RESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phosphosites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein by proteomic and phosphoproteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phosphosites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together, these findings highlight categories of phosphosites that merit further study and provide a phosphosite kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Doença de Huntington , Humanos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Temperatura Alta , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Fosforilação , Domínios Proteicos , ProteômicaRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the polyglutamine (polyQ) expansion in huntingtin (HTT) protein. The challenge of obtaining full-length HTT proteins with high purity limits the understanding of the HTT protein function. Here, we provide a protocol to generate and purify full-length recombinant human HTT proteins with various polyQ lengths, which is key to investigate the biochemical function of HTT proteins and the molecular mechanism underlying HD pathology. For complete details on the use and execution of this protocol, please refer to Jung et al. (2020).
Assuntos
Proteína Huntingtina/isolamento & purificação , Peptídeos/genética , Proteínas Recombinantes/isolamento & purificação , Animais , Baculoviridae/genética , Técnicas de Cultura de Células , Cromatografia de Afinidade/métodos , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9RESUMO
Inactivation of tumor suppressor Runt-related transcription factor 3 (RUNX3) plays an important role during early tumorigenesis. However, posttranslational modifications (PTM)-based mechanism for the inactivation of RUNX3 under hypoxia is still not fully understood. Here, we demonstrate a mechanism that G9a, lysine-specific methyltransferase (KMT), modulates RUNX3 through PTM under hypoxia. Hypoxia significantly increased G9a protein level and G9a interacted with RUNX3 Runt domain, which led to increased methylation of RUNX3 at K129 and K171. This methylation inactivated transactivation activity of RUNX3 by reducing interactions with CBFß and p300 cofactors, as well as reducing acetylation of RUNX3 by p300, which is involved in nucleocytoplasmic transport by importin-α1. G9a-mediated methylation of RUNX3 under hypoxia promotes cancer cell proliferation by increasing cell cycle or cell division, while suppresses immune response and apoptosis, thereby promoting tumor growth during early tumorigenesis. Our results demonstrate the molecular mechanism of RUNX3 inactivation by G9a-mediated methylation for cell proliferation and antiapoptosis under hypoxia, which can be a therapeutic or preventive target to control tumor growth during early tumorigenesis.
Assuntos
Carcinogênese/genética , Hipóxia Celular/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Metilação de DNA/genética , Acetilação , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Proteínas de Arabidopsis/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Western Blotting , Histona-Lisina N-Metiltransferase/genética , Cinética , Metilação , Mutação , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Plant immune response is initiated by Resistance proteins (R proteins). Toll/interleukin-1 receptor (TIR) domain in R proteins, which is responsible for the dimerization but has limited conservation in their primary structures. Suppressor of npr1-1, constitutive 1 (SNC1), a TIR-containing R protein, is involved in autoimmunity of plant, but the binding partner of SNC1 via the TIR domain and its specific cognate effector protein remain elusive. Here, we present the crystal structure of the TIR domain of Arabidopsis thaliana SNC1 (AtSNC1-TIR). The structure shows that AtSNC1-TIR domain is similar to those of other plant TIR domains including AtTIR, L6 and RPS4. Structural and sequence analysis on AtSNC1-TIR revealed that almost all conserved amino acids are located in the core of the structure, while the amino acids on the surface are highly variable, implicating that each TIR domain utilizes the variable surface for interacting its binding partner. In addition, the interaction between AtSNC1-TIR proteins in the crystal suggests two possible dimerization modes of AtSNC1-TIR domain. This study provides structural platform to investigate AtSNC1-TIR mediated signaling pathway of plant immune responses.
