RESUMO
Neuroglobin (Ngb) is expressed in the central and peripheral nervous system, cerebrospinal fluid, retina, and endocrine tissues where it is involved in binding O2 and other gasotransmitters. Several studies have highlighted its endogenous neuroprotective function. Huntington's disease (HD), a dominant hereditary disease, is characterized by the gradual loss of neurons in discrete areas of the central nervous system. We analyzed the expression of Ngb in the brain tissue of a mouse model of HD, in order to define the role of Ngb with respect to individual cell type vulnerability in HD and to gender and age of mice. Our results showed different expressions of Ngb among neurons of a specific region and between different brain regions. We evidenced a decreased intensity of Ngb at 13 weeks of age, compared to 7 weeks of age. The double immunofluorescence and fluorescence resonance energy transfer (FRET) experiments showed that the co-localization between Ngb and huntingtin at the subcellular level was not close enough to account for a direct interaction. We also observed a different expression of Ngb in the striatum, depending on the sex and age of animals. These findings provide the first experimental evidence for an adaptive response of Ngb in HD, suggesting that Ngb may exert neuroprotective effects in HD beyond its role in reducing sensitivity to oxidative stress.
Assuntos
Corpo Estriado/metabolismo , Regulação da Expressão Gênica/genética , Globinas/metabolismo , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/metabolismo , Fatores de Ribosilação do ADP , Animais , Toxinas Bacterianas , Linhagem Celular Tumoral , Colinesterases/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Feminino , Transferência Ressonante de Energia de Fluorescência , Proteína Huntingtina/genética , Doença de Huntington/genética , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Neuroglobina , Neurônios/metabolismo , Parvalbuminas/metabolismo , Fatores Sexuais , Fatores de TempoRESUMO
Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial (MITO) antiapoptotic heat-shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our group's use of mass-spectrometric analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses, and interact directly, as confirmed by fluorescence resonance energy transfer analysis. This is the first demonstration of TRAP1's presence in this cellular compartment. TRAP1 silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER-mitochondria crosstalk.
Assuntos
Neoplasias Colorretais/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação , ATPases Associadas a Diversas Atividades Celulares , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Inativação Gênica , Proteínas de Choque Térmico HSP90/genética , Humanos , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/genética , Dobramento de ProteínaRESUMO
In polarized epithelial cells, sorting of proteins and lipids to the apical or basolateral domain of the plasma membrane can occur via direct or indirect (transcytotic) pathways from the trans Golgi network (TGN). The 'rafts' hypothesis postulates that the key event for direct apical sorting of some transmembrane proteins and the majority of GPI-anchored proteins depends on their association with glycosphingolipid and cholesterol enriched microdomains (rafts). However, the mechanism of indirect sorting to the apical membrane is not clear. The polyimmunoglobulin receptor (pIgR) is one of the best studied proteins that follow the transcytotic pathway. It is normally delivered from the TGN to the basolateral surface of polarized Madin-Darby Canine Kidney (MDCK) cells from where it transports dIgA or dIgM to the apical surface. We have studied the intracellular trafficking of pIgR in Fischer rat thyroid cells (FRT), and have investigated the sorting machinery involved in transcytosis of this receptor in both FRT and MDCK cells. We found that, in contrast with MDCK cells, a significant amount (approximately 30%) of pIgR reaches the apical surface by a direct pathway. Furthermore, in both cell lines it does not associate with Triton X-100 insoluble microdomains, suggesting that at least in these cells 'rafts' are not involved in basolateral to apical transcytosis.