RESUMO
Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic "hairpin" structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.
Assuntos
Proteínas de Membrana/metabolismo , Mutação , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Colesterol/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Simulação de Dinâmica Molecular , Proibitinas , Ligação Proteica , Relação Estrutura-Atividade , Frações Subcelulares/metabolismoRESUMO
The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Aquaporina 1/metabolismo , Membrana Eritrocítica/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Proteínas de Membrana/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/química , Biofísica/métodos , Cromatografia de Afinidade/métodos , Reagentes de Ligações Cruzadas/farmacologia , Eritrócitos/citologia , Transportador de Glucose Tipo 1/química , Humanos , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Modelos Biológicos , Peptídeos/química , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de ProteínaRESUMO
Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Interferon gama/metabolismo , Neoplasias Mamárias Experimentais/terapia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Animais , Western Blotting , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Feminino , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Hemoglobinas/metabolismo , Histocitoquímica , Humanos , Linfócitos do Interstício Tumoral/citologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/metabolismo , Transdução Genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Unique among the retroviruses, mouse mammary tumor virus (MMTV) carries, in addition to the usual long terminal repeat (LTR) promoter, another promoter, P2, which is located in the central part of the proviral U3 sequence, within the LTR open reading frame (ORF). Using an in vitro reporter system based on a sensitive luciferase expression assay, we investigated the regulation of the P2 promoter in the context of the Mtv-2 and Mtv-8 genomes. Irrespective of the genomic source, the activity of the P2 promoter is regulated by a downstream-located enhancer and an upstream-located negative regulatory element (NRE), the activity of which overrides the activator. During this study, we unexpectedly detected another independent neighboring promoter that we called P3. The novel P3 promoter does not seem to be controlled by any NRE but is influenced by the same enhancer that modulates the P2 promoter. The respective transcription starts of the two promoters located in this tight cluster are only 61 bases apart. The transcripts originating from this promoter complex carry the same first intron, which is bound by canonical splice donor and splice acceptor sites located in the LTR. One novel doubly spliced transcript carrying a 459-nucleotide-long ORF was detected in several MMTV-carrying murine cells and could be successfully expressed in murine cells as a His-tagged fusion product. The novel viral protein, the function of which remains to be elucidated, has an apparent molecular mass of 20 kDa.
Assuntos
Regulação Viral da Expressão Gênica , Vírus do Tumor Mamário do Camundongo/genética , Regiões Promotoras Genéticas/genética , Sequências Repetidas Terminais , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes Reporter , Luciferases/análise , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética , Sítios de Splice de RNA , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
As for all retroviruses, the env mRNA is thought to be a singly spliced product of the full-length transcript from the P1 promoter in the MMTV provirus. However, we show that envelope proteins can be produced in an inducible manner in the absence of the P1 promoter from an otherwise complete provirus. Furthermore, we demonstrate in both reporter assays and the proviral context that the R region is necessary for protein production in transiently transfected cells and in a number of independent, stably transfected cell clones. Using 5' RACE, we show that a sequence within the R region functions as a TATA less initiator. The most distal part of the 5' LTR (first 804 bases of the U3 region) is required for the activity of the R-initiator element only when the provirus is integrated. Transfection with a full-length proviral DNA carrying a deletion of P1 in the 5' LTR resulted in the establishment of stable cell clones able to produce Env in a dexamethasone-dependent manner but not infectious virions. We therefore conclude that in the absence of P1, R can drive transcription of the spliced env mRNA but not genomic viral RNA.