. wrote the paper. protease 1 (Rip1), which cleaves three anti-sigma factors, The authors declare no conflict of interest. Anti-SigK (RskA), Anti-SigL (RslA), and Anti-SigM (RsmA) This article is a PNAS Direct Submission.M.S.W.isaguesteditorinvitedbythe (3), as well as PBP3 (16). However, there is substantial evi- Editorial Board. dence that S2Ps will cleave transmembrane segments non- 1To whom correspondence should be addressed. E-mail: [email protected]. fi speci callybothinvitroandinvivo.RsePwillcleavetheLacY This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. transmembrane domain both in vitro and in vivo (17), and 1073/pnas.1305934110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1305934110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 domain by performing a yeast two-hybrid screen of the Rip1- PDZ domain against a genome-wide M. tuberculosis protein library (28). Candidate Rip1-PDZ interactors were counterscreened against Ku, a cytoplasmic protein involved in the nonhomologous end-joining pathway of DNA repair (29, 30), in addition to the PDZ domains from PepA, PepD, and HtrA, three PDZ-con- taining proteases in the M. tuberculosis proteome (31). Of 7 × 104 candidate fusions screened, we identified four specific Rip1 PDZ interactors after counter screening. Two of these activation do- main (AD) fusions contained fusions to the uncharacterized proteins encoded by rv3333c and rv3439c, which we named Ppr1 and Ppr2, respectively. Both Ppr1 and Ppr2 interact with the Fig. 1. Membrane topology of Rip1 and its three anti-sigma factor sub- Rip1 PDZ, but not with Ku or the PDZ domains from PepA, strates. (A) Schematic representation of M. tuberculosis Rip1 illustrating the PepD, or HtrA (Fig. 2A). Ppr1 and Ppr2 are both proline- and four predicted transmembrane domains (TM1 through -4, in gray) and the alanine-rich proteins that share significant sequence similarity in PDZ domain between TM2 and 3 (in green). The vertical lines marked with their C-terminal regions (Fig. S1B). The M. tuberculosis yeast numbers 1–5 mark the fusion points to either β-galactosidase or alkaline two-hybrid library was constructed from partial digests of geno- phosphatase as follows: 1, Y89; 2, V145; 3, W218; 4, G361; and 5, L383. (B) mic DNA and therefore the AD-Ppr1 fusions identified in the Membrane topology of Rip1. lacZ (Upper) and phoA (Lower) fusions to Rip1 screen encoded truncated versions of the Ppr1 protein, all of at the positions indicated in A were expressed in M. smegmatis along with a vector control (vect) or a positive control (+)forlacZ (consisting of unfused which included the C terminus of Ppr1 beginning at amino acid lacZ)orphoA [an antigen85-phoA fusion (26)]. The upper image shows cells 131. The Ppr1 protein contains a predicted transmembrane do- bearing each β-galactosidase fusion cultured on media containing X-gal and main at its N terminus (amino acids 6–25) but has no identifiable the bottom panel shows each PhoA fusion cultured on media containing enzymatic function or conserved domains. BLAST searching of BCIP. (C) Membrane topology of Rip1 substrates. β-Gal or PhoA was fused to the M. tuberculosis Ppr1 protein sequence identified Ppr1 in the C terminus of Anti-SigK (RskA), Anti-SigL (RslA), or Anti-SigM (RsmA), pathogenic slow-growing mycobacteria, including Mycobacterium and the plasmids encoding these fusions were transformed into M. smegmatis bovis, Mycobacterium africanum, and Mycobacterium marinum, and cultured on media containing X-gal (Upper)orBCIP(Lower). The positive which contains three Ppr1 paralogs (Fig. S1A). We did not controls for X-gal and BCIP are the same as used in B.(D) Experimentally de- termined topology of Rip1 and its substrates. identify Ppr1 orthologs in M. smegmatis or in any other bacterial taxa outside of pathogenic mycobacteria.
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