Detection of wildtype Merkel cell polyomavirus genomic sequence and VP1 transcription in a subset of Merkel cell carcinoma.

AIMS: Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV). Characteristic for these virus-positive (VP) MCC is MCPyV integration into the host genome and truncation of the viral oncogene Large T antigen (LT), with full-length LT expression considered as incompatible with MCC growth. Genetic analysis of a VP-MCC/trichoblastoma combined tumour demonstrated that virus-driven MCC can arise from an epithelial cell. Here we describe two further cases of VP-MCC combined with an adnexal tumour, i.e. one trichoblastoma and one poroma. METHODS AND RESULTS: Whole-genome sequencing of MCC/trichoblastoma again provided evidence of a trichoblastoma-derived MCC. Although an MCC-typical LT-truncating mutation was detected, we could not determine an integration site and we additionally detected a wildtype sequence encoding full-length LT. Similarly, Sanger sequencing of the combined MCC/poroma revealed coding sequences for both truncated and full-length LT. Moreover, in situ RNA hybridization demonstrated expression of a late region mRNA encoding the viral capsid protein VP1 in both combined as well as in a few cases of pure MCC. CONCLUSION: The data presented here suggest the presence of wildtype MCPyV genomes and VP1 transcription in a subset of MCC.

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.

First Identification of a Large Set of Serine Hydrolases by Activity-Based Protein Profiling in Dibutyl Phthalate-Exposed Zebrafish Larvae.

Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposure to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120 h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120 h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches, such as ABPP, can, therefore, be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology.

Comprehensive Analysis of Transcript and Protein Relative Abundance During Blood Stages of Plasmodium falciparum Infection.

Plasmodium falciparum is the main causative agent of human malaria. During the intraerythrocytic development cycle, the P. falciparum morphology changes dramatically from circulating young rings to sequestered mature trophozoites and schizonts. Sequestered forms contribute to the pathophysiology of severe malaria as the infected erythrocytes obstruct the microvascular flow in deep organs and induce local inflammation. However, the sequestration mechanism limits the access to the corresponding parasitic form in the clinical samples from patients infected with P. falciparum. To complement this deficiency, we aimed to evaluate the relevance of mRNA study as a proxy of protein expression in sequestered parasites. To do so, we conducted a proteotranscriptomic analysis using five independent P. falciparum laboratory strain samples. RNA sequencing was performed, and the mRNA expression level was assessed on circulating ring-stage parasites. The level of protein expression were measured by LC-MS/MS on the corresponding sequestered mature forms after 18-24 h of maturation. Overall, our results showed a strong transcriptome/transcriptome and a very strong proteome/proteome correlation between samples. Moreover, positive correlations of mRNA and protein expression levels were found between ring-stage transcriptomes and mature form proteomes. However, twice more transcripts were identified at the ring stage than proteins at the mature trophozoite stage. A high level of transcript expression did not guarantee the detection of the corresponding protein. Finally, we pointed out discrepancies at the individual gene level. Taken together, our results show that transcript and protein expressions are overall correlated. However, mRNA abundance is not a perfect proxy of protein expression at the individual level. Importantly, our study shows limitations of the "blind" use of RNA-seq and the importance of multiomics approaches for P. falciparum blood stage study in clinical samples.

Proteome analysis of formalin-fixed paraffin-embedded colorectal adenomas reveals the heterogeneous nature of traditional serrated adenomas compared to other colorectal adenomas.

Traditional serrated adenoma (TSA) remains the least understood of all the colorectal adenomas, although these lesions have been associated with a significant cancer risk, twice that of the conventional adenoma (CAD) and of the sessile serrated adenoma (SSA/P). This study was performed to investigate the proteomic profiles of the different colorectal adenomas to better understand the pathogenesis of TSA. We performed a global quantitative proteome analysis using the label-free quantification (LFQ) method on 44 colorectal adenoma (12 TSAs, 15 CADs, and 17 SSA/Ps) and 17 normal colonic mucosa samples, archived as formalin-fixed paraffin-embedded blocks. Unsupervised consensus hierarchical clustering applied to the whole proteomic profile of the 44 colorectal adenomas identified four subtypes: C1 and C2 were well-individualized clusters composed of all the CADs (15/15) and most of the SSA/Ps (13/17), respectively. This is consistent with the fact that CADs and SSA/Ps are homogeneous and distinct colorectal adenoma entities. In contrast, TSAs were subdivided into C3 and C4 clusters, consistent with the more heterogeneous entity of TSA at the morphologic and molecular levels. Comparison of the proteome expression profile between the adenoma subtypes and normal colonic mucosa further confirmed the heterogeneous nature of TSAs, which overlapped either on CADs or SSA/Ps, whereas CADs and SSAs formed homogeneous and distinct entities. Furthermore, we identified LEFTY1 a new potential marker for TSAs that may be relevant for the pathogenesis of TSA. LEFTY1 is an inhibitor of the Nodal/TGFß pathway, which we found to be one of the most overexpressed proteins specifically in TSAs. This finding was confirmed by immunohistochemistry. Our study confirms that CADs and SSA/Ps form homogeneous and distinct colorectal adenoma entities, whereas TSAs are a heterogeneous entity and may arise from either SSA/Ps or from normal mucosa evolving through a process related to the CAD pathway. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

GPR50-Ctail cleavage and nuclear translocation: a new signal transduction mode for G protein-coupled receptors.

Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.

Identification of hemicatenane-specific binding proteins by fractionation of HeLa nuclei extracts.

DNA hemicatenanes (HCs) are four-way junctions in which one strand of a double-stranded helix is catenated with one strand of another double-stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, we sought to purify proteins capable of binding specifically HCs by fractionating nuclear extracts from HeLa cells. This approach identified three RNA-binding proteins: the Tudor-staphylococcal nuclease domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the paraspeckle protein component 1 and the splicing factor proline- and glutamine-rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in Escherichia coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited specificity for HCs, opening the interesting possibility of a link between the basic transcription machinery and HC structures via SND1.

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic.

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

Histamine releasing factor and elongation factor 1 alpha secreted via malaria parasites extracellular vesicles promote immune evasion by inhibiting specific T cell responses.

Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood stage-derived EVs of the proliferative response of CD4+ T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin-specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1α (EF-1α) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF- and EF-1α-deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLCγ1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF-1α alone or in combination with HRF conferred a long-lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei-derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV-mediated immune suppression in malaria-infected individuals.

Pairing MCL-1 inhibition with venetoclax improves therapeutic efficiency of BH3-mimetics in AML.

OBJECTIVES: Venetoclax combined with hypomethylating agents is a new therapeutic strategy frequently used for treating AML patients who are not eligible for conventional chemotherapy. However, high response rates are heterogeneous due to different mechanisms mediating resistance to venetoclax such as up-regulation of MCL-1 expression. We thus tested the anti-leukemic activity of S63845, a specific MCL-1 inhibitor. METHODS: Apoptosis induces by S63845 with or without venetoclax was evaluated in primary AML samples and in AML cell lines co-cultured or not with bone marrow (BM) mesenchymal stromal cells. Sensitivity of leukemic cells to S63845 was correlated to the expression level of BCL-2, MCL-1, and BCL-XL determined by Western Blot and mass spectrometry-based proteomics. RESULTS: We observed that even if MCL-1 expression is weak compared to BCL-2, S63845 induces apoptosis of AML cells and strongly synergizes with venetoclax. Furthermore, AML cells resistant to venetoclax are highly sensitive to S63845. Interestingly, the synergistic effect of S63845 toward venetoclax-mediated apoptosis of AML cells is still observed in a context of interaction with the BM microenvironment that intrinsically mediates resistance to BCL2 inhibition. CONCLUSION: These results are therefore of great relevance for clinicians as they provide the rational for combining BCL-2 and MCL-1 inhibition in AML.

PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells.

RNA-binding proteins (RBPs) have emerged as important regulators of invertebrate adult stem cells, but their activities remain poorly appreciated in mammals. Using a short hairpin RNA strategy, we demonstrate here that the 2 mammalian RBPs, PUMILIO (PUM)1 and PUM2, members of the PUF family of posttranscriptional regulators, are essential for hematopoietic stem/progenitor cell (HSPC) proliferation and survival in vitro and in vivo upon reconstitution assays. Moreover, we found that PUM1/2 sustain myeloid leukemic cell growth. Through a proteomic approach, we identified the FOXP1 transcription factor as a new target of PUM1/2. Contrary to its canonical repressive activity, PUM1/2 rather promote FOXP1 expression by a direct binding to 2 canonical PUM responsive elements present in the FOXP1-3' untranslated region (UTR). Expression of FOXP1 strongly correlates with PUM1 and PUM2 levels in primary HSPCs and myeloid leukemia cells. We demonstrate that FOXP1 by itself supports HSPC and leukemic cell growth, thus mimicking PUM activities. Mechanistically, FOXP1 represses the expression of the p21-CIP1 and p27-KIP1 cell cycle inhibitors. Enforced FOXP1 expression reverses shPUM antiproliferative and proapoptotic activities. Altogether, our results reveal a novel regulatory pathway, underscoring a previously unknown and interconnected key role of PUM1/2 and FOXP1 in regulating normal HSPC and leukemic cell growth.

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

Human Immunoglobulin Heavy Gamma Chain Polymorphisms: Molecular Confirmation Of Proteomic Assessment.

Immunoglobulin G (IgG) proteins are known for the huge diversity of the variable domains of their heavy and light chains, aimed at protecting each individual against foreign antigens. The IgG also harbor specific polymorphism concentrated in the CH2 and CH3-CHS constant regions located on the Fc fragment of their heavy chains. But this individual particularity relies only on a few amino acids among which some could make accurate sequence determination a challenge for mass spectrometry-based techniques.The purpose of the study was to bring a molecular validation of proteomic results by the sequencing of encoding DNA fragments. It was performed using ten individual samples (DNA and sera) selected on the basis of their Gm (gamma marker) allotype polymorphism in order to cover the main immunoglobulin heavy gamma (IGHG) gene diversity. Gm allotypes, reflecting part of this diversity, were determined by a serological method. On its side, the IGH locus comprises four functional IGHG genes totalizing 34 alleles and encoding the four IgG subclasses. The genomic study focused on the nucleotide polymorphism of the CH2 and CH3-CHS exons and of the intron. Despite strong sequence identity, four pairs of specific gene amplification primers could be designed. Additional primers were identified to perform the subsequent sequencing. The nucleotide sequences obtained were first assigned to a specific IGHG gene, and then IGHG alleles were deduced using a home-made decision tree reading of the nucleotide sequences. IGHG amino acid (AA) alleles were determined by mass spectrometry. Identical results were found at 95% between alleles identified by proteomics and those deduced from genomics. These results validate the proteomic approach which could be used for diagnostic purposes, namely for a mother-and-child differential IGHG detection in a context of suspicion of congenital infection.

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages.

Viruses often interfere with the DNA damage response to better replicate in their hosts. The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) protein has been reported to modulate the activity of the DNA repair structure-specific endonuclease subunit (SLX4) complex and to promote cell cycle arrest. Vpr also interferes with the base-excision repair pathway by antagonizing the uracil DNA glycosylase (Ung2) enzyme. Using an unbiased quantitative proteomic screen, we report that Vpr down-regulates helicase-like transcription factor (HLTF), a DNA translocase involved in the repair of damaged replication forks. Vpr subverts the DDB1-cullin4-associated-factor 1 (DCAF1) adaptor of the Cul4A ubiquitin ligase to trigger proteasomal degradation of HLTF. This event takes place rapidly after Vpr delivery to cells, before and independently of Vpr-mediated G2 arrest. HLTF is degraded in lymphocytic cells and macrophages infected with Vpr-expressing HIV-1. Our results reveal a previously unidentified strategy for HIV-1 to antagonize DNA repair in host cells.

Impact of hydroxycarbamide and interferon-α on red cell adhesion and membrane protein expression in polycythemia vera.

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.

Pharmacological targeting of apelin impairs glioblastoma growth.

Glioblastoma are highly aggressive brain tumours that are associated with an extremely poor prognosis. Within these tumours exists a subpopulation of highly plastic self-renewing cancer cells that retain the ability to expand ex vivo as tumourspheres, induce tumour growth in mice, and have been implicated in radio- and chemo-resistance. Although their identity and fate are regulated by external cues emanating from endothelial cells, the nature of such signals remains unknown. Here, we used a mass spectrometry proteomic approach to characterize the factors released by brain endothelial cells. We report the identification of the vasoactive peptide apelin as a central regulator for endothelial-mediated maintenance of glioblastoma patient-derived cells with stem-like properties. Genetic and pharmacological targeting of apelin cognate receptor abrogates apelin- and endothelial-mediated expansion of glioblastoma patient-derived cells with stem-like properties in vitro and suppresses tumour growth in vivo. Functionally, selective competitive antagonists of apelin receptor were shown to be safe and effective in reducing tumour expansion and lengthening the survival of intracranially xenografted mice. Therefore, the apelin/apelin receptor signalling nexus may operate as a paracrine signal that sustains tumour cell expansion and progression, suggesting that apelin is a druggable factor in glioblastoma.

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways.

DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA-protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity.

Identification of brain substrates of transglutaminase by functional proteomics supports its role in neurodegenerative diseases.

Transglutaminases are calcium-dependent enzymes that catalyze the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific glutamine and lysine residues. Some transglutaminase isoforms are present in the brain and are thought to participate in the protein aggregation characteristic of neurological diseases such as Huntington, Alzheimer's and Parkinson's disease. We have developed a functional proteomics strategy in which biotinylated amine-donor and amine-acceptor probes were used to identify the transglutaminase substrates present in brain. Bioinformatics analyses revealed that most of the 166 brain substrates identified interacted with huntingtin, the amyloid precursor protein or α-synuclein and that neurological disease was the most significant canonical pathway associated with the substrates. The physiological relevance of the substrates identified by mass spectrometry was confirmed by the fact that three of them (actin, ß-tubulin and a neurofilament subunit) were polymerized in neuronal cells when cytosolic calcium concentration was raised. We also showed by in-situ immunolabeling that some of the substrates were part of the protein aggregates found in neurological diseases. These results strongly support the idea that the crosslinking activity of brain transglutaminase participates in the formation of the protein aggregates found in diseases of the central nervous system.

T-cell factor 4 and ß-catenin chromatin occupancies pattern zonal liver metabolism in mice.

UNLABELLED: ß-catenin signaling can be both a physiological and oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20%-40% of hepatocellular carcinomas (HCCs) with specific metabolic features. We decipher the molecular determinants of ß-catenin-dependent zonal transcription using mice with ß-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T-cell factor (Tcf)-4 and ß-catenin, transcriptome, and metabolome. We find that Tcf-4 DNA bindings depend on ß-catenin. Tcf-4/ß-catenin binds Wnt-responsive elements preferentially around ß-catenin-induced genes. In contrast, genes repressed by ß-catenin bind Tcf-4 on hepatocyte nuclear factor 4 (Hnf-4)-responsive elements. ß-Catenin, Tcf-4, and Hnf-4α interact, dictating ß-catenin transcription, which is antagonistic to that elicited by Hnf-4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by ß-catenin, partly through xenobiotic nuclear receptors. CONCLUSIONS: ß-catenin patterns the zonal liver together with Tcf-4, Hnf-4α, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with ß-catenin mutational activation.