Molecular target of enteric VapC toxins and regulation of vapBC transcription by conditional cooperativity

The uibiquitous Type II toxin – antitoxin (TA) loci encode two proteins, a toxin and an antitoxin. The antitoxin combines with and neutralizes a cognate toxin. Usually, the TA genes form an operon that is transcribed by a single promoter located upstream of the genes. In most cases, the antitoxin autoregulates the TA operon via binding to operator sites in the promoter region. In almost all such cases, the toxin act as a co-repressor of transcription as the toxin enhances the DNA binding of the antitoxin. Recently, it has been shown that toxins play an additional role in stimulating transcription, as the antitoxin and toxin ratio is important for cooperative binding of the complex to DNA. The antitoxin is rapidly degraded by cellular proteases under conditions of stress and treatment with antibiotics, which leads to activation of the toxin. The toxins of TAs belong to different gene families. The most abundant TA gene family is vapBC that, in some organisms, have expanded into cohorts of genes. For example, the major human pathogen Mycobacterium tuberculosis contains at least 88 TAs, 45 of which are vapBC loci. VapC toxins encoded by vapBC loci are PIN domain proteins (PilT N-terminal). Eukaryotic PIN domain proteins are site-specific ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. From in vitro experiments it has been postulated that VapC toxins are RNases or DNases but their exact cellular target has remained elusive. Here I show that VapC encoded by Shigella flexneri 2a virulence plasmid pMYSH6000 and the chromosome of Salmonella enterica serovar Typhimurium LT2 are site-specific endoribonucleases that specifically cleave tRNAfMet in the anticodon stem-loop in vivo and in vitro. Furthermore, I show that VapC dependent depletion of tRNAfMet leads to bacteriostatic inhibition of global translation, which surprisingly induces low-level initiation of translation at elongator codons that are correctly positioned relative to a Shine & Dalgarno sequence. I also show that VapC forms a complex with VapB and acts as a co-repressor of vapBC transcription. During steady state growth VapB is in excess of VapC. However, nutrient stress or treatment with antibiotics leads to Lon protease dependent decrease in VapB levels. Furthermore, I show that VapC in excess of VapB directly interferes with cooperative DNA binding of the VapBC complex, which is dependent on the dimerisation of the VapC toxin. 2 In conclusion, I show that enteric VapCs not only regulate global cellular translation by tRNAfMet cleavage, but also regulate vapBC transcription by conditional cooperativity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

VapCs of <i>Mycobacterium tuberculosis</i> cleave RNAs essential for translation

The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin–Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify the cellular targets of 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribonucleases that cleave RNAs essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation

Toxin inhibition in <i>C. crescentus</i> VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo

Functional and structural plasticity co-express in a left premotor region during early bimanual skill learning

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Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide

Cerebellar and premotor activity during a non-fatiguing grip task reflects motor fatigue in relapsing-remitting multiple sclerosis

Fatigue is a common and highly disabling symptom of multiple sclerosis. Patients experience an effort-independent general subjective feeling of fatigue as well as excessive fatigability when engaging in physical or mental activity. Previous research using functional magnetic resonance imaging (fMRI) has revealed heterogeneous findings, but some evidence implicates the motor system. To identify brain correlates of fatigue, 44 mildly impaired patients with relapsing-remitting multiple sclerosis and 25 age- and gender-matched healthy controls underwent functional magnetic resonance imaging at 3 Tesla, while they performed alternating blocks of rest and a non-fatiguing precision grip task. We investigated neural correlates of fatigue using the motor subscore of Fatigue Scale for Motor and Cognitive Functions (FSMCMOTOR) using the bilateral motor cerebellum, putamen, and dorsal premotor cortex as regions of interest. Patients and healthy controls performed the grip force task equally well without being fatigued. In patients, task-related activity in lobule VI of right motor cerebellum changed in proportion with individual FSMCMOTOR scores. In right dorsal premotor cortex, linear increases in activity across consecutive task blocks scaled with individual FSMCMOTOR scores in healthy controls, but not in patients. In premotor and dorsomedial prefrontal areas, patients were impaired at upscaling task-related activity the more they were affected by motor fatigue. The results support the notion that increased sensorimotor processing in the cerebellum contributes to the experience of motor fatigue and fatigability in multiple sclerosis. Additionally, downscaling of motivational input or sensorimotor processing in prefrontal and premotor areas may constitute an additional pathophysiological factor