RESUMO
Parkin is an E3 ubiquitin ligase, which plays a key role in the development of Parkinson disease. Parkin defects also occur in numerous cancers, and a growing body of evidence indicates that Parkin functions as a tumor suppressor that impedes a number of cellular processes involved in tumorigenesis. Here, we generated murine and human models that closely mimic the advanced-stage tumors where Parkin deficiencies are found to provide deeper insights into the tumor suppressive functions of Parkin. Loss of Parkin expression led to aggressive tumor growth, which was associated with poor tumor antigen presentation and limited antitumor CD8+ T-cell infiltration and activation. The effect of Parkin deficiency on tumor growth was lost following depletion of CD8+ T cells. In line with previous findings, Parkin deficiency was linked with mitochondria-associated metabolic stress, PTEN degradation, and enhanced Akt activation. Increased Akt signaling led to dysregulation of antigen presentation, and treatment with the Akt inhibitor MK2206-2HCl restored antigen presentation in Parkin-deficient tumors. Analysis of data from patients with clear cell renal cell carcinoma indicated that Parkin expression was downregulated in tumors and that low expression correlated with reduced overall survival. Furthermore, low Parkin expression correlated with reduced patient response to immunotherapy. Overall, these results identify a role for Parkin deficiency in promoting tumor immune evasion that may explain the poor prognosis associated with loss of Parkin across multiple types of cancer. SIGNIFICANCE: Parkin prevents immune evasion by regulating tumor antigen processing and presentation through the PTEN/Akt network, which has important implications for immunotherapy treatments in patients with Parkin-deficient tumors.
Assuntos
Apresentação de Antígeno , Neoplasias , Animais , Humanos , Camundongos , Imunoterapia , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt , Evasão Tumoral , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Most genes associated with acute myeloid leukemia (AML) are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we find a set of splicing events that alter the expression of a subset of AML-associated genes independent of known somatic mutations. In particular, aberrant splicing triples the number of patients with reduced functional EZH2 compared with that predicted by somatic mutation alone. In addition, we unexpectedly find that the nonsense-mediated decay factor DHX34 exhibits widespread alternative splicing in sporadic AML, resulting in a premature stop codon that phenocopies the loss-of-function germline mutations observed in familial AML. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.
Assuntos
Processamento Alternativo , Leucemia Mieloide Aguda/genética , Mutação , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Genótipo , Humanos , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/genética , RNA Helicases/metabolismoRESUMO
Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that is responsible for host genome degradation and the processing of DNA ends for recombination-dependent DNA repair. In this chapter, we describe the ensemble methods currently employed by our laboratory to characterize the exonuclease activity of the T4 MR complex. DNA exonucleases play a vital role in maintaining the integrity of DNA through their participation in DNA repair pathways and as proofreaders for DNA polymerases. Methods for quantifying the general features of the exonuclease, and for determining steady-state kinetic parameters (Km, kcat), the polarity of exonuclease activity, and processivity are presented. These methods should be applicable to all DNA exonucleases, and to some extent endonucleases.
Assuntos
Bacteriófago T4/genética , DNA de Cadeia Simples/metabolismo , Ensaios Enzimáticos/métodos , Reparo de DNA por Recombinação , Proteínas Virais/metabolismo , Bacteriófago T4/metabolismo , Cromatografia em Camada Fina/instrumentação , Cromatografia em Camada Fina/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Eletroforese em Gel de Ágar/instrumentação , Eletroforese em Gel de Ágar/métodos , Ensaios Enzimáticos/instrumentação , Cinética , Coloração e Rotulagem/instrumentação , Coloração e Rotulagem/métodosRESUMO
A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). Mutagenesis analysis of ESDN's seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkCT10 regulator of kinase Crk-Like (CrkL) and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding.
