Biology subjects

Vertommen, D.

Publications and source records attributed to Vertommen, D..

24 records · Page 2Linked to original sources

bioRxiv2022

Cardiovirus leader proteins retarget RSK kinases toward alternative substrates to perturb nucleocytoplasmic traffic

Proteins from some unrelated pathogens, including viruses and bacteria can recruit and activate cellular p90-ribosomal protein S6 kinases (RSKs) through a common linear motif. Our data suggested that such pathogens proteins might act as adapters to dock the kinase toward specific substrates. We explored this hypothesis using the Cardiovirus leader protein (L) as a paradigm. L is known to trigger phenylalanine-glycine nucleoporins (FG-NUPs) hyperphosphorylation and nucleocytoplasmic trafficking perturbation. Using a biotin ligase fused to either RSK or to L, we identified FG-NUPs as primary partners of the L-RSK complex in infected cells. Mutant analysis shows that L uses distinct motifs to recruit RSK and to dock the L-RSK complex toward the FG-NUPs. Using an analog-sensitive RSK2 mutant kinase, we show that, in infected cells, L can trigger RSK to use NUP98 and NUP214 as direct substrates. Our data illustrate a novel virulence mechanism where pathogens proteins retarget cellular protein kinases toward specific substrates.

microbiology↗

bioRxiv2022

A molecular plugin rescues GroEL/ES substrates from pre-folding oxidation

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10) to GroEL induces CnoX release. Cryo-electron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal -helix. Furthermore, the identification of complexes in which CnoX, bound to GroEL, forms mixed-disulfides with GroEL substrates indicates that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, which suggests that Hsp60 molecular plugins have been conserved through evolution.

biochemistry↗

bioRxiv2022

Periplasmic oxidized-protein repair during copper stress in E. coli: a focus on the metallochaperone CusF.

Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveal that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows keeping copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins. AUTHOR SUMMARYOur study investigates the interconnection between the copper stress response and the methionine redox homeostasis in the Gram-negative bacterium Escherichia coli. We report that the copper-activation of the CusSR two-component system induces the expression of the periplasmic oxidized-protein repair system, MsrPQ. We demonstrate that MsrPQ is crucial for CusCFBA copper efflux pump activity under aerobic conditions as it maintains the periplasmic component CusF in its functional reduced form. Methionine emerges as a critical residue in copper trafficking proteins: however this naturally-selected advantage must be balanced by methionines high susceptibility to oxidation. Therefore the induction of MsrPQ by copper allows copper homeostasis under aerobic conditions, illustrating that E. coli has developed an integrated and dynamic circuit for sensing and counteracting stress caused by copper and oxidants, thus allowing bacteria to adapt to host cellular defences.

microbiology↗

bioRxiv2020

Exostosin-1 Glycosyltransferase Regulates Endoplasmic Reticulum Architecture and Dynamics

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including ER shape-dynamics-function relationships in mammalian cells. We have observed that, inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated to ER network extension. Our findings suggest that EXT1 drives glycosylation reactions involving ER structural proteins and high-energy nucleotide sugars, which might also apply to other organelles.

cell biology↗

bioRxiv2020

Dimeric Transmembrane Orientations of APP/C99 Regulate γ-Secretase Processing Line Impacting Signaling and Oligomerization

Amyloid precursor protein (APP) cleavage by the {beta} secretase produces the C99 transmembrane (TM) protein, which contains three dimerization-inducing Gly-x-x-x-Gly motifs. We demonstrate that dimeric C99 TM orientations regulate the precise cleavage lines by {gamma}-secretase. Of all possible dimeric orientations imposed by a coiled coil to the C99 TM-cytosolic domain, the dimer (cc-del7) containing 33Gly-x-x-x-Gly37 in the interface promoted A{beta}42 processing line and AICD (APP Intracellular Domain)-dependent gene transcription, including BACE1 mRNA induction, enhancing amyloidogenic signaling. Another orientation exhibiting 25Gly-x-x-x-Gly29 in the interface (cc-del6) favored processing to A{beta}43/40, induced significantly less gene transcription, while promoting formation of SDS-resistant "A{beta}-like" oligomers, reminiscent of A{beta} peptide oligomers. These required both Val24 of a pro-{beta} motif and the 25Gly-x-x-x-Gly29 interface. Thus, crossing angles imposed by dimeric orientations give access to {gamma}-secretase at A{beta}48 or A{beta}49, linking the former to enhanced signaling and A{beta}42. We discuss avenues of blocking amyloidogenic processing.

neuroscience↗

bioRxiv2018

SETD3 protein is the actin-specific histidine N-methyltransferase

Protein histidine methylation is rarely studied posttranslational modification of unknown biochemical importance. In vertebrates, only a few methylhistidne-containing proteins have been reported so far, including {beta}-actin as an essential example. The evolutionary conserved methylation of {beta}-actin H73 residue is catalyzed by a specific histidine N-methyltransferase that has never been identified molecularly. In the present investigation, we have purified actin-specific histidine N-methyltransferase from rat muscles about 1200-fold. Its activity was studied by the radiochemical assay employing either homogeneous recombinant human {beta}-actin produced in E. coli or its mutated form exhibiting substitution of H73 by Ala residue (H73A) as substrates. Three polypeptides of {approx}65, 75 and 90 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of SETD3 methyltransferase as the only plausible candidate. Rat SETD3 and its human ortholog were expressed in COS-7 cells, purified to homogeneity and shown to catalyze methylation of {beta}-actin at H73 residue as confirmed by mass spectrometry analysis. The SETD3 enzyme was active towards a synthetic peptide corresponding to residues 69-77 of {beta}-actin, but not to its mutated form exhibiting His-to-Ala substitution. Finally, Setd3-deficient HAP1 cells were devoid of methylated H73 in {beta}-actin and exhibited phenotypic changes, including a decrease in F-actin content and an increased glycolytic activity. We conclude that SETD3 is the actin-specific histidine N-methyltransferase. The data show for the first time the molecular identity of protein histidine N-methyltransferase in vertebrates and throw new light on the substrate specificity of SET-domain-containing enzymes.

biochemistry↗