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Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (?-TuRC) via Galectin-3-binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression. [ABSTRACT FROM AUTHOR]

In the environment, populations are exposed to different kinds of ionizing radiation. Little is known about their modes of action on non-human species, and whether or not they are similar for alpha, beta and gamma radiations, considered as the reference. In this context, tritium effects (beta emitter) under the form of tritiated water (HTO) were investigated in zebrafish, a common model in toxicology and ecotoxicology with a fully sequenced genome. Experiments were conducted on early life stages, considered to be highly sensitive to pollutants, by exposing eggs to 0.4 mGy/h of HTO until 10 days post fertilization. Tritium internalization was quantified, and effects were investigated using a combined approach of transcriptomic and proteomic analyses. Results highlighted similarities in the biological pathways affected by HTO by both techniques, such as defence response, muscle integrity and contraction, and potential visual alterations. These results correlated well with previous data obtained on earlier developmental stages (1 and 4 dpf). Interestingly, HTO effects were partly overlapping those obtained after gamma irradiation, underlying potential common modes of action. This study, therefore, brought a body of evidence on the effects of HTO observed at the molecular level on zebrafish larvae. Further studies could investigate if the effects persist in adult organisms. • Zebrafish eggs exposed for 7 and 10 days to HTO at 0.4 mGy/h. • Combination of transcriptomic and proteomic analysis was used to assess HTO effects. • Common modulation of genes and proteins was highlighted for several functions. • More pathways were affected by HTO in 10 dpf larvae compared to 7 dpf larvae. • Results align with previous findings on early stages exposed to HTO or gamma irradiation. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) remains a major cause of cancer related-death in developed countries. The mortality risk is correlated with the stage of CRC determined at the primary diagnosis and early diagnosis is associated with enhanced survival rate. Currently, only faecal occult blood tests are used to screen for CRC. Consequently, there is an incentive to identify specific markers of CRC. We used quantitative proteomic analysis of serum samples to characterize protein profiles in adenoma, CRC and healthy control samples. We identified 89 distinct proteins modulated between normal, colorectal adenoma and carcinoma patients. This list emphasizes proteins involved in enzyme regulator activities and in particular the serpin family. In serum samples, protein profiles of three members of the serpin family (SERPINA1, SERPINA3 and SERPINC1) were confirmed by ELISA assays. We obtained sensitivity/specificity values of 95%/95% for both SERPINA1 and SERPINC1, and 95%/55% for SERPINA3. This study supports the idea that serum proteins can discriminate adenoma and CRC patients from unaffected patients and reveals a panel of regulated proteins that might be useful for selecting patients for colonoscopy. By evaluating SERPINA1, SERPINA3 and SERPINC1, we highlight the potential role of the serpin family during the development and progression of CRC. Significance Colorectal cancer (CRC) remains a major cause of cancer mortality throughout the world. However, very few CRC biomarkers have satisfactory sensitivity and specificity in clinical practice. To the best of our knowledge our study is the first to profile sera proteomes between adenoma, CRC and healthy patients. We report a comprehensive list of proteins that may be used as early diagnostic biomarkers of CRC. It is noteworthy that 17% of these modulated proteins have been previously described as candidate biomarkers in CRC. Enzyme regulator activity was found to be the main molecular function among these proteins and, in particular, there was an enrichment of members of the serpin family. The subsequent verification on a new cohort by ELISA demonstrates that these serpins could be useful to discriminate healthy from colorectal carcinoma patients with a high sensitivity and specificity. The combination of these biomarkers should increase predictive powers of CRC diagnosis. The remaining candidates form a reserve for further evaluation of additional biomarkers for CRC diagnosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] We studied the early liver cell alterations due to CCHFV infection. [•] Protein profile of infected HepG2 cells was analyzed using 2D-DIGE and iTRAQ. [•] CCHFV modulated pathways including oxidative stress, apoptosis and virus entry. [•] We gave data on virus–host interactions potentially leading to CCHV pathogenesis. [•] We proposed possible targets for antiviral research. [Copyright &y& Elsevier]

Abstract: When in the vicinity of astrocytes, brain capillary endothelial cells (BCECs) develop the characteristic structural and functional features of the blood-brain barrier (BBB). The latter has low cellular permeability and restricts various compounds from entering the brain. We recently reported that the cytoskeleton-related proteins actin, gelsolin and filamin-A undergo the largest quantitative changes in bovine BCECs after re-induction of BBB functions by co-culture with glial cells. In the present study, we used an in-depth, proteomic approach to quantitatively compare differences in Triton-X-100-solubilized proteins from bovine BCECs with limited or re-induced BBB functions (i.e. cultured in the absence or presence of glial cells, respectively). The 81 protein spots of differing abundance were linked to 55 distinct genes. According to the Protein ANalysis THrough Evolutionary Relationships classification system and an Ingenuity Pathway Analysis, these quantitative changes mainly affected proteins involved in (i) cell structure and motility and (ii) protein metabolism and modification. The fold-changes affecting HSPB1, moesin and ANXA5 protein levels were confirmed by western blot analysis but were not accompanied by changes in the corresponding mRNA expression levels. Our results reveal that the bovine BCECs'' phenotype adaptation to variations in their environment involves the reorganization of the actin cytoskeleton. [Copyright &y& Elsevier]

The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease. [ABSTRACT FROM AUTHOR]

The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis, the etiological agent of cerebrospinal meningitis. crosses cellular barriers after adhering to host cells through type IV pili. Pilin glycosylation genes (pgl) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido-2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by "top-down" MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pili preparations originating from N. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacet-amido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glycer-amido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pgIBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) still remains the deadliest infectious disease worldwide with 1.5 million deaths in 2018, of which about 15% are attributed to resistant strains. Another significant example is Mycobacterium abscessus (M. abscessus), a nontuberculous mycobacteria (NTM) responsible for cutaneous and pulmonary infections, representing up to 95% of NTM infections in cystic fibrosis (CF) patients. M. abscessus is a new clinically relevant pathogen and is considered one of the most drug-resistant mycobacteria for which standardized chemotherapeutic regimens are still lacking. Together the emergence of M. tb and M. abscessus multi-drug resistant strains with ineffective and expensive therapeutics, have paved the way to the development of new classes of anti-mycobacterial agents offering additional therapeutic options. In this context, specific inhibitors of mycobacterial lipolytic enzymes represent novel and promising antibacterial molecules to address this challenging issue. The results highlighted here include a complete overview of the antibacterial activities, either in broth medium or inside infected macrophages, of two families of promising and potent anti-mycobacterial multi-target agents, i.e. oxadiazolone-core compounds (OX) and Cyclophostin & Cyclipostins analogs (CyC); the identification and biochemical validation of their effective targets (e.g., the antigen 85 complex and TesA playing key roles in mycolic acid metabolism) together with their respective crystal structures. To our knowledge, these are the first families of compounds able to target and impair replicating as well as intracellular bacteria. We are still impelled in deciphering their mode of action and finding new potential therapeutic targets against mycobacterial-related diseases. Image 1 • Overview of the antibacterial activities of lipolytic enzyme inhibitors. • Focus on two families of antimycobacterials with potent anti-lipolytic enzyme activity. • OX & CyC probes are attractive chemical tools to identify (Ser/Cys)-enzymes in mycobacteria. [ABSTRACT FROM AUTHOR]

Background: The epigenetic machinery is frequently altered in acute myeloid leukemia. Focusing on cytogenetically normal (CN) AML, we previously described an abnormal H3K27me3 enrichment covering 70 kb on the HIST1 cluster (6.p22) in CN-AML patient blasts. Here, we further investigate the molecular, functional, and prognosis significance of this epigenetic alteration named H3K27me3 HIST1 in NPM1-mutated (NPM1mut) CN-AML. Results: We found that three quarter of the NPM1mut CN-AML patients were H3K27me3 HIST1high. H3K27me3 HIST1high group of patients was associated with a favorable outcome independently of known molecular risk factors. In gene expression profiling, the H3K27me3 HIST1high mark was associated with lower expression of the histone genes HIST1H1D, HIST1H2BG, HIST1H2AE, and HIST1H3F and an upregulation of genes involved in myelomonocytic differentiation. Mass spectrometry analyses confirmed that the linker histone protein H1d, but not the other histone H1 subtypes, was downregulated in the H3K27me3 HIST1high group of patients. H1d knockdown primed ATRA-mediated differentiation of OCI-AML3 and U937 AML cell lines, as assessed on CD11b/CD11c markers, morphological and gene expression analyses. Conclusions: Our data suggest that NPM1mut AML prognosis depends on the epigenetic silencing of the HIST1 cluster and that, among the H3K27me3 silenced histone genes, HIST1H1D plays a role in AML blast differentiation. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) remains a major cause of cancer fatalities in developed countries. The risk of death is correlated to the stage of CRC during the primary diagnosis. Early diagnosis is closely associated with enhanced survival rate. We therefore investigated the AP-F13A1 as a potential protein marker of CRC. Methods: The protein expression of FXIII in 40 serum samples was evaluated by enzyme-linked immunosorbent assays. Additionally, targeted proteomic assays (LC-PRM) were used to evaluate the expression of the activation peptide of F13A1 (AP-F13A1) in a further 113 serum samples. Results were analyzed by the Wilcoxon test and receiver operating characteristic curves generated to assess statistical differences and diagnostic factors between CRC patients and controls. Results: AP-F13A1 was quantified in human serum samples using calibration curves with excellent linearity. AP-F13A1 was reduced in CRC patients using PRM assays from two distinct biobanks. The AUC for AP-F13A1 were 0.95 and 0.93. Sensitivity/specificity values for the two sets of patients were 75%/95% and 71%/95% respectively. Conclusion: We have presented the proof of principle that in vivo release of AP-F13A1 can be measured by PRM-based strategies in CRC serum samples. AP-F13A1 may be an effective serological biomarker as part of a screening program of CRC detection. [ABSTRACT FROM AUTHOR]

The metabolic process of purple sulphur bacteria's anoxygenic photosynthesis has been primarily studied in Allochromatium vinosum, a member of the Chromatiaceae family. However, the metabolic processes of purple sulphur bacteria from the Ectothiorhodospiraceae and Halorhodospiraceae families remain unexplored. We have analysed the proteome of Halorhodospira halophila, a member of the Halorhodospiraceae family, which was cultivated with various sulphur compounds. This analysis allowed us to reconstruct the first comprehensive sulphur-oxidative photosynthetic network for this family. Some members of the Ectothiorhodospiraceae family have been shown to use arsenite as a photosynthetic electron donor. Therefore, we analysed the proteome response of Halorhodospira halophila when grown under arsenite and sulphide conditions. Our analyses using ion chromatography-inductively coupled plasma mass spectrometry showed that thioarsenates are chemically formed under these conditions. However, they are more extensively generated and converted in the presence of bacteria, suggesting a biological process. Our quantitative proteomics revealed that the SoxAXYZB system, typically dedicated to thiosulphate oxidation, is overproduced under these growth conditions. Additionally, two electron carriers, cytochrome c 551 /c 5 and HiPIP III, are also overproduced. Electron paramagnetic resonance spectroscopy suggested that these transporters participate in the reduction of the photosynthetic Reaction Centre. These results support the idea of a chemically and biologically formed thioarsenate being oxidized by the Sox system, with cytochrome c 551 /c 5 and HiPIP III directing electrons towards the Reaction Centre., (© 2024 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

Drug development in oncology is highly challenging, with less than 5% success rate in clinical trials. This alarming figure points out the need to study in more details the multiple biological effects of drugs in specific contexts. Indeed, the comprehensive assessment of drug poly-pharmacology can provide insights into their therapeutic and adverse effects, to optimize their utilization and maximize the success rate of clinical trials. Recent technological advances have made possible in-depth investigation of drug poly-pharmacology. This review first highlights high-throughput methodologies that have been used to unveil new mechanisms of action of existing drugs. Then, we discuss how emerging chemo-proteomics strategies allow effectively dissecting the poly-pharmacology of drugs in an unsupervised manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative near-cognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

IMPRINTS-CETSA (Integrated Modulation of Protein Interaction States-Cellular Thermal Shift Assay) provides a highly resolved means to systematically study the interactions of proteins with other cellular components, including metabolites, nucleic acids and other proteins, at the proteome level, but no freely available and user-friendly data analysis software has been reported. Here, we report IMPRINTS.CETSA, an R package that provides the basic data processing framework for robust analysis of the IMPRINTS-CETSA data format, from preprocessing and normalization to visualization. We also report an accompanying R package, IMPRINTS.CETSA.app, which offers a user-friendly Shiny interface for analysis and interpretation of IMPRINTS-CETSA results, with seamless features such as functional enrichment and mapping to other databases at a single site. For the hit generation part, the diverse behaviors of protein modulations have been typically segregated with a two-measure scoring method, i.e. the abundance and thermal stability changes. We present a new algorithm to classify modulated proteins in IMPRINTS-CETSA experiments by a robust single-measure scoring. In this way, both the numerical changes and the statistical significances of the IMPRINTS information can be visualized on a single plot. The IMPRINTS.CETSA and IMPRINTS.CETSA.app R packages are freely available on GitHub at https://github.com/nkdailingyun/IMPRINTS.CETSA and https://github.com/mgerault/IMPRINTS.CETSA.app, respectively. IMPRINTS.CETSA.app is also available as an executable program at https://zenodo.org/records/10636134., (© The Author(s) 2024. Published by Oxford University Press.)

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The Kras G12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated Kras G12D . Mechanistically, Kras G12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-Kras G12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in Kras G12D -expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in Kras G12D -dependent tumors., (© 2024. The Author(s).)

Summary: DIAgui is an R package to simplify the processing of the report file from the DIA-NN software thanks to a Shiny application. It returns the quantification of either the precursors, the peptides, the proteins, or the genes thanks to the MaxLFQ algorithm. In addition, the latest version provides the Top3 and iBAQ quantification and the number of peptides used for the quantification. In the end, DIAgui produces ready-to-interpret files from the results of DIA mass spectrometry analysis and provides visualization and statistical tools that can be used in a user-friendly way., Availability and Implementation: Code and documentation are available on GitHub at https://github.com/marseille-proteomique/DIAgui. The package is written in R and also uses C++ code. A vignette shows its use in an R command line workflow., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

The crosstalk between cancer and stromal cells plays a critical role in tumor progression. Syntenin is a small scaffold protein involved in the regulation of intercellular communication that is emerging as a target for cancer therapy. Here, we show that certain aggressive forms of acute myeloid leukemia (AML) reduce the expression of syntenin in bone marrow stromal cells (BMSC). Stromal syntenin deficiency, in turn, generates a pro-tumoral microenvironment. From serial transplantations in mice and co-culture experiments, we conclude that syntenin-deficient BMSC stimulate AML aggressiveness by promoting AML cell survival and protein synthesis. This pro-tumoral activity is supported by increased expression of endoglin, a classical marker of BMSC, which in trans stimulates AML translational activity. In short, our study reveals a vicious signaling loop potentially at the heart of AML-stroma crosstalk and unsuspected tumor-suppressive effects of syntenin that need to be considered during systemic targeting of syntenin in cancer therapy., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Pancreatic ductal adenocarcinoma (PDAC) has been widely studied at multiomics level. However, little is known about its specific ubiquitination, a major post-translational modification (PTM). As PTMs regulate the final function of any gene, we decided to establish the ubiquitination profiles of 60 PDAC., Methods: We used specific proteomic tools to establish the ubiquitin dependent proteome (ubiquitinome) of frozen PDXs (Patients' derived xenographs). Then, we performed bioinformatics analysis to identify the possible associations of these ubiquitination profiles with tumour phenotype, patient survival and resistance to chemotherapies. Finally, we used proximity ligation assays (PLA) to detect and quantify the ubiquitination level of one identified marker., Findings: We identified 38 ubiquitination site profiles correlating with the transcriptomic phenotype of tumours and four had notable prognostic capabilities. Seventeen ubiquitination profiles displayed potential theranostic marker for gemcitabine, seven for 5-FU, six for oxaliplatin and thirteen for irinotecan. Using PLA, we confirmed the use of one ubiquitination profile as a drug-response marker, directly on paraffin embedded tissues, supporting the possible application of these biomarkers in the clinical setting., Interpretation: These findings bring new and important insights on the relationship between ubiquitination levels of proteins and different molecular and clinical features of PDAC patients. Markers identified in this study could have a potential application in clinical settings to help to predict response to chemotherapies thereby allowing the personalization of treatments., Funding: Fondation ARC (PJA 20181208270 and PGA 12021010002840_3562); INCa; Canceropôle PACA; DGOS; Amidex Foundation; Fondation de France; and INSERM., Competing Interests: Declaration of interests N.A.F., J.L.I. and N.J.D. have a pending patent entitled “Simple transcriptomic signatures to determine chemosensitivity for pancreatic ductal adenocarcinoma”. All other authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ruminski, Celis-Gutierrez, Jarmuzynski, Maturin, Audebert, Malissen, Camoin, Voisinne, Malissen and Roncagalli.)

A hallmark of Mycobacterium tuberculosis (M. tb), the aetiologic agent of tuberculosis, is its ability to metabolise host-derived lipids. However, the enzymes and mechanisms underlying such metabolism are still largely unknown. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogues, a new family of potent antimycobacterial molecules, react specifically and covalently with (Ser/Cys)-based enzymes mostly involved in bacterial lipid metabolism. Here, we report the synthesis of new CyC alkyne-containing inhibitors (CyC yne ) and their use for the direct fishing of target proteins in M. tb culture via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the capture and identification of a variety of enzymes, and many of them involved in lipid or steroid metabolisms. One of the captured enzymes, HsaD (Rv3569c), is required for the survival of M. tb within macrophages and is thus a potential therapeutic target. This prompted us to further explore and validate, through a combination of biochemical and structural approaches, the specificity of HsaD inhibition by the CyC analogues. We confirmed that the CyC bind covalently to the catalytic Ser 114 residue, leading to a total loss of enzyme activity. These data were supported by the X-ray structures of four HsaD-CyC complexes, obtained at resolutions between 1.6 and 2.6 Å. The identification of mycobacterial enzymes directly captured by the CyC yne probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life cycle., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Upregulation of the developmental Wnt planar cell polarity (Wnt/PCP) pathway is observed in many cancers and is associated with cancer development. We have recently shown that PRICKLE1, a core Wnt/PCP pathway component, is a marker of poor prognosis in triple-negative breast cancer (TNBC). PRICKLE1 is phosphorylated by the serine/threonine kinase MINK1 and contributes to TNBC cell motility and invasiveness. However, the identity of the substrates of MINK1 and the role of MINK1 enzymatic activity in this process remain to be addressed. We used a phosphoproteomic strategy to identify MINK1 substrates, including LL5β (also known as PHLDB2). LL5β anchors microtubules at the cell cortex through its association with CLASP proteins to trigger focal adhesion disassembly. LL5β is phosphorylated by MINK1, promoting its interaction with CLASP proteins. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in PRICKLE1-LL5β complex assembly and localization, as well as in cell migration. Analysis of gene expression data reveals that the concomitant upregulation of levels of mRNA encoding PRICKLE1 and LL5β, which are MINK1 substrates, is associated with poor metastasis-free survival in TNBC patients. Taken together, our results suggest that MINK1 may represent a potential target for treatment of TNBC., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

In pancreatic ductal adenocarcinoma (PDAC), signaling from stromal cells is implicated in metastatic progression. Tumor-stroma cross-talk is often mediated through extracellular vesicles (EVs). We previously reported that EVs derived from cancer-associated stromal fibroblasts (CAFs) that are abundant in annexin A6 (ANXA6 + EVs) support tumor cell aggressiveness in PDAC. Here, we found that the cell surface glycoprotein and tetraspanin CD9 is a key component of CAF-derived ANXA6 + EVs for mediating this cross-talk. CD9 was abundant on the surface of ANXA6 + CAFs isolated from patient PDAC samples and from various mouse models of PDAC. CD9 colocalized with CAF markers in the tumor stroma, and CD9 abundance correlated with tumor stage. Blocking CD9 impaired the uptake of ANXA6 + EVs into cultured PDAC cells. Signaling pathway arrays and further analyses revealed that the uptake of CD9 + ANXA6 + EVs induced mitogen-activated protein kinase (MAPK) pathway activity, cell migration, and epithelial-to-mesenchymal transition (EMT). Blocking either CD9 or p38 MAPK signaling impaired CD9 + ANXA6 + EV-induced cell migration and EMT in PDAC cells. Analysis of bioinformatic datasets indicated that CD9 abundance was an independent marker of poor prognosis in patients with PDAC. Our findings suggest that CD9-mediated stromal cell signaling promotes PDAC progression.

Endothelial-mesenchymal transition (EndMT) is an important source of cancer-associated fibroblasts (CAFs), which facilitates tumour progression. PDAC is characterised by abundant CAFs and tumour necrosis factor-α (TNF-α). Here, we show that TNF-α strongly induces human endothelial cells to undergo EndMT. Interestingly, TNF-α strongly downregulates the expression of the endothelial receptor TIE1, and reciprocally TIE1 overexpression partially prevents TNF-α-induced EndMT, suggesting that TNF-α acts, at least partially, through TIE1 regulation in this process. We also show that TNF-α-induced EndMT is reversible. Furthermore, TNF-α treatment of orthotopic mice resulted in an important increase in the stroma, including CAFs. Finally, secretome analysis identified TNFSF12, as a regulator that is also present in PDAC patients. With the aim of restoring normal angiogenesis and better access to drugs, our results support the development of therapies targeting CAFs or inducing the EndMT reversion process in PDAC.

Identification of protein networks becomes indispensable for determining the function of a given protein of interest. Some proteins harbor a PDZ binding motif (PDZBM) located at the carboxy-terminus end. This motif is necessary to recruit PDZ domain proteins which are involved in signaling, trafficking, and maintenance of cell architecture. In the present chapter, we present two complementary approaches (immunopurification and peptide-based purification procedures) followed by mass spectrometry analysis to identify PDZ domain proteins associated to a given protein of interest. As proof of example, we focus our attention on TANC1 which is a scaffold protein harboring a PDZBM at its carboxy-terminus. Using these two approaches, we identified several PDZ domain containing proteins. Some of them were found with both approaches, and some were specifically identified using peptide-based purification procedure. This exemplifies advantages and differences of both strategies to identify PDZ interactions.

Among the more than 160 PDZ containing proteins described in humans, the cytoplasmic scaffold Scribble stands out because of its essential role in many steps of cancer development and dissemination. Its fame has somehow blurred the importance of homologous proteins, Erbin and Lano, all belonging to the LRR and PDZ (LAP) protein family first described twenty years ago. In this review, we will retrace the history of LAP family protein research and draw attention to their contribution in cancer by detailing the features of its members at the structural and functional levels, and highlighting their shared-but also different-implication in the tumoral process.

The O-linked-N-acetyl-d-glucosaminylation (O-GlcNAcylation) modulates numerous aspects of cellular processes. Akin to phosphorylation, O-GlcNAcylation is highly dynamic, reversible, and responds rapidly to extracellular demand. Despite the absolute necessity to determine post-translational sites to fully understand the role of O-GlcNAcylation, it remains a high challenge for the major reason that unmodified proteins are in excess comparing to the O-GlcNAcylated ones. Based on a click chemistry approach, O-GlcNAcylated proteins were labelled with azido-GalNAc and coupled to agarose beads. The proteome extracted from C2C12 myotubes was submitted to an intensive fractionation prior to azide-alkyne click chemistry. This combination of fractionation and click chemistry is a powerful methodology to map O-GlcNAc sites; indeed, 342 proteins were identified through the identification of 620 peptides containing one or more O-GlcNAc sites. We localized O-GlcNAc sites on proteins involved in signalling pathways or in protein modification, as well as structural proteins. Considering the recent role of O-GlcNAcylation in the modulation of sarcomere morphometry and interaction between key structural protein, we focused on proteins involved in the cytoarchitecture of skeletal muscle cells. In particular, several O-GlcNAc sites were located into protein-protein interaction domains, suggesting that O-GlcNAcylation could be strongly involved in the organization and reorganization of sarcomere and myofibrils., Significance: O-GlcNAcylation is an atypical glycosylation involved in the regulation of almost all if not all cellular processes, but its precise role remains sometimes obscure because of the ignorance of the O-GlcNAc site localization; thus, it remains indispensable to precisely map the O-GlcNAcylated sites to fully understand the role of O-GlcNAcylation on a given protein. For this purpose, we combined extensive fractionation of skeletal muscle cells proteome with click chemistry to map O-GlcNAc sites without an a priori consideration. A total of 620 peptides containing one or more O-GlcNAc sites were identified; interestingly, several of them belong to low expressed proteins, in particular proteins involved in signalling pathways. We also focused on structural proteins in view of recent data supporting the role of O-GlcNAcylation in the modulation of sarcomere cytoarchitecture; importantly, some of the O-GlcNAc sites were mapped into protein-protein interaction domains, reinforcing the involvement of O-GlcNAcylation in the organization and reorganization of sarcomere, and in larger extent, of myofibrils., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Zearalenone (ZEA) is a mycotoxin produced by some Fusarium species. ZEA often occur as a contaminant in cereal grains and animal feeds. Human exposure occurs by ingestion of mycotoxin-contaminated products and can cause serious health problems. It was established that this mycotoxin have an hepato, haemato, immuno and genotoxic properties (Maaroufi et al., 1996; Lioi et al., 2004). While most ZEA toxic effects have been quite well investigated, more studies are required to elucidate its mechanisms of toxicity. In order to better understand the molecular mechanisms involved in ZEA toxicity, we used a proteomic approach, to assess the early changes in protein expression initiated by ZEA in HepG2 cells. Our results showed that, after 8h of exposure, cells were still viable and showed a significant change in a number of proteins involved in diverse cellular processes. These changes may provide the early affected functions and yield further insight into mechanisms underlying the involvement of mycotoxin-induced diseases., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The Wnt/beta-catenin signaling pathway has been increasingly implicated in liver development and physiology. Aberrant activation of this pathway is one of the major genetic events observed during the process of human HCC development. To gain insight into the mechanism underlying beta-catenin action in the liver, we conducted a quantitative differential proteomic analysis using 2-D DIGE combined with MS, in mice with liver-specific deletion of Apc resulting in acute activation of beta-catenin signaling (Apc(KOliv) mice). We identified 94 protein spots showing differential expression between mutant Apc(KOliv) and control mice, corresponding to 56 individual proteins. Most of the proteins identified were associated with metabolic pathways, such as ammonia and glucose metabolism. Our analysis showed an increase in lactate dehydrogenase activity together with a downregulation of two mitochondrial ATPase subunits (ATP5a1 and ATP5b). These observations indicate that beta-catenin signaling may induce a shift in the glucose metabolism from oxidative phosphorylation to glycolysis, known as the "Warburg effect". Imaging with (18)F-fluoro-2-deoxy-D-glucose-positron emission tomography suggests that the specific metabolic reprogramming induced by beta-catenin in the liver does not imply the first step of glycolysis. This observation may explain why some HCCs are difficult to assess by fluoro-2-deoxy-D-glucose-positron emission tomography imaging.