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1.
Cell ; 166(4): 935-949, 2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27477512

RESUMO

Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagia , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Proteína Huntingtina/metabolismo , Masculino , Camundongos , Doenças Neurodegenerativas/patologia , Agregados Proteicos , Dobramento de Proteína , Proteólise
2.
Nature ; 618(7965): 583-589, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37286596

RESUMO

Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut1. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.


Assuntos
Proteínas da Membrana Bacteriana Externa , Membrana Externa Bacteriana , Bacteroides thetaiotaomicron , Trato Gastrointestinal , Polissacarídeos , Humanos , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteroides thetaiotaomicron/enzimologia , Bacteroides thetaiotaomicron/metabolismo , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo
3.
EMBO J ; 41(23): e108970, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36281581

RESUMO

Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN-γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN-γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock-out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.


Assuntos
Infecções Bacterianas , Fagossomos , Ubiquitina-Proteína Ligases , Animais , Camundongos , Infecções Bacterianas/metabolismo , Interferon gama/metabolismo , Fagocitose , Fagossomos/metabolismo , Staphylococcus aureus , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
4.
Mol Cell Proteomics ; 23(6): 100778, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38679389

RESUMO

Trilaciclib, a cyclin-dependent kinase 4/6 inhibitor, was approved as a myeloprotective agent for protecting bone marrow from chemotherapy-induced damage in extensive-stage small cell lung cancer. This is achieved through the induction of a temporary halt in the cell cycle of bone marrow cells. While it has been studied in various cancer types, its potential in hematological cancers remains unexplored. This research aimed to investigate the efficacy of trilaciclib in hematological cancers. Utilizing mass spectrometry-based proteomics, we examined the alterations induced by trilaciclib in the chronic myeloid leukemia cell line, K562. Interestingly, trilaciclib promoted senescence in these cells rather than cell death, as observed in acute myeloid leukemia, acute lymphoblastic leukemia, and myeloma cells. In K562 cells, trilaciclib hindered cell cycle progression and proliferation by stabilizing cyclin-dependent kinase 4/6 and downregulating cell cycle-related proteins, along with the concomitant activation of autophagy pathways. Additionally, trilaciclib-induced senescence was also observed in the nonsmall cell lung carcinoma cell line, A549. These findings highlight trilaciclib's potential as a therapeutic option for hematological cancers and underscore the need to carefully balance senescence induction and autophagy modulation in chronic myeloid leukemia treatment, as well as in nonsmall cell lung carcinoma cell line.


Assuntos
Senescência Celular , Proteômica , Humanos , Senescência Celular/efeitos dos fármacos , Proteômica/métodos , Células K562 , Proliferação de Células/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Ciclo Celular/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos de Piridínio/farmacologia , Pirimidinas , Pirróis
5.
EMBO J ; 40(10): e106188, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33881780

RESUMO

Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.


Assuntos
Macrófagos/metabolismo , Fagossomos/metabolismo , Animais , Humanos , MAP Quinase Quinase Quinases/metabolismo , Fosforilação/fisiologia , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Staphylococcus aureus/metabolismo
6.
Expert Rev Mol Med ; 26: e6, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38604802

RESUMO

Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.


Assuntos
Descoberta de Drogas , Proteoma , Humanos , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , Descoberta de Drogas/métodos , Espectrometria de Massas , Desenvolvimento de Medicamentos
7.
J Proteome Res ; 22(8): 2629-2640, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37439223

RESUMO

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMTs) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukemia cells were treated with losmapimod, a known inhibitor of MAPK14 (p38α). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable of TMT-DDA in their ability to detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labeled quantitation in the TPP pipeline.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Proteoma , Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos
8.
EMBO J ; 38(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30804002

RESUMO

NEDD8 is a ubiquitin-like protein that activates cullin-RING E3 ubiquitin ligases (CRLs). Here, we identify a novel role for NEDD8 in regulating the activity of poly(ADP-ribose) polymerase 1 (PARP-1) in response to oxidative stress. We show that treatment of cells with H2O2 results in the accumulation of NEDD8 chains, likely by directly inhibiting the deneddylase NEDP1. One chain type, an unanchored NEDD8 trimer, specifically bound to the second zinc finger domain of PARP-1 and attenuated its activation. In cells in which Nedp1 is deleted, large amounts of tri-NEDD8 constitutively form, resulting in inhibition of PARP-1 and protection from PARP-1-dependent cell death. Surprisingly, these NEDD8 trimers are additionally acetylated, as shown by mass spectrometry analysis, and their binding to PARP-1 is reduced by the overexpression of histone de-acetylases, which rescues PARP-1 activation. Our data suggest that trimeric, acetylated NEDD8 attenuates PARP-1 activation after oxidative stress, likely to delay the initiation of PARP-1-dependent cell death.


Assuntos
Morte Celular , Proteína NEDD8/química , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Acetilação , Células Endoteliais da Veia Umbilical Humana , Humanos , Peróxido de Hidrogênio/farmacologia , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Oxidantes/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Multimerização Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
9.
EMBO J ; 38(11)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31028084

RESUMO

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization.


Assuntos
Inflamação , Interleucina-4/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Ativação de Macrófagos , Receptores Depuradores Classe A/agonistas , Receptores Depuradores Classe A/genética , Animais , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Células Cultivadas , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Lipólise/efeitos dos fármacos , Lipólise/genética , Lipoproteínas LDL/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Polissacarídeos/farmacologia , Processamento de Proteína Pós-Traducional/genética , Células RAW 264.7 , Receptores Depuradores Classe A/química , Receptores Depuradores Classe A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ubiquitinação/genética
10.
PLoS Pathog ; 17(3): e1009410, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33720986

RESUMO

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Macrófagos/microbiologia , Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose/microbiologia , Tropismo Viral/fisiologia , Animais , Bovinos , Células Gigantes , Humanos
11.
Nature ; 550(7677): 534-538, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29045385

RESUMO

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.


Assuntos
Aminopiridinas/química , Aminopiridinas/farmacologia , Indazóis/química , Indazóis/farmacologia , Fenóis/química , Fenóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismo , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Especificidade por Substrato , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/deficiência , Peptidase 7 Específica de Ubiquitina/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(34): 20836-20847, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32769205

RESUMO

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.


Assuntos
Staphylococcus aureus/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Família Multigênica/genética , Transporte Proteico/genética , Proteômica , Infecções Estafilocócicas/microbiologia , Toxinas Biológicas/metabolismo , Sistemas de Secreção Tipo VII/fisiologia , Virulência/genética , Peixe-Zebra/microbiologia
13.
J Hepatol ; 76(5): 1001-1012, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34942286

RESUMO

BACKGROUND & AIMS: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. METHODS: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. RESULTS: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. CONCLUSIONS: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Anticorpos Monoclonais , Dieta Hiperlipídica/efeitos adversos , Estudo de Associação Genômica Ampla , Humanos , Inflamação/metabolismo , Lipídeos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo
14.
EMBO J ; 37(10)2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29669860

RESUMO

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor-positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib-induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib-induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence-like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse-free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.


Assuntos
Neoplasias da Mama/enzimologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Piperazinas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma/análise , Piridinas/farmacologia , Temperatura , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células , Senescência Celular , Feminino , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/patologia , Fenótipo , Prognóstico , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteínas Quinases/farmacologia , Taxa de Sobrevida , Ubiquitina/metabolismo
15.
EMBO J ; 37(24)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30266825

RESUMO

Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5-regulators of microtubule and centrosome function-as cellular substrates of CDKL5. Antibodies against MAP1S phospho-Ser900 and CEP131 phospho-Ser35 confirmed CDKL5-dependent phosphorylation of these targets in human cells. The phospho-acceptor serine residues in MAP1S, CEP131 and DLG5 lie in the motif RPXSA, although CDKL5 can tolerate residues other than Ala immediately C-terminal to the phospho-acceptor serine. We provide insight into the control of CDKL5 activity and show that pathogenic mutations in CDKL5 cause a major reduction in CDKL5 activity in vitro and in cells. These data reveal the first cellular substrates of CDKL5, which may represent important biomarkers in the diagnosis and treatment of CDKL5 disorder, and illuminate the functions of this poorly characterized kinase.


Assuntos
Síndromes Epilépticas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espasmos Infantis/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Centrossomo/metabolismo , Proteínas do Citoesqueleto , Síndromes Epilépticas/genética , Síndromes Epilépticas/patologia , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteômica , Espasmos Infantis/genética , Espasmos Infantis/patologia , Proteínas Supressoras de Tumor/genética
16.
EMBO J ; 37(12)2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29789389

RESUMO

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Fagossomos/imunologia , Tuberculose/imunologia , Animais , Proteínas Relacionadas à Autofagia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Fagossomos/genética , Fagossomos/microbiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Tuberculose/genética
17.
Biochem J ; 478(23): 4119-4136, 2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34780645

RESUMO

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.


Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo
18.
Int J Mol Sci ; 23(23)2022 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-36499482

RESUMO

A reduction in FADD levels has been reported in precursor T-cell neoplasms and other tumor types. Such reduction would impact on the ability of tumor cells to undergo apoptosis and has been associated with poor clinical outcomes. However, FADD is also known to participate in non-apoptotic functions, but these mechanisms are not well-understood. Linking FADD expression to the severity of precursor T-cell neoplasms could indicate its use as a prognostic marker and may open new avenues for targeted therapeutic strategies. Using transcriptomic and clinical data from patients with precursor T-cell neoplasms, complemented by in vitro analysis of cellular functions and by high-throughput interactomics, our results allow us to propose a dual role for FADD in precursor T-cell neoplasms, whereby resisting cell death and chemotherapy would be a canonical consequence of FADD deficiency in these tumors, whereas deregulation of the cellular metabolism would be a relevant non-canonical function in patients expressing FADD. These results reveal that evaluation of FADD expression in precursor T-cell neoplasms may aid in the understanding of the biological processes that are affected in the tumor cells. The altered biological processes can be of different natures depending on the availability of FADD influencing its ability to exert its canonical or non-canonical functions. Accordingly, specific therapeutic interventions would be needed in each case.


Assuntos
Apoptose , Neoplasias , Humanos , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Apoptose/genética , Perfilação da Expressão Gênica , Morte Celular , Linfócitos T/metabolismo
19.
Acta Neuropathol ; 142(6): 961-984, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34514546

RESUMO

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a "pathological package" capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein-ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.


Assuntos
Ceramidas/metabolismo , Vesículas Extracelulares/metabolismo , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , alfa-Sinucleína/metabolismo , Glucosilceramidase/genética , Humanos , Mutação , Transtornos Parkinsonianos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo
20.
Mol Cell Proteomics ; 18(5): 909-922, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30808727

RESUMO

Dendritic cells (DCs) are professional phagocytes that use innate sensing and phagocytosis to internalize and degrade self as well as foreign material, such as pathogenic bacteria, within phagosomes. These intracellular compartments are equipped to generate antigenic peptides that serve as source for antigen presentation to T cells initiating adaptive immune responses. The phagosomal proteome of DCs is only partially studied and is highly dynamic as it changes during phagosome maturation, when phagosomes sequentially interact with endosomes and lysosomes. In addition, the activation status of the phagocyte can modulate the phagosomal composition and is able to shape phagosomal functions.In this study, we determined spatiotemporal changes of the proteome of DC phagosomes during their maturation and compared resting and lipopolysaccharide (LPS)-stimulated bone marrow-derived DCs by label-free, quantitative mass spectrometry. Ovalbumin-coupled latex beads were used as phagocytosis model system and revealed that LPS-treated DCs show decreased recruitment of proteins involved in phagosome maturation, such as subunits of the vacuolar proton ATPase, cathepsin B, D, S, and RAB7. In contrast, those phagosomes were characterized by an increased recruitment of proteins involved in antigen cross-presentation, e.g. different subunits of MHC I molecules, the proteasome and tapasin, confirming the observed increase in cross-presentation efficacy in those cells. Further, several proteins were identified that were not previously associated with phagosomal functions. Hierarchical clustering of phagosomal proteins demonstrated that their acquisition to DC phagosomes is not only dependent on the duration of phagosome maturation but also on the activation state of DCs. Thus, our study provides a comprehensive overview of how DCs alter their phagosome composition in response to LPS, which has profound impact on the initiation of efficient immune responses.


Assuntos
Células Dendríticas/metabolismo , Lipopolissacarídeos/farmacologia , Fagossomos/metabolismo , Proteoma/metabolismo , Animais , Células Dendríticas/efeitos dos fármacos , Cinética , Camundongos Endogâmicos C57BL , Fagossomos/efeitos dos fármacos , Proteômica , Fatores de Tempo
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