Erratum: 498 Structural Determination of the CqsR CACHE Domain and its Autoinducer - CORRIGENDUM.

[This corrects the article DOI: 10.1017/cts.2023.503.].

An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli.

Protein-protein interactions play important roles in regulating cellular functions. We present an optimized disulfide cross-linking protocol for testing predicted interactions of soluble or membrane proteins. Coexpression in E. coli of proteins with a single cysteine residue results in disulfide bond formation upon treating the cells with oxidants if the two proteins interact and the cysteine residues are near each other. Quantification of cross-linked proteins after immunoblot sensitively and reproducibly measures the interaction. For complete details on the use and execution of this protocol, please refer to Olenic et al. (2022).1.

Inhibitory proteins block substrate access by occupying the active site cleft of Bacillus subtilis intramembrane protease SpoIVFB.

Intramembrane proteases (IPs) function in numerous signaling pathways that impact health, but elucidating the regulation of membrane-embedded proteases is challenging. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that SpoIVFB inhibition requires BofA residues in and near a predicted transmembrane segment (TMS). This segment of BofA occupies the SpoIVFB active site cleft based on cross-linking experiments. SpoIVFB inhibition also requires SpoIVFA. The inhibitory proteins block access of the substrate N-terminal region to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. We built a structural model of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a TMS and a membrane-embedded C-terminal region. The model also predicts that SpoIVFA bridges the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of IP inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics.

Proteases are a type of protein that work by cutting up other proteins. The part of the protease that does the cutting is called the active site. Intramembrane proteases are a specific group of proteases that cut up the proteins within cell membranes. There is a lot of interest in learning how to control intramembrane proteases because they are important in regulating the signaling processes that cells use to communicate. SpoIVFB is an intramembrane protease from the bacterium Bacillus subtilis that is studied often as a model for these types of proteases. Bacillus subtilis uses SpoIVFB to produce spores, dormant reproductive cells that can survive extreme, harsh conditions for long periods with minimal energy. SpoIVFB is part of the system that allows spores to communicate with their 'parent cells', the cells they develop in. The activity of this protein is blocked by two other proteins called SpoIVFA and BofA. When these proteins are destroyed, SpoIVFB becomes active, but it is unclear exactly how SpoIVFA and BofA inhibit SpoIVFB. Understanding this relationship could help to reveal ways to regulate other intramembrane proteases. To address this question, Olenic et al. used genetic, biochemical and computer modelling techniques to study how SpoIVFB activity is regulated in Bacillus subtilis. The results show that a region of BofA blocks the area of SpoIVFB that cuts a protein called Pro-σ^K, which stops SpoIVFB from releasing active σ^K into the 'parent cell'. By making genetic variants of BofA, Olenic et al. identified three parts of BofA that are needed to fully inhibit SpoIVFB. A computer model predicts that these three parts give BofA the right shape to inhibit SpoIVFB, and that SpoIVFA helps by forming a bridge between BofA and SpoIVFB. This investigation reveals how the intramembrane protease SpoIVFB is regulated by SpoIVFA and BofA. This information could be useful in developing inhibitors for other intramembrane proteases. The next stage will be to make and test artificial inhibitors based on the structures studied here. If successful, these could have applications in areas such as medicine, agriculture, industry and environmental protection.

Conserved Proline Residues of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB Are Important for Substrate Interaction and Cleavage.

Intramembrane metalloproteases (IMMPs) regulate diverse biological processes by cleaving membrane-associated substrates within the membrane or near its surface. SpoIVFB is an intramembrane metalloprotease of Bacillus subtilis that cleaves Pro-σK during endosporulation. Intramembrane metalloproteases have a broadly conserved NPDG motif, which in the structure of an archaeal enzyme is located in a short loop that interrupts a transmembrane segment facing the active site. The aspartate residue of the NPDG motif acts as a ligand of the zinc ion involved in catalysis. The functions of other residues in the short loop are less well understood. We found that the predicted short loop of SpoIVFB contains two highly conserved proline residues, P132 of the NPDG motif and P135. Mutational analysis revealed that both proline residues are important for Pro-σK cleavage in Escherichia coli engineered to synthesize the proteins. Substitutions for either residue also impaired the Pro-σK interaction with SpoIVFB in copurification assays. Disulfide cross-linking experiments showed that the predicted short loop of SpoIVFB is in proximity to the N-terminal pro-sequence region (Proregion) of Pro-σK. Alanine substitutions for N129 and P132 of the SpoIVFB NPDG motif reduced cross-linking between its predicted short loop and the Proregion more than a P135A substitution. Conversely, the SpoIVFB P135A substitution reduced Pro-σK cleavage more than the N129A and P132A substitutions during sporulation of B. subtilis. We conclude that all three conserved residues of SpoIVFB are important for substrate interaction and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. IMPORTANCE Intramembrane metalloproteases (IMMPs) function in numerous signaling pathways. Bacterial IMMPs govern stress responses, including the sporulation of some species, thus enhancing the virulence and persistence of pathogens. Knowledge of IMMP-substrate interactions could aid therapeutic design, but structures of IMMP·substrate complexes are unknown. We examined the interaction of the IMMP SpoIVFB with its substrate Pro-σK, whose cleavage is required for Bacillus subtilis endosporulation. We found that conserved proline residues in a short loop predicted to interrupt a SpoIVFB transmembrane segment are important for Pro-σK binding and cleavage. The corresponding residues of the Escherichia coli IMMP RseP have also been shown to be important for substrate interaction and cleavage, suggesting that this is a broadly conserved feature of IMMPs, potentially suitable as a therapeutic target.

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL.IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

Liver-specific loss of Perilipin 2 alleviates diet-induced hepatic steatosis, inflammation, and fibrosis.

Hepatic inflammation and fibrosis are key elements in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive liver disease initiated by excess hepatic lipid accumulation. Lipid droplet protein Perilipin 2 (Plin2) alleviates dietary-induced hepatic steatosis when globally ablated; however, its role in the progression of NASH remains unknown. To investigate this further, we challenged Plin2 liver-specific knockout mice (designated L-KO) and their respective wild-type (WT) controls with a methionine-choline-deficient (MCD) diet for 15 days to induce a NASH phenotype of increased hepatic triglyceride levels through impaired phosphatidylcholine (PC) synthesis and very-low-density lipoprotein (VLDL) secretion. Results on liver weights, body weights, fat tissue mass, and histology in WT and L-KO mice fed the MCD diet revealed signs of hepatic steatosis, fibrosis, and inflammation; however, these effects were blunted in L-KO mice. In addition, levels of PC and VLDL were unchanged, and hepatic steatosis was reduced in L-KO mice fed the MCD diet, due in part to an increase in remodeling of PE to PC via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). These mice also exhibited decreased hepatic expression of proinflammatory markers cyclooxygenase 2, IL-6, TNF-α, IL-1ß, and reduced expression of endoplasmic reticulum (ER) stress proteins C/EBP homologous protein and cleaved caspase-1. Taken together, these results suggest that Plin2 liver-specific ablation alleviates diet-induced hepatic steatosis and inflammation via a PEMT-mediated mechanism that involves compensatory changes in proteins involved in phospholipid remodeling, inflammation, and ER stress that work to alleviate diet-induced NASH. Overall, these findings support a role for Plin2 as a target for NASH therapy.