Joëlle Vinh

A Thiol Peroxidase Is an H2O2 Receptor and Redox-Transducer in Gene Activation

The Yap1 transcription factor regulates hydroperoxide homeostasis in S. cerevisiae. Yap1 is activated by oxidation when hydroperoxide levels increase. We show that Yap1 is not directly oxidized by hydroperoxide. We identified the glutathione peroxidase (GPx)-like enzyme Gpx3 as a second component of the pathway, serving the role of sensor and transducer of the hydroperoxide signal to Yap1. When oxidized by H2O2, Gpx3 Cys36 bridges Yap1 Cys598 by a disulfide bond. This intermolecular disulfide bond is then resolved into a Yap1 intramolecular disulfide bond, the activated form of the regulator. Thioredoxin turns off the pathway by reducing both sensor and regulator. These data reveal a redox-signaling function for a GPx-like enzyme and elucidate a eukaryotic hydroperoxide-sensing mechanism. Gpx3 is thus a hydroperoxide receptor and redox-transducer.

Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5′ phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-β-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation.

The DEAD‐box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli

Ribosome assembly in Escherichia coli involves 54 ribosomal proteins and three RNAs. Whereas functional subunits can be reconstituted in vitro from the isolated components, this process requires long incubation times and high temperatures compared with the in vivo situation, suggesting that non‐ribosomal factors facilitate assembly in vivo. Here, we show that SrmB, a putative DEAD‐box RNA helicase, is involved in ribosome assembly. The deletion of the srmB gene causes a slow‐growth phenotype at low temperature. Polysome profile analyses of the corresponding cells reveal a deficit in free 50S ribosomal subunits and the accumulation of a new particle sedimenting around 40S. Analysis of the ribosomal RNA and protein contents of the 40S particle indicates that it represents a large subunit that is incompletely assembled. In particular, it lacks L13, one of the five ribosomal proteins that are essential for the early assembly step in vitro. Sucrose gradient fractionation also shows that, in wild‐type cells, SrmB associates with a pre50S particle. From our results, we propose that SrmB is involved in an early step of 50S assembly that is necessary for the binding of L13. This step may consist of a structural rearrangement that, at low temperature, cannot occur without the assistance of this putative RNA helicase.

A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots

Aquaporins are channel proteins that facilitate the diffusion of water across cell membranes. The genome of Arabidopsis thaliana encodes 35 full-length aquaporin homologues. Thirteen of them belong to the plasma membrane intrinsic protein (PIP) subfamily and predominantly sit at the plasma membrane (PM). In the present work we combine separations of membrane proteins (by one- and two-dimensional gel electrophoresis) with identification by MS (matrix-assisted laser-desorption ionization-time-of-flight and electrospray-ionization tandem MS) to take an inventory of aquaporin isoforms expressed in the PM of Arabidopsis thaliana roots. Our analysis provides direct evidence for the expression of five PIPs (PIP1;1, PIP1;5, PIP2;1, PIP2;2 and PIP2;7) in the root PM and suggests the presence of at least three other PIP isoforms. In addition, we show that the same PIP isoform can be present under several forms with distinct isoelectric points. More specifically, we identify phosphorylated aquaporins in the PIP1 and PIP2 subgroups and suggest the existence of other post-translational modifications. Their identification should provide clues to reveal novel molecular mechanisms for aquaporin regulation.

Abnormal recruitment of extracellular matrix proteins by excess Notch3ECD: a new pathomechanism in CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.

RACK1 Controls IRES-Mediated Translation of Viruses

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

Methylation of aquaporins in plant plasma membrane

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.

Distinct proteomic features of two fibrogenic liver cell populations: Hepatic stellate cells and portal myofibroblasts

In chronic liver diseases, the accumulation of extracellular matrix leading to fibrosis is caused by myofibroblasts, the origins of which are debatable. We performed a comparative proteomic study to identify markers and gain insight into distinct functions of myofibroblasts derived either from hepatic stellate cells (HSCs) or from portal mesenchymal cells. After isolation from normal liver and culture in similar conditions, myofibroblastic HSCs (MF‐HSCs) presented enlarged cytoplasms whereas portal myofibroblasts (PMFs) were more proliferative, and formed more stress fibers. The two cell types were subjected to comparative analyses by 2‐D MS/MS. Six proteins were overexpressed in PMFs, with myofibroblast‐related typical functions. Among them, cofilin‐1 showed the greatest difference in expression and a lower pI than expected. Immunoblot demonstrated higher levels of phosphorylation, a modification of the protein implicated in stress fiber formation. Eleven proteins, mostly involved in stress response, were overexpressed in MF‐HSCs. Cytoglobin had the highest level of overexpression, as confirmed by reverse transcription quantitative real‐time PCR, immunoblot and immunocytochemical analyses. These results identify cytoglobin as the best marker for distinguishing MF‐HSCs from PMFs and suggest different functions for the two cell populations in the liver wound healing response, with a prominent role for PMFs in scar formation.

Proteome screens for Cys residues oxidation: the redoxome.

The oxidation of the cysteine (Cys) residue to sulfenic (-S-OH), disulfide (-S-S-), or S-nitroso (S-NO) forms are thought to be a posttranslational modifications that regulate protein function. However, despite a few solid examples of its occurrence, thiol-redox regulation of protein function is still debated and often seen as an exotic phenomenon. A systematic and exhaustive characterization of all oxidized Cys residues, an experimental approach called redox proteomics or redoxome analysis, should help establish the physiological scope of Cys residue oxidation and give clues to its mechanisms. Redox proteomics still remains a technical challenge, mainly because of the labile nature of thiol-redox reactions and the lack of tools to directly detect the modified residues. Here we consider recent technical advances in redox proteomics, focusing on a gel-based fluorescent method and on the shotgun OxICAT technique.

Role of the Tau N-terminal region in microtubule stabilization revealed by new endogenous truncated forms

Tau is a central player in Alzheimers disease (AD) and related Tauopathies, where it is found as aggregates in degenerating neurons. Abnormal post-translational modifications, such as truncation, are likely involved in the pathological process. A major step forward in understanding the role of Tau truncation would be to identify the precise cleavage sites of the several truncated Tau fragments that are observed until now in AD brains, especially those truncated at the N-terminus, which are less characterized than those truncated at the C-terminus. Here, we optimized a proteomics approach and succeeded in identifying a number of new N-terminally truncated Tau species from the human brain. We initiated cell-based functional studies by analyzing the biochemical characteristics of two N-terminally truncated Tau species starting at residues Met11 and Gln124 respectively. Our results show, interestingly, that the Gln124-Tau fragment displays a stronger ability to bind and stabilize microtubules, suggesting that the Tau N-terminal domain could play a direct role in the regulation of microtubule stabilization. Future studies based on our new N-terminally truncated-Tau species should improve our knowledge of the role of truncation in Tau biology as well as in the AD pathological process.