RESUMO
OBJECTIVE: To contribute to clarify molecular mechanisms supporting senescence and de-differentiation of chondrocytes in chondrocyte pathologies such as osteoarthritis (OA). Specifically, we investigated the relationship between the nuclear lamina protein Lamin B1 and the negative regulator of chondrogenesis Slug transcription factor in osteoarthritic chondrocytes. METHODS: Lamin B1 and Slug proteins were analyzed in cartilage explants from normal subjects and OA patients by immunohistochemical technique. Their expression was confirmed on isolated chondrocytes both at passage 0 and passage 2 (de-differentiated chondrocytes) by immunofluorescence and western blot. Subsequently, we explored the "in vivo" binding of Slug on LMNB1 promoter by chromatin immunoprecipitation assay (ChIP). RESULTS: In this study we demonstrated that nuclear lamina protein Lamin B1 and anti-chondrogenic Slug transcription factor are upregulated in cartilage and OA chondrocytes. Furthermore, we found that Slug is "in vivo" recruited by LMNB1 gene promoter mostly when chondrocytes undergo de-differentiation or OA degeneration. CONCLUSIONS: We described for the first time a potential regulatory role of Slug on the LMNB1 gene expression in OA chondrocytes. These findings may have important implications for the study of premature senescence, and degeneration of cartilage, and may contribute to develop effective therapeutic strategies against signals supporting cartilage damage in different subsets of patients.
Assuntos
Condrócitos/metabolismo , Laminina/biossíntese , Osteoartrite do Joelho/metabolismo , Fatores de Transcrição/biossíntese , Idoso , Cartilagem Articular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Feminino , Humanos , Articulação do Joelho/metabolismo , Laminina/genética , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects.We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.
Assuntos
Proteínas de Membrana/fisiologia , Metaloendopeptidases/fisiologia , Proteínas Nucleares/metabolismo , Progéria/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Endopeptidases/metabolismo , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , Proteínas de Membrana/antagonistas & inibidores , Metaloendopeptidases/antagonistas & inibidores , Progéria/patologia , Prenilação de Proteína , Coelhos/imunologiaRESUMO
BACKGROUND: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms. OBJECTIVE: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells. METHODS: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery-Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres. RESULTS: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts. CONCLUSIONS: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.
Assuntos
Lamina Tipo A/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mioblastos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Diferenciação Celular , Linhagem Celular , Humanos , Insulina/metabolismo , Lamina Tipo A/genética , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular de Emery-Dreifuss/genética , Fosforilação , Transdução de SinaisRESUMO
The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnormal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin areas, suggesting a major involvement of emerin in pre-lamin A-mediated mechanisms of chromatin remodeling.
Assuntos
Montagem e Desmontagem da Cromatina , Heterocromatina , Lamina Tipo A , Membrana Nuclear/metabolismo , Senilidade Prematura/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia/genética , Lipodistrofia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mutação , Proteínas Nucleares , Timopoietinas/genética , Timopoietinas/metabolismoRESUMO
The effects of stabilising mutations on the folding process of common-type acylphosphatase have been investigated. The mutations were designed to increase the helical propensity of the regions of the polypeptide chain corresponding to the two alpha-helices of the native protein. Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism. The most substantial increase in helical content is found for the peptide carrying five mutations in the second alpha-helix. Acylphosphatase variants containing the corresponding mutations display, to different extents, enhanced conformational stabilities as indicated by equilibrium urea denaturation experiments monitored by changes of intrinsic fluorescence. All the protein variants studied here refold with apparent two-state kinetics. Mutations in the first alpha-helix are responsible for a small increase in the refolding rate, accompanied by a marked decrease in the unfolding rate. On the other hand, multiple mutations in the second helix result in a considerable increase in the refolding rate without any significant effect on the unfolding rate. Addition of trifluoroethanol was found to accelerate the folding of the acylphosphatase variants, the extent of the acceleration being inversely proportional to the intrinsic rate of folding of the corresponding mutant. The trifluoroethanol-induced acceleration is far less marked for those variants whose alpha-helical structure is efficiently stabilised by amino acid replacements. This observation suggests that trifluoroethanol acts in a similar manner to the stabilising mutations in promoting native-like secondary structure. Analysis of the kinetic data indicates that the second helix is fully consolidated in the transition state for folding of acylphosphatase, whereas the first helix is only partially formed. These data suggest that the second helix is an important element in the folding process of the protein.
Assuntos
Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Mutagênese Sítio-Dirigida/genética , Dobramento de Proteína , Hidrolases Anidrido Ácido/genética , Dicroísmo Circular , Relação Dose-Resposta a Droga , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Termodinâmica , Trifluoretanol/farmacologia , Ureia/farmacologia , AcilfosfataseRESUMO
The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.
Assuntos
Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Timopoietinas/metabolismo , Animais , Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Imunofluorescência , Immunoblotting , Imuno-Histoquímica , Masculino , Modelos Animais , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas Nucleares , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Regulação para CimaRESUMO
The HypF N-terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3-5 nm and 7-9 nm in width, whereas fibrils of 7-9 nm and 12-20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Benzotiazóis , Dicroísmo Circular , Clonagem Molecular , Corantes/farmacologia , Vermelho Congo/farmacologia , Escherichia coli/metabolismo , Corantes Fluorescentes/farmacologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Estrutura Terciária de Proteína , Tiazóis/farmacologia , Fatores de Tempo , Trifluoretanol/farmacologia , Ureia/farmacologiaRESUMO
Human metaphase chromosomes were isolated and digested in situ with HaeIII restriction enzyme to detect cytosine and guanine-rich sequences (CpG islands), which are known to be associated with most of the mammalian genes. Digested DNA was reconstructed by in situ nick translation employing digoxigenin-labeled nucleotides. The DNA sequences were revealed by antibodies conjugated either with fluorescein isothiocyanate or 1-nm colloidal gold. DNA was counterstained with propidium iodide. A sensitive, high resolution method for visualizing three signals, simultaneously excited by a single argon laser line of 488 nm has been developed. The green fluorescence of fluorescein isothiocyanate was detected in combination with the red fluorescence of propidium iodide, and the third signal was imaged by employing the reflectance mode of the confocal microscope after silver enhancement of the gold beads. The high reflectance intensity, the accurate localization and the non-fading properties of colloidal gold made the reaction a valuable tool for the detection of antigens and, as a consequence, of specific DNA sequences in chromosome preparations. Overlaying of three signals allowed the simultaneous observation of distinct structures: total DNA, as well as fluorescein- and gold-labeled sequences after in situ nick translation, or total DNA and centromeric sequences of two different chromosome pairs (17 and X) after in situ hybridization. The use of HaeIII restriction enzyme that cut CpG islands combined with in situ nick translation identified the chromosome sites where active, inactive or housekeeping genes can be located. In chromosomes, the fluorescent reaction pattern showed large areas of labeling, while a more defined staining, often organized in spot pairs that resembled an R-like banding, was detected when the reflected mode was used. These results are confirmed by the observation that R-like bands actually are multiple symmetrical spots localized on sister chromatids. In addition, some chromosomes, and in particular 1 and 9, displayed a C-negative banding due to the negativity of the centromeric areas. Reflectance confocal scanning microscopy and in situ nick translation represent a powerful tool to study the in situ genome organization.
Assuntos
Cromossomos Humanos/ultraestrutura , Ilhas de CpG , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Microscopia/métodos , Análise de Sequência de DNA/métodos , Cromossomos Humanos Par 17 , DNA Satélite/isolamento & purificação , Técnicas Genéticas , Humanos , Hibridização in Situ Fluorescente , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Cromossomo XRESUMO
Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.
Assuntos
Colágeno Tipo VI/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Colágeno Tipo VI/genética , Fibroblastos/citologia , Humanos , Camundongos , Camundongos Knockout , Camundongos NusRESUMO
Emery-Dreifuss muscular dystrophy (EMD) is an inherited myopathy characterised by muscle contractures, progressive muscle wasting and weakness, with humeroperoneal distribution. Cardiac arrhythmia and heart conduction block are also important characteristics of this disease. The X-linked form of EMD is caused by the absence of emerin, encoded by the STA gene (Xq28). Emerin is normally localized in muscle and other tissues at the nuclear rim. Currently, muscle and skin biopsies are used for the immunohistochemical diagnosis. We demonstrate that emerin is present in the cheek oral mucosa, in the exfoliating epithelial cells, and we propose the collection of these cells as a new method for the diagnosis of X-linked EMD patients and the detection of carriers by immunofluorescence techniques: smears from healthy subjects contained about 98% emerin-positive cells, those from X-linked EMD patients contained none and those from carriers contained about 45%. The technique is completely non-invasive, simple, repeatable and inexpensive.
Assuntos
Triagem de Portadores Genéticos , Ligação Genética , Mucosa Bucal/patologia , Distrofias Musculares/genética , Cromossomo X , Adolescente , Adulto , Estudos de Casos e Controles , Bochecha , Criança , Citodiagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Distrofias Musculares/patologia , Distrofia Muscular de Emery-DreifussRESUMO
The present review summarizes recent cytochemical findings on the functional organization of the nuclear domains, with a particular emphasis on the relation between nuclear envelope-associated proteins and chromatin. Mutations in two nuclear envelope-associated proteins, emerin and lamin A/C cause the Emery-Dreifuss muscular dystrophy; the cellular pathology associated with the disease and the functional role of emerin and lamin A/C in muscle cells are not well established. On the other hand, a large body of evidence indicates that nuclear envelope-associated proteins are involved in tissue-specific gene regulation. Moreover, chromatin remodeling complexes trigger gene expression by utilizing the nuclear matrix-associated actin, which is known to interact with both emerin and lamin A/C. It is thus conceivable that altered expression of these nuclear envelope-associated proteins can account for an impairment of gene expression mainly during cell differentiation as suggested by recent experimental findings on the involvement of emerin in myogenesis. The possibility that Emery-Deifuss muscular dystrophy pathogenesis could involve alteration of the signaling pathway is considered.
Assuntos
Imuno-Histoquímica , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/fisiopatologia , Membrana Nuclear/metabolismo , Expressão Gênica , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Membrana Nuclear/genética , Proteínas Nucleares , Transdução de Sinais , Timopoietinas/genética , Timopoietinas/metabolismoRESUMO
We analysed the temporal distribution of all the accidents occurred in the last 4 years among the workers engaged in tunnel digging for the new high speed railway network. The frequency and severity rates show significant differences according to work shifts and time of occurrence, with particular reference to hour of the day, hour of duty, day of the week, day of duty, and month of the year).
Assuntos
Acidentes de Trabalho/estatística & dados numéricos , Tolerância ao Trabalho Programado , Arquitetura de Instituições de Saúde , Humanos , ItáliaRESUMO
Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Anticolesterolemiantes/farmacologia , Diferenciação Celular , Células Cultivadas , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lamina Tipo A , Lovastatina/farmacologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Mioblastos/citologia , Mioblastos/metabolismo , Prenilação , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.
Assuntos
Autofagia/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Progéria/patologia , Precursores de Proteínas/metabolismo , Sirolimo/farmacologia , Antibacterianos/farmacologia , Western Blotting , Células Cultivadas , Criança , Cromatina/metabolismo , Humanos , Lamina Tipo A , Membrana Nuclear/efeitos dos fármacos , PrenilaçãoRESUMO
Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.
RESUMO
Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment.
Assuntos
Heterocromatina/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Progéria/tratamento farmacológico , Progéria/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Células Cultivadas , Criança , Metilação de DNA , Heterocromatina/ultraestrutura , Histonas/metabolismo , Humanos , Lamina Tipo A/deficiência , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lovastatina/farmacologia , Progéria/metabolismo , Progéria/patologia , Precursores de Proteínas/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.
Assuntos
Hidrolases Anidrido Ácido/química , Músculos/enzimologia , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Estabilidade Enzimática , Humanos , Cinética , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , AcilfosfataseRESUMO
The involvement of phosphatidylinositol 3-kinase (PI3K) in the induction of ornithine decarboxylase (ODC) was investigated by using specific PI3K inhibitors. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, treatment with LY294002 inhibited cell growth and provoked a complete block of the induction of ODC activity (IC50 approximately 2 microM) and ODC protein. Some reduction in the accumulation of ODC mRNA was also observed, whereas ODC turnover was not affected significantly. Wortmannin, another specific inhibitor of PI3K, structurally unrelated to LY294002, also inhibited ODC induction with an IC50 of about 10 nM. These results indicate that PI3K activity is required for the induction of ODC, possibly affecting both ODC mRNA level and translation. Since p70 S6 kinase (p70S6K) is considered an important mediator of PI3K action in several experimental systems, the effect of rapamycin, which can lead to selective inhibition of p70S6K, was also investigated. Rapamycin inhibited p70S6K activity and produced ODC inhibiting effects similar to those elicited by LY294002. However, LY294002 and wortmannin at concentrations which inhibited almost completely PI3K activity did not decrease p70S6K activity, suggesting that p70S6K does not mediate the PI3K effects on ODC, but may lie on a separate pathway in this experimental model.
Assuntos
Leucemia L1210/enzimologia , Ornitina Descarboxilase/biossíntese , Fosfatidilinositol 3-Quinases/fisiologia , Androstadienos/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Cromonas/farmacologia , Eflornitina/farmacologia , Indução Enzimática/efeitos dos fármacos , Leucemia L1210/genética , Leucemia L1210/patologia , Camundongos , Morfolinas/farmacologia , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Polienos/farmacologia , Sirolimo , WortmaninaRESUMO
The involvement of p44/42 mitogen-activated protein kinase (MAPK) in the induction of ornithine decarboxylase (ODC) was investigated by using PD98059, a specific MAPK-kinase (MEK1/2) inhibitor, and other signal-transduction inhibitors. In d,l-alpha-difluoromethylornithine (DFMO)-resistant L1210 cells stimulated to grow from quiescence, treatment with PD98059 inhibited p44/42 MAPK phosphorylation and the induction of ODC activity and protein. A marked reduction of the accumulation of mature ODC mRNA and its intron-containing precursor was observed, whereas ODC turnover was hardly affected. PD98059 also reduced the content of antizyme, but not that of antizyme mRNA. U0126, a novel and more potent inhibitor of MEK1/2, provoked a dose-dependent inhibition of ODC induction at lower concentrations with respect to PD98059. Other effective inhibitors of ODC induction proved to be genistein, manumycin A, herbimycin A, LY294002, wortmannin and KT5823, suggesting the involvement of other key proteins of signal-transduction pathways, i.e. Ras, Src, phosphatidylinositol 3-kinase and cGMP-dependent protein kinase, which may have a positive impact on MAPK. Cells kept in a DFMO-free medium, and thus containing high levels of putrescine and spermidine, showed enhanced MAPK phosphorylation and lower sensitivity to PD98059, compared with cells maintained in the presence of DFMO. In conclusion, these results indicate that the activation of p44/42 MAPK may favour the expression of ODC, and that polyamines, in turn, may affect the phosphorylation state of MAPK.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Leucemia Experimental/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Ornitina Descarboxilase/biossíntese , Transdução de Sinais , Animais , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Células Tumorais CultivadasRESUMO
Protein aggregation is a notable feature of various human disorders, including Parkinson's disease, Alzheimer's disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. alpha-synuclein, amyloid beta (Abeta) peptide, beta(2)-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu(2+) produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu(2+)-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu(2+)-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a beta structure introduced by the point mutation. The effect of Cu(2+) on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.