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1.
EMBO Mol Med ; 16(3): 475-505, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38360999

RESUMO

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 KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D 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-KrasG12D (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 KrasG12D-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 KrasG12D-dependent tumors.


Assuntos
Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Piperazinas , Tiazinas , Animais , Camundongos , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/genética , Caspase 3/genética , Caspase 3/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Grânulos de Estresse , Mutações Sintéticas Letais
2.
J Biol Chem ; 300(4): 106792, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38403249

RESUMO

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.


Assuntos
Proteínas de Transporte , Polaridade Celular , Proteínas Desgrenhadas , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas de Xenopus , Animais , Proteínas Desgrenhadas/metabolismo , Proteínas Desgrenhadas/genética , Humanos , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Xenopus laevis , Biossíntese de Proteínas
3.
EMBO Mol Med ; 15(11): e17570, 2023 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-37819151

RESUMO

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.


Assuntos
Leucemia Mieloide Aguda , Sinteninas , Animais , Camundongos , Sinteninas/genética , Sinteninas/metabolismo , Regulação para Baixo , Leucemia Mieloide Aguda/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Microambiente Tumoral
4.
BMC Biol ; 21(1): 164, 2023 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-37525144

RESUMO

BACKGROUND: Individual functional modifications shape the ability of wildlife populations to cope with anthropogenic environmental changes. But instead of adaptive response, human-altered environments can generate a succession of deleterious functional changes leading to the extinction of the population. To study how persistent anthropogenic changes impacted local species' population status, we characterised population structure, genetic diversity and individual response of gene expression in the tree frog Hyla orientalis along a gradient of radioactive contamination around the Chernobyl nuclear power plant. RESULTS: We detected lower effective population size in populations most exposed to ionizing radiation in the Chernobyl Exclusion Zone that is not compensated by migrations from surrounding areas. We also highlighted a decreased body condition of frogs living in the most contaminated area, a distinctive transcriptomics signature and stop-gained mutations in genes involved in energy metabolism. While the association with dose will remain correlational until further experiments, a body of evidence suggests the direct or indirect involvement of radiation exposure in these changes. CONCLUSIONS: Despite ongoing migration and lower total dose rates absorbed than at the time of the accident, our results demonstrate that Hyla orientalis specimens living in the Chernobyl Exclusion Zone are still undergoing deleterious changes, emphasizing the long-term impacts of the nuclear disaster.


Assuntos
Acidente Nuclear de Chernobyl , Animais , Humanos , Densidade Demográfica , Animais Selvagens , Radiação Ionizante , Anuros/genética
5.
EBioMedicine ; 92: 104634, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37257316

RESUMO

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.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Prognóstico , Medicina de Precisão , Proteômica , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Ubiquitinação , Neoplasias Pancreáticas
6.
Front Immunol ; 14: 1139123, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37006259

RESUMO

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.


Assuntos
Transdução de Sinais , Linfócitos T , Linfócitos T/metabolismo , Transdução de Sinais/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Mapas de Interação de Proteínas
7.
J Environ Radioact ; 261: 107141, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36878054

RESUMO

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.


Assuntos
Monitoramento de Radiação , Poluentes Químicos da Água , Animais , Peixe-Zebra/metabolismo , Transcriptoma , Trítio/metabolismo , Proteômica , Larva/metabolismo , Poluentes Químicos da Água/metabolismo
8.
Mol Ther ; 31(2): 471-486, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35965411

RESUMO

The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.


Assuntos
Oligonucleotídeos Antissenso , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Neoplasias de Próstata Resistentes à Castração/terapia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , RNA Mensageiro/uso terapêutico , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/uso terapêutico , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética
9.
FEBS J ; 290(6): 1563-1582, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36197115

RESUMO

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 (CyCyne ) 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 Ser114 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 CyCyne probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life cycle.


Assuntos
Antituberculosos , Proteínas de Bactérias , Hidrolases , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis , Compostos Organofosforados , Humanos , Antituberculosos/síntese química , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Macrófagos/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Tuberculose/tratamento farmacológico , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Compostos Organofosforados/química , Cristalografia por Raios X , Hidrolases/antagonistas & inibidores , Hidrolases/química , Simulação por Computador
10.
J Cell Sci ; 135(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35971817

RESUMO

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 LL5ß (also known as PHLDB2). LL5ß anchors microtubules at the cell cortex through its association with CLASP proteins to trigger focal adhesion disassembly. LL5ß 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-LL5ß 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 LL5ß, 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.


Assuntos
Proteínas Serina-Treonina Quinases , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Movimento Celular , Humanos , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Serina/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
11.
Sci Signal ; 15(745): eabg8191, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35917363

RESUMO

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.


Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Vesículas Extracelulares , Neoplasias Pancreáticas , Animais , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Vesículas Extracelulares/metabolismo , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas
12.
Commun Biol ; 5(1): 732, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35869257

RESUMO

Proteomic, cellular and biochemical analysis of the stress protein NUPR1 reveals that it binds to PARP1 into the nucleus and inhibits PARP1 activity in vitro. Mutations on residues Ala33 or Thr68 of NUPR1 or treatment with its inhibitor ZZW-115 inhibits this effect. PARylation induced by 5-fluorouracil (5-FU) treatment is strongly enhanced by ZZW-115 and associated with a decrease of NAD+/NADH ratio and rescued by the PARP inhibitor olaparib. Cell death induced by ZZW-115 treatment of pancreas cancer-derived cells is rescued by olaparib and improved with PARG inhibitor PDD00017273. The mitochondrial catastrophe induced by ZZW-115 treatment or by genetic inactivation of NUPR1 is associated to a hyperPARylation of the mitochondria, disorganization of the mitochondrial network, mitochondrial membrane potential decrease, and with increase of superoxide production, intracellular level of reactive oxygen species (ROS) and cytosolic levels of Ca2+. These features are rescued by olaparib or NAD+ precursor nicotinamide mononucleotide in a dose-dependent manner and partially by antioxidants treatments. In conclusion, inactivation of NUPR1 induces a hyperPARylation, which in turn, induces a mitochondrial catastrophe and consequently a cell death through a non-canonical Parthanatos, since apoptosis inducing-factor (AIF) is not translocated out of the mitochondria.


Assuntos
NAD , Tiazinas , Morte Celular , NAD/metabolismo , Piperazinas , Proteômica
13.
EMBO J ; 41(9): e110466, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35307861

RESUMO

Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that ß-hydroxybutyrate (ßOHB) is an alternative cell-intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG-CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage-independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while ßOHB stimulates metastatic dissemination to the liver. These findings suggest that ßOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.


Assuntos
Corpos Cetônicos , Neoplasias Pancreáticas , Ácido 3-Hidroxibutírico/metabolismo , Animais , Corpos Cetônicos/metabolismo , Camundongos , Oxo-Ácido-Liases , Pâncreas/metabolismo
14.
Nucleic Acids Res ; 50(5): 2667-2680, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35166826

RESUMO

The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSß (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSß and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA , Endonucleases , Proteína 2 Homóloga a MutS , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína 2 Homóloga a MutS/metabolismo
15.
EMBO Mol Med ; 14(2): e11814, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34957688

RESUMO

Resistance to BRAF/MEK inhibitor therapy in BRAFV600 -mutated advanced melanoma remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapy; however, the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MMDR) in response to BRAFV600 pathway inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MMDR is mediated by drug-induced linear clustering of phosphorylated DDR1 and DDR2, two tyrosine kinase collagen receptors. Depletion and pharmacological targeting of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF-targeted therapy. In xenografts, targeting DDR with imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling, and delays tumor relapse. Mechanistically, DDR-dependent MMDR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. These findings reveal a novel role for a collagen-rich matrix and DDR in tumor cell adaptation and resistance. They also provide important insights into environment-mediated drug resistance and a preclinical rationale for targeting DDR signaling in combination with targeted therapy in melanoma.


Assuntos
Receptor com Domínio Discoidina 1 , Receptor com Domínio Discoidina 2 , Melanoma , Humanos , Melanoma/patologia , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas B-raf , Receptores Mitogênicos/química , Microambiente Tumoral
16.
J Cell Biol ; 220(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34705028

RESUMO

iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Proteínas Repressoras/metabolismo , Fuso Acromático/metabolismo , Motivos de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosina Tipo I/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Proteínas Repressoras/química
17.
Sci Adv ; 7(39): eabc7371, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34559557

RESUMO

Ubiquitin-fold modifier 1 (UFM1) is involved in neural and erythroid development, yet its biological roles in these processes are unknown. Here, we generated zebrafish models deficient in Ufm1 and Ufl1 that exhibited telomere shortening associated with developmental delay, impaired hematopoiesis and premature aging. We further report that HeLa cells lacking UFL1 have instability of telomeres replicated by leading-strand synthesis. We uncover that MRE11 UFMylation is necessary for the recruitment of the phosphatase PP1-α leading to dephosphorylation of NBS1. In the absence of UFMylation, NBS1 remains phosphorylated, thereby reducing MRN recruitment to telomeres. The absence of MRN at telomeres favors the formation of the TRF2-Apollo/SNM1 complex consistent with the loss of leading telomeres. These results suggest that MRE11-UFMylation may serve as module to recruit PP1-α. Last, zebrafish expressing Mre11 that cannot be UFMylated phenocopy Ufm1-deficient zebrafish, demonstrating that UFMylation of MRE11 is a previously undescribed evolutionarily conserved mechanisms regulating telomere length.

18.
Biomedicines ; 9(7)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34356858

RESUMO

The tumor suppressor menin has dual functions, acting either as a tumor suppressor or as an oncogene/oncoprotein, depending on the oncological context. Triple-negative breast cancer (TNBC) is characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2/HER2) and is often a basal-like breast cancer. TNBC is associated with a dismal prognosis and an insufficient response to chemotherapies. Previously, menin was shown to play a proliferative role in ER-positive breast cancer; however, the functions of menin in TNBC remain unknown. Here, we have demonstrated that menin is expressed in various TNBC subtypes with the strongest expression in the TNBC Hs 578T cells. The depletion of menin by an antisense oligonucleotide (ASO) inhibits cell proliferation, enhances apoptosis in Hs 578T cells, highlighting the oncogenic functions of menin in this TNBC model. ASO-based menin silencing also delays the tumor progression of TNBC xenografts. Analysis of the menin interactome suggests that menin could drive TNBC tumorigenesis through the regulation of MLL/KMT2A-driven transcriptional activity, mRNA 3'-end processing and apoptosis. The study provides a rationale behind the use of ASO-based therapy, targeting menin in monotherapy or in combination with chemo or PARP inhibitors for menin-positive TNBC treatments.

19.
J Cell Sci ; 134(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34350965

RESUMO

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.


Assuntos
Citoesqueleto , Septinas , Actinas , Animais , Citoesqueleto/metabolismo , Humanos , Mamíferos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Septinas/genética , Septinas/metabolismo
20.
Mol Microbiol ; 116(4): 1099-1112, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34411374

RESUMO

Penicillin-binding proteins (PBPs) are crucial enzymes of peptidoglycan assembly and targets of ß-lactam antibiotics. However, little is known about their regulation. Recently, membrane proteins were shown to regulate the bifunctional transpeptidases/glycosyltransferases aPBPs in some bacteria. However, up to now, regulators of monofunctional transpeptidases bPBPs have yet to be revealed. Here, we propose that TseB could be such a PBP regulator. This membrane protein was previously found to suppress tetracycline sensitivity of a Bacillus subtilis strain deleted for ezrA, a gene encoding a regulator of septation ring formation. In this study, we show that TseB is required for B. subtilis normal cell shape, tseB mutant cells being shorter and wider than wild-type cells. We observed that TseB interacts with PBP2A, a monofunctional transpeptidase. While TseB is not required for PBP2A activity, stability, and localization, we show that the overproduction of PBP2A is deleterious in the absence of TseB. In addition, we showed that TseB is necessary not only for efficient cell wall elongation during exponential phase but also during spore outgrowth, as it was also observed for PBP2A. Altogether, our results suggest that TseB is a new member of the elongasome that regulates PBP2A function during cell elongation and spore germination.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Bacillus subtilis/citologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação
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