Site-2 protease substrate specificity and coupling in trans by a PDZ-substrate adapter protein.

Site-2 proteases (S2Ps) are intramembrane metalloproteases that cleave transmembrane substrates in all domains of life. Many S2Ps, including human S2P and Mycobacterium tuberculosis Rip1, have multiple substrates in vivo, which are often transcriptional regulators. However, S2Ps will also cleave transmembrane sequences of nonsubstrate proteins, suggesting additional specificity determinants. Many S2Ps also contain a PDZ domain, the function of which is poorly understood. Here, we identify an M. tuberculosis protein, PDZ-interacting protease regulator 1 (Ppr1), which bridges between the Rip1 PDZ domain and anti-sigma factor M (Anti-SigM), a Rip1 substrate, but not Anti-SigK or Anti-SigL, also Rip1 substrates. In vivo analyses of Ppr1 function indicate that it prevents nonspecific activation of the Rip1 pathway while coupling Rip1 cleavage of Anti-SigM, but not Anti-SigL, to site-1 proteolysis. Our results support a model of S2P substrate specificity in which a substrate-specific adapter protein tethers the S2P to its substrate while holding the protease inactive through its PDZ domain.

The Rip1 protease of Mycobacterium tuberculosis controls the SigD regulon.

Regulated intramembrane proteolysis of membrane-embedded substrates by site-2 proteases (S2Ps) is a widespread mechanism of transmembrane signal transduction in bacteria and bacterial pathogens. We previously demonstrated that the Mycobacterium tuberculosis S2P Rip1 is required for full virulence in the mouse model of infection. Rip1 controls transcription in part through proteolysis of three transmembrane anti-sigma factors, anti-SigK, -L, and -M, but there are also Rip1-dependent, SigKLM-independent pathways. To determine the contribution of the sigma factors K, L, and M to the Δrip1 attenuation phenotype, we constructed an M. tuberculosis ΔsigKΔ sigL ΔsigM mutant and found that this strain fails to recapitulate the marked attenuation of Δrip1 in mice. In a search for additional pathways controlled by Rip1, we demonstrated that the SigD regulon is positively regulated by the Rip1 pathway. Rip1 cleavage of transmembrane anti-SigD is required for expression of SigD target genes. In the absence of Rip1, proteolytic maturation of RsdA is impaired. These findings identify RsdA/SigD as a fourth arm of the branched pathway controlled by Rip1 in M. tuberculosis.

Function of site-2 proteases in bacteria and bacterial pathogens.

Site-2 proteases (S2Ps) are a class of intramembrane metalloproteases named after the founding member of this protein family, human S2P, which control cholesterol and fatty acid biosynthesis by cleaving Sterol Regulatory Element Binding Proteins which control cholesterol and fatty acid biosynthesis. S2Ps are widely distributed in bacteria and participate in diverse pathways that control such diverse functions as membrane integrity, sporulation, lipid biosynthesis, pheromone production, virulence, and others. The most common signaling mechanism mediated by S2Ps is the coupled degradation of transmembrane anti-Sigma factors to activate ECF Sigma factor regulons. However, additional signaling mechanisms continue to emerge as more prokaryotic S2Ps are characterized, including direct proteolysis of membrane embedded transcription factors and proteolysis of non-transcriptional membrane proteins or membrane protein remnants. In this review we seek to comprehensively review the functions of S2Ps in bacteria and bacterial pathogens and attempt to organize these proteases into conceptual groups that will spur further study. This article is part of a Special Issue entitled: Intramembrane Proteases.

M. tuberculosis intramembrane protease Rip1 controls transcription through three anti-sigma factor substrates.

Regulated intramembrane proteolysis (RIP) is a mechanism of transmembrane signal transduction that functions through intramembrane proteolysis of substrates. We previously reported that the RIP metalloprotease Rv2869c (Rip1) is a determinant of Mycobacterium tuberculosis (Mtb) cell envelope composition and virulence, but the substrates of Rip1 were undefined. Here we show that Rip1 cleaves three transmembrane anti-sigma factors: anti-SigK, anti-SigL and anti-SigM, negative regulators of Sigma K, L and M. We show that transcriptional activation of katG in response to phenanthroline requires activation of SigK and SigL by Rip1 cleavage of anti-SigK and anti-SigL. We also demonstrate a Rip1-dependent pathway that activates the genes for the mycolic acid biosynthetic enzyme KasA and the resuscitation promoting factor RpfC, but represses the bacterioferritin encoding gene bfrB. Regulation of these three genes by Rip1 is not reproduced by deletion of Sigma K, L or M, either indicating a requirement for multiple Rip1 substrates or additional arms of the Rip1 pathway. These results identify a branched proteolytic signal transduction system in which a single intramembrane protease cleaves three anti-sigma factor substrates to control multiple downstream pathways involved in lipid biosynthesis and defence against oxidative stress.

Mining the metabiome: identifying novel natural products from microbial communities.

Microbial-derived natural products provide the foundation for most of the chemotherapeutic arsenal available to contemporary medicine. In the face of a dwindling pipeline of new lead structures identified by traditional culturing techniques and an increasing need for new therapeutics, surveys of microbial biosynthetic diversity across environmental metabiomes have revealed enormous reservoirs of as yet untapped natural products chemistry. In this review, we touch on the historical context of microbial natural product discovery and discuss innovations and technological advances that are facilitating culture-dependent and culture-independent access to new chemistry from environmental microbiomes with the goal of reinvigorating the small molecule therapeutics discovery pipeline. We highlight the successful strategies that have emerged and some of the challenges that must be overcome to enable the development of high-throughput methods for natural product discovery from complex microbial communities.