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Xavier Roucou

Publications and source records attributed to Xavier Roucou.

16 recordsLinked to original sources
PubMed2026

Expanding the human proteome with microproteins and peptideins.

A major scientific drive is to characterize the protein-coding genome, which is a primary basis for studying human health. But the fundamental question remains of what has been missed in previous analyses. Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states1-3, with major implications for biomedical science. However, a key gap in knowledge has been which ncORFs produce small microproteins or alternative protein molecules that contribute to the human proteome. Here we report the collaborative efforts of the TransCODE Consortium4 to produce a consensus landscape of protein-level evidence for ncORFs. We show that about 25% of a set of 7,264 ncORFs gives rise to detectable peptides in a large-scale analysis of 95,520 proteomics experiments. We develop an annotation framework for ncORF-encoded microproteins as human proteins and codify the new conceptual model of 'peptideins' as microproteins that have indeterminate potential as functional proteins. To probe the biological implications of peptideins, we create an evolutionary analysis approach, termed ORF relative branch length (ORBL), and determine that evolutionary constraint is common and associates with observation of ncORF-derived peptides. We then characterize a pan-essential cellular phenotype for one peptidein from the OLMALINC long non-coding RNA. Overall, we generate public research tools supported by GENCODE and PeptideAtlas and advance biomedical discovery for understudied components of the human proteome.

Humans↗
PubMed2025

High-quality peptide evidence for annotating non-canonical open reading frames as human proteins.

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

GENCODE↗
PubMed2024

Deciphering the ghost proteome in ovarian cancer cells by deep proteogenomic characterization.

Proteogenomics is becoming a powerful tool in personalized medicine by linking genomics, transcriptomics and mass spectrometry (MS)-based proteomics. Due to increasing evidence of alternative open reading frame-encoded proteins (AltProts), proteogenomics has a high potential to unravel the characteristics, variants, expression levels of the alternative proteome, in addition to already annotated proteins (RefProts). To obtain a broader view of the proteome of ovarian cancer cells compared to ovarian epithelial cells, cell-specific total RNA-sequencing profiles and customized protein databases were generated. In total, 128 RefProts and 30 AltProts were identified exclusively in SKOV-3 and PEO-4 cells. Among them, an AltProt variant of IP_715944, translated from DHX8, was found mutated (p.Leu44Pro). We show high variation in protein expression levels of RefProts and AltProts in different subcellular compartments. The presence of 117 RefProt and two AltProt variants was described, along with their possible implications in the different physiological/pathological characteristics. To identify the possible involvement of AltProts in cellular processes, cross-linking-MS (XL-MS) was performed in each cell line to identify AltProt-RefProt interactions. This approach revealed an interaction between POLD3 and the AltProt IP_183088, which after molecular docking, was placed between POLD3-POLD2 binding sites, highlighting its possibility of the involvement in DNA replication and repair.

Humans↗
PubMed2006

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells.

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.

Animals↗
PubMed2006

Prion protein prevents Bax-mediated cell death in the absence of other Bcl-2 family members in Saccharomyces cerevisiae.

Although there is no consensus regarding the normal function of the prion protein, increasing evidence points towards a role in cellular protection against cell death. We have previously shown that prion protein is a potent inhibitor of Bax-induced apoptosis in human primary neurons and in the breast carcinoma MCF-7 cells. Here, we used the yeast Saccharomyces cerevisiae to investigate if the neuroprotective function of prion protein requires other members of the Bcl-2 family given that S. cerevisiae lacks Bcl-2 genes but undergoes a mitochondrial-dependent apoptotic cell death upon exogenous expression of Bax protein. We show that Bax induces cell death and growth inhibition in S. cerevisiae. Prion protein prevents Bax-mediated cell death. Prion protein overcomes Bax-mediated growth arrest in S phase but cannot overcome population growth inhibition because the cells then accumulate in G(2)/M phase. We conclude that prion protein does not require other Bcl-2 family proteins to protect against Bax-mediated cell death.

Animals↗
PubMed2004

Cellular prion protein neuroprotective function: implications in prion diseases.

Prion protein can display two conformations: a normal cellular conformation (PrP) and a pathological conformation associated with prion diseases (PrP(Sc)). Three complementary strategies are used by researchers investigating how PrP is involved in the pathogenesis of prion diseases: elucidation of the normal function of PrP, determination of how PrP(Sc) is toxic to neurons, and unraveling the mechanism for the conversion of PrP to PrP(Sc). We review the normal function of PrP as an antioxidant and an antiapoptotic protein in vivo and in vitro. This review also addresses contrasting evidence that PrP is cytotoxic. Finally, we discuss the implication of the neuroprotective role of PrP in prion diseases.

Animals↗
PubMed2004

Neuroprotective functions of prion protein.

The normal function of prion protein (PrP) is usually disregarded at the expense of the more fascinating role of PrP in transmissible prion diseases. However, the normal PrP may play an important role in cellular function in the central nervous system, since PrP is highly expressed in neurons and motifs in the sequence of PrP are conserved in evolution. The finding that prion null mice do not have a significant overt phenotype suggests that the normal function of PrP is of minor importance. However, the absence of PrP in cells or in vivo contributes to an increased susceptibility to oxidative stress or apoptosis-inducing insults. An alternative explanation is that the PrP normal function is so important that it is redundant. Probing into the characteristics of PrP has revealed a number of features that could mediate important cellular functions. The neuroprotective actions so far identified with PrP are initiated through cell surface signaling, antioxidant activity, or anti-Bax function. Here, we review the characteristics of the PrP and the evidence that PrP protects against neurodegeneration and neuronal cell death.

Animals↗
PubMed2003

p75 neurotrophin receptor protects primary cultures of human neurons against extracellular amyloid beta peptide cytotoxicity.

The cytotoxicity of extracellular amyloid beta peptide (Abeta) has been clearly demonstrated in many cell types. In contrast, primary human neurons in culture are resistant to extracellular Abeta-mediated toxicity. Here, we investigate the involvement of p75 neurotrophin receptor (p75NTR) in Abeta-treated human neurons. We find that Abeta1-40 and Abeta1-42, but not the reverse control peptide, Abeta40-1, rapidly increase the levels of p75NTR in a specific and dose-dependent manner. In contrast to observations in cell lines, enhanced expression of p75NTR in human neurons via a herpes simplex virus amplicon vector does not increase the susceptibility of neurons to Abeta. Unexpectedly, inhibition of p75NTR expression with an antisense expression construct or incubation of the cells with an antibody to the extracellular domain of p75NTR sensitizes human neurons to extracellular nonfibrillar or fibrillar Abeta1-42 cytotoxicity. Unlike intracellular Abeta, extracellular Abeta toxicity is independent of p53 and Bax activity. However, Abeta toxicity is inhibited by caspase inhibitors and the glycogen synthase kinase 3beta inhibitor lithium. Neuroprotection against Abeta is phosphatidylinositide 3-kinase dependent but Akt independent. These results are consistent with a neuroprotective role for p75NTR against extracellular Abeta toxicity in human neurons.

Amyloid beta-Peptides↗
PubMed2003

Cytosolic prion protein is not toxic and protects against Bax-mediated cell death in human primary neurons.

Recently, it was observed that reverse-translocated cytosolic PrP and PrP expressed in the cytosol induce rapid death in neurons (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). In this study, we investigated whether accumulation of prion protein (PrP) in the cytosol is toxic to human neurons in primary culture. We show that in these neurons, a single PrP isoform lacking signal peptide accumulates in the cytosol of neurons treated with epoxomicin, a specific proteasome inhibitor. Therefore, endogenously expressed PrP is subject to the endoplasmic reticulum-associated degradation (ERAD) pathway and is degraded by the proteasome in human primary neurons. In contrast to its toxicity in N2a cells, reverse-translocated PrP (ERAD-PrP) is not toxic even when neurons are microinjected with cDNA constructs to overexpress either wild-type PrP or mutant PrPD178N. We found that ERAD-PrP in human neurons remains detergent-soluble and proteinase K-sensitive, in contrast to its detergent-insoluble and proteinase K-resistant state in N2a cells. Furthermore, not only is microinjection of a cDNA construct expressing CyPrP not toxic, it protects these neurons against Bax-mediated cell death. We conclude that in human neurons, ERAD-PrP is not converted naturally into a form reminiscent of scrapie PrP and that PrP located in the cytosol retains its protective function against Bax. Thus, it is unlikely that simple accumulation of PrP in the cytosol can cause neurodegeneration in prion diseases.

Blotting, Western↗
PubMed2003

The molecular neighborhood of subunit 8 of yeast mitochondrial F1F0-ATP synthase probed by cysteine scanning mutagenesis and chemical modification.

The detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F(1)-F(0) interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F(1)F(0)-ATP synthase.

Adenosine Triphosphatases↗
PubMed2002

Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein.

In response to various apoptotic stimuli, Bax, a pro-apoptotic member of the Bcl-2 family, is oligomerized and permeabilizes the mitochondrial outer membrane to apoptogenic factors, including cytochrome c. Bax oligomerization can also be induced by incubating isolated mitochondria containing endogenous Bax with recombinant tBid (caspase-8-cleaved Bid) in vitro. The mechanism by which Bax oligomerizes under these conditions is still unknown. To address this question, recombinant human full-length Bax was purified as a monomeric protein. Bax failed to oligomerize spontaneously in isolated mitochondria or in liposomes composed of either cardiolipin or lipids extracted from mitochondria. However, in the presence of tBid, the protein formed large complexes in mitochondrial membranes and induced the release of cytochrome c. tBid also induced Bax oligomerization in isolated mitochondrial outer membranes, but not in other membranes, such as plasma membranes or microsomes. Moreover, tBid-induced Bax oligomerization was inhibited when mitochondria were pretreated with protease K. The presence of the voltage-dependent anion channel was not required either for Bax oligomerization or for Bax-induced cytochrome c release. Finally, Bax oligomerization was reconstituted in proteoliposomes made from mitochondrial membrane proteins. These findings imply that tBid is necessary but not sufficient for Bax oligomerization; a mitochondrial protein is also required.

Animals↗
PubMed2002

Bid induces cytochrome c-impermeable Bax channels in liposomes.

Bax is a proapoptotic member of the Bcl-2 family of proteins. The Bax protein is dormant in the cytosol of normal cells and is activated upon induction of apoptosis. In apoptotic cells, Bax gets translocated to mitochondria, inserts into the outer membrane, oligomerizes and triggers the release of cytochrome c, possibly by channel formation. The BH3 domain-only protein Bid induces a conformational change in Bax before its insertion into the outer membrane. The mechanism by which Bid promotes Bax activation is not understood, and whether Bid is the only protein required for Bax activation is unclear. Here we report that recombinant full-length Bax (Bax(FL)) does not form channels in lipid bilayers when purified as a monomer. In contrast, in the presence of Bid cut with caspase 8 (cut Bid), Bax forms ionic channels in liposomes and planar bilayers. This channel-forming activity requires an interaction between cut Bid and Bax, and is inhibited by Bcl-x(L). Moreover, in the absence of the putative transmembrane C-terminal domain, Bax does not form ionic channels in the presence of cut Bid. Cut Bid does not induce Bax oligomerization in liposomes and the Bax channels formed in the presence of cut Bid are not large enough to permeabilize vesicles to cytochrome c. In conclusion, our results suggest that monomeric Bax(FL) can form channels only in the presence of cut Bid. Cut Bid by itself is unable to induce Bax oligomerization in lipid membranes. It is suggested that another factor that might be present in mitochondria is required for Bax oligomerization.

Animals↗
PubMed2002

On the release of cytochrome c from mitochondria during cell death signaling.

Mitochondria play key roles in apoptosis, a central step being the release of cytochrome c (cyt c) across the outer mitochondrial membrane into the cytoplasm. We review this process in terms of the influences that induce mitochondria to release cyt c, the possible mechanisms of such release and the downstream consequences for caspase activation. The contributions of members of the Bcl-2 family in regulating mitochondrial activities relevant to apoptotic signaling are considered. Antiapoptotic members, such as Bcl-2 itself, are antagonistic to other family members, which prominently include Bax amongst a host of other proapoptotic proteins homologous to Bcl-2. Focus is placed on technical methods of determining cyt c release, which encompass cell fractionation, biochemistry, immunochemistry and confocal microscopy [including observations of release in real time using cyt c-green fluorescent protein (GFP) fusion proteins]. The advantages and potential pitfalls of the various approaches are discussed, with some emphasis on the use of cyt c-GFP fusions and the determination of the characteristics of the putative outer membrane pore through which cyt c and other mitochondrial proteins with proapoptotic functions may pass. The richness of this field relating to mitochondria and cell death is brought out by consideration of studies carried out in mammalian and yeast cells.

Animals↗