Biology subjects

Kraft, C.

Publications and source records attributed to Kraft, C..

5 recordsLinked to original sources

bioRxiv2024

Lymphostatin: Structure of a large multi-functional virulence factor

Enteropathogenic and Enterohaemorrhagic Escherichia coli are enteric pathogens of global importance and human infections can be life-threatening. Lymphostatin is a key virulence factor of these bacteria, being required for intestinal colonisation and a potent inhibitor of the mitogen- and antigen-activated proliferation of lymphocytes and proinflammatory responses. In some strains, it also mediates adherence to host cells and influences actin nucleation at sites of attachment. This 365 kDa protein requires glycosyltransferase and cysteine protease motifs for activity against lymphocytes, but high-resolution structural information has proven elusive and the molecular mechanisms by which it acts remain unclear. Here, we describe the structure of lymphostatin from the prototype O127:H6 enteropathogenic E. coli strain determined by electron cryo-microscopy. Our results reveal two glycosyltransferase domains, one PaTox-like protease domain, an ADP-ribosyltransferase domain, and a delivery domain. Long linkers act to hold these domains together. These linkers occlude the catalytic sites of the N-terminal glycosyltransferase and protease domains. In this dormant state, lymphostatin binds to HEK-293T cells, where it forms large clusters before being taken up and sequestered into cytosolic foci. With more functional domains than any other known large bacterial toxin, lymphostatin can be regarded as the multifunctional Swiss army knife of pathogenic Escherichia coli, enabling complex interactions with the host cells in different environments.

biophysics↗

bioRxiv2024

Codon Pair-Specific Translation Defects Trigger Ribosome-Associated Quality Control to Avoid Proteotoxic Stress

tRNA modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding, but how codon optimality defects trigger cellular phenotypes remains unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs, triggering ribosome collisions and inducing a coordinated and hierarchical response of cellular quality control pathways. Ribosome profiling reveals an unexpected functional diversity for wobble-uridine (U34) modifications during decoding. The same modification can have different effects at the A and P sites. Furthermore, modification-dependent stalling codon pairs induce ribosome collisions, triggering ribosome-associated quality control (RQC) to prevent protein aggregation by degrading aberrant nascent peptides and mRNAs. RQC inactivation stimulates the expression of molecular chaperones to remove protein aggregates. Our results show that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs and reveal the coordinated regulation and adaptability of cellular surveillance systems to ensure efficient and accurate protein synthesis and maintain protein homeostasis.

biochemistry↗

bioRxiv2023

Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus

The year 2022 was marked by the mpox outbreak caused by human monkeypox virus (MPXV), which is about 98 % identical to vaccinia virus (VACV) at the sequence level regarding the proteins involved in DNA replication. We present the strategy for the production of the VACV DNA polymerase holoenzyme composed of the E9 polymerase associated with its co-factor, the A20-D4 heterodimer, which led to the 3.8 [A] cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. Model building used high-resolution structures of components of the complex and the A20 structure predicted by AlphaFold 2. The structure of E9 does not change in context of the holoenzyme compared to the crystal structure. As for the MPXV holoenzyme, a contact between E9 and D4 is mediated by a cluster of hydrophobic residues. The holoenzyme structure is quite compact and surprisingly similar to the MPXV holoenzyme in presence of a DNA template, with the exception of a movement of the finger domain and the thumb domain, which becomes ordered in presence of DNA. Even in absence of DNA, the VACV holoenzyme structure is too compact for an agreement with SAXS data. This suggests the presence of more open conformations in solution, which are also predicted by Alphafold 2 indicating hinge regions located within A20. Using biolayer interferometry we showed that indeed, the E9-D4 interaction is weak and transient although very important as it has not been possible to obtain viable viruses carrying mutations of key residues in the E9-D4 interface. Author SummaryThe 2022 outbreak of mpox is caused by monkeypox virus closely related to the best studied model, vaccinia virus. Genome replication, which takes place largely autonomously in the cytosol of the infected cell, is still not really understood. Viral DNA synthesis involves a DNA repair enzyme, the uracil-DNA glycosylase D4 linked to the structural protein A20 forming the processivity factor, which in turn binds to E9 forming the complex required for processive DNA synthesis. Here we present the first structure of the vaccinia virus polymerase holoenzyme E9-A20-D4 at 3.8 [A] obtained by cryo-electron microscopy. This structure, together with several recent structures from monkeypox virus, provide a static view of the complex with a previously undescribed contact between E9 and D4. Our small-angle scattering data show that other conformations, taking advantage of 2 hinge regions in A20, exist in solution. Using site-directed mutagenesis and binding studies we show that the contact between E9 and D4, which serves to encircle the template strand, is important, but transient. Thus the current model of the orientation of the holoenzyme on the replication fork may not be the only one possible.

molecular biology↗

bioRxiv2022

mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria

Signal-sequence dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and facilitated by dedicated protein targeting factors, such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins, but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors, but is dependent on the SecYEG-translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.

microbiology↗

bioRxiv2022

LRBA balances antigen presentation and T-cell responses by facilitating autophagy through the binding to PIK3R4 and FYCO1

Reduced autophagy is associated with the aberrant humoral response observed in lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency; however, the exact molecular mechanism and its impact on T-cell responses remain unknown. We identified two novel LRBA interactors, phosphoinositide 3-kinase regulatory subunit 4 (PIK3R4) and FYVE And Coiled-Coil Domain Autophagy Adaptor 1 (FYCO1). Both proteins play essential roles in different stages of autophagy. PIK3R4 facilitates the production of phosphatidylinositol-3 phosphate (PI(3)P) required for autophagosome formation and autophagosome-lysosome fusion, whereas FYCO1 allows autophagosome movement. LRBA-KO cells showed an impaired PI(3)P production, a delayed autophagosome-lysosome fusion, an accumulation of enlarged autophagosomes, and an atypical lysosomal positioning. These abnormalities led to decreased cargo material degradation and prolonged antigen presentation to T-cells via autophagy, resulting in increased production of proinflammatory cytokines, as autophagy is a major intracellular degradation system for major histocompatibility class II complex (MHCII) loading. Aberrant autophagosome formation, cargo degradation and antigen presentation were rescued by ectopic expression of WT-LRBA. In summary, we identified a novel function of LRBA that is crucial for T-cell-driven response through the interaction with two proteins of the autophagy machinery. These observations may contribute to the exacerbated T-cell dysregulation observed in LRBA-deficient patients.

immunology↗