RESUMO
The eukaryotic translation initiation factor eIF2B is a multi-subunit complex with a crucial role in the regulation of global protein synthesis in the cell. The complex comprises five subunits, termed α through ε in order of increasing size, arranged as a heterodecamer with two copies of each subunit. Regulation of the co-stoichiometric expression of the eIF2B subunits is crucial for the proper function and regulation of the eIF2B complex in cells. We have investigated the control of stoichiometric eIF2B complexes through mutual stabilization of eIF2B subunits. Our data show that the stable expression of the catalytic eIF2Bε subunit in human cells requires co-expression of eIF2Bγ. Similarly, stable expression of eIF2Bδ requires both eIF2Bß and eIF2Bγ+ε. The expression of these subunits decreases despite there being no change in either the levels or the translation of their mRNAs. Instead, these subunits are targeted for degradation by the ubiquitin-proteasome system. The data allow us to propose a model for the formation of stoichiometric eIF2B complexes which can ensure their stoichiometric incorporation into the holocomplex.
Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Células HEK293 , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Mutação , Polirribossomos/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , Transfecção , UbiquitinaçãoRESUMO
The eukaryotic translation initiation factor (eIF) eIF2B is a key regulator of mRNA translation, being the guanine nt exchange factor (GEF) responsible for the recycling of the heterotrimeric G-protein, eIF2, which is required to allow translation initiation to occur. Unusually for a GEF, eIF2B is a multi-subunit protein, comprising five different subunits termed α through ε in order of increasing size. eIF2B is subject to tight regulation in the cell and may also serve additional functions. Here we review recent insights into the subunit organization of the mammalian eIF2B complex, gained both from structural studies of the complex and from studies of mutations of eIF2B that result in the neurological disorder leukoencephalopathy with vanishing white matter (VWM). We will also discuss recent data from yeast demonstrating a novel function of the eIF2B complex key for translational regulation.
Assuntos
Fator de Iniciação 2B em Eucariotos/genética , Leucoencefalopatias/genética , Mutação , Biossíntese de Proteínas/genética , Animais , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/metabolismo , Predisposição Genética para Doença/genética , Humanos , Leucoencefalopatias/metabolismo , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
BACKGROUND: Leukoencephalopathy with Vanishing White Matter (VWM) is an autosomal recessive disorder caused by germline mutations in the genes EIF2B1-5, which encode the 5 subunits of the eukaryotic translation initiation factor eIF2B. To date, analysis of the biochemical effects of mutations in the EIF2B2-5 genes has been carried out, but no study has been performed on mutations in the EIF2B1 gene. This gene encodes eIF2Bα, the smallest subunit in eIF2B which has an important role in both the structure and regulation of the eIF2B complex. METHODS: eIF2B subunits were overexpressed in HEK293 cells and isolated from the resulting cell lysates by affinity chromatography. Formation of the eIF2B complex and binding of its substrate, eIF2, was assessed by western blot. Assays of the guanine nucleotide exchange (GEF) activity were also carried out. RESULTS: Of the 5 eIF2Bα mutations studied, we found 3 that showed loss or reduction of binding of eIF2Bα to the rest of the complex, one with increased GEF activity, and one where no effects on activity or complex formation were observed. CONCLUSIONS: This is the first study on eIF2Bα VWM mutations. We show that some mutations cause expected decreases in GEF activity or complex formation, similar to a majority of observed VWM mutations. However, we also observe some unexpected changes which hint at other effects of these mutations on as yet undescribed functions of eIF2B.
Assuntos
Fator de Iniciação 2B em Eucariotos/genética , Leucoencefalopatias/genética , Modelos Moleculares , Western Blotting , Cromatografia de Afinidade , Fator de Iniciação 2B em Eucariotos/química , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação/genética , Plasmídeos/genética , Ligação Proteica/genética , Subunidades Proteicas/genéticaRESUMO
Eukaryotic initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for eIF2 and a critical regulator of protein synthesis, (e.g., as part of the integrated stress response). Certain mutations in the EIF2B genes cause leukoencephalopathy with vanishing white matter (VWM), an often serious neurological disorder. Comprising 5 subunits, α-ε (eIF2Bε being the catalytic one), eIF2B has always been considered an αßγδε heteropentamer. We have analyzed the subunit interactions within mammalian eIF2B by using a combination of mass spectrometry and in vivo studies of overexpressed complexes to gain further insight into the subunit arrangement of the complex. Our data reveal that eIF2B is actually decameric, a dimer of eIF2B(ßγδε) tetramers stabilized by 2 copies of eIF2Bα. We also demonstrate a pivotal role for eIF2Bδ in the formation of eIF2B(ßγδε) tetramers. eIF2B(αßγδε)2 decamers show greater binding to eIF2 than to eIF2B(ßγδε) tetramers, which may underlie the increased activity of the former. We examined the levels of eIF2B subunits in a panel of different mouse tissues and identified different levels of eIF2B subunits, particularly eIF2Bα, which implies heterogeneity in the cellular proportions of eIF2B(αßγδε) and eIF2B(ßγδε) complexes, with important implications for the regulation of translation in individual cell types.
Assuntos
Fator de Iniciação 2B em Eucariotos/química , Regulação da Expressão Gênica , Sequência de Aminoácidos , Animais , Catálise , Citoplasma/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteômica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Eukaryotic initiation factor 2B (eIF2B) plays a key role in protein synthesis and in its control. It comprises five different subunits, α-ε, of which eIF2Bε contains the catalytic domain. Formation of the complete complex is crucial for full activity and proper control of eIF2B. Mutations in the genes for eIF2B cause an often severe neurological disorder, "vanishing white matter." eIF2Bγ and eIF2Bε contain homologous and conserved domains with sequence similarity to nucleotidyl transferases (NTs) and acyl transferases and can form a binary complex. The latter contain a hexad repeat that mainly comprises isoleucyl residues (hence termed the "I-patch" region). These data reveal that certain residues in the NT domains of eIF2Bγ/ε, which are highly conserved throughout eukaryotes, play key roles in the interactions between subunits in the eIF2B complex. Our data show that the I-patch regions are important in the interactions between the catalytic eIF2Bγε complex and the other subunits. We also studied the functional effects of vanishing white matter mutations in the NT and I-patch domains. Lastly, our data show that eIF2Bγ promotes the expression of eIF2Bε, providing a mechanism for achieving correct stoichiometry of these eIF2B subunits in the cell.
Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexos Multienzimáticos/metabolismo , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/genética , Células HEK293 , Humanos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/genética , Mapeamento de Peptídeos , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
Autosomal recessive mutations in eukaryotic initiation factor 2B (eIF2B) cause leukoencephalopathy vanishing white matter with a wide clinical spectrum. eIF2B comprises five subunits (α-ε; genes EIF2B1, 2, 3, 4 and 5) and is the guanine nucleotide-exchange factor (GEF) for eIF2. It plays a key role in protein synthesis. Here, we have studied the functional effects of selected VWM mutations in EIF2B2-5 by coexpressing mutated and wild-type subunits in human cells. The observed functional effects are very diverse, including defects in eIF2B complex integrity; binding to the regulatory α-subunit; substrate binding; and GEF activity. Activity data for recombinant eIF2B complexes agree closely with those for patient-derived cells with the same mutations. Some mutations do not affect these parameters even though they cause severe disease. These findings are important for three reasons; they demonstrate that measuring eIF2B activity in patients' cells has limited value as a diagnostic test; they imply that severe disease can result from alterations in eIF2B function other than defects in complex integrity, substrate binding or GEF activity, and last, the diversity of functional effects of VWM mutations implies that seeking agents to manage or treat VWM should focus on downstream effectors of eIF2B, not restoring eIF2B activity.
Assuntos
Fator de Iniciação 2B em Eucariotos/deficiência , Fator de Iniciação 2B em Eucariotos/metabolismo , Leucoencefalopatias/genética , Complexos Multiproteicos/metabolismo , Bioensaio , Extratos Celulares , Fator de Iniciação 2B em Eucariotos/química , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Homologia de Sequência de AminoácidosRESUMO
CDC4/FBXW7 is part of a ubiquitin ligase complex which targets molecules such as cyclin E, c-myc, and c-jun for destruction. CDC4 mutations occur in several cancer types and are best described in colorectal tumors. Knockout of CDC4 in vitro in colorectal cancer cells causes changes suggestive of chromosomal instability (CIN). In p53(+/-) mice, radiation-induced lymphomas show deletion or mutation of one copy of CDC4 and knockdown of CDC4 leads to increased aneuploidy in mouse fibroblasts. We screened 244 colorectal tumors and 40 cell lines for CDC4 mutations and allelic loss. Six percent (18 of 284) of tumors, including near-diploid (CIN-) lesions, harbored CDC4 mutations and there was no association between mutation and CIN (polyploidy). The CDC4 mutation spectrum in colorectal tumors was heavily biased towards C:G > T:A changes, either missense mutations at critical arginine residues or nonsense changes in the 5' half of the gene. The reasons for this odd mutation spectrum were unclear but C:G > T:A changes were not found more often than expected at APC, K-ras, or p53 in the same tumors and we found no specific defects in DNA repair to account for the observations. No colorectal tumor was found to carry two CDC4 mutations predicted to abolish protein function; partial loss of CDC4 function may therefore cause tumorigenesis. The in vitro studies, therefore, did not assess the functional effects of mutant alleles which are found in vivo. CDC4 mutations may be selected primarily to drive progression through the cell cycle although CIN might be an important secondary effect in some cancers.
Assuntos
Proteínas de Ciclo Celular/genética , Instabilidade Cromossômica , Neoplasias Colorretais/genética , Proteínas F-Box/genética , Perda de Heterozigosidade , Mutação/genética , Ubiquitina-Proteína Ligases/genética , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adenoma/genética , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , DNA de Neoplasias , Proteína 7 com Repetições F-Box-WD , Genes ras/genética , Humanos , Repetições de Microssatélites , Ploidias , Polimorfismo Conformacional de Fita Simples , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Uterine fibroids are some of the most common tumours of females, but relatively little is known about their molecular basis. Several studies have suggested that deletions on chromosome 7q could have a role in fibroid formation. We analysed 165 sporadic uterine fibroids to define a small 3.2 megabase (Mb) commonly deleted region on 7q22.3-q31.1, flanked by clones AC005070 and AC007567. We also used oligonucleotide microarrays to compare the expression profiles of 10 samples of normal myometrium and 15 fibroids, nine of which displayed 7q-deletions. Activating transcription factor 3, patched homolog (Drosophila), homeo box A5, death-associated protein kinase 1, and retinoic acid receptor responder 3 were downregulated, and excision repair crosscomplementing 3, transcription factor AP-2 gamma and protein kinase C beta 1 were upregulated in fibroids. New pathways were discovered related to fibroid formation. The presence or absence of 7q-deletions did not dramatically affect the global expression pattern of the tumours; changes, however, were observed in genes related to vesicular transport and nucleic acid binding.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 7 , Leiomioma/genética , Neoplasias Uterinas/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização In Situ/métodos , Leiomioma/patologia , Perda de Heterozigosidade , Miométrio/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Valores de Referência , Neoplasias Uterinas/patologiaRESUMO
CONTEXT: Leydig cell tumors (LCTs) are the most common non-germ-cell neoplasms of the testis. LCTs are often hormonally active and can result in precocious virilization or in adult feminization. We identified an LCT in an affected individual from a kindred with hereditary leiomyomatosis and renal cell cancer (HLRCC) and a germline fumarate hydratase (FH) mutation (N64T). OBJECTIVE: Our objective was to investigate the role of FH mutations in predisposition to LCTs. DESIGN: We tested for pathogenic effects of the N64T mutation and screened an additional 29 unselected adult LCTs for FH alterations. We also tested these LCTs for mutations in two genes, the LH/choriogonadotropin receptor (LHCGR) and the guanine nucleotide-binding protein alpha (GNAS) that had been implicated in LCT tumorigenesis. RESULTS: No mutations were found in GNAS, and one tumor had a LHCGR somatic substitution. In addition to the HLRCC case with the N64T germline FH mutation, we identified one other LCT with a previously unreported FH mutation (M411I). Both LCTs from these patients showed loss of the wild-type FH allele. Immunohistochemical and in situ hybridization analyses demonstrated activation of the hypoxia/angiogenesis pathway not only in the tumors belonging to the FH mutation carriers but also in several other mutation-negative LCTs. CONCLUSIONS: Our study shows that some LCTs are caused by FH mutations and represents one of the first reports of germline mutations in any type of adult testicular tumor.
Assuntos
Fumarato Hidratase/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Tumor de Células de Leydig/genética , Neoplasias Testiculares/genética , Sequência de Bases , Cromograninas , DNA Complementar/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Imuno-Histoquímica , Hibridização In Situ , Tumor de Células de Leydig/química , Masculino , Modelos Moleculares , Neovascularização Patológica , Receptores do LH/genética , Análise de Sequência de DNA , Neoplasias Testiculares/química , Fator A de Crescimento do Endotélio Vascular/análiseAssuntos
Fator de Iniciação 2B em Eucariotos/genética , Leucoencefalopatias/genética , Degeneração Neural/genética , Adulto , Idade de Início , Substituição de Aminoácidos , Sequência de Bases , Feminino , Humanos , Leucoencefalopatias/epidemiologia , Leucoencefalopatias/patologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/fisiologia , Degeneração Neural/patologia , Isoformas de Proteínas/genéticaRESUMO
Dercum's disease (adiposis dolorosa, lipomatosis dolorosa morbus Dercum), is a rare disorder resulting in painful fatty deposits around the upper legs, trunk, and upper arms. The portrait painted of Dercum's disease is very complicated, with many other disorders seen associated with the disease. There are no clear pathological mechanisms known, although it is suspected that there is either a metabolic or autoimmune component involved. Here, the authors review the literature to date, including some information from their own studies. In particular, the authors will look at the different strands of evidence pointing to the pathological mechanism of the disorder.
Assuntos
Adipose Dolorosa/diagnóstico , Adipose Dolorosa/terapia , Adipose Dolorosa/etiologia , HumanosRESUMO
Somatic interstitial deletions of chromosome segment 7q22-q31 in uterine leiomyomas are a frequent event, thought to be indicative of a tumor suppressor gene in the region. Previous LOH and CGH studies have refined this region to 7q22.3-q31, although the target gene has not been identified. Here, we have used tiling-path resolution microarray CGH to further refine the region and to identify homozygous deletions in fibroids. Furthermore, we have screened all manually annotated genes in the region for mutations. We have refined the minimum deleted region at 7q22.3-q31 to 2.79 Mbp and identified a second region of deletion at 7q34. However, we identified no pathogenic coding variation.
Assuntos
Cromossomos Humanos Par 7 , Dano ao DNA , Leiomioma/genética , Análise de Sequência com Séries de Oligonucleotídeos , Deleção de Sequência , Neoplasias Uterinas/genética , Mapeamento Cromossômico , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Humanos , Histerectomia , Leiomioma/cirurgia , Proteínas de Neoplasias/genética , Hibridização de Ácido Nucleico , Neoplasias Uterinas/cirurgiaRESUMO
OBJECTIVE: To examine differences between sporadic and familial uterine leiomyomata related to expression of apoptosis-related proteins and tumor ultrastructure. DESIGN: Expression of apoptosis-related proteins was measured by immunohistochemistry. Tumor ultrastructure was evaluated by transmission electron microscopy. SETTING: Human genetics laboratory. PATIENT(S): Patients confirmed for hereditary leiomyomatosis and renal cell carcinoma (HLRCC), and anonymous archival sporadic leiomyoma patients. INTERVENTION(S): Samples for electron microscopy were collected from myomectomy and hysterectomy with informed consent. Other samples were archival. MAIN OUTCOME MEASURE(S): Intensity of immunohistochemistry staining and evaluation of electron micrographs. RESULT(S): Immunohistochemistry revealed increases in expression of antiapoptotic Bcl-2 and the proliferation factor proliferating cell nuclear antigen (PCNA) in both sporadic and HLRCC uterine leiomyomata. Furthermore, we observed an increase in antiapoptotic Bcl-x and a concurrent decrease in proapoptotic Bak solely in HLRCC leiomyomas. We also observed ultrastructural alterations in HLRCC and sporadic leiomyomas, particularly pertaining to extracellular matrix and intermediate filament aggregation. CONCLUSION(S): The observed alterations in expression of apoptosis-related proteins indicate a shift in both HLRCC and sporadic leiomyomas to increased resistance to apoptosis compared with myometrium, which appears to be stronger in HLRCC leiomyomas. The changes observed in HLRCC leiomyomas appear to be related to activation of the hypoxia pathways. The results suggest not only a partial overlap in the pathogenic mechanism of the two tumor types, but also intriguing differences.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/ultraestrutura , Leiomioma/metabolismo , Leiomioma/ultraestrutura , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/ultraestrutura , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/ultraestrutura , Leiomioma/congênito , Distribuição TecidualRESUMO
The Mendelian tumour syndromes hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary paragangliomatosis with phaeochromocytomas (HPGL) result from mutations in nuclear genes (FH and SDHB/C/D, respectively) that encode Krebs cycle enzymes. HPGL tumours are highly vascular and there is evidence that inactivation of SDH leads to activation of the hypoxia/angiogenesis pathway. In contrast, uterine leiomyomas are not generally regarded as particularly vascular lesions. In order to test the possibility that activation of the hypoxia/angiogenesis pathway contributes to tumourigenesis in HLRCC, increased vascularity and hypoxia pathway activation were searched for in HLRCC tumours. Microvessel density was markedly higher in uterine leiomyomas from HLRCC than in the surrounding myometrium; it was notable that sporadic uterine leiomyomas were actually less vascular than normal myometrium. In HLRCC tumours, there was increased expression of transcripts from the hypoxia-responsive genes vascular endothelial growth factor (VEGF) and BNIP3; sporadic uterine leiomyomas did not show these changes. All uterine leiomyomas showed decreased expression of thrombospondin 1. Although sporadic and HLRCC uterine leiomyomas appear to have identical morphology, their pathways of tumourigenesis may be fundamentally different. As is the case in HPGL, it is probable that failure of the Krebs cycle in HLRCC tumours causes inappropriate signalling that the cell is in a hypoxic state, leading to angiogenesis and perhaps directly to clonal expansion and tumour growth through some uncharacterized, cell-autonomous effect.
Assuntos
Carcinoma de Células Renais/irrigação sanguínea , Neoplasias Renais/irrigação sanguínea , Leiomiomatose/irrigação sanguínea , Síndromes Neoplásicas Hereditárias/patologia , Neovascularização Patológica/patologia , Adulto , Carcinoma de Células Renais/patologia , Hipóxia Celular , Feminino , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Neoplasias Renais/patologia , Leiomiomatose/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/patologia , Neoplasias Uterinas/irrigação sanguínea , Neoplasias Uterinas/patologiaRESUMO
It is well documented that disturbances in mitochondrial function are associated with rare childhood disorders and possibly with many common diseases of ageing, such as Parkinson's disease and dementia. There has also been increasing evidence linking mitochondrial dysfunction with tumorigenesis. Recently, heterozygous germline mutations in two enzymes of the Krebs tricarboxylic acid cycle (TCA cycle) have been shown to predispose individuals to tumours. The two enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), are ubiquitously expressed, playing a vital role in adenosine triphosphate (ATP) production through the mitochondrial respiratory chain. Germline mutations in FH are associated with leiomyomatosis and renal cell carcinoma, whilst SDH mutations are associated with predisposition to paraganglioma (PGL) and phaeochromocytoma (PCC). At present, there are few data to explain the pathway(s) involved in this predisposition to neoplasia through TCA cycle defects. We shall review the mechanisms by which mutations in FH and SDH might play a role in tumorigenesis. These include pseudo-hypoxia, mitochondrial dysfunction and impaired apoptosis, oxidative stress and anabolic drive. All of these mechanisms are currently poorly defined. To date, FH and SDH mutations have not been reported in non-familial leiomyomata, renal cancers, PCCs or PGLs. It remains entirely possible, however, that the underlying mechanisms of tumorigenesis in these sporadic tumours are the same as those in the Mendelian syndromes.
Assuntos
Ciclo do Ácido Cítrico , Fumarato Hidratase/genética , Neoplasias/etiologia , Succinato Desidrogenase/genética , Fumarato Hidratase/metabolismo , Mutação em Linhagem Germinativa , Humanos , Hipóxia/complicações , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Neoplasias/enzimologia , Neoplasias/genética , Estresse Oxidativo , Succinato Desidrogenase/metabolismoRESUMO
Igsf8 is an immunoglobulin protein that binds to the tetraspanin molecules, CD81 and CD9. We describe the genomic organization of mouse and human Igsf8, and reveal a dynamic expression pattern during embryonic and fetal development. Igsf8 is first expressed at E9.5 in a ventral domain of the neural tube, with dorsal expression apparent at E10.5. We show that the ventral, but not the dorsal, domain of neural tube expression is dependent on Shh signaling. From E11.5, Igsf8 is expressed at the lateral edge of the ventricular zone, in early postmitotic neuroblasts, and in dorsal root and cranial ganglia. Igsf8 is also expressed in the branchial arches, dorsal pancreatic primordium, neural retina, olfactory epithelium, gut, kidney, and lung.