Structure and function of prodrug-activating peptidases.

Bacteria protect themselves from the toxicity of antimicrobial metabolites they produce through several strategies. In one resistance mechanism, bacteria assemble a non-toxic precursor on an N-acyl-d-asparagine prodrug motif in the cytoplasm, then export it to the periplasm where a dedicated d-amino peptidase hydrolyzes the prodrug motif. These prodrug-activating peptidases contain an N-terminal periplasmic S12 hydrolase domain and C-terminal transmembrane domains (TMDs) of varying lengths: type I peptidases contain three transmembrane helices, and type II peptidases have an additional C-terminal ABC half-transporter. We review studies which have addressed the role of the TMD in function, the substrate specificity, and the biological assembly of ClbP, the type I peptidase that activates colibactin. We use modeling and sequence analyses to extend those insights to other prodrug-activating peptidases and ClbP-like proteins which are not part of prodrug resistance gene clusters. These ClbP-like proteins may play roles in the biosynthesis or degradation of other natural products, including antibiotics, may adopt different TMD folds, and have different substrate specificity compared to prodrug-activating homologs. Finally, we review the data supporting the long-standing hypothesis that ClbP interacts with transporters in the cell and that this association is important for the export of other natural products. Future investigations of this hypothesis as well as of the structure and function of type II peptidases will provide a complete account of the role of prodrug-activating peptidases in the activation and secretion of bacterial toxins.

A small molecule inhibitor prevents gut bacterial genotoxin production.

The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin's biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis.

Structural basis of colibactin activation by the ClbP peptidase.

Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.

COVID GEAS: COVID-19 National Survey in Patients With Systemic Autoimmune Diseases.

OBJECTIVES: COVID-19 outcomes in population with systemic autoimmune diseases (SAD) remain poorly understood. The aim was to examine demographic and clinical factors associated with COVID-19 infection in people with rheumatic disease. METHODS: Two phases cross-sectional survey of individuals with rheumatic disease in April 2020 and October 2020. COVID infection, severity of disease, age, sex, smoking status, underlying rheumatic disease diagnosis, comorbidities and rheumatic disease medications taken immediately prior to infection were analyzed. RESULTS: A total of 1,529 individuals with autoimmunity disease diagnosis were included. Out of 50 positive patients, 21 required telephone medical assistance, 16 received assessment by primary care physician, 9 were evaluated in Emergency Department and 4 patient required hospitalization. Multivariate analysis was performed without obtaining differences in any of the systemic autoimmune diseases. Regarding the treatments, significant differences were found (p 0.011) in the treatment with anti-TNF-alpha agents with OR 3.422 (1.322-8.858) and a trend to significance (p 0.094) was observed in patients receiving mycophenolate treatment [OR 2.016 (0.996-4-081)]. CONCLUSIONS: Anti-TNF-alpha treatment was associated with more than 3-fold risk of suffering from SARS-CoV-2 infection, although in all cases infection was mild. Cumulative incidence in patients with SAD was up to 5 times higher than general population but with great differences between autoimmune diseases.

Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis.

OBJECTIVE: To identify the genetic cause of disease in a form of congenital spinal muscular atrophy and arthrogryposis (CSMAA). METHODS: A 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis, and diminished lower limb movement. Whole-exome sequencing (WES) was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identified variant. RESULTS: WES revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. Functional studies by Western blot and immunohistochemical analysis show that p.S94L increased TRPV4 activity-based cytotoxicity and resultant decreased TRPV4 expression levels, therefore involves a gain-of-function mechanism. CONCLUSIONS: This study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of CSMAA.

Clinical outcomes of minimally invasive versus open approach for one-level transforaminal lumbar interbody fusion at the 3- to 4-year follow-up.

PURPOSE: Supporters of minimally invasive approaches for transforaminal lumbar interbody fusion (TLIF) have reported short-term advantages associated with a reduced soft tissue trauma. Nevertheless, mid- and long-term outcomes and specifically those involving physical activities have not been adequately studied. The aim of this study was to compare the clinical outcomes of mini-open versus classic open surgery for one-level TLIF, with an individualized evaluation of the variables used for the clinical assessment. METHODS: A prospective cohort study was conducted of 41 individuals with degenerative disc disease who underwent a one-level TLIF from January 2007 to June 2008. Patients were randomized into two groups depending on the type of surgery performed: classic open (CL-TLIF) group and mini-open approach (MO-TLIF) group. The visual analog scale (VAS), North American Spine Society (NASS) Low Back Pain Outcome instrument, Oswestry Disability Index (ODI) and the Short Form 36 Health Survey (SF-36) were used for clinical assessment in a minimum 3-year follow-up (36-54 months). RESULTS: Patients of the MO-TLIF group presented lower rates of lumbar (p = 0.194) and sciatic pain (p = 0.427) and performed better in daily life activities, especially in those requiring mild efforts: lifting slight weights (p = 0.081), standing (p = 0.097), carrying groceries (p = 0.033), walking (p = 0.069) and dressing (p = 0.074). Nevertheless, the global scores of the clinical questionnaires showed no statistical differences between the CL-TLIF and the MO-TLIF groups. CONCLUSIONS: Despite an improved functional status of MO-TLIF patients in the short term, the clinical outcomes of mini-open TLIF at the 3- to 4-year follow-up showed no clinically relevant differences to those obtained with open TLIF.