Biology subjects

Schwabe, J.

Publications and source records attributed to Schwabe, J..

5 recordsLinked to original sources

bioRxiv2025

A single-domain response regulator activates exopolysaccharide synthesis by interacting with the initiating phosphoglycosyl transferase

Exported polysaccharides have crucial functions in bacteria. Polysaccharide biosynthesis in the ubiquitous Wzx/Wzy- and ABC-transporter-dependent pathways starts with the transfer of a sugar-1-phosphate from a nucleotide-sugar donor to undecaprenyl phosphate, a reaction catalyzed by a phosphoglycosyl transferase (PGT). Both reaction substrates are limited and shared among multiple glycoconjugate pathways, raising the question of how bacteria regulate these pathways. In Myxococcus xanthus, EpsZ, which belongs to the family of large monotopic PGTs (monoPGTs), starts the Wzx/Wzy-dependent exopolysaccharide (EPS) biosynthesis. The Dif chemosensory system regulates EPS biosynthesis by an unknown mechanism via the phosphorylated single-domain response regulator EpsW (EpsW[~]P). Here, we show that EpsW[~]P stimulates EPS biosynthesis at the post-translational level. Moreover, MiniTurbo-based proximity labeling experiments suggest that EpsW[~]P interacts directly with EpsZ. Additionally, heterologous expression of these two proteins in Salmonella enterica demonstrates that EpsW stimulates EpsZ enzymatic activity. S. enterica WbaP, the prototype large monoPGT, forms a functional homodimer, with dimerization involving a distinct cytoplasmic {beta}-hairpin. However, AlphaFold-based structural modeling shows that EpsZ lacks this {beta}-hairpin, suggesting an alternative mechanism for dimerization. Our structural modeling of a EpsZ-EpsW heterocomplex suggests that EpsW[~]P, by direct interaction, promotes the formation of the stable EpsZ dimer. These findings uncover a new model for the regulation of polysaccharide biosynthesis in which EpsW[~]P allosterically facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating EPS biosynthesis at its initial step. Genomics and structural modeling suggest that the regulation of large monoPGTs by a single-domain response regulator is widespread in myxobacteria and potentially beyond. SignificanceBacteria produce various polysaccharides with important biological functions and biotechnological applications. Polysaccharide synthesis is energy-costly and requires substrates that are in limited supply raising the question of how bacteria regulate these pathways. Here, we explored the regulation of exopolysaccharide biosynthesis in Myxococcus xanthus. We demonstrate that the phosphorylated single-domain response regulator EpsW activates exopolysaccharide biosynthesis at the post-translational level by stimulating the activity of the phosphoglycosyl transferase EpsZ. By directly interacting with EpsZ, phosphorylated EpsW facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating exopolysaccharide biosynthesis at its initial step. We propose that this previously unrecognized regulatory mechanism is broadly conserved, not only in myxobacteria but also beyond.

microbiology↗

bioRxiv2024

Identification of EcpK, a bacterial tyrosine pseudokinase important for exopolysaccharide biosynthesis in Myxococcus xanthus

Bacteria synthesize and export chemically diverse polysaccharides that function in many physiological processes and are widely used in industrial applications. In the ubiquitous Wzx/Wzy-dependent biosynthetic pathways for these polysaccharides, the polysaccharide co-polymerase (PCP) facilitates the polymerization of repeat units in the periplasm, and in Gram-negative bacteria, also polysaccharide translocation across the outer membrane. These PCPs are integral inner membrane proteins with extended periplasmic domains, and functionally depend on alternating between different oligomeric states. The oligomeric state, in turn, is determined by a cognate cytoplasmic bacterial tyrosine kinase (BYK), which is either part of the PCP or a stand-alone protein. Interestingly, BYK-like proteins, which lack key catalytic residues and/or the phosphorylated Tyr residues, have been described. In Myxococcus xanthus, the exopolysaccharide (EPS) is synthesized and exported via the Wzx/Wzy-dependent EPS pathway in which EpsV serves as the PCP. Here, we confirm that EpsV lacks the BYK domain. Using phylogenomics, experiments and computational structural biology, we identify EcpK as important for EPS biosynthesis and show that it structurally resembles canonical BYKs but lacks residues important for catalysis and Tyr phosphorylation. Using proteomic analyses, two-hybrid assays and structural modeling, we demonstrate that EcpK directly interacts with EpsV. Based on these findings, we suggest that EcpK is a BY pseudokinase and functions as a scaffold, which by direct protein-protein interactions, rather than by Tyr phosphorylation, facilitates EpsV function. EcpK and EpsV homologs are present in other bacteria, suggesting a broad conservation of this mechanism. ImportanceBacteria produce a variety of polysaccharides with important biological functions. In Wzx/Wzy-dependent pathways for the biosynthesis of secreted and capsular polysaccharides in Gram-negative bacteria, the polysaccharide co-polymerase (PCP) is a key protein that facilitates repeat unit polymerization and polysaccharide translocation across the outer membrane. PCP function depends on assembly/disassembly cycles that are determined by the phosphorylation/dephosphorylation cycles of an associated bacterial tyrosine kinase (BYK). Here, we identify the BY pseudokinase EcpK as essential for exopolysaccharide biosynthesis in Myxococcus xanthus. Based on experiments and computational structural biology, we suggest that EcpK is a scaffold protein, guiding the assembly/disassembly cycles of the partner PCP via binding/unbinding cycles independently of Tyr phosphorylation/dephosphorylation cycles. We suggest that this novel mechanism is broadly conserved.

microbiology↗

bioRxiv2024

Damaging mutations in LXRα uncouple lipogenesis from hepatotoxicity and implicate hepatic cholesterol sensing in human liver health

The nuclear receptor Liver X Receptor- (LXR) activates lipogenic gene expression in hepatocytes. Its inhibition has therefore been proposed as a strategy to treat metabolic-dysfunction-associated steatotic liver disease (MASLD). In order to understand the impact of reducing LXR activity on human health we first examined the association between the carriage of rare loss of function mutations in NR1H3 (encoding LXR) and metabolic and hepatic phenotypes. We identified 63 rare predicted damaging variants in the ligand binding domain of LXR in 454,787 participants in UK Biobank. On functional characterisation, 42 of these were found to be severely impaired. Consistent with loss of the lipogenic actions of LXR, carriers of damaging mutations in LXR had reduced serum triglycerides ({beta}=-0.13 s.d. {+/-}0.03, P=2.7x10-5, N(carriers)=971). Surprisingly, these carriers also had elevated concentrations of serum liver enzymes (e.g. ALT: {beta}=0.17s.d. {+/-}0.03, P=1.1x10-8, N(carriers)=972) with a 35% increased risk of clinically significant elevations in ALT (OR=1.32, 95%CI:1.15-1.53, P=1.2x10-4, N(carriers)=972), suggestive of hepatotoxicity. We generated a knock-in mouse carrying one of the most severely damaging mutations (Nr1h3 p.W441R) which we demonstrated to have dominant negative properties. Homozygous knock-in mice rapidly developed severe hepatitis and fibrotic liver injury following exposure to western diet despite markedly reduced steatosis, liver triglycerides and lipogenic gene expression. This phenotype was completely rescued by viral over-expression of wildtype LXR specifically in hepatocytes, indicating a cell-autonomous effect of the mutant on hepatocyte health. While homozygous LXR knockout mice showed some evidence of hepatocyte injury under similar dietary conditions, the phenotype of the LXRW441R/W441R mouse was much more severe, suggesting that dominant negative mutations that actively co-repress target genes can result in pathological impacts significantly more severe than those seen with simple absence of the receptor. In summary, our results show that loss of function mutations in LXR occur in at least 1/450 people and are associated with evidence of liver dysfunction. These findings implicate LXR in the maintenance of human liver health, identify a new murine model of rapidly progressive fibrotic liver disease and caution against LXR antagonism as a therapeutic strategy for MASLD.

genetics↗

bioRxiv2022

Evidence for a widespread third system for bacterial polysaccharide export across the outer membrane comprising a composite OPX/β-barrel translocon

In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein WzaE. coli is an octamer, in which the eight C-terminal domains form an -helical OM pore, and the eight copies of the three N-terminal domains (D1-D3) a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18- stranded {beta}-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments and computational structural biology, we characterize EpsX as an OM 18-stranded {beta}-barrel protein important for EPS synthesis and identify AlgE, a {beta}-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, only consists of the periplasmic D1 and D2 domains and lacks the domain for spanning the OM (henceforth D1D2OPX protein). In vivo, EpsX and EpsY mutually stabilize each other, supporting their direct interaction. Based on these observations, we propose a model whereby EpsY and EpsX make up a novel type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning translocon together with the periplasmic D1D2OPX protein EpsY. Based on computational genomics, similar composite systems are present widespread in Gram-negative bacteria. This model provides a framework for these proteins future experimental characterization.

microbiology↗

bioRxiv2019

Claudin-12 deficiency causes nerve barrier breakdown, mechanical hypersensitivity and painful neuropathy

Peripheral nerves and their axons are shielded by the blood-nerve and the myelin barrier, but understanding of how these barriers impact nociception is limited. Here, we identified a regulatory axis of the tight junction protein claudin-12, sex-dependently controlling perineurial and myelin barrier integrity. In nerve biopsies, claudin-12 in Schwann cells was lost in male and postmenopausal female patients with painful but not painless polyneuropathy. Global Cldn12 gene-knockout selectively increased perineurial/myelin barrier leakage, damaged tight junction protein expression and morphology, increased proinflammatory cytokines and induced mechanical hypersensitivity in naive and neuropathic male mice, respectively. Other barriers and neurological function remained intact. In vitro transfection studies documented claudin-12 plasma membrane localisation without interaction with other tight junction proteins or intrinsic sealing properties. Rather, claudin-12 had a regulatory tight junction protein function on the myelin barrier via the morphogen SHH in vivo in Cldn12-KO and after local siRNA knockdown. Fertile female mice were completely protected. Collectively, these studies reveal the critical role of claudin-12 maintaining the myelin barrier and highlight restoration of the claudin-12/SHH pathway as a potential target for painful neuropathy.

neuroscience↗