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1.
Sci Rep ; 12(1): 395, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013445

RESUMO

The aetiology of Amyotrophic Lateral Sclerosis (ALS) is still poorly understood. The discovery of genetic forms of ALS pointed out the mechanisms underlying this pathology, but also showed how complex these mechanisms are. Excitotoxicity is strongly suspected to play a role in ALS pathogenesis. Excitotoxicity is defined as neuron damage due to excessive intake of calcium ions (Ca2+) by the cell. This study aims to find a relationship between the proteins coded by the most relevant genes associated with ALS and intracellular Ca2+ accumulation. In detail, the profile of eight proteins (TDP-43, C9orf72, p62/sequestosome-1, matrin-3, VCP, FUS, SOD1 and profilin-1), was analysed in three different cell types induced to raise their cytoplasmic amount of Ca2+. Intracellular Ca2+ accumulation causes a decrease in the levels of TDP-43, C9orf72, matrin3, VCP, FUS, SOD1 and profilin-1 and an increase in those of p62/sequestosome-1. These events are associated with the proteolytic action of two proteases, calpains and caspases, as well as with the activation of autophagy. Interestingly, Ca2+ appears to both favour and hinder autophagy. Understanding how and why calpain-mediated proteolysis and autophagy, which are physiological processes, become pathological may elucidate the mechanisms responsible for ALS and help discover new therapeutic targets.

2.
Neurobiol Aging ; 103: 130.e1-130.e7, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33637330

RESUMO

We report a case of childhood-onset ALS with a FUS gene mutation presenting cognitive impairment and a rapid clinical progression. The patient, an 11-year-old girl, presented with right distal upper limb weakness and mild intellectual disability at the Griffith Mental Development Scales. The disease rapidly worsened and the patient became tetraplegic and bed-ridden 2 years after symptom onset. A c.1509_1510delAG mutation in exon 14 of the FUS gene was detected, resulting in a predicted truncated protein, p.G504Wfs*12, lacking the nuclear localization signal. The levels of FUS mRNA in the proband were not significantly different compared to controls. Western immunoblot analysis showed that one antibody (500-526) detected in the proband ~50% of the amount of FUS protein compared to controls, while 3 other antibodies (2-27, 400-450 and FUS C-terminal), which recognize both wild type and the mutated FUS, detected 60% to 75% of the amount of the protein. These findings indicate that p.G504Wfs*12 FUS is more prone to undergo post-translational modification respect to wild type FUS.

3.
Neurobiol Aging ; 72: 189.e11-189.e17, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30236613

RESUMO

We report an apparently sporadic amyotrophic lateral sclerosis patient carrying a heterozygous novel frameshift SOD1 mutation (p.Ser108LeufsTer15), predicted to cause a premature protein truncation. RT-PCR analysis of SOD1 mRNA and SDS-PAGE/Western blot analysis of PBMC demonstrated that mRNA from the mutant allele is expressed at levels similar to those of the wild-type allele, but the truncated protein is undetectable also in the insoluble fraction and after proteasome inhibition. Accordingly, the dismutation activity in erythrocytes is halved. Thus, the pathogenic mechanism associated with this mutation might be based on an insufficient activity of SOD1 that would make motor neurons more vulnerable to oxidative injury. However, it cannot be excluded that p.Ser108LeufsTer15 SOD1 is present in the nervous tissue and, being less charged and hence having less repulsive forces than the wild-type protein, may trigger toxic mechanisms as a consequence of its propensity to aggregate.


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico , Esclerose Amiotrófica Lateral/genética , Superóxido Dismutase-1/genética , Idoso , Feminino , Mutação da Fase de Leitura , Humanos
4.
Biochim Biophys Acta ; 1843(4): 725-34, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24440855

RESUMO

Accumulation of transactive response DNA binding protein (TDP-43) fragments in motor neurons is a post mortem hallmark of different neurodegenerative diseases. TDP-43 fragments are the products of the apoptotic caspases-3 and -7. Either excessive or insufficient cellular Ca(2+) availability is associated with activation of apoptotic caspases. However, as far as we know, it is not described whether activation of caspases, due to restricted intracellular Ca(2+), affects TDP-43 cleavage. Here we show that in various cell lineages with restricted Ca(2+) availability, TDP-43 is initially cleaved by caspases-3 and -7 and then, also by caspases-6 and -8 once activated by caspase-3. Furthermore, we disclose the existence of a TDP-43 caspase-mediated fragment of 15kDa, in addition to the well-known fragments of 35 and 25kDa. Interestingly, with respect to the other two fragments this novel fragment is the major product of caspase activity on murine TDP-43 whereas in human cell lines the opposite occurs. This outcome should be considered when murine models are used to investigate TDP-43 proteinopathies.


Assuntos
Apoptose/genética , Cálcio/metabolismo , Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos
5.
Acta Neuropathol ; 121(5): 611-22, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21120508

RESUMO

TDP-43, encoded by TARDBP, is a ubiquitously expressed, primarily nuclear protein. In recent years, TDP-43 has been identified as the major pathological protein in ALS due to its mislocalisation in the cytoplasm of motor neurons of patients with and without TARDBP mutations and expression in forms that do not match its predicted molecular weight. In this study, the TDP-43 profile was investigated using western immunoblot analysis in whole lysates, nuclei and cytoplasm of circulating lymphomonocytes from 16 ALS patients, 4 with (ALS/TDP+) and 12 without (ALS/TDP-) TARDBP mutations in the protein C-terminal domain, and thirteen age-matched, healthy donors (controls). Three disease-unaffected first-degree relatives of an ALS/TDP+ patient were also included: one carried the parent mutation (Rel/TDP+) whereas the other two did not (Rel/TDP-). In all ALS patients, relatives and controls, TDP-43 retained the predicted molecular weight in whole cell lysates and nuclei, but in the cytoplasm its molecular weight was slightly smaller than expected. In quantitative terms, TDP-43 was expressed at approximately the same levels in whole cell lysates of ALS patients, relatives and controls. In contrast, TDP-43 accumulated in the cytoplasm with concomitant nuclear depletion in all ALS/TDP+ patients, in about 50% of ALS/TDP- patients and in the Rel/TDP+ subject compared to the controls. In the remaining ALS/TDP- patients and in the two Rel/TDP- subjects, TDP-43 matched the control levels in both subcellular compartments. Were these findings further confirmed, circulating lymphomonocytes could be informative of TDP-43 mislocalisation in nervous tissue and used as a biomarker for future disease risk.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Subpopulações de Linfócitos/metabolismo , Subpopulações de Linfócitos/patologia , Monócitos/metabolismo , Monócitos/patologia , Adulto , Idoso , Esclerose Amiotrófica Lateral/genética , Citoplasma/patologia , Proteínas de Ligação a DNA/química , Feminino , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética
6.
Mol Neurobiol ; 41(2-3): 314-40, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20127207

RESUMO

Sphingolipids are polar membrane lipids present as minor components in eukaryotic cell membranes. Sphingolipids are highly enriched in nervous cells, where they exert important biological functions. They deeply affect the structural and geometrical properties and the lateral order of cellular membranes, modulate the function of several membrane-associated proteins, and give rise to important intra- and extracellular lipid mediators. Sphingolipid metabolism is regulated along the differentiation and development of the nervous system, and the expression of a peculiar spatially and temporarily regulated sphingolipid pattern is essential for the maintenance of the functional integrity of the nervous system: sphingolipids in the nervous system participate to several signaling pathways controlling neuronal survival, migration, and differentiation, responsiveness to trophic factors, synaptic stability and synaptic transmission, and neuron-glia interactions, including the formation and stability of central and peripheral myelin. In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal sphingolipid patterns and altered membrane organization that participate to several events related to the pathogenesis of these diseases. The most impressive consequence of this deregulation is represented by anomalous sphingolipid-protein interactions that are at least, in part, responsible for the misfolding events that cause the fibrillogenic and amyloidogenic processing of disease-specific protein isoforms, such as amyloid beta peptide in Alzheimer's disease, huntingtin in Huntington's disease, alpha-synuclein in Parkinson's disease, and prions in transmissible encephalopathies. Targeting sphingolipid metabolism represents today an underexploited but realistic opportunity to design novel therapeutic strategies for the intervention in these diseases.


Assuntos
Membrana Celular/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Esfingolipídeos/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Membrana Celular/química , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Bainha de Mielina/química , Bainha de Mielina/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Priônicas/patologia , Doenças Priônicas/fisiopatologia , Esfingolipidoses/patologia , Esfingolipidoses/fisiopatologia , Esfingolipídeos/química
7.
Brain Pathol ; 20(2): 431-40, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19725832

RESUMO

Previously, myelin from cerebral white matter (CWM) of two subjects of a family with orthochromatic adult-onset autosomal-dominant leukodystrophy (ADLD) was disclosed to exhibit defective large isoform of myelin-associated glycoprotein (L-MAG) and patchy distribution only in the elder subject. L-MAG and neural cell adhesion molecule (N-CAM) (N-CAM 180, 140, and 120) are structurally related and concur to myelin/axon interaction. In early developmental stages, in neurons and glia N-CAM is converted into polysialylated (PSA)-NCAM by two sialyltransferases sialyltransferase-X (STX) and polysialyltransferase-1 (PST). Notably, PSA-NCAM disrupts N-CAM adhesive properties and is nearly absent in the adult brain. Here, CWM extracts and myelin of the two subjects were searched for the expression pattern of the N-CAM isoforms and PSA-NCAM, and their CWM was evaluated for N-CAM, STX and PST gene copy number and gene expression as mRNA. Biochemically, we disclosed that in CWM extracts and myelin from both subjects, PSA-NCAM accumulates, N-CAM 180 considerably increases, N-CAM 140 is modestly modified and N-CAM 120 remarkably decreases; duplication of genes encoding N-CAM, STX and PST was not revealed, whereas PST mRNA was clearly increased. Immunohistochemically, in CWM of both subjects, we found an unusually diffuse accumulation of PSA-NCAM without inflammation markers. PSA-NCAM persistence, up-regulated PST mRNA and previously uncovered defective L-MAG may be early pathogenetic events in this ADLD form.


Assuntos
Cérebro/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Leucoencefalopatias/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Adulto , Idade de Início , Western Blotting , Família , Dosagem de Genes , Regulação da Expressão Gênica , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Imuno-Histoquímica , Leucoencefalopatias/genética , Pessoa de Meia-Idade , Bainha de Mielina/metabolismo , Glicoproteína Associada a Mielina/genética , Fibras Nervosas Mielinizadas/metabolismo , Molécula L1 de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/genética , Reação em Cadeia da Polimerase , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ácidos Siálicos/genética , Sialiltransferases/genética
8.
Brain Pathol ; 20(2): 351-60, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19338576

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder consisting of progressive loss of motor neurons. TDP-43 has been identified as a component of ubiquitin-immunoreactive inclusions of motor neurons in ALS. We focused on the diffuse cytoplasmic TDP-43 immunoreactivity in ALS neurons, and quantitatively assessed it in comparison with skein/round TDP-43 and ubiquitin immunostaining in motor neurons of 30 sporadic ALS cases. The percentage of spinal motor neurons with cytoplasmic TDP-43 immunoreactivity was higher than that of ubiquitin-immunoreactive ones. The percentage of TDP-43-positive motor neurons was independent of neuron counts in anterior horns, while the percentage of ubiquitinated neurons was inversely correlated. Aiming to define the cytosolic localization of TDP-43, the immunoblot analysis of spinal cord and frontal cortex showed that full-length TDP-43, the 45 kDa form and ubiquitinated TDP-43 are found in the soluble inclusion-free fraction. The present data suggest that delocalization, accumulation and ubiquitination of TDP-43 in the cytoplasm of motor neurons are early dysfunctions in the cascade of the events leading to motor neuron degeneration in ALS, preceding the formation of insoluble inclusion bodies. Being cytoplasmic accumulation an ongoing event during the course of the illness, a therapeutic approach to this incurable disease can be envisaged.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Medula Espinal/metabolismo , Adulto , Idoso , Esclerose Amiotrófica Lateral/patologia , Contagem de Células , Progressão da Doença , Feminino , Lobo Frontal/metabolismo , Hipocampo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios Motores/patologia , Neurônios/metabolismo , Neurônios/patologia , Medula Espinal/patologia , Lobo Temporal/metabolismo , Fatores de Tempo , Ubiquitina/metabolismo , Ubiquitinação
9.
J Neurochem ; 109(1): 105-15, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19187445

RESUMO

Niemann-Pick disease (NPD) type A is a neurodegenerative disorder caused by sphingomyelin (SM) accumulation in lysosomes relying on reduced or absent acid sphingomyelinase (ASM) activity. NPD-A patients develop progressive neurodegeneration including cerebral and cerebellar atrophy, relevant Purkinje cell and myelin deficiency with death within 3 years. ASM'knock-out' (ASMKO) mice, an animal model of NPD-A, develop a phenotype largely mimicking that of NPD-A. The mechanisms underlying myelin formation are poorly documented in ASMKO mice. In this study we determined the content of four myelin-specific proteins, myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin associated glycoprotein (MAG) and proteolipid protein (PLP), and that of myelin-enriched sphingolipids in the brains of ASMKO and wild-type mice in early stages of post-natal (pn) life. Protein and mRNA analysis revealed that in ASMKO mice beginning from 4 post-natal weeks (wk-pn), the expression levels of MAG, CNP, and MBP were below those observed in wild-type mice and the same applied to PLP at 10 wk-pn. Moreover, at 4 wk-pn the expression of SOX10, one of the transcription factors involved in oligodendrocyte development and maintenance was lower in ASMKO mice. Lipid analysis showed that SM and the gangliosides GM3 and GM2 accumulated in the brains of ASMKO mice, as opposed to galactocerebroside and galactosulfocerebroside that, in parallel with the mRNAs of UDP-galactose ceramide galactosyltransferase and galactose-3-O-sulfotransferase 1, the two transferases involved in their synthesis, decreased. Myelin lipid analysis showed a progressive sphingomyelin accumulation in ASMKO mice; noteworthy, of the two sphingomyelin species known to be resolved by TLC, only that with the lower Rf accumulated. The immunohistochemical analysis showed that the reduced expression of myelin specific proteins in ASMKO mice at 10 wk-pn was not restricted to the Purkinje layer of the cerebellar cortex but involved the cerebral cortex as well. In conclusion, reduced oligodendrocyte metabolic activity is likely to be the chief cause of myelin deficiency in ASMKO mice, thus shedding light on the molecular dysfunctions underlying neurodegeneration in NPD-A.


Assuntos
Encéfalo/metabolismo , Proteínas da Mielina/metabolismo , Doença de Niemann-Pick Tipo A/metabolismo , Fatores de Transcrição SOXE/deficiência , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/deficiência , Animais , Encéfalo/enzimologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Proteínas da Mielina/genética , Doença de Niemann-Pick Tipo A/genética , Fatores de Transcrição SOXE/biossíntese , Fatores de Transcrição SOXE/genética , Esfingolipídeos/genética , Esfingomielina Fosfodiesterase/genética
10.
J Nephrol ; 19(6): 710-6, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17173242

RESUMO

BACKGROUND: Proteasomes are 'proteolytic machineries' implicated in many cellular functions, including protein turnover, inflammatory response and immunosurveillance. They exist in various forms sharing the same catalytic core - the 20S proteasome. This core consists of 28 subunits codified by 14 different genes, 3 of which - beta 1, beta 2 and beta 5 - are catalytically active and show peptidyl-glutamyl peptide hydrolyzing (PGPH), trypsin-like and chymo-trypsin-like activities, respectively. Under IFN- delta and TNF- alfa stimuli, the 3 active constitutive subunits are replaced by the corresponding ones - i.e., LMP2, MECL-1, LMP7 - known as inducible subunits, thus resulting in the constitution of the 'immunoproteasome' that is specifically implicated in MHC class I-presented peptide generation. This process is enhanced when the proteasome is associated with the polymeric protein 11S regulator/PA28 made up of 4 alfa and 3 beta subunits. METHODS: The 20S proteasome was purified from post mortem specimens of human kidney cortex by chromatographic and ultracentrifugation techniques. It was then characterized on the basis of (i) multicatalytic activity evaluated using specific fluorogenic peptides, (ii) electrophoretic mobility on non-denaturating polyacrylamide gels followed by in-gel visualization by fluorogenic peptide overlaying and Coomassie blue staining and (iii) subunit composition as ascertained by SDS-PAGE and 2-dimensional electrophoresis followed by silver staining or Western immunoblotting using specific antibodies against the proteasome subunits. The 20S proteasome was also studied for its association with the 11S regulator by Western immunoblotting using an antibody to the regulator alfa subuniT. RESULTS: T he purified proteasome was shown to have PGPH, trypsin-like and chymotrypsin-like activities. Furthermore, it incorporated the inducible subunits and was associated with the 11S regulator. CONCLUSIONS: The features we observed make renal cells susceptible to an over-expression of inflammatory response to immunological challenges.


Assuntos
Córtex Renal/enzimologia , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Humanos , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Córtex Renal/imunologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologia
11.
Circ Res ; 98(1): 45-54, 2006 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-16339483

RESUMO

During angiogenesis, a combined action between newly secreted extracellular matrix proteins and the repertoire of integrins expressed by endothelial cells contributes in the regulation of their biological functions. Extracellular matrix-engaged integrins influence tyrosine kinase receptors, thus promoting a regulatory cross-talk between adhesive and soluble stimuli. For instance, vitronectin has been reported to positively regulate VEGFR-2. Here, we show that collagen I downregulates VEGF-A-mediated VEGFR-2 activation. This activity requires the tyrosine phosphatase SHP2, which is recruited to the activated VEGFR-2 when cells are plated on collagen I, but not on vitronectin. Constitutive expression of SHP2(C459S) mutant inhibits the negative role of collagen I on VEGFR-2 phosphorylation. VEGFR-2 undergoes internalisation, which is associated with dynamin II phosphorylation. Expression of SHP2(C459S) impairs receptor internalisation suggesting that SHP2-dependent dephosphorylation regulates this process. These findings demonstrate that collagen I in provisional extracellular matrix surrounding nascent capillaries triggers a signaling pathway that negatively regulates angiogenesis.


Assuntos
Colágeno Tipo I/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais/fisiologia , Humanos , Neovascularização Fisiológica , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Transdução de Sinais , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Vitronectina/farmacologia
12.
Anticancer Res ; 25(5): 3203-10, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16101128

RESUMO

Proteasomes are multisubunit proteases involved in many cellular processes, including tumorigenesis and immune surveillance. In their catalytic core, the 20S proteasome, the beta1, beta2 and beta5 subunits show peptidylglutamyl peptide hydrolyzing (PGPH), trypsin-like and chymotrypsin-like activities, respectively. By IFN-gamma and TNFalpha stimulus, these subunits are replaced by their counterparts LMP2, MECL-1 and LMP7, defined inducible subunits, thus originating the immunoproteasome, and expression of the proteasome activator PA28 is enhanced. These modifications strengthen MHC-class I restricted peptide generation. The 20S proteasome has been detected immunohistochemically in formalin-fixed samples purified from fresh surgical specimens of 18 tumors (G20S) and from 8 samples of normal peritumoral tissue. The G20S, LMP2, MECL-1 and LMP7 increased in only 12 cases, along with unvaried trypsin-like and decreased PGPH and chymotrypsin-like activities; PA28 was unvaried in all 18 samples. The immunoproteasome alterations may represent an anomalous immunological attitude of glioblastomas.


Assuntos
Neoplasias do Sistema Nervoso Central/enzimologia , Glioblastoma/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Cisteína Endopeptidases/metabolismo , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Linfócitos/enzimologia , Linfócitos/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Microglia/enzimologia , Microglia/patologia , Complexos Multienzimáticos/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/isolamento & purificação
13.
Brain Pathol ; 15(2): 116-23, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15912883

RESUMO

Adult-onset dominant leukodystrophies are a heterogeneous group of rare disorders, whose etiology, pathogenesis and molecular background are still unknown. We report the neuropathological and biochemical investigations of the brains and their myelin proteins components in 2 members of an Italian family affected by an adult-onset autosomal dominant leukoencephalopathy. Clinical signs included spastic paraparesis, pseudobulbar syndrome, action tremor of head and hands, and moderate memory impairment. No mental deterioration or neuropathy was present. Onset was subacute (range 42-53 years) and progression spanned 4 to 7 years. The neuropathological phenotype overlapped that of orthochromatic leukodystrophies. The biochemical analysis revealed an abnormal myelin-associated glycoprotein (MAG); the defect was localized at the C-terminal domain of the L-MAG isoform, resulting in a protein approximately 5 kDa shorter than the normal counterpart. No mutation in the MAG gene-coding regions was uncovered, and linkage analysis formally excluded the entire MAG locus. We show that the identified MAG protein alteration is probably due to an abnormal post-translational event. Considering MAG function in the maintenance of myelin, the abnormal protein may have a role in the pathogenesis of this disease. This is the first report of a possible pathogenetic role of MAG in a hereditary disease affecting the central white matter.


Assuntos
Encéfalo/patologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Glicoproteína Associada a Mielina/metabolismo , Processamento de Proteína Pós-Traducional , Adulto , Idade de Início , Western Blotting , Encéfalo/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Leucodistrofia de Células Globoides/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Glicoproteína Associada a Mielina/química , Glicoproteína Associada a Mielina/genética , Linhagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Curr Drug Targets ; 4(8): 657-71, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14577657

RESUMO

The ubiquitin-proteasome pathway plays a role in the degradation of the bulk of proteins in the cytoplasmic and nuclear compartments. In this pathway proteins are targeted for degradation by covalent ligation with ubiquitin, a reaction that requires ATP. Following the binding of the first ubiquitin molecule with the epsilon-amino group of a lysine residue of the substrate protein, a polyubiquitin chain is usually formed, in which the C-terminus of each ubiquitin unit is linked to a specific Lys residue of the previous ubiquitin. Central to this pathway is the 26S proteasome, a high molecular mass multifunctional protease which requires ATP for its catalytic activity. Substrates of the 26S proteasome are not only old or damaged proteins, but also short lived proteins functioning as regulatory factors in a large array of cellular processes, such as cell cycle progression, cell growth and gene expression, inflammatory response and immune surveillance. A number of inhibitors of the catalytic activity of proteasomes have been developed and successfully employed in the study of their functional and structural properties, as well as of their involvement in different cellular processes. Some of these molecules due to their toxicity are used only as experimental research tools; others instead are now in clinical trials for treatment of a variety of hematologic malignancies and solid tumors and of reperfusion injury occurring after cerebral ischemia and myocardial infarction. Furthermore, proteasome inhibitors are described to interfere with HIV maturation, budding and aggressiveness, and cytostatic drugs, as well as antiretroviral agents used in HAART, have been shown to behave in vitro and in cultured cell lines as inhibitors of proteasome proteolytic activity at therapeutic dosages.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Complexos Multienzimáticos/antagonistas & inibidores , Proteínas/antagonistas & inibidores , Ubiquitinas/antagonistas & inibidores , Animais , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/administração & dosagem , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas/metabolismo , Ubiquitinas/metabolismo
15.
J Neuroimmunol ; 135(1-2): 135-40, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12576233

RESUMO

In most tissues expressing MHC class I molecules, proteasomes incorporating IFN-gamma-inducible subunits, defined immuno-proteasomes, exist together with constitutive proteasomes. In physiological conditions, the central nervous system expresses neither MHC class I molecules nor TAP1 and TAP2 transporters but besides being constitutive, it is unknown whether immuno-proteasomes are also present in this tissue. We present evidence that in human brain, the two types of proteasome exist suggesting that under physiological conditions, the mechanisms regulating expression of IFN-gamma-inducible subunits as well as of MHC class I molecules and TAP1 and TAP2 transporters in nervous tissue, are not entirely coordinated.


Assuntos
Encéfalo/enzimologia , Interferon gama/farmacologia , Complexos Multienzimáticos/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Apresentação do Antígeno , Humanos , Rim/enzimologia , Peso Molecular , Subunidades Proteicas
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