RESUMEN
Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.
Asunto(s)
Arginina , Cisteína , Proteína 1 Asociada A ECH Tipo Kelch , Neoplasias Pulmonares , Proteoma , Humanos , Cisteína/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteoma/metabolismo , Arginina/metabolismo , Mutación , Argininosuccinato Sintasa/metabolismo , Argininosuccinato Sintasa/genética , Cisplatino/farmacología , Línea Celular Tumoral , Proteómica/métodos , Regulación Neoplásica de la Expresión Génica , Supervivencia Celular/efectos de los fármacos , ARN de Transferencia/metabolismo , ARN de Transferencia/genéticaRESUMEN
mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis. Despite protein quality control mechanisms, amino acid shortage in melanoma induces aberrant proteins by ribosomal frameshifting. The extent and the underlying mechanisms related to this phenomenon are yet unknown. Here, we show that tryptophan depletion-induced ribosomal frameshifting is a widespread phenomenon in cancer. We termed this event sloppiness and strikingly observed its association with MAPK pathway hyperactivation. Sloppiness is stimulated by RAS activation in primary cells, suppressed by pharmacological inhibition of the oncogenic MAPK pathway in sloppy cells, and restored in cells with acquired resistance to MAPK pathway inhibition. Interestingly, sloppiness causes aberrant peptide presentation at the cell surface, allowing recognition and specific killing of drug-resistant cancer cells by T lymphocytes. Thus, while oncogenes empower cancer progression and aggressiveness, they also expose a vulnerability by provoking the production of aberrant peptides through sloppiness.
Asunto(s)
Neoplasias/genética , Oncogenes , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Linfocitos T/citología , Animales , Carcinogénesis , Membrana Celular/metabolismo , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Mutación del Sistema de Lectura , Sistema de Lectura Ribosómico , Humanos , Inmunoterapia/métodos , Sistema de Señalización de MAP Quinasas , Melanoma/metabolismo , Ratones , Neoplasias/metabolismo , Péptidos/química , Inhibidores de Proteínas Quinasas , Ribosomas/metabolismo , Linfocitos T/metabolismo , Triptófano/química , Triptófano/metabolismoRESUMEN
Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1-4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity.
Asunto(s)
Triptófano-ARNt Ligasa , Triptófano , Codón/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma , Neoplasias/inmunología , Fenilalanina , Linfocitos T , Triptófano/metabolismo , Triptófano Oxigenasa/genética , Triptófano Oxigenasa/metabolismo , Triptófano-ARNt Ligasa/genética , Triptófano-ARNt Ligasa/metabolismoRESUMEN
Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.
Asunto(s)
Presentación de Antígeno , Mutación del Sistema de Lectura , Melanoma/inmunología , Péptidos/genética , Péptidos/inmunología , Biosíntesis de Proteínas/inmunología , Linfocitos T/inmunología , Línea Celular , Codón/genética , Sistema de Lectura Ribosómico/efectos de los fármacos , Sistema de Lectura Ribosómico/genética , Sistema de Lectura Ribosómico/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/inmunología , Interferón gamma/farmacología , Melanoma/patología , Péptidos/química , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Proteoma , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Triptófano/deficiencia , Triptófano/genética , Triptófano/metabolismoRESUMEN
Perturbation of protein phosphorylation represents an attractive approach to cancer treatment. Besides kinase inhibitors, protein phosphatase inhibitors have been shown to have anti-cancer activity. A prime example is the small molecule LB-100, an inhibitor of protein phosphatases 2A/5 (PP2A/PP5), enzymes that affect cellular physiology. LB-100 has proven effective in pre-clinical models in combination with immunotherapy, but the molecular underpinnings of this synergy remain understood poorly. We report here a sensitivity of the mRNA splicing machinery to phosphorylation changes in response to LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Altered phosphorylation endows LB-100-treated colorectal adenocarcinoma cells with differential splicing patterns. In PP2A-inhibited cells, over 1000 events of exon skipping and intron retention affect regulators of genomic integrity. Finally, we show that LB-100-evoked alternative splicing leads to neoantigens that are presented by MHC class 1 at the cell surface. Our findings provide a potential explanation for the pre-clinical and clinical observations that LB-100 sensitizes cancer cells to immune checkpoint blockade.
Asunto(s)
Neoplasias del Colon , Empalme del ARN , Humanos , Empalme Alternativo/efectos de los fármacos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Inhibidores Enzimáticos/farmacología , Fosforilación , Proteína Fosfatasa 2/metabolismo , Empalme del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/genética , Piperazinas/farmacologíaRESUMEN
Ubiquitination has crucial roles in many cellular processes, and dysregulation of ubiquitin machinery enzymes can result in various forms of pathogenesis. Cells only have a limited set of ubiquitin-conjugating (E2) enzymes to support the ubiquitination of many cellular targets. As individual E2 enzymes have many different substrates and interactions between E2 enzymes and their substrates can be transient, it is challenging to define all in vivo substrates of an individual E2 and the cellular processes it affects. Particularly challenging in this respect is UBE2D3, an E2 enzyme with promiscuous activity in vitro but less defined roles in vivo. Here, we set out to identify in vivo targets of UBE2D3 by using stable isotope labeling by amino acids in cell culture-based and label-free quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with the levels of proteins from metabolic pathways, in particular retinol metabolism, being the most affected. However, the impact of UBE2D3 depletion on the ubiquitinome was much more prominent. Interestingly, molecular pathways related to mRNA translation were the most affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control, is dependent on UBE2D3. We show by Targets of Ubiquitin Ligases Identified by Proteomics 2 methodology that RPS10 and RPS20 are direct targets of UBE2D3 and demonstrate that the catalytic activity of UBE2D3 is required to ubiquitinate RPS10 in vivo. In addition, our data suggest that UBE2D3 acts at multiple levels in autophagic protein quality control. Collectively, our findings show that depletion of an E2 enzyme in combination with quantitative diGly-based ubiquitinome profiling is a powerful tool to identify new in vivo E2 substrates, as we have done here for UBE2D3. Our work provides an important resource for further studies on the in vivo functions of UBE2D3.
Asunto(s)
Proteoma , Ubiquitina , Proteoma/metabolismo , Ubiquitinación , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The vitronectin receptor integrin αVß5 can reside in two distinct adhesion structures - focal adhesions (FAs) and flat clathrin lattices (FCLs). Here, we investigate the mechanism that regulates the subcellular distribution of ß5 in keratinocytes and show that ß5 has approximately 7- and 5-fold higher affinity for the clathrin adaptors ARH (also known as LDLRAP1) and Numb, respectively, than for the talin 1 (TLN1); all proteins that bind to the membrane-proximal NPxY motif of the ß5 cytoplasmic domain. Using mass spectrometry, we identified ß5 interactors, including the Rho GEFs p115Rho-GEF and GEF-H1 (also known as ARHGEF1 and ARHGEF2, respectively), and the serine protein kinase MARK2, depletion of which diminishes the clustering of ß5 in FCLs. Replacement of two serine residues (S759 and S762) in the ß5 cytoplasmic domain with phospho-mimetic glutamate residues causes a shift in the localization of ß5 from FAs into FCLs without affecting the interactions with MARK2, p115Rho-GEF or GEF-H1. Instead, we demonstrate that changes in the actomyosin-based cellular contractility by ectopic expression of activated Rho or disruption of microtubules regulates ß5 localization. Finally, we present evidence that ß5 in either FAs or FCLs functions to promote adhesion to vitronectin, cell spreading, and proliferation.
Asunto(s)
Clatrina , Receptores de Vitronectina , Adhesión Celular/fisiología , Proliferación Celular , Clatrina/metabolismo , Adhesiones Focales/metabolismo , Receptores de Vitronectina/metabolismo , Serina/metabolismoRESUMEN
As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gßγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression, protein-protein associations, or genetic interaction profiles, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.
Asunto(s)
Canales de Potasio/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Análisis de la Célula Individual/métodos , Células Cultivadas , Haploidia , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Interferones/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis , Fenotipo , Fosforilación/genética , Canales de Potasio/deficiencia , Canales de Potasio/genética , Proteolisis , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vía de Señalización WntRESUMEN
The family of integrin transmembrane receptors is essential for the normal function of multicellular organisms by facilitating cell-extracellular matrix adhesion. The vitronectin-binding integrin αVß5 localizes to focal adhesions (FAs) as well as poorly characterized flat clathrin lattices (FCLs). Here, we show that, in human keratinocytes, αVß5 is predominantly found in FCLs, and formation of the αVß5-containing FCLs requires the presence of vitronectin as ligand, Ca2+, and the clathrin adaptor proteins ARH (also known as LDLRAP1), Numb and EPS15/EPS15L1. Integrin chimeras, containing the extracellular and transmembrane domains of ß5 and the cytoplasmic domains of ß1 or ß3, almost exclusively localize in FAs. Interestingly, lowering actomyosin-mediated contractility promotes integrin redistribution to FLCs in an integrin tail-dependent manner, while increasing cellular tension favors αVß5 clustering in FAs. Our findings strongly indicate that clustering of integrin αVß5 in FCLs is dictated by the ß5 subunit cytoplasmic domain, cellular tension and recruitment of specific adaptor proteins to the ß5 subunit cytoplasmic domains.
Asunto(s)
Clatrina/metabolismo , Receptores de Vitronectina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Calcio/metabolismo , Células Cultivadas , Adhesiones Focales/metabolismo , Humanos , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vitronectina/metabolismoRESUMEN
Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.
Asunto(s)
Aminoácidos/metabolismo , Dipeptidasas/metabolismo , Lactatos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Aminoácidos/sangre , Citosol/metabolismo , Ejercicio Físico/fisiología , Células HEK293 , Humanos , Lactatos/sangre , Metaboloma , ProteolisisRESUMEN
c-Myc is one of the major human proto-oncogenes and is often associated with tumor aggression and poor clinical outcome. Paradoxically, Myc was also reported as a suppressor of cell motility, invasiveness, and metastasis. Among the direct targets of Myc are many components of the protein synthesis machinery whose induction results in an overall increase in protein synthesis that empowers tumor cell growth. At present, it is largely unknown whether beyond the global enhancement of protein synthesis, Myc activation results in translation modulation of specific genes. Here, we measured Myc-induced global changes in gene expression at the transcription, translation, and protein levels and uncovered extensive transcript-specific regulation of protein translation. Particularly, we detected a broad coordination between regulation of transcription and translation upon modulation of Myc activity and showed the connection of these responses to mTOR signaling to enhance oncogenic transformation and to the TGFß pathway to modulate cell migration and invasiveness. Our results elucidate novel facets of Myc-induced cellular responses and provide a more comprehensive view of the consequences of its activation in cancer cells.
Asunto(s)
Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica , Metástasis de la Neoplasia/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Apoptosis , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Perfilación de la Expresión Génica , Genes myc , Humanos , Biosíntesis de Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, autoinhibited and an open, active conformation. Although the open conformation of mDia2 induces actin assembly thereby controlling many cellular processes, mDia2 possesses also actin-independent and conformation-insensitive scaffolding roles related to microtubules and p53, respectively. Thus, we hypothesize that mDia2 may have other unappreciated functions and regulatory modes. Here we identify and validate proteasome and Ubiquitin as mDia2-interacting partners using stable isotope labeling with amino acids in cell culture-based quantitative proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated, binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome substrate. Surprisingly, knockdown of mDia2 increases the activity of the proteasome in vitro, whereas mDia2 overexpression has opposite effects only when it adopts the open conformation and cannot induce actin assembly. Consistently, a combination of candidate and unbiased proteome-wide analyses indicates that mDia2 regulates the cellular levels of proteasome substrate ß-catenin and a number of ubiquitinated actin-regulatory proteins. Hence, these findings add more complexity to the mDia2 activity cycle by showing that the open conformation may control actin dynamics also through actin-independent regulation of the proteasome.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , NADPH Deshidrogenasa/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica/métodos , Actinas/metabolismo , Animales , Marcaje Isotópico , Ratones , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/fisiología , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/fisiología , Conformación Proteica , Mapeo de Interacción de Proteínas , Ubiquitina/metabolismoRESUMEN
OBJECTIVE: Platelet activation and subsequent protein release play an important role in healthy hemostasis and inflammatory responses, yet the identity and quantity of proteins in the platelet releasate are still debated. Here, we present a reversed releasate proteomics approach to determine unambiguously and quantitatively proteins released from activated platelets. APPROACH AND RESULTS: Isolated platelets were mock and fully stimulated after which the released proteins in the supernatant were removed. Using high-end proteomics technology (2D chromatography, stable isotope labeling, electron transfer dissociation, and high collision dissociation fragmentation) allowed us to quantitatively discriminate the released proteins from uncontrolled lysis products. Monitoring the copy numbers of ≈ 4500 platelet proteins, we observed that after stimulation via thrombin and collagen, only 124 (<3%) proteins were significantly released (P<0.05). The released proteins span a concentration range of ≥ 5 orders, as confirmed by ELISA. The released proteins were highly enriched in secretion tags and contained all known factors at high concentrations (>100 ng/mL, eg, thrombospondin, von Willebrand factor, and platelet factor 4). Interestingly, in the lower concentration range of the releasate many novel factors were identified. CONCLUSIONS: Our reversed releasate dataset forms the first unambiguous, in depth repository for molecular factors released by platelets.
Asunto(s)
Plaquetas/metabolismo , Activación Plaquetaria , Proteínas/metabolismo , Proteómica/métodos , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Colágeno/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Marcaje Isotópico , Espectrometría de Masas en Tándem , Trombina/metabolismoRESUMEN
OBJECTIVE: Platelet adhesion to subendothelial collagen is dependent on the integrin α2ß1 and glycoprotein VI (GPVI) receptors. The major signaling routes in collagen-dependent platelet activation are outlined; however, crucial detailed knowledge of the actual phosphorylation events mediating them is still limited. Here, we explore phosphotyrosine signaling events downstream of GPVI with site-specific detail. APPROACH AND RESULTS: Immunoprecipitations of phosphotyrosine-modified peptides from protein digests of GPVI-activated and resting human platelets were compared by stable isotope-based quantitative mass spectrometry. We surveyed 214 unique phosphotyrosine sites over 2 time points, of which 28 showed a significant increase in phosphorylation on GPVI activation. Among these was Tyr370 of oligophrenin-1 (OPHN1), a Rho GTPase-activating protein. To elucidate the function of OPHN1 in platelets, we performed an array of functional platelet analyses within a small cohort of patients with rare oligophrenia. Because of germline mutations in the OPHN1 gene locus, these patients lack OPHN1 expression entirely and are in essence a human knockout model. Our studies revealed that among other unaltered properties, patients with oligophrenia show normal P-selectin exposure and αIIbß3 activation in response to GPVI, as well as normal aggregate formation on collagen under shear conditions. Finally, the major difference in OPHN1-deficient platelets turned out to be a significantly reduced collagen-induced filopodia formation. CONCLUSIONS: In-depth phosphotyrosine screening revealed many novel signaling recipients downstream of GPVI activation uncovering a new level of detail within this important pathway. To illustrate the strength of such data, functional follow-up of OPHN1 in human platelets deficient in this protein showed reduced filopodia formation on collagen, an important parameter of platelet hemostatic function.
Asunto(s)
Plaquetas/metabolismo , Proteínas del Citoesqueleto/sangre , Proteínas Activadoras de GTPasa/sangre , Errores Innatos del Metabolismo/sangre , Proteínas Nucleares/sangre , Glicoproteínas de Membrana Plaquetaria/metabolismo , Seudópodos/metabolismo , Transducción de Señal , Estudios de Casos y Controles , Niño , Colágeno/metabolismo , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Proteínas Activadoras de GTPasa/deficiencia , Proteínas Activadoras de GTPasa/genética , Hemostasis , Humanos , Inmunoprecipitación , Masculino , Espectrometría de Masas , Errores Innatos del Metabolismo/genética , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Selectina-P/sangre , Fosforilación , Adhesividad Plaquetaria , Pruebas de Función Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteómica/métodos , Estrés Mecánico , Factores de Tiempo , TirosinaRESUMEN
Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Reparación del ADN por Unión de Extremidades , Transducción de Señal , Proteína 28 que Contiene Motivos Tripartito , Proteína 1 de Unión al Supresor Tumoral P53 , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Ubiquitinación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Fosforilación , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Células HEK293 , Telómero/metabolismo , Daño del ADN , Cromatina/metabolismo , AnimalesRESUMEN
Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias , Humanos , Linfocitos T CD8-positivos , Neoplasias/genéticaRESUMEN
Mammalian carboxylesterase 1 enzymes can hydrolyze many xenobiotic chemicals and endogenous lipids. We here identified and characterized a mouse strain (FVB/NKI) in which three of the eight Ces1 genes were spontaneously deleted, removing Ces1c and Ces1e partly, and Ces1d entirely. We studied the impact of this Ces1c/d/e deficiency on drug and lipid metabolism and homeostasis. Ces1c/d/e-/- mice showed strongly impaired conversion of the anticancer prodrug irinotecan to its active metabolite SN-38 in plasma, spleen and lung. Plasma hydrolysis of the oral anticancer prodrug capecitabine to 5-DFCR was also profoundly reduced in Ces1c/d/e-/- mice. Our findings resolved previously unexplained FVB/NKI pharmacokinetic anomalies. On a medium-fat diet, Ces1c/d/e-/- female mice exhibited moderately higher body weight, mild inflammation in gonadal white adipose tissue (gWAT), and increased lipid load in brown adipose tissue (BAT). Ces1c/d/e-/- males showed more pronounced inflammation in gWAT and an increased lipid load in BAT. On a 5-week high-fat diet exposure, Ces1c/d/e deficiency predisposed to developing obesity, enlarged and fatty liver, glucose intolerance and insulin resistance, with severe inflammation in gWAT and increased lipid load in BAT. Hepatic proteomics analysis revealed that the acute phase response, involved in the dynamic cycle of immunometabolism, was activated in these Ces1c/d/e-/- mice. This may contribute to the obesity-related chronic inflammation and adverse metabolic disease in this strain. While Ces1c/d/e deficiency clearly exacerbated metabolic syndrome development, long-term (18-week) high-fat diet exposure overwhelmed many, albeit not all, observed phenotypic differences.
Asunto(s)
Hidrolasas de Éster Carboxílico , Síndrome Metabólico , Profármacos , Animales , Femenino , Masculino , Ratones , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Inflamación , Irinotecán , Lípidos , Mamíferos , Obesidad/metabolismoRESUMEN
Targeting the PI3K-AKT-mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K-AKT-mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies.
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Neoplasias de la Mama , Humanos , Animales , Ratones , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores mTOR , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Serina-Treonina Quinasas TORRESUMEN
Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a two-phase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC-MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC-MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients.