RESUMO
The extracellular matrix N-glycoprotein periostin is thought to enhance tumor invasion. In this study, the expression patterns of periostin and its splice isoforms were investigated in renal cell carcinoma (RCC). Periostin mRNA expression patterns were characterized in 30 fresh-frozen RCCs in normal fetal and adult renal tissues by both isoform-specific and nonspecific RT-PCR and by gene expression array analysis. Its protein expression was analyzed by immunohistochemistry, using tissue microarrays with tissue from 1007 RCC patients. Periostin mRNA in RCC was increased, as observed in both RT-PCR and gene microarray analyses, with significantly higher expression in the clear cell than in the papillary subtype. Four of eight periostin isoforms, identified in fetal kidney by direct sequencing, have not been described to date. Three isoforms could be detected in both RCC and matched non-neoplastic tissue, and one of them was expressed more frequently in RCC. Periostin protein was detected in both mesenchymal cells of the tumor stroma and epithelial tumor cells. Greater amounts of periostin in tumor epithelia correlated with the presence of sarcomatoid differentiation, higher tumor stage, lymph node metastases, and poor overall survival in the clear cell subtype. In conclusion, periostin expression in tumor epithelia may contribute to sarcomatoid differentiation and more aggressive behavior of RCC. The presence of a tumor-associated periostin isoform suggests splice-specific regulation in RCC tissue.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/diagnóstico , Moléculas de Adesão Celular/metabolismo , Neoplasias Renais/diagnóstico , Proteínas de Neoplasias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/metabolismo , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/metabolismo , Processamento de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Biologic treatment options such as tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of inflammatory diseases, including rheumatoid arthritis. Recent data suggest, however, that full and long-lasting responses to TNF inhibitors are limited because of the activation of the pro-inflammatory TH17/interleukin (IL)-17 pathway in patients. Therefore, dual TNF/IL-17A inhibition is an attractive avenue to achieve superior efficacy levels in such diseases. Based on the marketed anti-TNF antibody adalimumab, we generated the bispecific TNF/IL-17A-binding FynomAb COVA322. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. COVA322 was characterized in detail and showed a remarkable ability to inhibit TNF and IL-17A in vitro and in vivo. Through its unique mode-of-action of inhibiting simultaneously TNF and the IL-17A homodimer, COVA322 represents a promising drug candidate for the treatment of inflammatory diseases. COVA322 is currently being tested in a Phase 1b/2a study in psoriasis ( ClinicalTrials.gov Identifier: NCT02243787).
Assuntos
Interleucina-17/antagonistas & inibidores , Psoríase/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/imunologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Feminino , Humanos , Interleucina-17/imunologia , Masculino , Camundongos , Psoríase/imunologia , Fator de Necrose Tumoral alfa/imunologiaAssuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/farmacocinética , Complexo CD3/metabolismo , Imunoglobulina G/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.
Assuntos
Retículo Endoplasmático/fisiologia , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas/fisiologia , Glicosilação , Manose/metabolismo , Oligossacarídeos/biossíntese , Oligossacarídeos/metabolismo , alfa-Glucosidases/metabolismoRESUMO
In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.
Assuntos
Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas/metabolismo , Desenho de Fármacos , Retículo Endoplasmático/ultraestrutura , Humanos , Proteínas de Membrana/fisiologia , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/etiologia , Chaperonas Moleculares/uso terapêutico , Proteínas/genéticaRESUMO
Immature and nonnative proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery. Folding-incompetent glycoproteins are eventually targeted for ER-associated protein degradation (ERAD). EDEM1 (ER degradation-enhancing alpha-mannosidase-like protein 1), a putative mannose-binding protein, targets misfolded glycoproteins for ERAD. We report that endogenous EDEM1 exists mainly as a soluble glycoprotein. By high-resolution immunolabeling and serial section analysis, we find that endogenous EDEM1 is sequestered in buds that form along cisternae of the rough ER at regions outside of the transitional ER. They give rise to approximately 150-nm vesicles scattered throughout the cytoplasm that are lacking a recognizable COPII coat. About 87% of the immunogold labeling was over the vesicles and approximately 11% over the ER lumen. Some of the EDEM1 vesicles also contain Derlin-2 and the misfolded Hong Kong variant of alpha-1-antitrypsin, a substrate for EDEM1 and ERAD. Our results demonstrate the existence of a vesicle budding transport pathway out of the rough ER that does not involve the canonical transitional ER exit sites and therefore represents a previously unrecognized passageway to remove potentially harmful misfolded luminal glycoproteins from the ER.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/fisiologia , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Glicoproteínas/química , Glicosilação , Humanos , Proteínas de Membrana/metabolismo , Desnaturação Proteica , Dobramento de Proteína , RatosRESUMO
The prion protein (PrP) is crucially involved in transmissible spongiform encephalopathies (TSE), but neither its exact role in disease nor its physiological function are known. Here we show for mice, using histological, immunochemical and PCR-based methods, that stimulation of innate resistance was followed by appearance of numerous endogenous retroviruses and ensuing PrP up-regulation in germinal centers of the spleen. Subsequently, the activated retroviruses disappeared in a PrP-dependent manner. Our results reveal the regular involvement of endogenous retroviruses in murine immune responses and provide evidence for an essential function of PrP in the control of the retroviral activity. The interaction between PrP and ubiquitous endogenous retroviruses may allow new interpretations of TSE pathophysiology and explain the evolutionary conservation of PrP.