Biology subjects

Belghazi, M.

Publications and source records attributed to Belghazi, M..

8 recordsLinked to original sources

bioRxiv2025

A hyperglycosylated form of Kv1.2 upregulated in LGI1 knockout mice

Kv1 voltage-gated potassium channels determine key functional neuronal properties. Their activity is modulated by subunit composition and post-translational modifications such as phosphorylation and glycosylation. Using an antibody directed against a phosphotyrosine (Y458) located in the C-terminal tail of Kv1.2, we identified yet unreported high molecular weight forms of Kv1.2 among them, a phosphorylated and heavily glycosylated 100 kDa form. Owing to the significant downregulation of Kv1.2 in LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, we investigated, in total brain and the hippocampal formation of both WT and Lgi1-/- mice, the distribution of phosphoY458 Kv1.2 and we compared their respective proteomic interactomes with those of Kv1.2. In addition to major differences between the interactomes of pY458Kv1.2 and Kv1.2 in WT and Lgi1-/-, we found a major reshaping of pY458 Kv1.2 molecular neighbourhood between WT and Lgi1-/- as well as a significant upregulation of the glycosylated form in Lgi1-/-.

neuroscience↗

bioRxiv2025

Quantitative mapping of methionine sensitivity to oxidation in the copper-bound PcuC chaperone

Copper is typically coordinated by histidine, cysteine, or methionine in proteins, and these residues are particularly sensitive to oxidation. However, it remains unclear whether copper-coordinating residues are more prone to oxidation than non-coordinating ones, and how their susceptibility changes between the apo and copper-bound states. The copper chaperone PcuC, important for cytochrome c oxidase assembly in bacteria, contains a canonical binding site composed of two histidines and two methionines (H51xnM63x22H86xM88), as well as a disordered C-terminal extension enriched in methionine and histidine. To quantify methionine oxidation sensitivity in both apo- and Cu-bound PcuC, we used a methionine-specific oxaziridine probe combined with mass spectrometry and compared labeling patterns to those generated by 18O-labeled hydrogen peroxide. We show that methionine residues display distinct oxidation sensitivities in the apoprotein, and that the oxaziridine reacts similarly to H218O2. Importantly, this probe enables quantification of methionine oxidation independently of hydroxyl radicals generated by copper-driven Fenton chemistry, which lacks residue specificity. In the copper-bound form, Cu binding strongly alters methionine reactivity, with a marked increase in oxidation of the coordinating Met63 and Met88. Structural analysis revealed that two copper ions occupy the canonical site, while the C-terminal extension does not contribute to coordination. Comparison of structural features and oxidation values showed that methionine sensitivity correlates with solvent exposure in the folded domain, but with local positive charge in the disordered region. These findings demonstrate that copper coordination modulates methionine oxidation, and that oxaziridine-based probes provide powerful tools for mapping oxidation sensitivity in (metallo)proteins.

biochemistry↗

bioRxiv2025

Bacterial Hsp90 promotes virulence factor production through maintenance of NRPS megaenzymes

Pathogenic bacteria produce virulence factors critical to host infection. Here, we demonstrate the crucial role of the bacterial Hsp90 chaperone in the production of two major virulence factors, the colibactin genotoxin in Escherichia coli and the pyoverdine siderophore in Pseudomonas aeruginosa. Colibactin, a hybrid polyketide/non-ribosomal peptide (PK-NRP), and pyoverdine, a non-ribosomal peptide (NRP), are metabolites produced by complex biosynthetic pathways involving large cytoplasmic enzymes called megasynthases. Using comparative proteomics, we found that megasynthase abundance was markedly reduced in hsp90 deletion mutants of E. coli and P. aeruginosa compared to wild-type strains. This reduction was independent of transcriptional or translational regulation. We further revealed an interplay between Hsp90 and the HslUV protease in controlling megasynthase levels. Remarkably, we found that Hsp90 stabilizes additional NRP and PK-NRP megasynthases, suggesting a general role for Hsp90 as a chaperone of these enzymes. These findings open new avenues for enhancing the biosynthesis of complex metabolites for biotechnological applications through proteostasis modulation, and may also have implications for combating bacterial infections.

microbiology↗

bioRxiv2025

Remodeling of RNA-Binding Proteome and RNA-mediated regulation as a new layer of control of sporulation

Sporulation allows certain bacteria to survive extreme conditions for extended periods posing challenges to public health and food safety. Transcriptional level of regulation relying on {sigma} factors has been well studied in the non-pathogenic model bacterium Bacillus subtilis, while post-transcriptional control remains poorly understood. RNA-binding proteins (RBPs) and small non-coding RNAs (sRNAs) modulate gene expression by affecting mRNA stability or translation. Recent studies suggest that RNA-mediated regulation plays a role in sporulation, but its networks and interactions remain largely uncharacterized, highlighting the need for further investigation. To address this knowledge gap, we adapted OOPS (Orthogonal Organic Phase Separation), a high-throughput method to specifically purify RNA binding proteome (RBPome). By monitoring its dynamics, we observed RBPome is highly remodeled during B. subtilis process. Our approach led to the identification of novel RBPs and potential RNA-mediated post-transcriptional regulators of sporulation. This work provides important insights into the interplay between RNAs and RBPs, advancing the understanding of post-transcriptional regulation in Gram-positive spore-forming bacteria. It also offers new resources for exploring the molecular mechanisms that govern sporulation. IMPORTANCEUnderstanding how bacteria survive extreme conditions is key to tackling challenges in health, food safety, and industry. This study reveals a previously unexplored layer of control in Bacillus subtilis, a model organism for spore-forming bacteria--those that can produce spores, which are highly resistant, dormant cells able to endure harsh environments. By mapping, for the first time, the full set of proteins that interact with RNA during sporulation, this work uncovers how bacteria fine-tune their internal programs. The identification of novel RNA-binding proteins sheds light on how bacteria adapt at the molecular level and lays a valuable foundation for future mechanistic studies that will deepen our understanding of bacterial adaptation and resilience.

microbiology↗

bioRxiv2023

Molecular Basis of Kv1 Channel Downregulation and Its Epileptogenic Implications in Lgi1 Knock-out Mice

The Kv1 members (KCNA, Shaker) of the voltage-gated potassium channels are implicated in determining key functional neuronal properties from spike generation at axonal initial segments to the control of synaptic strength at nerve terminals. In animal models of LGI1-dependent autosomal dominant lateral temporal lobe epilepsy (ADTLE), Kv1 channels are downregulated, suggesting their crucial involvement in epileptogenesis. The molecular basis of Kv1 channel-downregulation in LGI1 knock-out mice has not been elucidated and how the absence of this extracellular protein induces an important modification in the expression of Kv1 remains unknown. In this study we analyse by immunofluorescence the detailed modifications in neuronal Kv1.1 and Kv1.2 distribution throughout the hippocampal formation of LGI1 knock-out mice. We show that Kv1 downregulation is not restricted to the axonal compartment, but also takes place in the somatodendritic region and is accompanied by a drastic decrease in Kv2 expression levels. Moreover, we find that the downregulation of these Kv channels is associated with an important increase in bursting patterns. Finally, mass spectrometry uncovered key important modifications in the Kv1 interactome that highlight the epileptogenic implication of Kv1 downregulation in LGI1 knock-out animals.

neuroscience↗

bioRxiv2023

Convergent origin and accelerated evolution of vesicle-associated RhoGAP proteins in two unrelated parasitoid wasps

Animal venoms and other protein-based secretions that perform a variety of functions, from predation to defense, are highly complex cocktails of bioactive compounds. Gene duplication, accompanied by modification of the expression and/or function of one of the duplicates under the action of positive selection, followed by further duplication to produce multigene families of toxins is a well-documented process in venomous animals. This evolutionary model has been less described in parasitoid wasps, which use maternal fluids, including venom, to protect their eggs from encapsulation by the host immune system. Here, we evidence the convergent recruitment and accelerated evolution of two multigene families of RhoGAPs presumably involved in virulence in two unrelated parasitoid wasp species, Leptopilina boulardi (Figitidae) and Venturia canescens (Icheumonidae). In both species, these RhoGAPs are associated with vesicles that act as transport systems to deliver virulence factors, but are produced in different tissues: the venom gland in Leptopilina sp. and the ovarian calyx in V. canescens. We show that the gene encoding the cellular RacGAP1 is at the origin of the virulent RhoGAP families found in Leptopilina sp. and V. canescens. We also show that both RhoGAP families have undergone evolution under positive selection and that almost all of these RhoGAPs lost their GAP activity and GTPase binding ability due to substitutions in key amino acids. These results suggest an accelerated evolution and functional diversification of these vesicle-associated RhoGAPs in the two phylogenetically distant parasitoid species. The potential new function(s) and the exact mechanism of action of these proteins in host cells remain to be elucidated.

evolutionary biology↗

bioRxiv2020

A Sequence determinant in 3'UTR of mRNAs for Nuclear Retention by Paraspeckles

Paraspeckles are nuclear membraneless structures composed of a long non-coding RNA, Nuclear-Enriched-Abundant-Transcript-1 and RNA binding proteins, which associate numerous mRNAs. It is therefore believed that their cellular function is to sequester in the nucleus their associated proteins and/or target mRNAs. However, little is known about the molecular determinant in mRNA targets that allow their association to paraspeckles except that inverted repeats of Alu sequences (IRAlu) present in 3UTR of mRNAs may allow this association. While in a previous study we established the list of paraspeckle target RNAs in a rat pituitary cell line, we didnt find however, inverted repeated SINEs, the rat equivalent of primate IRAlus in 3UTR of these RNAs. By developing a candidate gene strategy, we selected a paraspeckle target gene, namely calreticulin mRNA, and we searched for other potential RNA recruitment elements in its 3UTR, since 3UTRs usually contain the sequence recognition for nuclear localization. We found a 15-nucleotide sequence, present as a tandem repeat in 3UTR of this mRNA, which is involved in the nuclear retention by paraspeckles. While being not overrepresented in the 3UTR of the paraspeckle target RNAs, this recruitment element was present in near 30% of all 3UTR mRNAs. In addition, since an oligonucleotide containing this sequence binds the paraspeckle protein component HNRNPK, it is proposed that HNRNPK may constitute a bridging protein between paraspeckles and target mRNAs.

molecular biology↗

bioRxiv2019

Functional insights from the GC-poor genomes of two aphid parasitoids, Aphidius ervi and Lysiphlebus fabarum

BackgroundParasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biocontrol, and infecting aphids requires overcoming both aphid defenses and their defensive endosymbionts. ResultsWe present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp), highly syntenic, and the most AT-rich reported thus far for any arthropod (GC content: 25.8% and 23.8%). This nucleotide bias is accompanied by skewed codon usage, and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and age-specific energy demands. We identify expansions of F-box/Leucine-rich-repeat proteins, suggesting that diversification in this gene family may be associated with their broad host range or with countering defenses from aphids endosymbionts. The absence of some immune genes (Toll and Imd pathways) resembles similar losses in their aphid hosts, highlighting the potential impact of symbiosis on both aphids and their parasitoids. ConclusionsThese findings are of fundamental interest for insect evolution and beyond. This will provide a strong foundation for further functional studies including coevolution with respect to their hosts, the basis of successful infection, and biocontrol. Both genomes are available at https://bipaa.genouest.org.

genomics↗