RESUMEN
Bariatric surgery is often the preferred method to resolve obesity and diabetes, with â¼800,000 cases worldwide yearly and high outcome variability. The ability to predict the long-term body mass index (BMI) change following surgery has important implications for individuals and the health care system in general. Given the tight connection between eating habits, sugar consumption, BMI, and the gut microbiome, we tested whether the microbiome before any treatment is associated with different treatment outcomes, as well as other intakes (high-density lipoproteins [HDL], triglycerides, etc.). A projection of the gut microbiome composition of obese (sampled before and after bariatric surgery) and lean patients into principal components was performed, and the relation between this projection and surgery outcome was studied. The projection revealed three different microbiome profiles belonging to lean, obese, and obese individuals who underwent bariatric surgery, with the postsurgery microbiome more different from the lean microbiome than the obese microbiome. The same projection allowed for a prediction of BMI loss following bariatric surgery, using only the presurgery microbiome. The microbial changes following surgery were an increase in the relative abundance of Proteobacteria and Fusobacteria and a decrease in Firmicutes. The gut microbiome can be decomposed into main components depicting the patient's development and predicting in advance the outcome. Those may be translated into the better clinical management of obese individuals planning to undergo metabolic surgery. IMPORTANCE BMI and diabetes can affect the gut microbiome composition. Bariatric surgery has large variabilities in the outcome. The microbiome was previously shown to be a good predictor for multiple diseases. We analyzed here the gut microbiome before and after bariatric surgery and showed the following. (i) The microbiome before surgery can be used to predict surgery outcomes. (ii) The postsurgery microbiome drifts further away from the lean microbiome than the microbiome of the presurgery obese patients. These results can lead to a microbiome-based presurgery decision whether to perform surgery.
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The potential of circulating tumor cells (CTCs) in the diagnosis and prognosis of cancer patients has become increasingly attractive. However, molecular analysis of CTCs is hindered by low sensitivity and a high level of background leukocytes in CTC enrichment technologies. We have developed a novel protocol using a microfluidic device, which enriches and retrieves CTCs from blood samples. The principle of CTC capturing is that tumor cells are larger and less deformable than normal blood cells. To evaluate the potential of utilizing Celsee PREP100 in CTC molecular analysis, we prepared prostate cancer cell lines PC3 and LNCaP, retrieved the captured cells and analyzed them using PCR amplicon sequencing. We were able to recover an average of 79% of 1101,100 PC3 and 601,500 LNCaP cells, and detect the p.K139fs*3 deletion of the p53 gene in PC3 cells and p.T877A mutation of the androgen receptor gene in LNCaP cells. Next, we spiked these two types of cells into normal donor blood samples, captured the cells and analyzed them using PCR amplicon sequencing. The PC3 and LNCaP cells were captured and retrieved with the ratio of captured CTCs to the background leukocytes reaching 1:1.5 for PC3 and 1:2.9 for LNCaP cells. We further revealed that the p.K139fs*3 deletion and p.T877A mutation can be detected in the captured PC3 and LNCaP cells, respectively. We successfully validated this approach using clinical blood samples from patients with metastatic prostate cancer. Our results demonstrated a novel approach for CTC enrichment and illustrated the potential of CTC molecular characterization for diagnosis, prognosis and treatment selection of patients with metastatic malignancy.
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Biomarcadores de Tumor/genética , Separación Celular/instrumentación , Mutación , Células Neoplásicas Circulantes/patología , Neoplasias de la Próstata/genética , Línea Celular Tumoral , Detección Precoz del Cáncer , Humanos , Dispositivos Laboratorio en un Chip , Masculino , Células Neoplásicas Circulantes/química , Pronóstico , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Receptores Androgénicos/genética , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5' end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3' end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts.
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Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Endocitosis/fisiología , Aparato de Golgi/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mioblastos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Citoesqueleto de Actina/metabolismo , Empalme Alternativo , Animales , Línea Celular , Citoesqueleto/metabolismo , Distonina , Ratones , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/metabolismoRESUMEN
The microtubule-actin crosslinking factor 1 (MACF1) is a ubiquitous cytoskeletal linker protein with multiple spliced isoforms expressed in different tissues. The MACF1a isoform contains microtubule and actin-binding regions and is expressed at high levels in the nervous system. Macf1-/- mice are early embryonic lethal and hence the role of MACF1 in the nervous system could not be determined. We have specifically knocked out MACF1a in the developing mouse nervous system using Cre/loxP technology. Mutant mice died within 24-36h after birth of apparent respiratory distress. Their brains displayed a disorganized cerebral cortex with a mixed layer structure, heterotopia in the pyramidal layer of the hippocampus, disorganized thalamocortical and corticofugal fibers, and aplastic anterior and hippocampal commissures. Embryonic neurons showed a defect in traversing the cortical plate. Our data suggest a critical role for MACF1 in neuronal migration that is dependent on its ability to interact with both microfilaments and microtubules.
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Encéfalo/anomalías , Encéfalo/metabolismo , Proteínas de Microfilamentos/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Encéfalo/fisiopatología , Diferenciación Celular/genética , Movimiento Celular/genética , Corteza Cerebral/anomalías , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Hipocampo/anomalías , Hipocampo/metabolismo , Hipocampo/fisiopatología , Ratones , Ratones Noqueados , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/fisiopatología , Vías Nerviosas/anomalías , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiopatología , Neurogénesis/genéticaRESUMEN
Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy that has been linked to mutations in multiple genes. Mutations in the neurofilament light (NFL) chain gene lead to the CMT2E form whereas mutations in the myotubularin-related protein 2 and 13 (MTMR2 and MTMR13) genes lead to the CMT4B form. These two forms share characteristic pathological hallmarks on nerve biopsies including concentric sheaths ('onion bulbs') and, in at least one case, myelin loops. In addition, MTMR2 protein has been shown to interact physically with both NFL and MTMR13. Here, we present evidence that CMT-linked mutations of MTMR2 can cause NFL aggregation in a cell line devoid of endogenous intermediate filaments, SW13vim(-). Mutations in the protein responsible for X-linked myotubular myopathy (myotubularin, MTM1) also induced NFL abnormalities in these cells. We also show that two MTMR2 mutant proteins, G103E and R283W, are unable to form dimers and undergo phosphorylation in vivo, implicating impaired complex formation in myotubularin-related pathology.
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Enfermedad de Charcot-Marie-Tooth/metabolismo , Proteínas de Neurofilamentos/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Animales , Células COS , Línea Celular Tumoral , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Chlorocebus aethiops , Dimerización , Humanos , Mutagénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas no Receptoras/química , Serina/metabolismo , TransfecciónRESUMEN
BACKGROUND: Giant axonal neuropathy (GAN) is a hereditary neurological disorder that affects both central and peripheral nerves. The main pathological hallmark of the disease is abnormal accumulations of intermediate filaments (IFs) in giant axons and other cell types. Mutations in the GAN gene, encoding gigaxonin, cause the disease. Gigaxonin is important in controlling protein degradation via the ubiquitin-proteasome system. The goal of this study was to examine global alterations in gene expression in fibroblasts derived from newly identified GAN families compared with normal cells. RESULTS: We report the characterization of fibroblast explants obtained from two unrelated GAN patients. We identify three novel putative mutant GAN alleles and show aggregation of vimentin IFs in these fibroblasts. By microarray analysis, we also demonstrate that the expression of lipid metabolism genes of the GAN fibroblasts is disrupted, which may account for the abnormal accumulations of lipid droplets in these cells. CONCLUSION: Our findings suggest that aberrant lipid metabolism in GAN patients may contribute to the progression of the disease.
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Codón sin Sentido , Proteínas del Citoesqueleto/genética , Fibroblastos/metabolismo , Metabolismo de los Lípidos/genética , Lípidos/análisis , Mutación Missense , Alelos , Axones/ultraestructura , Línea Celular/metabolismo , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/fisiología , Fibroblastos/ultraestructura , Perfilación de la Expresión Génica , Genotipo , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/ultraestructura , Intrones/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Vimentina/análisisRESUMEN
The molecules that mediate neuron death in Alzheimer's disease (AD) are largely unknown. We report that beta-amyloid (Abeta), a death-promoting peptide implicated in the pathophysiology of AD, induces the proapoptotic protein Bcl-2 interacting mediator of cell death (Bim) in cultured hippocampal and cortical neurons. We further find that Bim is an essential mediator of Abeta-induced neurotoxicity. Our examination of postmortem AD human brains additionally reveals upregulation of Bim in vulnerable entorhinal cortical neurons, but not in cerebellum, a region usually unaffected by AD. Accumulating evidence links inappropriate induction/activation of cell cycle-related proteins to AD, but their roles in the disease have been unclear. We find that the cell cycle molecule cyclin-dependent kinase 4 (cdk4) and its downstream effector B-myb, are required for Abeta-dependent Bim induction and death in cultured neurons. Moreover, neurons that overexpress Bim in AD brains also show elevated levels of the cell cycle-related proteins cdk4 and phospho-Rb. Our observations indicate that Bim is a proapoptotic effector of Abeta and of dysregulated cell cycle proteins in AD and identify both Bim and cell cycle elements as potential therapeutic targets.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/fisiología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/fisiología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/fisiología , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/toxicidad , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Supervivencia Celular/fisiología , Células Cultivadas , Hipocampo/metabolismo , Humanos , Proteínas de la Membrana/genética , Neuronas/patología , Proteínas Proto-Oncogénicas/genética , RatasRESUMEN
Dystonia musculorum (dt) is an inherited autosomal recessive neuropathy in mice. Homozygous animals display primarily sensory neurodegeneration resulting in a severe loss of coordination. Several dt strains exist, including spontaneous mutants dt-Alb (Albany), dt-J (Jackson Labs), and dt-Frk (Frankel), and a transgene insertion mutant, Tg4. They contain mutations in the gene encoding Bullous Pemphigoid Antigen 1 (BPAG1), or dystonin. BPAG1 is a member of the plakin family of cytolinker proteins. BPAG1 is alternatively spliced to produce several isoforms, including the major brain-specific isoform, BPAG1a. The neurological phenotype observed in dt-Alb mice is thought to result from the absence of BPAG1a protein in the developing nervous system. The goal of this study was to determine the precise molecular nature of the dt-Alb mutation and examine residual BPAG1 expression in homozygous dt-Alb mice. A combination of molecular biological strategies revealed that the dt-Alb lesion is a deletion-insertion eliminating a large part of the coding region of BPAG1a. The molecular lesion in the dt-Alb BPAG1 allele is expected to render it completely non-functional. Although transcripts corresponding to BPAG1 segments still remaining in homozygous dt-Alb mice could be detected by RT-PCR, there was no positive signal for BPAG1 in the brain of dt-Alb mice by Northern blotting. Western blotting with polyclonal anti-BPAG1 antibodies confirmed the absence of functional BPAG1 protein (full-length or truncated) in the dt-Alb brain. Our identification of the 5' junction of the dt-Alb insertion makes it possible to genotype dt-Alb animals by standard PCR.
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Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , Encéfalo/metabolismo , Distonina , Expresión Génica/genética , Genotipo , Ratones , Ratones Mutantes Neurológicos , Ratones Transgénicos , Biología Molecular/métodos , Fenotipo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1(-/-) mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3(-/-) and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, beta-catenin, GSK3beta, and APC. Upon Wnt stimulation, MACF1 appeared to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of beta-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/beta-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/beta-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway.
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Proteínas de Microfilamentos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Proteína Axina , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Distonina , Regulación del Desarrollo de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Mesodermo/patología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Complejos Multiproteicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Represoras/metabolismo , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins that is involved in cross-linking the cytoskeletal elements and attaching them to cell junctions. BPAG1 null mice develop severe degeneration of sensory neurons that was attributed in part due to the absence of a splice variant called BPAG1a that harbors an actin-binding domain at the N-terminus. Additional alternative splicing also results in BPAG1a isoforms with different first exons, leading to three additional types of BPAG1a called isoforms 1, 2 and 3 (or BPAG1a1, BPAG1a2, and BPAG1a3). These unique N-terminal extensions of the BPAG1a isoforms are of variable length. In this study, we characterized these N-terminal isoforms and evaluated the influence of these unique N-terminal sequences to the actin-binding properties. The unique N-terminal region of isoform 1 is very short and was not expected to affect the property of the ABD that followed it. In contrast, transfection studies and mutagenesis analyses signified that the N-terminal sequences of isoform 2 had the ability to bundle actin filaments and the N-terminal region that contained isoform 3 showed cortical localization. Isoforms 1, 2 and 3 also displayed differential tissue expression profiles. Taken together, these data suggested that the unique N-terminal regions of these isoforms have different roles that may be tailored to meet tissue specific functions.
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Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Actinas/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Distonina , Exones , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína , Alineación de Secuencia , TransfecciónAsunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Mutación , Proteínas de Neurofilamentos/genética , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente Indirecta , Duplicación de Gen , Humanos , Masculino , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas de Neurofilamentos/metabolismo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
Intermediate filaments (IFs) are found in most eukaryotic cells and are made up of various IF proteins. IFs are highly insoluble in conventional extraction buffers and are therefore commonly purified under denaturing condition. Purified IF proteins can be reassembled into filaments by dialysis. At least 65 IF proteins are found in humans, and the procedures for the purification of each subunit vary somewhat, although many basic steps are similar. To illustrate the isolation of IFs, a detailed protocol is described for purifying neurofilament proteins (NFL, NFM, and NFH subunits) from bovine spinal cord. These three proteins form the predominant IF network in mature neurons. An alternative method for the purification of NFL from a prokaryotic expression system is also included. The isolation of recombinant proteins from bacteria is quite straightforward and may therefore be the method of choice for producing and purifying IFs. Finally, there is a discussion of the purification methods of other IF proteins.
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Filamentos Intermedios , Proteínas de Neurofilamentos/aislamiento & purificación , Animales , Bovinos , Cromatografía por Intercambio Iónico , Médula Espinal/químicaRESUMEN
Although, in principle, gene expression profiling is well suited to isolate pathogenic molecules associated with Alzheimer's disease (AD), techniques such as microarray present unique analytic challenges when applied to disorders of the brain. Here, we addressed these challenges by first constructing a spatiotemporal model, predicting a priori how a molecule underlying AD should behave anatomically and over time. Then, guided by the model, we generated gene expression profiles of the entorhinal cortex and the dentate gyrus, harvested from the brains of AD cases and controls covering a broad age span. Among many expression differences, the retromer trafficking molecule VPS35 best conformed to the spatiotemporal model of AD. Western blotting confirmed the abnormality, establishing that VPS35 levels are reduced in brain regions selectively vulnerable to AD. VPS35 is the core molecule of the retromer trafficking complex and further analysis revealed that VPS26, another member of the complex, is also downregulated in AD. Cell culture studies, using small interfering RNAs or expression vectors, showed that VPS35 regulates Abeta peptide levels, establishing the relevance of the retromer complex to AD. Reviewing our findings in the context of recent studies suggests how downregulation of the retromer complex in AD can regulate local levels of Abeta peptide.
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Enfermedad de Alzheimer/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Artefactos , Ácido Aspártico Endopeptidasas , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Persona de Mediana Edad , Transporte de Proteínas/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. We have identified a novel alternatively spliced isoform of MACF1. We named this isoform MACF1b and renamed the original isoform MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. We studied the subcellular localization of MACF1b proteins in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex.
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Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Microfilamentos/metabolismo , Plaquinas/metabolismo , Animales , Células COS , Compartimento Celular/fisiología , Chlorocebus aethiops , Regulación hacia Abajo/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica/fisiología , Humanos , Membranas Intracelulares/ultraestructura , Pulmón/metabolismo , Pulmón/ultraestructura , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Plaquinas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/fisiología , ARN Interferente PequeñoRESUMEN
Peripherin is a neuronal intermediate filament protein that is expressed chiefly in motor neurons and other nerve cells that project into the peripheral nervous system. Transgenic mice that over-express peripherin develop motor neuron degeneration, suggesting that mutations in peripherin could contribute to the development of motor neuron disease. In this paper, we report the identification of a homozygous mutation in the peripherin gene (PRPH) in a patient with amyotrophic lateral sclerosis (ALS). The mutation resulted in a substitution of aspartate with tyrosine at amino acid position 141, which is located within the first linker region of the rod domain. Immunocytochemical analysis of the spinal cord of the patient upon autopsy revealed distinctive large aggregates within the cell bodies of residual spinal motor neurons that contained peripherin and was also immunoreactive with antibodies to the neurofilament proteins. In order to study the effect of the mutation on peripherin assembly, we performed transient transfections. Unlike wild-type peripherin, which self-assembles to form a filamentous network, the mutant peripherin was prone to form aggregates in transfected cells, indicating that the mutation adversely affects peripherin assembly. Moreover, the neurofilament light (NF-L) protein was not able to rescue the mutant protein from forming aggregates. These data imply that mutation of PRPH is a contributing factor for ALS.
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Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Médula Espinal/patología , Adulto , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Secuencia de Bases , Western Blotting , Encéfalo/patología , Células Cultivadas , Genotipo , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Datos de Secuencia Molecular , Neuronas Motoras/patología , Proteínas de Neurofilamentos/metabolismo , Periferinas , Mutación Puntual , Médula Espinal/metabolismo , TransfecciónRESUMEN
Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11-q21 (ref. 2). Here we report a missense mutation in the gene encoding 27-kDa small heat-shock protein B1 (HSPB1, also called HSP27) that segregates in the family with CMT2F. Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (distal HMNs) confirmed the previously observed mutation and identified four additional missense mutations. We observed the additional HSPB1 mutations in four families with distal HMN and in one individual with CMT neuropathy. Four mutations are located in the Hsp20-alpha-crystallin domain, and one mutation is in the C-terminal part of the HSP27 protein. Neuronal cells transfected with mutated HSPB1 were less viable than cells expressing the wild-type protein. Cotransfection of neurofilament light chain (NEFL) and mutant HSPB1 resulted in altered neurofilament assembly in cells devoid of cytoplasmic intermediate filaments.
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Enfermedad de Charcot-Marie-Tooth/genética , Proteínas de Choque Térmico/genética , Neuropatía Hereditaria Motora y Sensorial/genética , Mutación Missense , Proteínas de Neoplasias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Femenino , Proteínas de Choque Térmico HSP27 , Humanos , Masculino , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Degeneración Nerviosa/genética , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , TransfecciónAsunto(s)
Proteínas del Citoesqueleto/metabolismo , Actinas/metabolismo , Animales , Far-Western Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes , Células COS , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Humanos , Técnicas In Vitro , Microtúbulos/metabolismo , Nocodazol , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Coloración y Etiquetado , Tiazoles , Tiazolidinas , Transfección , Técnicas del Sistema de Dos HíbridosAsunto(s)
Regulación de la Expresión Génica/genética , Filamentos Intermedios/fisiología , Glicoproteínas de Membrana , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Animales , Axones/fisiología , Tamaño de la Célula/fisiología , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas de Filamentos Intermediarios/genética , Filamentos Intermedios/ultraestructura , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , PeriferinasRESUMEN
The human Gas2-related gene on chromosome 22 (hGAR22) encodes two alternatively spliced mRNA species. The longer mRNA encodes a protein with a deduced molecular mass of 36.3 kDa (GAR22alpha), whereas the shorter mRNA encodes a larger protein with a deduced molecular mass of 72.6 kDa (GAR22beta). We show that both hGAR22 proteins contain a calponin homology actin-binding domain and a Gas2-related microtubule-binding domain. Using rapid amplification of cDNA ends, we have cloned the mouse orthologue of hGAR22, mGAR22, and found its protein products to be extremely well conserved. We also report the cDNA cloning of a human Gas2-related gene on chromosome 17 (hGAR17). hGAR17 also encodes two protein isoforms. The overall cytoskeletal binding properties of the hGAR17 and hGAR22 proteins are remarkably similar. hGAR17 mRNA expression is limited to skeletal muscle. Although hGAR22 and mGAR22 mRNAs are expressed nearly ubiquitously, mGAR22 protein can only be detected in testis and brain. Furthermore, only the beta isoform is present in these tissues. GAR22beta expression is induced in a variety of cultured cells by growth arrest. The absolute amounts of GAR22beta protein expressed are low. The beta isoforms of hGAR17 and hGAR22 appear to be able to crosslink microtubules and microfilaments in transfected cells. This finding suggests that the physiological functions of these proteins may involve integration of these two components of the cytoskeleton.