RESUMO
The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream, playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS); however, the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression, compromised barrier integrity, and increased P-glycoprotein transporter activity. In addition, mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover, our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally, C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.
Assuntos
Esclerose Lateral Amiotrófica , Barreira Hematoencefálica , Células Endoteliais , Humanos , Esclerose Lateral Amiotrófica/genética , Barreira Hematoencefálica/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Células Endoteliais/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to â¼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS.
RESUMO
Dipeptide repeat (DPR) proteins are aggregation-prone polypeptides encoded by the pathogenic GGGGCC repeat expansion in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic ß-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Dipeptídeos , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Neurônios Motores/metabolismoRESUMO
Disruption to protein homeostasis caused by lysosomal dysfunction and associated impairment of autophagy is a prominent pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The most common genetic cause of ALS/FTD is a G4C2 hexanucleotide repeat expansion in C9orf72 (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 repeat transcripts gives rise to dipeptide repeat (DPR) proteins that have been shown to be toxic and may contribute to disease etiology. Genetic variants in TMEM106B have been associated with frontotemporal lobar degeneration with TDP-43 pathology and disease progression in C9ALS/FTD. TMEM106B encodes a lysosomal transmembrane protein of unknown function that is involved in various aspects of lysosomal biology. How TMEM106B variants affect C9ALS/FTD is not well understood but has been linked to changes in TMEM106B protein levels. Here, we investigated TMEM106B function in the context of C9ALS/FTD DPR pathology. We report that knockdown of TMEM106B expression exacerbates the accumulation of C9ALS/FTD-associated cytotoxic DPR proteins in cell models expressing RAN-translated or AUG-driven DPRs as well as in C9ALS/FTD-derived iAstrocytes with an endogenous G4C2 expansion by impairing autophagy. Loss of TMEM106B caused a block late in autophagy by disrupting autophagosome to autolysosome maturation which coincided with impaired lysosomal acidification, reduced cathepsin activity, and juxtanuclear clustering of lysosomes. Lysosomal clustering required Rab7A and coincided with reduced Arl8b-mediated anterograde transport of lysosomes to the cell periphery. Increasing Arl8b activity in TMEM106B-deficient cells not only restored the distribution of lysosomes, but also fully rescued autophagy and DPR protein accumulation. Thus, we identified a novel function of TMEM106B in autophagosome maturation via Arl8b. Our findings indicate that TMEM106B variants may modify C9ALS/FTD by regulating autophagic clearance of DPR proteins. Caution should therefore be taken when considering modifying TMEM106B expression levels as a therapeutic approach in ALS/FTD.
RESUMO
Astrocytes are highly specialised cells, responsible for CNS homeostasis and neuronal activity. Lack of human in vitro systems able to recapitulate the functional changes affecting astrocytes during ageing represents a major limitation to studying mechanisms and potential therapies aiming to preserve neuronal health. Here, we show that induced astrocytes from fibroblasts donors in their childhood or adulthood display age-related transcriptional differences and functionally diverge in a spectrum of age-associated features, such as altered nuclear compartmentalisation, nucleocytoplasmic shuttling properties, oxidative stress response and DNA damage response. Remarkably, we also show an age-related differential response of induced neural progenitor cells derived astrocytes (iNPC-As) in their ability to support neurons in co-culture upon pro-inflammatory stimuli. These results show that iNPC-As are a renewable, readily available resource of human glia that retain the age-related features of the donor fibroblasts, making them a unique and valuable model to interrogate human astrocyte function over time in human CNS health and disease.
Assuntos
Astrócitos/metabolismo , Fibroblastos/metabolismo , Envelhecimento , Sistema Nervoso Central , HumanosRESUMO
It is important to understand how the disease process affects the metabolic pathways in amyotrophic lateral sclerosis and whether these pathways can be manipulated to ameliorate disease progression. To analyse the basis of the metabolic defect in amyotrophic lateral sclerosis we used a phenotypic metabolic profiling approach. Using fibroblasts and reprogrammed induced astrocytes from C9orf72 and sporadic amyotrophic lateral sclerosis cases we measured the production rate of reduced nicotinamide adenine dinucleotides (NADH) from 91 potential energy substrates simultaneously. Our screening approach identified that C9orf72 and sporadic amyotrophic lateral sclerosis induced astrocytes have distinct metabolic profiles compared to controls and displayed a loss of metabolic flexibility that was not observed in fibroblast models. This loss of metabolic flexibility, involving defects in adenosine, fructose and glycogen metabolism, as well as disruptions in the membrane transport of mitochondrial specific energy substrates, contributed to increased starvation induced toxicity in C9orf72 induced astrocytes. A reduction in glycogen metabolism was attributed to loss of glycogen phosphorylase and phosphoglucomutase at the protein level in both C9orf72 induced astrocytes and induced neurons. In addition, we found alterations in the levels of fructose metabolism enzymes and a reduction in the methylglyoxal removal enzyme GLO1 in both C9orf72 and sporadic models of disease. Our data show that metabolic flexibility is important in the CNS in times of bioenergetic stress.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Proteína C9orf72/metabolismo , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Adulto , Idoso , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Progressão da Doença , Metabolismo Energético , Feminino , Glicogênio Fosforilase/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Peptides are of increasing interest as therapeutics in a wide range of diseases, including metabolic diseases such as diabetes and obesity. In the latter, peptide hormones such as peptide YY (PYY) and pancreatic peptide (PP) are important templates for drug design. Characteristic for these peptides is that they contain a C-terminal that is α-amidated, and this amidation is crucial for biological function. A challenge is to generate such peptides by recombinant means and particularly in a production scale. Here, we have examined an intein-mediated approach to generate a PYY derivative in a larger scale. Initially, we experienced challenges with hydrolysis of the intein fusion protein, which was reduced by a T3C mutation in the intein. Subsequently, we further engineered the intein to decrease the absolute size and improve the relative yield of the PYY derivative, which was achieved by substituting 54 residues of the 198 amino acid intein with an eight amino acid linker. The optimized intein construct was used to produce the PYY derivative under high cell density cultivation conditions, generating the peptide thioester precursor in good yields and subsequent amidation provided the target peptide.
Assuntos
Amidas/química , Inteínas , Peptídeos/química , Peptídeos/síntese química , Engenharia de Proteínas , Inteínas/genética , Modelos Moleculares , Mutação , Peptídeos/genéticaRESUMO
D-Alanine is a central component of the cell wall in most prokaryotes. D-Alanine synthesis in Escherichia coli is carried out by two different alanine racemases encoded by the alr and dadX genes. Deletion of alr and dadX from the E. coli genome results in a D-alanine auxotrophic phenotype. However, we have observed growth of prototrophic phenotypic revertants during routine culturing of a D-alanine auxotrophic strain. We present a detailed comparison of the proteome and transcriptome profiles of the D-alanine auxotroph and a prototrophic revertant strain. Most noticeably, a general upregulation of genes involved in methionine synthesis in the revertant strain was detected. The appearance of the revertant phenotype was genetically linked to point mutations in the methionine repressor gene (metJ). Our results reveal an alternative metabolic pathway which can supply essential d-alanine for peptidoglycan synthesis of alr- and dadX-deficient E. coli mutants and provide evidence for significant alanine racemase coactivity of the E. coli cystathionine beta-lyase (MetC).
Assuntos
Alanina Racemase/genética , Alanina Racemase/metabolismo , Alanina/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Liases/metabolismo , Regulação para Cima/fisiologia , Alanina/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Liases/genéticaRESUMO
Backbone cyclic insulin was designed and prepared by reverse proteolysis in partial organic solvent of a single-chain precursor expressed in yeast. The precursor contains two loops to bridge the two chains of native insulin. The cyclisation method uses Achromobacter lyticus protease and should be generally applicable to proteins with C-terminal lysine and proximal N-terminal. The presence of the ring-closing bond and the native insulin disulfide patterns were documented by LC-MS peptide maps. The cyclic insulin was shown to be inert towards degradation by CPY, but was somewhat labile towards chymotrypsin. Intravenous administration of the cyclic insulin to Wistar rats showed the compounds to be equipotent to HI despite much lower insulin receptor affinity.
Assuntos
Insulina/análogos & derivados , Peptídeos Cíclicos/síntese química , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Ciclização , Insulina/química , Insulina/farmacologia , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Wistar , Receptor de Insulina/metabolismoRESUMO
PEGylation modification has been used to improve the pharmacokinetic properties of protein-based drugs. For example, PEGylated human growth hormone (hGH) has been shown to exhibit better pharmacokinetic profiles than the unmodified hGH. Unlike chemical PEGylation of hGH that is difficult to be controlled to result in homogeneity, microbial transglutaminase (mTGase) only conjugates poly(ethelene glycol) (PEG) on glutamine-40 (Q40) and glutamine-141 (Q141) of hGH, the only glutamine residues exposed. Yet, an mTGase that can selectively conjugate PEG to only 1 glutamine residue is more desirable to control the homogeneity of the product. In this study, the authors have developed a novel high-throughput assay, with which they have identified 5 mTGase mutants that are highly specific for conjugating PEG to Q141 of hGH. In this scintillation proximity assay (SPA)-based method, the authors have (1) achieved a high expression level of active mTGase, which is toxic to the living cell, directly from Escherichia coli (0.2 U/mL/OD600) by in vivo activation; (2) developed a high-throughput affinity purification method to eliminate the strong interference of cellular protein to mTGase reaction; and (3) used therapeutic protein as the substrate. This method is highly sensitive, is easily automated, and could be generally applied to screening mTGases with desired specificity targeting on different therapeutic proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Glicina/química , Ensaios de Triagem em Larga Escala , Hormônio do Crescimento Humano/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Eletroforese Capilar , Escherichia coli/genética , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/genética , Humanos , Cinética , Dados de Sequência Molecular , Mutação , Plasmídeos , Sensibilidade e Especificidade , Streptomyces/enzimologia , Especificidade por Substrato/genéticaRESUMO
The protein composition and N-terminal sequences of proteins in the outer membrane of Chlamydia trachomatis L2 were analysed following isolation of N-terminal peptides using combined fractional diagonal chromatography and identification by liquid chromatography tandem MS. Acetylation of primary amino groups of in vivo generated proteolytic cleavage sites facilitated identification of such sites in known outer membrane proteins (MOMPs). Our results further support a proposed prediction of the topology of the MOMPs. Furthermore, a previously unknown MOMP, CTL0626 (Ct372), was assigned as an MOMP with a carbohydrate-selective porin (OprB) family motif, and the presence of CTL0626 was confirmed using antibodies raised against the protein.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Chlamydia trachomatis/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Sequência de Bases , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Células Epiteliais/química , Células Epiteliais/microbiologia , Células HeLa , Humanos , Dados de Sequência Molecular , Porinas/análise , Porinas/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.
Assuntos
Antagonistas da Insulina/farmacologia , Peptídeos/farmacologia , Receptor de Insulina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Humanos , Insulina/metabolismo , Insulina/farmacologia , Antagonistas da Insulina/química , Antagonistas da Insulina/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Ratos , Ratos Zucker , Receptor de Insulina/metabolismoRESUMO
Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D-PAGE profiles of [35S]-labelled Chlamydia proteins from whole lysates of infected cells to 2D-PAGE profiles of proteins from purified Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the 'chlamydial protease- or proteasome-like activity factor' (CPAF) and were clearly present in 2D-PAGE profiles of whole lysates of infected cells but absent from purified Chlamydia. CPAF was recently identified by Zhong and colleagues as a secreted protease which cleaves host cell transcription factors essential for MHC class I and II antigen presentation. The identification of CPAF in this paper verifies the applicability of the described method for the identification of secreted proteins. We extend the findings by Zhong et al. by proteome studies of expression and turnover of C. trachomatis CPAF showing that the degradation of C. trachomatis D CPAF in the host cell is very limited. Furthermore, we show that two fragments of CPAF exist in C. pneumoniae as well as in C. trachomatis.
Assuntos
Chlamydia trachomatis/enzimologia , Chlamydophila pneumoniae/enzimologia , Endopeptidases/metabolismo , Sequência de Aminoácidos , Anticorpos Antibacterianos , Sequência de Bases , Linhagem Celular , Chlamydia trachomatis/genética , Chlamydia trachomatis/imunologia , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/imunologia , Clonagem Molecular , DNA Bacteriano/genética , Eletroforese em Gel Bidimensional , Endopeptidases/genética , Endopeptidases/imunologia , Endopeptidases/isolamento & purificação , Células HeLa , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Inibidores de Proteases/farmacologia , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.
Assuntos
Proteínas de Bactérias/análise , Chlamydia trachomatis/química , Chlamydia trachomatis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydophila pneumoniae/química , Chlamydophila pneumoniae/genética , Eletroforese em Gel Bidimensional , Técnica Indireta de Fluorescência para Anticorpo , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , ProteomaRESUMO
Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.