RESUMO
Protein-polymer conjugates are widely used in many clinical and industrial applications, but lack of experimental data relating protein-polymer interactions to improved protein stability prevents their rational design. Advances in synthetic chemistry have expanded the palette of polymer designs, including development of nonlinear architectures, novel monomer chemical scaffolds, and control of hydrophobicity, but more experimental data are needed to transform advances in chemistry into next generation conjugates. Using an integrative biophysical approach, we investigated the molecular basis for polymer-based thermal stabilization of a human galectin protein, Gal3C, conjugated with polymers of linear and nonlinear architectures, different degrees of polymerization, and varying hydrophobicities. Independently varying the degree of polymerization and polymer architecture enabled delineation of specific polymer properties contributing to improved protein stability. Insights from NMR spectroscopy of the polymer-conjugated Gal3C backbone revealed patterns of protein-polymer interactions shared between linear and nonlinear polymer architectures for thermally stabilized conjugates. Despite large differences in polymer chemical scaffolds, protein-polymer interactions resulting in thermal stabilization appear conserved. We observed a clear relation between polymer length and protein-polymer thermal stability shared among chemically different polymers. Our data indicate a wide range of polymers may be useful for engineering conjugate properties and provide conjugate design criteria.
RESUMO
Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = 006) and iNOS expression (P = 003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = 04 and P = 02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , ômega-N-Metilarginina/uso terapêutico , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Metaplasia , Camundongos , Taxa de Mutação , Transplante de Neoplasias , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Nitritos/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/metabolismo , Ubiquitina C/metabolismo , ômega-N-Metilarginina/farmacologiaRESUMO
The functional conversion of white adipose tissue (WAT) into a tissue with brown adipose tissue (BAT)-like activity, often referred to as "browning," represents an intriguing strategy for combating obesity and metabolic disease. We demonstrate that thyroid hormone receptor (TR) activation by a synthetic agonist markedly induces a program of adaptive thermogenesis in subcutaneous WAT that coincides with a restoration of cold tolerance to cold-intolerant mice. Distinct from most other browning agents, pharmacological TR activation dissociates the browning of WAT from activation of classical BAT. TR agonism also induces the browning of white adipocytes in vitro, indicating that TR-mediated browning is cell autonomous. These data establish TR agonists as a class of browning agents, implicate the TRs in the browning of WAT, and suggest a profound pharmacological potential of this action.