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1.
Mol Cell ; 81(17): 3481-3495.e7, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34358446

RESUMO

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.


Assuntos
Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/fisiologia , Animais , Linhagem Celular Tumoral , Citoplasma/metabolismo , Feminino , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Canais Iônicos/metabolismo , Masculino , Metilação , Camundongos , Camundongos Nus , Proteínas Nucleares/metabolismo , Peptídeos/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Spliceossomos/metabolismo
2.
Nature ; 586(7829): 412-416, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33029011

RESUMO

An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory1-4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories5-10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF211. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds11-13. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically11 and pharmacologically inhibiting the ISR kinases14-17, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB11, enhances long-term memory in health and disease18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Hipocampo/citologia , Consolidação da Memória , Neurônios/metabolismo , Somatostatina/metabolismo , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Fator de Iniciação 2 em Eucariotos/deficiência , Fator de Iniciação 2 em Eucariotos/genética , Potenciais Pós-Sinápticos Excitadores , Hipocampo/fisiologia , Potenciação de Longa Duração , Masculino , Memória de Longo Prazo , Camundongos , Camundongos Endogâmicos C57BL , Inibição Neural , Plasticidade Neuronal , Parvalbuminas , Fosforilação , Células Piramidais/fisiologia , Transmissão Sináptica
3.
Mol Cell Proteomics ; 22(10): 100639, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37657519

RESUMO

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.


Assuntos
Fosfopeptídeos , Proteínas Proto-Oncogênicas c-akt , Fosforilação , Fosfopeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fosfoproteínas/metabolismo
4.
Chembiochem ; 24(19): e202300141, 2023 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-37088717

RESUMO

Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.


Assuntos
Neoplasias , Quimera de Direcionamento de Proteólise , Camundongos , Animais , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química
5.
PLoS Biol ; 18(11): e3000981, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33253182

RESUMO

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.


Assuntos
Acetilcoenzima A/biossíntese , Nucléolo Celular/metabolismo , ATP Citrato (pro-S)-Liase/deficiência , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetatos/metabolismo , Acetilação , Linhagem Celular , Nucléolo Celular/ultraestrutura , Expressão Gênica , Técnicas de Inativação de Genes , Células HCT116 , Histona Desacetilases/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo
6.
Mol Cell Proteomics ; 20: 100154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34592423

RESUMO

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites are necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ϵ-GG peptides are enriched with anti-K-ε-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 µg input per sample processed in ∼2 h. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility, and significantly reduced processing time. The automated method also significantly reduced variability across process replicates compared with the manual method. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of the method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.


Assuntos
Proteômica/métodos , Proteínas Ubiquitinadas/metabolismo , Animais , Anticorpos/imunologia , Automação , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Células Jurkat , Fenômenos Magnéticos , Neoplasias Mamárias Experimentais/metabolismo , Espectrometria de Massas , Camundongos , Peptídeos , Sefarose , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/imunologia , Ubiquitinação , Fluxo de Trabalho
7.
Blood ; 133(11): 1171-1185, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30587525

RESUMO

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.


Assuntos
Carcinogênese/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proliferação de Células , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Citarabina/farmacologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Nus , Fosforilação , Biossíntese de Proteínas , Proteoma/análise , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
bioRxiv ; 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38979143

RESUMO

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

9.
Nat Commun ; 15(1): 6744, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39112447

RESUMO

Demyelination is a common pathological feature in a wide range of diseases, characterized by the loss of myelin sheath and myelin-supporting oligodendrocytes. These losses lead to impaired axonal function, increased vulnerability of axons to damage, and result in significant brain atrophy and neuro-axonal degeneration. Multiple pathomolecular processes contribute to neuroinflammation, oligodendrocyte cell death, and progressive neuronal dysfunction. In this study, we use the cuprizone mouse model of demyelination to investigate long-term non-invasive gamma entrainment using sensory stimulation as a potential therapeutic intervention for promoting myelination and reducing neuroinflammation in male mice. Here, we show that multisensory gamma stimulation mitigates demyelination, promotes oligodendrogenesis, preserves functional integrity and synaptic plasticity, attenuates oligodendrocyte ferroptosis-induced cell death, and reduces brain inflammation. Thus, the protective effects of multisensory gamma stimulation on myelin and anti-neuroinflammatory properties support its potential as a therapeutic approach for demyelinating disorders.


Assuntos
Cuprizona , Doenças Desmielinizantes , Modelos Animais de Doenças , Bainha de Mielina , Oligodendroglia , Animais , Cuprizona/toxicidade , Masculino , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/terapia , Doenças Desmielinizantes/patologia , Camundongos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Bainha de Mielina/metabolismo , Camundongos Endogâmicos C57BL , Ferroptose , Plasticidade Neuronal , Encéfalo/patologia , Encéfalo/metabolismo , Doenças Neuroinflamatórias/induzido quimicamente , Doenças Neuroinflamatórias/patologia
10.
Blood ; 117(6): 1828-33, 2011 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-21051557

RESUMO

Older patients with acute myeloid leukemia (AML) have limited treatment options and a poor prognosis, thereby warranting novel therapeutic strategies. We evaluated the efficacy of lenalidomide as front-line therapy for older AML patients. In this phase 2 study, patients 60 years of age or older with untreated AML received high-dose (HD) lenalidomide at 50 mg daily for up to 2 28-day cycles. If patients achieved a complete remission (CR)/CR with incomplete blood count recovery (CRi) or did not progress after 2 cycles of HD lenalidomide, they received low-dose lenalidomide (10 mg daily) until disease progression, an unacceptable adverse event, or completion of 12 cycles. Thirty-three AML patients (median age, 71 years) were enrolled with intermediate (55%), unfavorable (39%), or unknown (6%) cytogenetic risk. Overall CR/CRi rate was 30%, and 53% in patients completing HD lenalidomide. The CR/CRi rate was significantly higher in patients presenting with a low (< 1000/µL) circulating blast count (50%, P = .01). The median time to CR/CRi was 30 days, and duration of CR/CRi was 10 months (range, 1- ≥ 17 months). The most common grades ≥ 3 toxicities were thrombocytopenia, anemia, infection, and neutropenia. HD lenalidomide has evidence of clinical activity as initial therapy for older AML patients, and further study of lenalidomide in AML and MDS is warranted. This study is registered at www.clinicaltrials.gov as #NCT00546897.


Assuntos
Antineoplásicos/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Talidomida/análogos & derivados , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Contagem de Células Sanguíneas , Análise Citogenética , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Lenalidomida , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Indução de Remissão , Talidomida/administração & dosagem , Talidomida/efeitos adversos , Fatores de Tempo , Resultado do Tratamento
11.
Methods Mol Biol ; 2365: 203-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34432246

RESUMO

Ubiquitination is a post-translational modification that affects protein degradation as well as a variety of cellular processes. Methods that globally profile ubiquitination are powerful tools to better understand these processes. Here we describe an updated method for identification and quantification of thousands of sites of ubiquitination from cells, tissues, or other biological materials. The method involves cell lysis and digestion to peptides, immunoaffinity enrichment with an antibody recognizing di-glycine remnants left behind at ubiquitinated lysines, and liquid chromatography-tandem mass spectrometry analysis of the enriched peptides.


Assuntos
Espectrometria de Massas , Anticorpos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação
12.
Am J Prev Med ; 43(6 Suppl 5): S425-34, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23157761

RESUMO

BACKGROUND: Callers to 2-1-1 have greater need for and lesser use of cancer control services than other Americans. Integrating cancer risk assessment and referrals to preventive services into 2-1-1 systems is both feasible and acceptable to callers. PURPOSE: To determine whether callers will act on these referrals. METHODS: In a randomized trial, 2-1-1 callers (n=1200) received standard service and those with at least one cancer risk factor or need for screening were assigned to receive verbal referrals only, verbal referrals + a tailored reminder mailed to their home, or verbal referrals + a telephone health coach/navigator. All data were collected from June 2010 to March 2012 and analyzed in March and April 2012. RESULTS: At 1-month follow-up, callers in the navigator condition were more likely to report having contacted a cancer control referral than those receiving tailored reminders or verbal referrals only (34% vs 24% vs 18%, respectively; n=772, p<0.0001). Compared to verbal referrals only, navigators were particularly effective in getting 2-1-1 callers to contact providers for mammograms (OR=2.10, 95% CI=1.04, 4.22); Paps (OR=2.98, 95% CI=1.18, 7.54); and smoking cessation (OR=2.07, 95% CI=1.14, 3.74). CONCLUSIONS: Given the extensive reach of 2-1-1s and the elevated risk profile of their callers, even modest response rates could have meaningful impact on population health if proactive health referrals were implemented nationally.


Assuntos
Serviços de Informação/organização & administração , Neoplasias/diagnóstico , Serviços Preventivos de Saúde/métodos , Encaminhamento e Consulta/organização & administração , Adulto , Estudos de Viabilidade , Feminino , Seguimentos , Humanos , Masculino , Mamografia/estatística & dados numéricos , Programas de Rastreamento/estatística & dados numéricos , Neoplasias/prevenção & controle , Medição de Risco/métodos , Fatores de Risco , Abandono do Hábito de Fumar/estatística & dados numéricos , Telefone
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