Biology subjects

Fabre, B.

Publications and source records attributed to Fabre, B..

7 recordsLinked to original sources

bioRxiv2026

TurboID-based proteomic profiling reveals proxitome of the IRT1 metal transporter and new insight into metal uptake regulation in plants

IRT1 is the major root iron transporter responsible for broad spectrum metal absorption in Arabidopsis root epidermal cells. Non-iron metal substrates of IRT1 were recently shown to regulate IRT1 cell surface levels by endocytosis and vacuolar degradation. TurboID-based proximity labeling was recently developed to detect protein:protein interactions and thus shed light on the intricate regulation of proteins in living cells. Although TurboID-based proximity labeling technology has been successfully established in mammals, its application in plant systems remains limited and inexistent for highly hydrophobic multispann transmembrane proteins. Here, we established TurboID for proximity labeling of IRT1 and identified 494 IRT1-specific proximal proteins, including the previously reported FYVE1 IRT1 interacting protein. To showcase the biological relevance of identified IRT1 proximal proteins, we characterized further the NHX5 Na+(K+)/H+ antiporter and the RGLG2 E3 ubiquitin ligase. We validated both IRT1 proximal proteins as IRT1 partners using several orthogonal assays. We also highlight the contribution of NHX-type antiporters and RGLG-type E3 ligases in plants responses to non-iron metal nutrition and IRT1 endocytosis. Overall, our work showcases the power of TurboID to identify new interacting proteins for plant transporters, expanding the application of this technology to proteins notoriously difficult to work with.

plant biology↗

bioRxiv2025

Antagonistic insulin mimetics lock the insulin receptor in an alternative apo-state

Despite significant advancements in high-resolution structural analysis of activated human insulin receptor (IR), the molecular mechanisms underlying its conformational plasticity that govern the transition from the apo state to the activated state are still not well understood. This leaves critical aspects of IR regulation unclear. Here, we reveal the mechanism by which the insulin mimetics Ada, Trim, and S661 fully inhibit the insulin receptor. The receptor is stabilized in a yet structurally un-described {cap}-shaped conformation which is induced by antagonist binding between the L1 and FnIII-1 domains. In contrast to insulin-bound IR structures, the -CT helix is not observable in the {cap} conformation, and the membrane-proximal regions of the FnIII-3 domains are >10 nm apart, which prohibits transmembrane signal transduction and kinase domain activation. Analysis of apo-IR electron cryo-microscopy data indicates that the {cap}-shaped state is one of several metastable apo-IR conformations. These findings underscore the intrinsic conformational dynamics of apo-IR and its role in integrating insulin binding and receptor activation.

molecular biology↗

bioRxiv2025

Drosophila melanogaster miPEP8 regulates cell size through its interaction with ref(2)P/p62

MiPEPs are microproteins encoded by primary transcripts of microRNAs (pri-miRNAs). Initially identified in plants, we recently characterized a miPEP in Drosophila melanogaster, named miPEP8, which is involved in the regulation of wing size. However, mechanisms at play are unknown. In the present study, we take advantage of the drosophila cell line Schneider 2 (S2) to further investigate miPEP8 function at the molecular level. Overexpressing miPEP8 in S2 cells induced a reduction of cell size as well as an increase of the proportion of cells in the G1 phase of the cell cycle and an increase of the autophagic flux. A proteomics analysis revealed that miPEP8 overexpression in S2 cells induces the upregulation of several proteins including the autophagosome cargo protein ref(2)P (the orthologue of the human p62/Sequestosome 1 protein). The interactome of miPEP8 was generated and revealed interactions between this miPEP8 and the mTORC1/autophagy pathway. Bioinformatics analysis identified a short linear motif (SLiM) on miPEP8 sequence. Mutation of this SLiM prevented the interaction between ref(2)P/p62 and miPEP8. Mutation of the SLiM also reverted the smaller cell size phenotype observed when overexpressing miPEP8 in S2 cells. Finally, the cell size phenotype was reversed when cells were treated with RNA interference targeting ref(2)P/p62, suggesting that this protein plays a role in regulating the cell size in Drosophila.

biochemistry↗

bioRxiv2022

In depth exploration of the alternative proteome of Drosophila melanogaster

Recent studies have shown that hundreds of small proteins were occulted when protein coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocystronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome wide exploration of the alternative proteome is still limited to a few species. In the present article we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains and short linear motifs were predicted for 235 of the alternative proteins identified and point towards specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed fourteen alternative proteins with developmentally regulated expression patterns and ten induced upon heat shock treatment of embryos, demonstrating stage or stress specific production of alternative proteins.

biochemistry↗

bioRxiv2020

Functional analysis of caspase cleavable proteoforms from the Drosophila GSK-3 gene shaggy.

The Drosophila shaggy (sgg) gene encodes the major fly orthologue of Glycogen Synthase Kinase -3 (GSK-3), a key highly conserved kinase at the heart of many signalling pathways. The sgg locus is complex, encoding multiple protein isoforms that are expressed in distinct temporal and tissue-specific patterns across development. Its isoforms predominantly differ at the carboxy and amino termini due to the use of different transcriptional start sites and alternative splicing events that include internal and terminal exons. One interesting class of proteins isoforms is represented by the Sgg-PD class (Sgg46), three proteoforms that contain a large 582 amino acid N-terminal domain which contains recognition sites for caspase-mediated cleavage. Regulated cleavage at these sites by non-apoptotic caspases has previously been implicated in the regulation of Sgg activity in adult bristle development. Here, we take a genome engineering approach to introduce specific tags into this unique Sgg-PD exon and utilise these for localisation and protein interaction studies. We also generated new loss of function alleles and specific mutations in the caspase cleavage motifs. We find that loss of functions Sgg-PD class alleles are viable and fertile, but exhibit adult locomotor and bristle defects. Expression analysis of lines carrying tags on both sides of the caspase cleavage sites indicates that the cleavage is developmentally regulated during embryogenesis. Surprisingly, we found that in some cells, particularly embryonic hemocytes, the N-terminal domain released by caspase cleavage is retained while the polypeptide containing the conserved kinase domain is apparently lost. Transcriptomic analysis of embryos homozygous for the new caspase-insensitive allele indicates a role for Sgg-PD in the regulation of cytoskeletal and cell junction functions, which is supported by proteomics analysis using specific in locus tags to identify common and unique protein interaction partners with N- and C-terminal domains. Taken together, our work identifies new activities for the Sgg protein and uncovers unexpected roles for caspase cleavage in Sgg biology.

developmental biology↗

bioRxiv2020

RNF5 Regulation of RBBP4 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we found that increased expression and abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patients correlated with poor prognosis. RNF5 inhibition decreased AML cell growth in culture and in vivo, and blocked development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition led to transcriptional changes that overlapped with those seen upon HDAC1 inhibition. RNF5 induced the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment and subsequent epigenetic regulation of genes involved in AML development and maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhanced the sensitivity of AML cells to histone deacetylase (HDAC) inhibitors. Notably, low expression of RNF5 and HDAC coincided with a favorable prognosis. Our studies identified ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML while highlighting RNF5/RBBP4 as markers to stratify patients for treatment with HDAC inhibitors.

cancer biology↗

bioRxiv2019

Characterisation of protein isoforms encoded by the Drosophila Glycogen Synthase Kinase 3 gene shaggy

The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. We used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. We performed proteomic analysis of the two major proteoform classes and generated mutant lines for both of these for transcriptomic and phenotypic analysis. We uncovered distinct tissue-specific localization patterns for all of the tagged proteoforms we examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoform classes we examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. Our findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3 and GSK-3{beta} encoded by independent vertebrate genes. Our analysis suggests that different protein isoforms generated by alternative splicing perform distinct functions.

genetics↗