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1.
Biochemistry ; 60(37): 2773-2780, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34469142

RESUMO

The prevailing opinion is that prefibrillar ß-amyloid (Aß) species, rather than end-stage amyloid fibrils, cause neuronal dysfunction in Alzheimer's disease, although the mechanisms behind Aß neurotoxicity remain to be elucidated. Luminescent conjugated oligothiophenes (LCOs) exhibit spectral properties upon binding to amyloid proteins and have previously been reported to change the toxicity of Aß1-42 and prion protein. In a previous study, we showed that an LCO, pentamer formyl thiophene acetic acid (p-FTAA), changed the toxicity of Aß1-42. Here we investigated whether an LCO, heptamer formyl thiophene acetic acid (h-FTAA), could change the toxicity of Aß1-42 by comparing its behavior with that of p-FTAA. Moreover, we investigated the effects on toxicity when Aß with the Arctic mutation (AßArc) was aggregated with both LCOs. Cell viability assays on SH-SY5Y neuroblastoma cells demonstrated that h-FTAA has a stronger impact on Aß1-42 toxicity than does p-FTAA. Interestingly, h-FTAA, but not p-FTAA, rescued the AßArc-mediated toxicity. Aggregation kinetics and binding assay experiments with Aß1-42 and AßArc when aggregated with both LCOs showed that h-FTAA and p-FTAA either interact with different species or affect the aggregation in different ways. In conclusion, h-FTAA protects against Aß1-42 and AßArc toxicity, thus showing h-FTAA to be a useful tool for improving our understanding of the process of Aß aggregation linked to cytotoxicity.


Assuntos
Acetatos/química , Precursor de Proteína beta-Amiloide/metabolismo , Tiofenos/química , Acetatos/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/fisiologia , Precursor de Proteína beta-Amiloide/toxicidade , Proteínas Amiloidogênicas/química , Corantes Fluorescentes/química , Humanos , Cinética , Luminescência , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/fisiologia , Coloração e Rotulagem/métodos , Tiofenos/metabolismo
2.
Cells ; 10(9)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34572142

RESUMO

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.


Assuntos
Poro Nuclear/metabolismo , Pressão Osmótica/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina , Citoplasma/metabolismo , Humanos , Membrana Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Agregados Proteicos/fisiologia , Proteínas/metabolismo , Estresse Fisiológico/fisiologia
3.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445649

RESUMO

Protein aggregation is associated with a growing list of human diseases. A substantial fraction of proteins in eukaryotic proteomes constitutes a proteostasis network-a collection of proteins that work together to maintain properly folded proteins. One of the overarching functions of the proteostasis network is the prevention or reversal of protein aggregation. How proteins aggregate in spite of the anti-aggregation activity of the proteostasis machinery is incompletely understood. Exposed hydrophobic patches can trigger degradation by the ubiquitin-proteasome system, a key branch of the proteostasis network. However, in a recent study, we found that model glycine (G)-rich or glutamine/asparagine (Q/N)-rich prion-like domains differ in their susceptibility to detection and degradation by this system. Here, we expand upon this work by examining whether the features controlling the degradation of our model prion-like domains generalize broadly to G-rich and Q/N-rich domains. Experimentally, native yeast G-rich domains in isolation are sensitive to the degradation-promoting effects of hydrophobic residues, whereas native Q/N-rich domains completely resist these effects and tend to aggregate instead. Bioinformatic analyses indicate that native G-rich domains from yeast and humans tend to avoid degradation-promoting features, suggesting that the proteostasis network may act as a form of selection at the molecular level that constrains the sequence space accessible to G-rich domains. However, the sensitivity or resistance of G-rich and Q/N-rich domains, respectively, was not always preserved in their native protein contexts, highlighting that proteins can evolve other sequence features to overcome the intrinsic sensitivity of some LCDs to degradation.


Assuntos
Agregados Proteicos/fisiologia , Proteoma/metabolismo , Proteostase , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
4.
J Med Chem ; 64(16): 12003-12021, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34351166

RESUMO

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.


Assuntos
Encéfalo/diagnóstico por imagem , Compostos Heterocíclicos com 3 Anéis/química , Proteína Huntingtina/metabolismo , Agregados Proteicos/fisiologia , Piridinas/química , Compostos Radiofarmacêuticos/química , Doença de Alzheimer , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Feminino , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Piridinas/síntese química , Piridinas/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade
5.
Sci Rep ; 11(1): 15934, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354200

RESUMO

A non-invasive and sensitive blood test has long been a goal for early stage disease diagnosis and treatment for Alzheimer's disease (AD) and other proteinopathy diseases. We previously reported that preeclampsia (PE), a severe pregnancy complication, is another proteinopathy disorder with impaired autophagy. We hypothesized that induced autophagy deficiency would promote accumulation of pathologic protein aggregates. Here, we describe a novel, sensitive assay that detects serum protein aggregates from patients with PE (n = 33 early onset and 33 late onset) and gestational age-matched controls (n = 77) as well as AD in both dementia and prodromal mild cognitive impairment (MCI, n = 24) stages with age-matched controls (n = 19). The assay employs exposure of genetically engineered, autophagy-deficient human trophoblasts (ADTs) to serum from patients. The aggregated protein complexes and their individual components, including transthyretin, amyloid ß-42, α-synuclein, and phosphorylated tau231, can be detected and quantified by co-staining with ProteoStat, a rotor dye with affinity to aggregated proteins, and respective antibodies. Detection of protein aggregates in ADTs was not dependent on transcriptional upregulation of these biomarkers. The ROC curve analysis validated the robustness of the assay for its specificity and sensitivity (PE; AUC: 1, CI: 0.949-1.00; AD; AUC: 0.986, CI: 0.832-1.00). In conclusion, we have developed a novel, noninvasive diagnostic and predictive assay for AD, MCI and PE.


Assuntos
Doença de Alzheimer/sangue , Análise Química do Sangue/métodos , Pré-Eclâmpsia/sangue , Adulto , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Disfunção Cognitiva/diagnóstico , Feminino , Testes Hematológicos/métodos , Humanos , Imuno-Histoquímica , Fragmentos de Peptídeos , Pré-Eclâmpsia/diagnóstico , Gravidez , Agregados Proteicos/fisiologia , Curva ROC , Trofoblastos/efeitos dos fármacos , alfa-Sinucleína , Proteínas tau
6.
Cell Mol Life Sci ; 78(17-18): 6105-6117, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34297165

RESUMO

Transthyretin (TTR) is an extracellular protein mainly produced in the liver and choroid plexus, with a well-stablished role in the transport of thyroxin and retinol throughout the body and brain. TTR is prone to aggregation, as both wild-type and mutated forms of the protein can lead to the accumulation of amyloid deposits, resulting in a disease called TTR amyloidosis. Recently, novel activities for TTR in cell biology have emerged, ranging from neuronal health preservation in both central and peripheral nervous systems, to cellular fate determination, regulation of proliferation and metabolism. Here, we review the novel literature regarding TTR new cellular effects. We pinpoint TTR as major player on brain health and nerve biology, activities that might impact on nervous systems pathologies, and assign a new link between TTR and angiogenesis and cancer. We also explore the molecular mechanisms underlying TTR activities at the cellular level, and suggest that these might go beyond its most acknowledged carrier functions and include interaction with receptors and activation of intracellular signaling pathways.


Assuntos
Amiloidose/etiologia , Pré-Albumina/metabolismo , Amiloidose/metabolismo , Sistema Nervoso Central/metabolismo , Humanos , Neurônios/citologia , Neurônios/metabolismo , Pré-Albumina/química , Pré-Albumina/genética , Agregados Proteicos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tiroxina/química , Tiroxina/metabolismo , Vitamina A/química , Vitamina A/metabolismo
7.
Biochim Biophys Acta Mol Basis Dis ; 1867(10): 166205, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214607

RESUMO

Proteolysis mediated by lysosomal cathepsin proteases maintains a physiological flow in autophagy, phagocytosis and endocytosis. Neuronal Ceroid Lipofuscinosis (NCL) is a childhood neurodegenerative disorder characterized by disturbed autophagic flow and pathological accumulation of proteins. We demonstrated a therapeutic clearance of protein aggregates after dosing NCL10 mice with recombinant human pro-cathepsin-D. Prompted by these results and speculating that cathepsins may act in a redundant and in an hierarchical manner we envisaged that a treatment with human recombinant cysteine proteases pro-cathepsin-L (proCTSL) and pro-cathepsin-B (proCTSB) could similarly be used to induce protein degradation. Both enzymes were taken up by mannose 6-phosphate receptor- and LRP-receptor-mediated endocytosis and processed to the lysosomal mature cathepsins. In murine NCL10 astrocytes an abnormal increase in LAMP1 and saposin expression was revealed. Although proCTSB application did not improve this phenotype, proCTSL treatment led to reduced saposin-C levels in this model as well as in an acute brain slice model. Intracerebral dosing in a NCL10 mouse model revealed cellular and lysosomal uptake of both enzymes. Only proCTSL mildly reduced saposin-C levels and attenuated reactive astrogliosis. Application of both proteases did not improve weight loss and mortality of mutant mice. Our data reveal that although recombinant lysosomal proteases can be efficiently delivered to neuronal lysosomes cysteine proteases are less efficient in protein aggregates clearance as compared to the cathepsin-D treatment. Our data including in vitro degradation assays support the idea that bulk proteolysis requires a hierarchical process in which both aspartyl and cysteine hydrolases play a role.


Assuntos
Catepsina B/metabolismo , Catepsina L/metabolismo , Lisossomos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Neurônios/metabolismo , Agregados Proteicos/fisiologia , Proteínas/metabolismo , Animais , Autofagia/fisiologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Gliose/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteólise
8.
Biochim Biophys Acta Proteins Proteom ; 1869(9): 140682, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34102324

RESUMO

Protein aggregation has two aspects, namely, mechanistic and kinetics. Understanding protein aggregation kinetics is critical for prediction of progression of diseases caused by amyloidosis, accumulation of aggregates in biotherapeutics during storage and engineering commercial nano-biomaterials. In this work, we have collected experimentally determined absolute protein aggregation rates and developed an SVM based regression model to predict absolute rates of protein and peptide aggregation near-physiological conditions. The regression model achieved a correlation coefficient of 0.72 with MAE of 0.91 (natural log of kapp, where kapp is in hour-1) using leave-one-out cross-validation on a dataset of 82 non-redundant proteins/peptides. The model accounts for the experimental conditions (such as temperature, pH, ionic and protein concentration) and sequence-based properties. The amino acid sequence features revealed by this model as being important for aggregation kinetics, are also associated with the aggregation mechanism. In particular, inherent aggregation propensity of the protein/peptide sequence and number of aggregation prone regions (APRs) unpunctuated by the gatekeeping residues, were found to play important roles in the prediction of the absolute aggregation rates. This analysis shows that mechanism and kinetics of protein aggregation are coupled via common sequence attributes. The aggregation kinetic prediction method developed in this work is available at https://web.iitm.ac.in/bioinfo2/absolurate-pred/index.html.


Assuntos
Biologia Computacional/métodos , Previsões/métodos , Agregados Proteicos/fisiologia , Algoritmos , Amiloide/química , Simulação por Computador , Bases de Dados de Proteínas , Cinética , Modelos Químicos , Peptídeos/química , Proteínas/química , Análise de Regressão
9.
Biochim Biophys Acta Mol Basis Dis ; 1867(10): 166202, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34144092

RESUMO

Onset of protein aggregation reflects failure of the cellular folding machinery to keep aggregation-prone protein from misfolding and accumulating into a non-degradable state. FRET based analysis and biochemical data reveal that cytosolic prion (cyPrP) and httQ-103 interact with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) leading to few detectable aggregates in GAPDH-over expressing cells.The preventive effect of GAPDH suggests that this abundant and long-lived cytoplasmic protein has an active role in the shielding and maintenance, in soluble form of proteins as heterogeneous as huntingtin and cyPrP.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Agregados Proteicos/fisiologia , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HeLa , Humanos
10.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064208

RESUMO

In Parkinson's disease, aggregates of α-synuclein within Lewy bodies and Lewy neurites represent neuropathological hallmarks. However, the cellular and molecular mechanisms triggering oligomeric and fibrillary α-synuclein aggregation are not fully understood. Recent evidence indicates that oxidative stress induced by metal ions and post-translational modifications such as phosphorylation, ubiquitination, nitration, glycation, and SUMOylation affect α-synuclein conformation along with its aggregation propensity and neurotoxic profiles. In addition, proteolytic cleavage of α-synuclein by specific proteases results in the formation of a broad spectrum of fragments with consecutively altered and not fully understood physiological and/or pathological properties. In the present review, we summarize the current knowledge on proteolytical α-synuclein cleavage by neurosin, calpain-1, cathepsin D, and matrix metalloproteinase-3 in health and disease. We also shed light on the contribution of the same enzymes to proteolytical processing of pathogenic proteins in Alzheimer's disease and report potential cross-disease mechanisms of pathogenic protein aggregation.


Assuntos
alfa-Sinucleína/metabolismo , Doença de Alzheimer/metabolismo , Animais , Humanos , Doença de Parkinson/metabolismo , Peptídeo Hidrolases/metabolismo , Agregados Proteicos/fisiologia , Proteólise
11.
Diabetes ; 70(6): 1229-1241, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34016598

RESUMO

Insulin-producing pancreatic ß-cells are central to glucose homeostasis, and their failure is a principal driver of diabetes development. To preserve optimal health ß-cells must withstand both intrinsic and extrinsic stressors, ranging from inflammation to increased peripheral insulin demand, in addition to maintaining insulin biosynthesis and secretory machinery. Autophagy is increasingly being appreciated as a critical ß-cell quality control system vital for glycemic control. Here we focus on the underappreciated, yet crucial, roles for selective and organelle-specific forms of autophagy as mediators of ß-cell health. We examine the unique molecular players underlying each distinct form of autophagy in ß-cells, including selective autophagy of mitochondria, insulin granules, lipid, intracellular amyloid aggregates, endoplasmic reticulum, and peroxisomes. We also describe how defects in selective autophagy pathways contribute to the development of diabetes. As all forms of autophagy are not the same, a refined view of ß-cell selective autophagy may inform new approaches to defend against the various insults leading to ß-cell failure in diabetes.


Assuntos
Autofagia/fisiologia , Células Secretoras de Insulina/fisiologia , Animais , Diabetes Mellitus/etiologia , Diabetes Mellitus/patologia , Diabetes Mellitus/fisiopatologia , Humanos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiopatologia , Mitofagia/fisiologia , Agregados Proteicos/fisiologia , Fatores de Transcrição/fisiologia , Ubiquitina-Proteína Ligases/fisiologia
12.
Biochemistry ; 60(21): 1658-1669, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34009955

RESUMO

The microtubule-associated protein tau promotes the stabilization of the axonal cytoskeleton in neurons. In several neurodegenerative diseases, such as Alzheimer's disease, tau has been found to dissociate from microtubules, leading to the formation of pathological aggregates that display an amyloid fibril-like structure. Recent structural studies have shown that the tau filaments isolated from different neurodegenerative disorders have structurally distinct fibril cores that are specific to the disease. These "strains" of tau fibrils appear to propagate between neurons in a prion-like fashion that maintains their initial template structure. In addition, the strains isolated from diseased tissue appear to have structures that are different from those made by the most commonly used in vitro modeling inducer molecule, heparin. The structural differences among strains in different diseases and in vitro-induced tau fibrils may contribute to recent failures in clinical trials of compounds designed to target tau pathology. This study identifies an isoquinoline compound (ANTC-15) isolated from the fungus Aspergillus nidulans that can both inhibit filaments induced by arachidonic acid (ARA) and disassemble preformed ARA fibrils. When compared to a tau aggregation inhibitor currently in clinical trials (LMTX, LMTM, or TRx0237), ANTC-15 and LMTX were found to have opposing inducer-specific activities against ARA and heparin in vitro-induced tau filaments. These findings may help explain the disappointing results in translating potent preclinical inhibitor candidates to successful clinical treatments.


Assuntos
Isoquinolinas/farmacologia , Tauopatias/fisiopatologia , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/química , Aspergillus nidulans/metabolismo , Fungos/metabolismo , Humanos , Isoquinolinas/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Príons/metabolismo , Agregados Proteicos/fisiologia , Agregação Patológica de Proteínas/metabolismo , Relação Estrutura-Atividade , Tauopatias/metabolismo , Proteínas tau/fisiologia
13.
J Vis Exp ; (170)2021 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-33970128

RESUMO

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.


Assuntos
Doenças Neurodegenerativas/fisiopatologia , Agregados Proteicos/fisiologia , Espectrometria de Fluorescência/métodos , Humanos
14.
Methods Mol Biol ; 2322: 3-16, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34043187

RESUMO

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein (αSyn). They include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In each disease, it has been proposed that aggregates of αSyn represent different conformational strains of αSyn, leading to self-propagation and spreading from cell to cell. It has been considered that αSyn aggregates grow by seeded polymerization mechanisms. Previously, the mechanism of seed conversion in prion protein aggregation has been exploited by real-time quaking-induced conversion (RT-QuIC) assay. It was further refined by incorporating the fluorescent dye thioflavin-T, which enabled the real-time monitoring of kinetic changes with a highly sensitive detection of seed aggregates present at an extremely low level. In an application for diagnostics, it has been reported that αSyn RT-QuIC exhibits specificity between 82% and 100%, while its sensitivity varies between 70% and 100%, on the basis of a study in which this assay was performed at multiple different laboratories. Furthermore, it has been suggested that the αSyn RT-QuIC method can be applied to study the biochemical characteristics of different αSyn strains among synucleinopathies. In this article, we describe the detailed protocols for αSyn RT-QuIC assays.


Assuntos
Sinucleinopatias/metabolismo , alfa-Sinucleína/metabolismo , Benzotiazóis/metabolismo , Bioensaio/métodos , Encéfalo/metabolismo , Humanos , Cinética , Proteínas Priônicas/metabolismo , Agregados Proteicos/fisiologia
15.
Chem Phys Lipids ; 237: 105083, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33887213

RESUMO

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic peptide found in pancreatic islets of type-2 diabetes (T2D) patients. Under certain conditions, hIAPP is able to form amyloid fibrils that play a role in the progression of T2D. hIAPP is synthesized in the ß-cell of the pancreas and stored in the secretory granules before being released into the extracellular compartment. It has been suggested that natural stabilizing agents, such as insulin or zinc present in the secretory granules with hIAPP could prevent hIAPP fibril formation. The difference in the amino acid sequences of IAPP among species strongly correlates with amyloidogenicity and toxicity. The residue histidine at position 18 is known to be important in modulating the fibril formation, membrane leakage and toxicity. In this study, we have synthesized four analogues of hIAPP (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and characterized their aggregation with either insulin or zinc in order to determine the effect of the residue-18 on the insulin-IAPP and zinc-IAPP interactions using a variety of biophysical experiments including thioflavin-T fluorescence, transmission electron microscopy imaging, circular dichroism, and NMR spectroscopy. We show that insulin reduced hIAPP fibril formation both in solution and in the presence of membrane and hIAPP-membrane damage and that the interactions are somewhat mediated by the residue-18. In addition, our results reveal that zinc affects the process of hIAPP fibril formation in solution but not in the presence of membrane. Our results indicate that the nature of the residue-18 is important for zinc binding. Based on this observation, we hypothesize that zinc binds to the residues in the N-terminal region of hIAPP, which is not accessible in the presence of membrane due to its strong interaction with lipids.


Assuntos
Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Agregados Proteicos/fisiologia , Lipossomas Unilamelares/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Microscopia Eletrônica de Transmissão , Ligação Proteica , Espectrometria de Fluorescência , Lipossomas Unilamelares/química
16.
Chem Phys Lipids ; 237: 105085, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33895131

RESUMO

Currently, Alzheimer's Disease (AD) is a complex neurodegenerative condition, with limited therapeutic options. Several factors, like Amyloid ß (Aß) aggregation, tau protein hyperphosphorylation, bio-metals dyshomeostasis and oxidative stress contribute to AD pathogenesis. These pathogenic processes might occur in the aqueous phase but also on neuronal membranes. Thus, investigating the connection between Aß and biomembranes, becomes important for unveiling the molecular mechanism underlying Aß amyloidosis as a critical event in AD pathology. In this work, the interaction of two peptides, made up with hybrid sequences from Tau protein 9-16 (EVMEDHAG) or 26-33 (QGGYTMHQ) N-terminal domain and Aß16-20 (KLVFF) hydrophobic region, with full length Aß40 or Aß42 peptides is reported. The studied "chimera" peptides Ac-EVMEDHAGKLVFF-NH2 (τ9-16-KL) and Ac-QGGYTMHQKLVFF-NH2 (τ26-33-KL) are endowed with Aß recognition and metal ion interaction capabilities provided by the tau or Aß sequences, respectively. These peptides were characterized in previous study along with their metal dependent interaction and amyloidogenesis, either in the presence or absence of metal ion and artificial membranes made up with Total Lipid Brain Extract (TLBE) components, (Sciacca et al., 2020). In the present paper, the ability of the two peptides to inhibit Aß aggregation is studied using composite experimental conditions including aqueous solution, the presence of metal ions (Cu or Zn), the presence of lipid vesicles mimicking neuronal membranes as well as the co-presence of metals and TLBE artificial membranes. We used Thioflavine-T (ThT) fluorescence or MALDI-TOF spectrometry analysis of Aß limited proteolysis to respectively monitor the Aß aggregation kinetic or validation of the Aß interacting regions. We demonstrate that τ9-16-KL and τ26-33-KL peptides differently affect Aß aggregation kinetics, with the tau sequence playing a crucial role. The results are discussed in terms of chimera's peptides hydrophobicity and electrostatic driven interactions at the aqueous/membrane interface.


Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Fragmentos de Peptídeos/química , Peptídeos/química , Agregados Proteicos/fisiologia , Lipossomas Unilamelares/química , Zinco/química , Proteínas tau/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Humanos , Cinética , Peptídeos/metabolismo , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925935

RESUMO

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas Citotóxicas Formadoras de Poros/química , Agregados Proteicos/fisiologia , Sequência de Aminoácidos/genética , Proteínas de Anfíbios/química , Animais , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Secreções Corporais/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ranidae/metabolismo , Estresse Salino , Pele/metabolismo
18.
Bioorg Med Chem Lett ; 40: 127914, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33691165

RESUMO

Amyloids have long been associated with a variety of human degenerative diseases. Discoveries indicate, however, that there are several amyloids that serve functional roles in the human body. These amyloids are involved in a variety of biological processes ranging from storage of peptide hormones to necroptosis of cells. Additionally, there are distinct differences between toxic amyloids and their functional counterparts including kinetics of assembly/disassembly and structural features. This digest article surveys the biological roles of functional amyloids found in the human body, key differences between functional and toxic amyloids, and potential therapeutic applications.


Assuntos
Amiloide/química , Amiloide/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Doenças Neurodegenerativas/terapia , Animais , Hormônios/metabolismo , Corpo Humano , Humanos , Cinética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Peptídeos/química , Agregados Proteicos/fisiologia , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais
19.
Int J Biol Macromol ; 177: 40-47, 2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33607130

RESUMO

Materials based on ordered protein aggregates have recently received a lot of attention for their application as drug carriers, due to their biocompatibility and their ability to sequester many biological fluids. Bovine serum albumin (BSA) is a good candidate for this use due to its high availability and tendency to aggregate and gel under acidic conditions. In the present work, we employ spectroscopic techniques to investigate the heat-induced BSA aggregation at the molecular scale, in the 12-84 °C temperature range, at pH = 5 where two different isoforms of the protein are stable. Samples at low and high protein concentration are examined. With the advantage of the combined use of FTIR and CD, we recognize the aggregation-prone species and the different distribution of secondary structures, conformational rearrangements and types of aggregates, of millimolar compared to micromolar BSA solutions. Further, as a new tool, we use the Maximum Entropy Method to fit the kinetic curves to investigate the distribution of kinetic constants of the complex hierarchical aggregation process. Finally, we characterize the activation energy of the initial self-assembling step to observe that the formation of both small and large aggregates is driven by the same interactions.


Assuntos
Soroalbumina Bovina/química , Temperatura Alta , Ponto Isoelétrico , Cinética , Agregados Proteicos/fisiologia , Estrutura Secundária de Proteína/fisiologia , Análise Espectral/métodos
20.
Toxicology ; 453: 152736, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33631298

RESUMO

Cisplatin-induced ototoxicity is one of the important reasons that limit the drug's clinical application, and its mechanism has not been fully elucidated so far. The aim of this study was to explore the attenuate effect of tauroursodeoxycholic acid (TUDCA), a proteostasis promoter, on cisplatin-induced ototoxicity in vivo and in vitro, and to explore its possible mechanism. Auditory brainstem response (ABR) was measured to identify the attenuate effects of TUDCA administered subcutaneously [500 mg/kg/d × 3d, cisplatin: 4.6 mg/kg/d × 3d, intraperitoneal injection (i.p.)] or trans-tympanically (0.5 mg/mL, cisplatin: 12 mg/kg, i.p. with a pump) in Sprague-Dawley (SD) rats subjected to cisplatin-induced hearing loss. The cochlear explants of neonatal rats and OC1 auditory hair cell-like cell lines cultured in vitro were used to observe the number of apoptotic cells and the endoplasmic reticulum (ER) stress in the control, cisplatin (5 µM for 48 h for cochlear explants, 10 µM for 24 h for OC1 cells), and cisplatin + TUDCA (1 mM for 24 h for cochlear explants, 1.6 mM for 24 h for OC1 cells) groups. Differences in the expression of key proteins in the ER protein quality control (ERQC) system were detected. The changes in the attenuate effect of TUDCA on cisplatin-induced ototoxicity after down-regulating calreticulin (CRT), UDP-glucose ceramide glucosyltransferase-like 1 (UGGT1), and OS9 ER lectin (OS9) were also measured. The effect of TUDCA (10 mM) on stabilizing unfolded or misfolded proteins (UFP/MFP) was analyzed in a cell-free 0.2 % bovine serum albumin (BSA) aggregation system in vitro. Both the subcutaneous and trans-tympanic TUDCA administration alleviated cisplatin-induced increase in ABR thresholds in rats. TUDCA was able to reduce cisplatin-induced apoptosis and alleviate ER stress in cochlear explants and OC1 cells. Under the cisplatin treatment, the expression levels of CRT, UGGT1, and OS9 in the auditory hair cell increased, and the expression of total ubiquitinated proteins decreased. TUDCA attenuated the effect of cisplatin on UGGT1 and OS9, and recovered the protein ubiquitination levels. After down-regulating CRT, UGGT1, or OS9, the protective effect of TUDCA decreased. In the cell-free experimental system, TUDCA inhibited the aggregation of denatured BSA molecules. In summary, TUDCA can attenuate cisplatin-induced ototoxicity, possibly by inhibiting the accumulation and aggregation of UFP/MFP and the associated ER stress.


Assuntos
Cisplatino/toxicidade , Retículo Endoplasmático/efeitos dos fármacos , Ototoxicidade/prevenção & controle , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/uso terapêutico , Animais , Antineoplásicos/toxicidade , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/patologia , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Masculino , Ototoxicidade/patologia , Agregados Proteicos/fisiologia , Ratos , Ratos Sprague-Dawley , Ácido Tauroquenodesoxicólico/farmacologia
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