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1.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360826

RESUMO

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.


Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Eritrócitos/metabolismo , Glicômica , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Glicosilação , Humanos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
2.
Biomedicines ; 9(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34440152

RESUMO

Mutations in Cu/Zn Superoxide Dismutase (SOD1) gene represent one of the most common causes of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder that specifically affects motor neurons (MNs). The dismutase-active SOD1 G93A mutant is responsible for the formation of toxic aggregates onto the mitochondrial surface, using the Voltage-Dependent Anion Channel 1 (VDAC1) as an anchor point to the organelle. VDAC1 is the master regulator of cellular bioenergetics and by binding to hexokinases (HKs) it controls apoptosis. In ALS, however, SOD1 G93A impairs VDAC1 activity and displaces HK1 from mitochondria, promoting organelle dysfunction, and cell death. Using an ALS cell model, we demonstrate that a small synthetic peptide derived from the HK1 sequence (NHK1) recovers the cell viability in a dose-response manner and the defective mitochondrial respiration profile relative to the ADP phosphorylation. This correlates with an unexpected increase of VDAC1 expression and a reduction of SOD1 mutant accumulation at the mitochondrial level. Overall, our findings provide important new insights into the development of therapeutic molecules to fight ALS and help to better define the link between altered mitochondrial metabolism and MNs death in the disease.

3.
Front Physiol ; 12: 708695, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34421651

RESUMO

VDACs are pore-forming proteins, coating the mitochondrial outer membrane, and playing the role of main regulators for metabolites exchange between cytosol and mitochondria. In mammals, three isoforms have evolutionary originated, VDAC1, VDAC2, and VDAC3. Despite similarity in sequence and structure, evidence suggests different biological roles in normal and pathological conditions for each isoform. We compared Homo sapiens and Mus musculus VDAC genes and their regulatory elements. RNA-seq transcriptome analysis shows that VDAC isoforms are expressed in human and mouse tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3, with the exception of reproductive system. Numerous transcript variants for each isoform suggest specific context-dependent regulatory mechanisms. Analysis of VDAC core promoters has highlighted that, both in a human and a mouse, VDAC genes show features of TATA-less ones. The level of CG methylation of the human VDAC genes revealed that VDAC1 promoter is less methylated than other two isoforms. We found that expression of VDAC genes is mainly regulated by transcription factors involved in controlling cell growth, proliferation and differentiation, apoptosis, and bioenergetic metabolism. A non-canonical initiation site termed "the TCT/TOP motif," the target for translation regulation by the mTOR pathway, was identified in human VDAC2 and VDAC3 and in every murine VDACs promoter. In addition, specific TFBSs have been identified in each VDAC promoter, supporting the hypothesis that there is a partial functional divergence. These data corroborate our experimental results and reinforce the idea that gene regulation could be the key to understanding the evolutionary specialization of VDAC isoforms.

4.
Biomolecules ; 11(5)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064816

RESUMO

Alpha-Synuclein (αSyn) is a protein whose function is still debated, as well as its role in modulation of mitochondrial function in both physiological and pathological conditions. Mitochondrial porins or Voltage-Dependent Anion Channel (VDAC) proteins are the main gates for ADP/ATP and various substrates towards the organelle. Furthermore, they act as a mitochondrial hub for many cytosolic proteins, including αSyn. This review analyzes the main aspects of αSyn-mitochondria interaction, focusing on the role of VDAC and its emerging involvement in the pathological processes.


Assuntos
Citosol/metabolismo , Mitocôndrias/patologia , Degeneração Neural/patologia , Doença de Parkinson/patologia , Canais de Ânion Dependentes de Voltagem/metabolismo , alfa-Sinucleína/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Degeneração Neural/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo
5.
iScience ; 24(4): 102323, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33889819

RESUMO

Glycosylation is a fundamental post-translational modification of proteins that boosts their structural diversity providing subtle and specialized biological properties and functions. All those genetic diseases due to a defective glycan biosynthesis and attachment to the nascent glycoproteins fall within the wide area of congenital disorders of glycosylation (CDG), mostly causing multisystem involvement. In the present paper, we detailed the unique serum N-glycosylation of a CDG-candidate patient with an unexplained neurological phenotype and liver adenomatosis harboring a recurrent pathogenic HNF1α variant. Serum transferrin isoelectric focusing showed a surprising N-glycosylation pattern consisting on hyposialylation, as well as remarkable hypersialylation. Mass spectrometry-based glycomic analyses of individual serum glycoproteins enabled to unveil hypersialylated complex N-glycans comprising up to two sialic acids per antenna. Further advanced MS analysis showed the additional sialic acid is bonded through an α2-6 linkage to the peripheral N-acetylglucosamine residue.

6.
Allergy ; 76(8): 2500-2509, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33583051

RESUMO

PURPOSE: Tear fluid N-Glycome from patients affected with vernal (VKC) and atopic keratoconjunctivitis (AKC) was investigated to identify specific changes in tears and to recognize possible glyco-biomarkers. METHODS: The analysis of the N-glycans was performed using matrix-assisted laser desorption ionization mass spectrometry on single tear samples. Tears from control normal subjects (CTRL), VKC and AKC patients were processed and treated with peptide N-glycosidase F (PNGase F) to deglycosylate N-glycoproteins. Released N-glycans were purified, permethylated, and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry (MALDI-TOF MS and MALDI-TOF MS/MS). RESULTS: More than 150 complex N-glycans, including highly fucosylated biantennary, triantennary, tetra-antennary, and bisecting species, were observed in our spectra. Three distinct patterns for CTRL, VKC, and AKC patients were identified in terms of relative intensities for some N-glycans structures. Major variations involved bisecting and hyperfucosylated glycoforms. The most intense ions were associated with species at m/z 1907.0 (asialo, agalacto, bisected, biantennary structure-NGA2B) in CTRL MS profiles, at m/z 2605.3 and 2966.5 in VKC, and at m/z 2792.4 in AKC corresponding to a well-known biantennary, disialylated N-glycan. Several peaks were associated with structures bearing one or two Lewis X epitopes. Structures were confirmed by MS/MS analysis. Quantitative differences among the three groups were statistically significant. CONCLUSIONS: Tear MS profiles are rich in specific glycoforms, particularly those with a high fucosylation degree, indicating both core and peripheral decoration. Tear N-glycome analysis provided important information for a better comprehension of VKC and AKC alterations at the molecular level.


Assuntos
Conjuntivite Alérgica , Ceratoconjuntivite , Conjuntivite Alérgica/diagnóstico , Glicômica , Humanos , Polissacarídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Lágrimas
7.
Glycoconj J ; 38(2): 201-211, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32915358

RESUMO

N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supporting diagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) preferentially coupled to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously time-consuming, some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analytical capabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of total serum and single serum glycoproteins from specific patients with CDG (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). This strategy, accomplished by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extending the current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled to differentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported applications demonstrated that RFMS method, coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.

8.
Antioxidants (Basel) ; 9(12)2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33276691

RESUMO

Mitochondria from affected tissues of amyotrophic lateral sclerosis (ALS) patients show morphological and biochemical abnormalities. Mitochondrial dysfunction causes oxidative damage and the accumulation of ROS, and represents one of the major triggers of selective death of motor neurons in ALS. We aimed to assess whether oxidative stress in ALS induces post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific PTMs of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neuron model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High-Resolution ESI-MS/MS. We found selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1WT or NSC34 cells. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not from NSC34 control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons. Data are available via ProteomeXchange with identifier .

9.
Int J Mol Sci ; 21(19)2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33036380

RESUMO

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs' presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon-intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1-3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.


Assuntos
Regulação da Expressão Gênica , Canais de Ânion Dependentes de Voltagem/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Biologia Computacional/métodos , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Células HeLa , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Família Multigênica , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Isoformas de Proteínas , Fatores de Transcrição/metabolismo , Ativação Transcricional , Canais de Ânion Dependentes de Voltagem/metabolismo
10.
Int J Mol Sci ; 21(21)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105548

RESUMO

MPP+ is the active metabolite of MPTP, a molecule structurally similar to the herbicide Paraquat, known to injure the dopaminergic neurons of the nigrostriatal system in Parkinson's disease models. Within the cells, MPP+ accumulates in mitochondria where it inhibits complex I of the electron transport chain, resulting in ATP depletion and neuronal impairment/death. So far, MPP+ is recognized as a valuable tool to mimic dopaminergic degeneration in various cell lines. However, despite a large number of studies, a detailed characterization of mitochondrial respiration in neuronal cells upon MPP+ treatment is still missing. By using high-resolution respirometry, we deeply investigated oxygen consumption related to each respiratory state in differentiated neuroblastoma cells exposed to the neurotoxin. Our results indicated the presence of extended mitochondrial damage at the inner membrane level, supported by increased LEAK respiration, and a drastic drop in oxygen flow devoted to ADP phosphorylation in respirometry measurements. Furthermore, prior to complex I inhibition, an enhancement of complex II activity was observed, suggesting the occurrence of some compensatory effect. Overall our findings provide a mechanistic insight on the mitochondrial toxicity mediated by MPP+, relevant for the standardization of studies that employ this neurotoxin as a disease model.


Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doença de Parkinson/patologia , 1-Metil-4-fenilpiridínio/toxicidade , Difosfato de Adenosina/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Oxigênio/metabolismo , Respiração
11.
Biochim Biophys Acta Bioenerg ; 1861(12): 148289, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810507

RESUMO

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Regiões Promotoras Genéticas , Canal de Ânion 1 Dependente de Voltagem/genética , Sítios de Ligação , Hipóxia Celular/genética , Sobrevivência Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Biogênese de Organelas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo
12.
Front Cell Dev Biol ; 8: 397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582695

RESUMO

Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed.

13.
Cell Mol Life Sci ; 77(16): 3195-3213, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31655859

RESUMO

The Voltage-Dependent Anion-selective Channel (VDAC) is the pore-forming protein of mitochondrial outer membrane, allowing metabolites and ions exchanges. In Saccharomyces cerevisiae, inactivation of POR1, encoding VDAC1, produces defective growth in the presence of non-fermentable carbon source. Here, we characterized the whole-genome expression pattern of a VDAC1-null strain (Δpor1) by microarray analysis, discovering that the expression of mitochondrial genes was completely abolished, as consequence of the dramatic reduction of mtDNA. To overcome organelle dysfunction, Δpor1 cells do not activate the rescue signaling retrograde response, as ρ0 cells, and rather carry out complete metabolic rewiring. The TCA cycle works in a "branched" fashion, shunting intermediates towards mitochondrial pyruvate generation via malic enzyme, and the glycolysis-derived pyruvate is pushed towards cytosolic utilization by PDH bypass rather than the canonical mitochondrial uptake. Overall, Δpor1 cells enhance phospholipid biosynthesis, accumulate lipid droplets, increase vacuoles and cell size, overproduce and excrete inositol. Such unexpected re-arrangement of whole metabolism suggests a regulatory role of VDAC1 in cell bioenergetics.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Genes Mitocondriais/genética , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Oxirredução , Porinas/genética , Porinas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Canal de Ânion 1 Dependente de Voltagem/genética
14.
Curr Dev Nutr ; 3(9): nzz081, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31598580

RESUMO

Background: The mean intake of vitamin A of Australians aged 2 y and older was 300 µg retinol equivalents lower in the 2011-2012 national nutrition survey than in 1995 and decreases preponderated in adults rather than young children. Objective: This aim of this study was to identify the foods associated with this change and to examine how the method used to adjust for within-person variability affects the estimated prevalence of inadequate intakes in both surveys. Methods: Foods contributing to vitamin A intake were calculated from the first day of data. The prevalence of inadequate intakes was calculated using a 2-d average, the Iowa State University method, and the National Cancer Institute (NCI) method and either taken from the published reports or calculated using Food Standards Australia New Zealand's in-house software. Results: In adults, lower consumption of liver, yellow fat spreads, milk products, and carrots and similar root vegetables accounted for most of the change in intake. Vitamin A intake data were less right-skewed in 2011-2012 than in 1995. The prevalence of inadequate vitamin A intake depended on the adjustment method chosen: for example, in 2011-2012 it ranged between 3% and 55% in men aged 19-30 y. The NCI method prevalence (21% for this group) is taken as the preferred estimate of inadequacy because the method adjusts around the mean and accounts for several other sources of variance. However, the NCI method could not be used to analyze the 1995 survey. Conclusions: The lower vitamin A intake in Australia was related to changes in retinol intake rather than carotenoid intake and to lower consumption of several different types of food. The estimated prevalence of inadequate intake depends on the statistical method chosen for analysis. A direct measure of vitamin A status is needed to allow conclusions about the implications of the decreasing intake of this vitamin.

15.
Glycoconj J ; 36(6): 461-472, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31529350

RESUMO

Congenital disorders of glycosylation (CDG) are genetic diseases characterized by deficient synthesis (CDG type I) and/or abnormal processing (CDG type II) of glycan moieties linked to protein and lipids. The impact of the molecular defects on protein glycosylation and in turn on the clinical phenotypes of patients with CDG is not yet understood. ALG12-CDG is due to deficiency of ALG12 α1,6-mannosyltransferase that adds the eighth mannose residue on the dolichol-PP-oligosaccharide precursor in the endoplasmic reticulum. ALG12-CDG is a severe multisystem disease associated with low to deficient serum immunoglobulins and recurrent infections. We thoroughly investigated the glycophenotype in a patient with novel ALG12 variants and immunodeficiency. We analyzed serum native transferrin, as first line test for CDG and we profiled serum IgG and total serum N-glycans by a combination of consolidated (N-glycan analysis by MALDI MS) and innovative mass spectrometry-based protocols, such as GlycoWorks RapiFluor N-glycan analysis coupled with LC-ESI MS. Intact serum transferrin showed, as expected for a CDG type I defect, underoccupancy of N-glycosylation sites. Surprisingly, total serum proteins and IgG N-glycans showed some specific changes, consisting in accumulating amounts of definite high-mannose and hybrid structures. As a whole, ALG12-CDG behaves as a dual CDG (CDG-I and II defects) and it is associated with distinct, abnormal glycosylation of total serum and IgG N-glycans. Glycan profiling of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Deficiência de IgG/genética , Imunoglobulinas/genética , Manosiltransferases/genética , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Glicoproteínas/sangue , Glicosilação , Humanos , Deficiência de IgG/sangue , Deficiência de IgG/metabolismo , Deficiência de IgG/patologia , Imunoglobulinas/sangue , Imunoglobulinas/deficiência , Lactente , Masculino , Manosiltransferases/sangue , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Polissacarídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transferrina/genética , Transferrina/metabolismo , Sequenciamento Completo do Exoma
16.
Front Cell Neurosci ; 13: 346, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474832

RESUMO

Mutations in superoxide dismutase (SOD1) are the second most common cause of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the death of motor neurons in the brain and spinal cord. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles, such as mitochondria and ER, disrupting their functions. Here, we demonstrate that mutant SOD1 binds specifically to the N-terminal domain of the voltage-dependent anion channel (VDAC1), an outer mitochondrial membrane protein controlling cell energy, metabolic and survival pathways. Mutant SOD1G93A and SOD1G85R, but not wild type SOD1, directly interact with VDAC1 and reduce its channel conductance. No such interaction with N-terminal-truncated VDAC1 occurs. Moreover, a VDAC1-derived N-terminal peptide inhibited mutant SOD1-induced toxicity. Incubation of motor neuron-like NSC-34 cells expressing mutant SOD1 or mouse embryonic stem cell-derived motor neurons with different VDAC1 N-terminal peptides resulted in enhanced cell survival. Taken together, our results establish a direct link between mutant SOD1 toxicity and the VDAC1 N-terminal domain and suggest that VDAC1 N-terminal peptides targeting mutant SOD1 provide potential new therapeutic strategies for ALS.

17.
Methods Mol Biol ; 2044: 255-272, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432418

RESUMO

CSF diagnostics has proved to be a formidable testing ground for N-glycoproteomic analysis of neurological diseases. To characterize specific N-glycan profiles of CSF in early and advanced phases of Alzheimer's disease, as well as in lysosomal storage disorders such as Tay-Sachs disease, we set up in our lab a robust and feasible protocol by coupling bioanalytical methods and mass spectrometry analysis.Starting from a few microliters of CSF, after protein denaturation, reduction, and alkylation, N-glycans are released from glycoproteins using the peptide-N-glycosidase F (PNGase F) and purified. The analysis of permethylated N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS allowed us to identify specific glyco-structures and also to distinguish between isobaric N-glycans.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Glicoproteínas/líquido cefalorraquidiano , Glicoproteínas/química , Polissacarídeos/líquido cefalorraquidiano , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Doença de Tay-Sachs/líquido cefalorraquidiano , Idoso , Gangliosídeo G(M2)/metabolismo , Humanos , Íons/química , Polissacarídeos/análise , Polissacarídeos/isolamento & purificação
18.
FEBS Open Bio ; 9(7): 1184-1193, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31206247

RESUMO

Voltage-dependent anion channel isoform 2 of the yeast Saccharomyces cerevisiae (yVDAC2) was believed for many years to be devoid of channel activity. Recently, we isolated yVDAC2 and showed that it exhibits channel-forming activity in the planar lipid bilayer system when in its so-called native form. Here, we describe an alternative strategy for yVDAC2 isolation, through heterologous expression in bacteria and refolding in vitro. Recombinant yVDAC2, like its native form, is able to form voltage-dependent channels. However, some differences between native and recombinant yVDAC2 emerged in terms of voltage dependence and ion selectivity, suggesting that, in this specific case, the recombinant protein might be depleted of post-translational modification(s) that occur in eukaryotic cells.


Assuntos
Engenharia de Proteínas/métodos , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Canal de Ânion 2 Dependente de Voltagem/fisiologia , Sequência de Aminoácidos , Fenômenos Eletrofisiológicos , Membranas Intracelulares/metabolismo , Bicamadas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo
19.
Methods Mol Biol ; 1750: 75-91, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29512066

RESUMO

In this chapter, we present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid-phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Glicômica/métodos , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glicoproteínas/líquido cefalorraquidiano , Glicosilação , Humanos , Polissacarídeos/líquido cefalorraquidiano
20.
Biochim Biophys Acta Bioenerg ; 1859(4): 270-279, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29408701

RESUMO

The yeast Saccharomyces cerevisiae genome is endowed with two distinct isoforms of Voltage-Dependent Anion Channel (VDAC). The isoform yVDAC2 is currently understudied with respect to the best known yVDAC1. Yet, since the discovery, the function of yVDAC2 was unclear, leading to the hypothesis that it might be devoid of a channel function. In this work we have elucidated, by bioinformatics modeling and electrophysiological analysis, the functional activity of yVDAC2. The conformation of yVDAC2 and, for comparison, of yVDAC1 were modeled using a multiple template approach involving mouse, human and zebrafish structures and both showed to arrange the sequences as the typical 19-stranded VDAC ß-barrel. Molecular dynamics simulations showed that yVDAC2, in comparison with yVDAC1, has a different number of permeation paths of potassium and chloride ions. yVDAC2 protein was over-expressed in the S. cerevisiae cells depleted of functional yVDAC1 (Δpor1 mutant) and, after purification, it was reconstituted in artificial membranes (planar lipid bilayer (PLB) system). The protein displayed channel-forming activity and the calculated conductance, voltage-dependence and ion selectivity values were similar to those of yVDAC1 and other members of VDAC family. This is the first time that yVDAC2 channel features are detected and characterized.


Assuntos
Mitocôndrias/química , Membranas Mitocondriais/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 2 Dependente de Voltagem/química , Animais , Sítios de Ligação , Cloretos/química , Cloretos/metabolismo , Biologia Computacional , Expressão Gênica , Humanos , Transporte de Íons , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Potássio/química , Potássio/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Canal de Ânion 2 Dependente de Voltagem/genética , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Peixe-Zebra
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