Les cadres ouverts de lecture alternatifs contribuent significativement au protéome des eucaryotes

Un défi majeur de l’ère post-génomique est de définir l’ensemble des protéines encodées par le génome : le protéome. Un ARNm mature est en général associé à un seul cadre ouvert de lecture (ORF, open reading frame en anglais) de référence (RefORF) codant pour une protéine. Des ORFs alternatifs (AltORFs) sont cependant présents dans les régions non-traduites (UTRs), ou chevauchant le RefORF dans les cadres de lecture alternatifs +2 et +3. Les AltORFs offrnt le potentiel d’augmenter la diversité protéique, mais leur réelle contribution au protéome est peu caractérisée. Par des techniques de biologie moléculaire, de biochimie, et de biologie cellulaire, j'ai tout d’abord mis en évidence chez plusieurs mammifères supérieurs, l’expression endogène d'une protéine alternative appelée AltPrP à partir du gène PRNP. La découverte d'AltPrP devrait améliorer notre compréhension des rôles pathologiques et physiologiques de ce gène. Suite à la découverte d'AltPrP, et basé sur ce modèle d’AltORF chevauchant un RefORF, j'ai entrepris d’investiguer l’étendue de l’utilisation de ces AltORFs comme source de diversité protéique, chez l’humain en particulier. Par des méthodes computationnelles, j'ai participé à la création d'une base de données d'AltORFs prédits dans les ARNm humains (HA1tORF, pour Human Alternative ORFs). HA1tORF est consultable et interrogeable en ligne. Elle facilitera et accélérera la découverte et l’étude des AltORFs. J'ai ensuite mis au point une approche protéomique afin d'apporter des preuves expérimentales de l’utilisation à grande échelle des AltORFs. Une base de données d’AltORFs, mise à jour pour inclure ceux chevauchant en tout ou partie les UTRs, a été créée et utilisée pour déterminer par spectrométrie de masse la contribution des protéines alternatives au protéome humain. J'ai validé l’expression de 1259 protéines alternatives prédites à travers différents échantillons. J'ai aussi démontré que l’expression d'AltORFs impliquait d’importants biais dans les dessins expérimentaux (transfections ou criblage de banques d’ADNc par exemple). Enfin, un grand nombre de protéines alternatives semblent conservées à travers l’évolution, suggérant leur importance fonctionnelle. En conclusion, mes travaux de doctorat ont permis de mettre en évidence que les AltORFs conduisent à l’expression de nouvelles protéines jusqu’alors ignorées. Ces résultats redéfinissent notre vision du protéome, remettent en question notre compréhension de la structure et de la fonction des gènes eucaryotes, et ouvrent la voie vers l’étude fonctionnelle des protéines alternatives

Les cadres ouverts de lecture alternatifs contribuent significativement au protéome des eucaryotes

Un défi majeur de l’ère post-génomique est de définir l’ensemble des protéines encodées par le génome : le protéome. Un ARNm mature est en général associé à un seul cadre ouvert de lecture (ORF, open reading frame en anglais) de référence (RefORF) codant pour une protéine. Des ORFs alternatifs (AltORFs) sont cependant présents dans les régions non-traduites (UTRs), ou chevauchant le RefORF dans les cadres de lecture alternatifs +2 et +3. Les AltORFs offrnt le potentiel d’augmenter la diversité protéique, mais leur réelle contribution au protéome est peu caractérisée. Par des techniques de biologie moléculaire, de biochimie, et de biologie cellulaire, j'ai tout d’abord mis en évidence chez plusieurs mammifères supérieurs, l’expression endogène d'une protéine alternative appelée AltPrP à partir du gène PRNP. La découverte d'AltPrP devrait améliorer notre compréhension des rôles pathologiques et physiologiques de ce gène. Suite à la découverte d'AltPrP, et basé sur ce modèle d’AltORF chevauchant un RefORF, j'ai entrepris d’investiguer l’étendue de l’utilisation de ces AltORFs comme source de diversité protéique, chez l’humain en particulier. Par des méthodes computationnelles, j'ai participé à la création d'une base de données d'AltORFs prédits dans les ARNm humains (HA1tORF, pour Human Alternative ORFs). HA1tORF est consultable et interrogeable en ligne. Elle facilitera et accélérera la découverte et l’étude des AltORFs. J'ai ensuite mis au point une approche protéomique afin d'apporter des preuves expérimentales de l’utilisation à grande échelle des AltORFs. Une base de données d’AltORFs, mise à jour pour inclure ceux chevauchant en tout ou partie les UTRs, a été créée et utilisée pour déterminer par spectrométrie de masse la contribution des protéines alternatives au protéome humain. J'ai validé l’expression de 1259 protéines alternatives prédites à travers différents échantillons. J'ai aussi démontré que l’expression d'AltORFs impliquait d’importants biais dans les dessins expérimentaux (transfections ou criblage de banques d’ADNc par exemple). Enfin, un grand nombre de protéines alternatives semblent conservées à travers l’évolution, suggérant leur importance fonctionnelle. En conclusion, mes travaux de doctorat ont permis de mettre en évidence que les AltORFs conduisent à l’expression de nouvelles protéines jusqu’alors ignorées. Ces résultats redéfinissent notre vision du protéome, remettent en question notre compréhension de la structure et de la fonction des gènes eucaryotes, et ouvrent la voie vers l’étude fonctionnelle des protéines alternatives

The Neuroprotective Role of Protein Quality Control in Halting the Development of Alpha-Synuclein Pathology

Synucleinopathies are a family of neurodegenerative disorders that comprises Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. Each of these disorders is characterized by devastating motor, cognitive, and autonomic consequences. Current treatments for synucleinopathies are not curative and are limited to improvement of quality of life for affected individuals. Although the underlying causes of these diseases are unknown, a shared pathological hallmark is the presence of proteinaceous inclusions containing the α-synuclein (α-syn) protein in brain tissue. In the past few years, it has been proposed that these inclusions arise from the self-templated, prion-like spreading of misfolded and aggregated forms of α-syn throughout the brain, leading to neuronal dysfunction and death. In this review, we describe how impaired protein homeostasis is a prominent factor in the α-syn aggregation cascade, with alterations in protein quality control (PQC) pathways observed in the brains of patients. We discuss how PQC modulates α-syn accumulation, misfolding and aggregation primarily through chaperoning activity, proteasomal degradation, and lysosome-mediated degradation. Finally, we provide an overview of experimental data indicating that targeting PQC pathways is a promising avenue to explore in the design of novel neuroprotective approaches that could impede the spreading of α-syn pathology and thus provide a curative treatment for synucleinopathies

MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells

Selective transport of pyruvate across the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) is a fundamental step that couples cytosolic and mitochondrial metabolism. The recent molecular identification of the MPC complex has revealed two interacting subunits, MPC1 and MPC2. Although in yeast, an additional subunit, MPC3, can functionally replace MPC2, no alternative MPC subunits have been described in higher eukaryotes. Here, we report for the first time the existence of a novel MPC subunit termed MPC1-like (MPC1L), which is present uniquely in placental mammals. MPC1L shares high sequence, structural, and topological homology with MPC1. In addition, we provide several lines of evidence to show that MPC1L is functionally equivalent to MPC1: 1) when co-expressed with MPC2, it rescues pyruvate import in a MPC-deleted yeast strain; 2) in mammalian cells, it can associate with MPC2 to form a functional carrier as assessed by bioluminescence resonance energy transfer; 3) in MPC1 depleted mouse embryonic fibroblasts, MPC1L rescues the loss of pyruvate-driven respiration and stabilizes MPC2 expression; and 4) MPC1- and MPC1L-mediated pyruvate imports show similar efficiency. However, we show that MPC1L has a highly specific expression pattern and is localized almost exclusively in testis and more specifically in postmeiotic spermatids and sperm cells. This is in marked contrast to MPC1/MPC2, which are ubiquitously expressed throughout the organism. To date, the biological importance of this alternative MPC complex during spermatogenesis in placental mammals remains unknown. Nevertheless, these findings open up new avenues for investigating the structure-function relationship within the MPC complex

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

International audienceThe transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

<div><p>A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.</p></div

Summary of LC-MS/MS analyses of human samples.

<p>Summary of LC-MS/MS analyses of human samples.</p

Co-expression of alternative and reference proteins in cDNA transfection experiments is common.

<p>(<i>A</i>) Distribution of the number of predicted RefORFs-contained AltORFs per gene in the human genome. Top, schematic representation of a mRNA with a RefORF (grey)-containing AltORF (green). By definition, RefORFs are present in the +1 reading frame and AltORFs are present in the non-canonical +2 and +3 reading frames. (<i>B</i>) Strategy to detect the co-expression of reference and alternative proteins in cDNA transfection experiments. HA and GFP tags permit the detection of reference and alternative proteins, respectively. Top, graphical representation of a mRNA with a RefORF-contained AltORF. Middle, typical cDNA construct used in transfection experiments. Bottom, representation of constructs used in (<i>C</i>). (<i>C</i>) Western blot analyses of HA-tagged LGALS3BP (Lectin galactoside-binding soluble 3 binding protein), VEGFC (vascular endothelium growth factor), p53 (cellular tumor antigen p53), CDC42 (cell division cycle 42), BDKRB2 (bradykinin receptor), and SRSF1 (serine/arginine-rich splicing factor 1), and their respective GFP-tagged alternative proteins using anti-HA and anti-GFP antibodies (top panels). Bottom panels show the cellular distribution of alternative proteins by confocal fluorescence microscopy (differential interference contrast and Hoechst, left panels; GFP, right panels). Scale bar: 10 μm.</p

A database to predict AltORFs in human mRNAs.

<p>(<i>A</i>) A canonical mRNA and its possible AltORFs. The RefORF is the main protein coding ORF annotated in current nucleotide databases. An AltORF is a nucleotide region comprised between an AUG codon and a stop codon distinct from the RefORF and is predicted to encode an alternative protein. AltORFs may be localized in 5′ UTRs, overlapping the 5′UTR and the RefORF, overlapping the RefORF, overlapping the RefORF and the 3′UTR, or in the 3′UTR. (<i>B</i>) Representation of the database generation process. Distinct AltORFs number indicates the total number of predicted AltORFs that encode alternative proteins with unique amino acid sequences. Since an AltORF may be present in several transcripts, the total number of AltORFs in the transcriptome exceeds the number of distinct AltORFs. (<i>C</i>) Distribution in % of AltORFs. (<i>D,E</i>) Distribution of the number of predicted AltORFs per mRNA in (<i>D</i>) and the size distribution of AltORFs (empty bars, left and bottom scale) compared to RefORFs (grey bars, right and top scale) in (<i>E</i>). Boxes and arrows indicate the median.</p

Transfection of tagged constructs validate the expression and translation initiation site prediction of alternative proteins detected by LC-MS/MS.

<p>Top diagrams represent the constructs used to detect the co-expression of HA-tagged reference and GFP-tagged alternative proteins by western blot analyses of HeLa cell lysates. GFP is inserted before the alternative stop codon in frame with the AltORF. The black line represents a specific region of the endogenous mRNA. For AltORFs located in 5′UTRs of <i>ZNF83</i> and <i>SLC35A4</i>, the constructs do not contain the RefORF since the insertion of GFP may prevent the expression of the downstream RefORF. For AltORFs overlapping the 5′UTR and the RefORF (<i>IDH3B</i>), and for AltORFs overlapping the RefORF (<i>BDH2</i>, <i>NIPA1</i>, <i>SCARB2</i>), the HA tag was introduced before the GFP tag in frame with the RefORFs. Western blots show the co-expression of reference and corresponding alternative proteins in cell lysates with anti-HA and anti-GFP antibodies, respectively. The left and right lanes are cell lysates from cells expressing a construct with a normal alternative initiation AUG codon or with an inactivated alternative initiation AAG codon, respectively. NPTII, encoded in the expression plasmid, was used as a transfection control. Molecular weight markers in kDa are indicated on the right. Bottom panels show confocal/DIC images with the various cellular distributions of GFP-tagged alternative proteins. Nuclei were stained with Hoechst. Scale bar: 10 μm. * The reference protein was not detected due to the small size (<3 kDa) of the truncated HA-tagged reference IDH3B protein.</p