RESUMO
Amyloid assemblies of Tau are associated with Alzheimer's disease (AD). In AD Tau undergoes several abnormal post-translational modifications, including hyperphosphorylation and glycosylation, which impact disease progression. N-glycosylated Tau was reported to be found in AD brain tissues but not in healthy counterparts. This is surprising since Tau is a cytosolic protein whereas N-glycosylation occurs in the ER-Golgi. Previous in vitro studies indicated that N-glycosylation of Tau facilitated its phosphorylation and contributed to maintenance of its Paired Helical Filament structure. However, the specific Tau residue(s) that undergo N-glycosylation and their effect on Tau-engendered pathology are unknown. High-performance liquid chromatography and mass spectrometry (LC-MS) analysis indicated that both N359 and N410 were N-glycosylated in wild-type (WT) human Tau (hTau) expressed in human SH-SY5Y cells. Asparagine to glutamine mutants, which cannot undergo N-glycosylation, at each of three putative N-glycosylation sites in hTau (N167Q, N359Q, and N410Q) were generated and expressed in SH-SY5Y cells and in transgenic Drosophila. The mutants modulated the levels of hTau phosphorylation in a site-dependent manner in both cell and fly models. Additionally, N359Q ameliorated, whereas N410Q exacerbated various aspects of hTau-engendered neurodegeneration in transgenic flies.
Assuntos
Doença de Alzheimer/genética , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Proteínas tau/genética , Doença de Alzheimer/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Drosophila/genética , Drosophila/metabolismo , Glicosilação , Humanos , Longevidade/genética , Doenças Neurodegenerativas/metabolismo , Fosforilação , Proteínas tau/metabolismoRESUMO
Neurofibrillary tangles of the Tau protein and plaques of the amyloid ß peptide are hallmarks of Alzheimer's disease (AD), which is characterized by the conversion of monomeric proteins/peptides into misfolded ß-sheet rich fibrils. Halting the fibrillation process and disrupting the existing aggregates are key challenges for AD drug development. Previously, we performed in vitro high-throughput screening for the identification of potent inhibitors of Tau aggregation using a proxy model, a highly aggregation-prone hexapeptide fragment 306VQIVYK311 (termed PHF6) derived from Tau. Here we have characterized a hit molecule from that screen as a modulator of Tau aggregation using in vitro, in silico, and in vivo techniques. This molecule, an anthraquinone derivative named Purpurin, inhibited ~ 50% of PHF6 fibrillization in vitro at equimolar concentration and disassembled pre-formed PHF6 fibrils. In silico studies showed that Purpurin interacted with key residues of PHF6, which are responsible for maintaining its ß-sheets conformation. Isothermal titration calorimetry and surface plasmon resonance experiments with PHF6 and full-length Tau (FL-Tau), respectively, indicated that Purpurin interacted with PHF6 predominantly via hydrophobic contacts and displayed a dose-dependent complexation with FL-Tau. Purpurin was non-toxic when fed to Drosophila and it significantly ameliorated the AD-related neurotoxic symptoms of transgenic flies expressing WT-FL human Tau (hTau) plausibly by inhibiting Tau accumulation and reducing Tau phosphorylation. Purpurin also reduced hTau accumulation in cell culture overexpressing hTau. Importantly, Purpurin efficiently crossed an in vitro human blood-brain barrier model. Our findings suggest that Purpurin could be a potential lead molecule for AD therapeutics.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antraquinonas/farmacologia , Oligopeptídeos/genética , Agregados Proteicos/efeitos dos fármacos , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Animais , Animais Geneticamente Modificados/genética , Barreira Hematoencefálica/efeitos dos fármacos , Modelos Animais de Doenças , Drosophila melanogaster/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fosforilação/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder which is characterized by the deposits of intra-cellular tau protein and extra-cellular amyloid-ß (Aß) peptides in the human brain. Understanding the mechanism of protein aggregation and finding compounds that are capable of inhibiting its aggregation is considered to be highly important for disease therapy. METHODS: We used an in vitro High-Throughput Screening for the identification of potent inhibitors of tau aggregation using a proxy model; a highly aggregation-prone hexapeptide fragment 306VQIVYK311 derived from tau. Using ThS fluorescence assay we screened a library of 2401 FDA approved, bio-active and natural compounds in attempt to find molecules which can efficiently modulate tau aggregation. RESULTS: Among the screened compounds, palmatine chloride (PC) alkaloid was able to dramatically reduce the aggregation propensity of PHF6 at sub-molar concentrations. PC was also able to disassemble preformed aggregates of PHF6 and reduce the amyloid content in a dose-dependent manner. Insights obtained from MD simulation showed that PC interacted with the key residues of PHF6 responsible for ß-sheet formation, which could likely be the mechanism of inhibition and disassembly. Furthermore, PC could effectively inhibit the aggregation of full-length tau and disassemble preformed aggregates. CONCLUSIONS: We found that PC possesses "dual functionality" towards PHF6 and full-length tau, i.e. inhibit their aggregation and disassemble pre-formed fibrils. GENERAL SIGNIFICANCE: The "dual functionality" of PC is valuable as a disease modifying strategy for AD, and other tauopathies, by inhibiting their progress and reducing the effect of fibrils already present in the brain.
Assuntos
Alcaloides de Berberina/farmacologia , Fragmentos de Peptídeos/efeitos dos fármacos , Proteínas tau/efeitos dos fármacos , Neoplasias das Glândulas Suprarrenais/patologia , Amiloide/efeitos dos fármacos , Amiloide/ultraestrutura , Dicroísmo Circular , Simulação por Computador , Ensaios de Triagem em Larga Escala , Humanos , Técnicas In Vitro , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Feocromocitoma/patologia , Agregação Patológica de Proteínas , Células Tumorais Cultivadas , Proteínas tau/químicaRESUMO
According to World Health Organization WHO, Tuberculosis (TB) is the second cause of death from infectious disease worldwide. During 2021, 10.6 million people were infected with TB, and 1.6 million people died. TB is caused by pathogens belonging to the Mycobacterium tuberculosis complex (MTBC), mainly by Mycobacterium tuberculosis (M.tb). Members of this complex are acid-fast bacilli, which can cause intrapulmonary and extra pulmonary TB, and can be divided into various lineages, based on genomic markers. The main public health threat comes from drug resistant M.tb strains, which are responsible for about 25% of TB death and treatment failure worldwide. Treating drug resistant TB patients significantly raises the costs of TB treatment. This study provides an overview of the demographic and drug susceptibility characteristics of newly diagnosed TB patients in Israel in 2021. The State of Israel has a very low level of TB endemicity and is at a pre-elimination phase. Notably, only 11.7% of the newly diagnosed TB patients were born in Israel. In this report, of the 154 new laboratory-confirmed TB patients, 66.7% had pulmonary TB, while 16% had extrapulmonary TB. Males accounted for 52% of the patients, with the most prevalent age group being 21-40. Most patients were citizens of Israel (53.9%), while 37.7% had no Israeli citizenship. Among non-citizens, there was a predominance of males and patients aged 21-40. The susceptibility profile showed a high resistance rate to streptomycin (18.2%) and to a lower extent to isoniazid (13.6%), pyrazinamide (8.4%), rifampicin (7.8%), and ethambutol (3.2%). Only 2 cases of XDR-TB and 10 MDR-TB strains were detected in Israel in 2021, with both XDR strains and 5 out of 10 MDR strains belonging to the Beijing lineage. Most of Beijing isolates were resistant to at least one tested drug. Genomic sequencing of 134 out of 156 strains and bioinformatics analysis using the MTBseq program and WHO mutation catalogue shows a good match with only 9 discrepancies between phenotypic and genotypic susceptibility profiles in first line drugs. The most common lineage is Delhi-Cas (23%) followed by the Beijing lineage (17%). Most patients from the Delhi-Cas lineage were born in Africa, while patients with Beijing isolates were born in different countries. Minimum spanning tree analysis identified 15 clusters. The study highlights the need for ongoing surveillance of TB using molecular and phenotypic tools to further decreasing the spreading level of the disease and develop effective treatment strategies.
Assuntos
Mycobacterium tuberculosis , Tuberculose Extrapulmonar , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Masculino , Humanos , Feminino , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Israel/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose/tratamento farmacológico , Tuberculose/epidemiologia , Genômica , Demografia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla/genéticaRESUMO
Coinfection of HIV and multidrug-resistant tuberculosis (MDR-TB) presents significant challenges in terms of the treatment and prognosis of tuberculosis, leading to complexities in managing the disease and impacting the overall outcome for TB patients. This study presents a remarkable case of a patient with MDR-TB and HIV coinfection who survived for over 8 years, despite poor treatment adherence and comorbidities. Whole genome sequencing (WGS) of the infecting Mycobacterium tuberculosis (Mtb) strain revealed a unique genomic deletion, spanning 18 genes, including key genes involved in hypoxia response, intracellular survival, immunodominant antigens, and dormancy. This deletion, that we have called "Del-X," potentially exerts a profound influence on the bacterial physiology and its virulence. Only few similar deletions were detected in other non-related Mtb genomes worldwide. In vivo evolution analysis identified drug resistance and metabolic adaptation mutations and their temporal dynamics during the patient's treatment course.
RESUMO
Alzheimer's disease (AD) is the most common neurodegenerative disorder and has no disease-modifying treatment yet. The hallmarks of AD are two amyloidogenic proteins: tau and amyloid ß (Aß). Tau undergoes several posttranslational modifications, including N-glycosylation. Tau was reported to be N-glycosylated in AD brains, but not in healthy counterparts, which may affect AD etiology. Here, we aimed to examine the effect of N-glycosylation on aggregation propensity of tau. To that end, a novel SH-SY5Y cell-based model was generated in which recombinant human tau (htau) is forced to be secreted from the cells. Secreted htau was found to localize in the secretory pathway compartments and to undergo N-glycosylation. Following N-glycan cleavage of the secreted htau, various biophysical results collectively indicated that the untreated N-glycosylated secreted htau is markedly less aggregative, contains thinner and shorter fibrils, as compared to treated de-glycosylated secreted htau. This finding shows that N-glycans attached to htau may affect its aggregation. This could help to better understand the effect of N-glycosylated htau on AD progression.
Assuntos
Doença de Alzheimer/metabolismo , Modelos Biológicos , Agregação Patológica de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas tau/biossíntese , Linhagem Celular Tumoral , Glicosilação , HumanosRESUMO
The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., ß-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. Although deviations from normal protein glycosylation have been documented in AD, their role in disease pathology has been barely explored. Here our analysis of available expression data sets indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled us to predict the occurrence of AD with remarkable accuracy in a test cohort and identify a set of key genes whose expression determines this classification. We then studied in vivo the effect of reducing expression of homologs of 6 of these genes in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. Our results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology.