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1.
Chem Commun (Camb) ; 56(11): 1633-1636, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31939462

RESUMO

We utilized solution-phase biopanning to obtain a de novo peptide (LA12) that specifically bound to the core region of the human amylin monomer. LA12 stabilized the random coil conformation of amylin to suppress aggregation in a dose-dependent manner with the highest suppression effect of 78% and reduced the cytotoxicity of amylin.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Peptídeos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Animais , Bioprospecção/métodos , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Peptídeos/toxicidade , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Ratos
2.
Biochim Biophys Acta Biomembr ; 1862(2): 183108, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672548

RESUMO

Increasing lines of evidence show that the oligomeric intermediates of amyloid peptides/proteins are toxic to biological membranes. However, the structural features of the oligomers that are closely associated with the ability to damage biological membranes are far from understanding. In this study, we constructed two species of oligomers using hIAPP18-27 peptide and its d,l-alternating isomer, examined the disruptive ability of the oligomers to POPC/POPG 4:1 vesicles by leakage assay and 31P NMR spectroscopy, and characterized the structural features of the oligomers by CD, TEM, 1H NMR and fluorescence quenching experiments. We found that the d,l-alternating peptide oligomers are more disruptive than the all-L peptide oligomers to the lipid membrane. The characterization of the secondary structure revealed that the d,l-alternating peptide adopts an extended polyproline type-II (PPII) conformation, while the all-L peptide adopts a random coil conformation in oligomers. Compared with the all-L peptide oligomers, the d,l-alternating peptide oligomers are less compact and keep more hydrophobic groups water exposed. Both the changes from PPII to α-sheet in the structure of d,l-alternating peptide and from random coil to ß-sheet in the structure of all-L peptide reduce the ability of the peptide oligomers to disrupt the lipid membrane. Our results suggest that an oligomer with extended peptide chains could be more potent in membrane disruption than an oligomer with folded peptide chains and an increase in peptide-peptide interaction could decrease the disruptive ability of oligomer.


Assuntos
Membrana Celular/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Lipossomos/química , Fragmentos de Peptídeos/química , Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Isomerismo , Fragmentos de Peptídeos/farmacologia , Fosfolipídeos/química , Conformação Proteica em Folha beta , Multimerização Proteica
3.
J Nat Med ; 74(1): 247-251, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31267354

RESUMO

Alzheimer's diseases (AD) and type 2 diabetes (T2D) are two age-related diseases characterized by amyloid fibrillogenesis. Prevention of amyloid aggregation is a promising therapeutic strategy for AD and T2D. Two spermine alkaloids, kukoamines A and B, isolated from Lycii Cortex (LyC) were investigated for their inhibitory effect on amyloid aggregation. Both kukoamines A and B inhibited aggregation of amyloid ß (Aß) and human islet amyloid polypeptide (hIAPP) in a dose-dependent manner. Kukoamine B showed stronger inhibitory activity than kukoamine A. These results on the inhibitory activity of kukoamines A and B on Aß and hIAPP indicate that the catechol moiety is essential for inhibition of amyloid aggregation.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Ácidos Cafeicos/metabolismo , Diabetes Mellitus Tipo 2/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Espermina/análogos & derivados , Catecóis/química , Humanos , Espermina/metabolismo
4.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140283, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31526870

RESUMO

The abnormal aggregation of human islet amyloid polypeptide (hIAPP) is a crucial pathogenic factor associated with type 2 diabetes (T2D). The development of effective inhibitors to prevent hIAPP aggregation is a common therapeutic strategy against T2D. Lithospermic acid (LA) is a natural compound with diversified biological activities. In this study, electrospray ionization coupled with ion mobility-mass spectrometry, thioflavin T fluorescence assay, Congo red binding assay, Nile red fluorescence assay, circular dichroism spectroscopy, transmission electron microscopy, cell toxicity, lactate dehydrogenase assay (LDH) assay and molecular docking were combined to explore the influence of LA on hIAPP aggregation. Results showed that LA had favorable binding affinity to hIAPP and formed hIAPP-LA complexes, which could alter the relative abundance of the compact and extended conformers and promoted the transition of extended structures to compact conformers. LA also displayed strong inhibitory actions on fibrillation and potential protective effects against hIAPP-induced cell toxicity. Therefore, the obtained results were useful to understand the possible inhibitory mechanism of LA on hIAPP aggregation and provided valuable reference for the screening of potent amyloid inhibitors.


Assuntos
Amiloide/metabolismo , Benzofuranos/farmacologia , Depsídeos/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Simulação de Acoplamento Molecular , Conformação Proteica , Ratos
5.
BMC Gastroenterol ; 19(1): 228, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31883514

RESUMO

BACKGROUND: Compounds in clinical development for nonalcoholic steatohepatitis (NASH) improve liver histopathology in diet-induced obese mouse models of biopsy-confirmed NASH. Since the biopsy section used for histopathological evaluation represents only < 1% of the whole mouse liver, we evaluated how well biopsy-based quantitative image analyses correlate to stereology-based whole-liver quantitative changes upon drug treatment. METHODS: Male leptin-deficient Lepob/Lepob mice were fed the Amylin liver NASH (AMLN) diet for 16 weeks before stratification into treatment groups using a biopsy-based evaluation of type I collagen αI (col1a1) levels. Mice were treated for 8 weeks with either vehicle (PO, QD), liraglutide (0.4 mg/kg, SC, QD), elafibranor (30 mg/kg, PO, QD) or INT-767 (10 mg/kg, PO, QD). Terminal quantitative histological assessment of liver lipid (hematoxylin-eosin staining), inflammation (galectin-3 immunohistochemistry (IHC); gal-3), and fibrosis (col1a1 IHC) was performed on terminal liver biopsies and compared with stereologically sampled serial sections spanning the medial, left and right lateral lobe of the liver. RESULTS: The distribution of liver lipid and fibrosis was markedly consistent across lobes, whereas inflammation showed some variability. While INT-767 and liraglutide significantly reduced total liver weight by 20 and 48%, respectively, elafibranor tended to exacerbate hepatomegaly in Lepob/Lepob-NASH mice. All three compounds markedly reduced biopsy-based relative liver lipid content. Elafibranor and INT-767 significantly reduced biopsy-based relative gal-3 levels (P < 0.001), whereas INT-767 and liraglutide tended to reduce relative col1a1 levels. When changes in liver weight was accounted for, both INT-767 and liraglutide significantly reduced biopsy-based total col1a1 content. Although minor differences in absolute and relative liver lipid, inflammation and fibrosis levels were observed across lobes, the interpretation of drug-induced effects were consistent with biopsy-based conclusions. Notably, the incorporation of changes in total liver mass revealed that liraglutide's efficacy reached statistical significances for all analyzed parameters. CONCLUSIONS: In conclusion, in-depth analyses of liver homogeneity demonstrated that drug-induced improvement in liver biopsy-assessed histopathology is representative for overall liver effects assessed using stereology. Importantly, these findings reveal how changes in whole-liver mass should be considered to provide a deeper understanding of apparent drug treatment efficacy in preclinical NASH studies.


Assuntos
Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/complicações , Animais , Ácidos e Sais Biliares/uso terapêutico , Biópsia , Peso Corporal/efeitos dos fármacos , Chalconas/uso terapêutico , Colágeno Tipo I/análise , Dieta Hiperlipídica , Galectina 3/análise , Polipeptídeo Amiloide das Ilhotas Pancreáticas/administração & dosagem , Leptina/deficiência , Lipídeos/análise , Liraglutida/uso terapêutico , Fígado/química , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Tamanho do Órgão/efeitos dos fármacos , PPAR alfa/agonistas , PPAR delta/agonistas , Propionatos/uso terapêutico , Reprodutibilidade dos Testes
6.
Protein Eng Des Sel ; 32(2): 95-102, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31769491

RESUMO

Islet amyloid is a pathologic feature of type 2 diabetes (T2D) that is associated with ß-cell loss and dysfunction. These amyloid deposits form via aggregation of the ß-cell secretory product islet amyloid polypeptide (IAPP) and contain other molecules including the heparan sulfate proteoglycan perlecan. Perlecan has been shown to bind amyloidogenic human IAPP (hIAPP) via its heparan sulfate glycosaminoglycan (HS GAG) chains and to enhance hIAPP aggregation in vitro. We postulated that reducing the HS GAG content of perlecan would also decrease islet amyloid deposition in vivo. hIAPP transgenic mice were crossed with Hspg2Δ3/Δ3 mice harboring a perlecan mutation that prevents HS GAG attachment (hIAPP;Hspg2Δ3/Δ3), and male offspring from this cross were fed a high fat diet for 12 months to induce islet amyloid deposition. At the end of the study body weight, islet amyloid area, ß-cell area, glucose tolerance and insulin secretion were analyzed. hIAPP;Hspg2Δ3/Δ3 mice exhibited significantly less islet amyloid deposition and greater ß-cell area compared to hIAPP mice expressing wild type perlecan. hIAPP;Hspg2Δ3/Δ3 mice also gained significantly less weight than other genotypes. When adjusted for differences in body weight using multiple linear regression modeling, we found no differences in islet amyloid deposition or ß-cell area between hIAPP transgenic and hIAPP;Hspg2Δ3/Δ3 mice. We conclude that loss of perlecan exon 3 reduces islet amyloid deposition in vivo through indirect effects on body weight and possibly also through direct effects on hIAPP aggregation. Both of these mechanisms may promote maintenance of glucose homeostasis in the setting of T2D.


Assuntos
Peso Corporal , Proteoglicanas de Heparan Sulfato/deficiência , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Animais , Contagem de Células , Humanos , Camundongos , Camundongos Transgênicos
7.
Chem Commun (Camb) ; 55(95): 14359-14362, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31720593

RESUMO

Human islet amyloid polypeptide (hIAPP) oligomers are transient due to rapid aggregation rate in vitro, but play an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). Here we report an easy and robust method to generate toxic hIAPP oligomers, which are stable for at least 8 hours. The toxic hIAPP oligomers are quickly transformed from α-helix to ß-sheet by membrane phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, exhibiting distinct nanomechanical features from the hIAPP oligomers or pristine fibrils. DOPC liposomes significantly block the cytotoxicity induced by the hIAPP oligomers, which has the potential for new treatment.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Nanotecnologia , Fosfatidilcolinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Lipossomos/química , Lipossomos/farmacologia , Imagem Óptica , Fosfatidilcolinas/química
8.
Protein Eng Des Sel ; 32(2): 87-93, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31768548

RESUMO

The polypeptide amylin is responsible for islet amyloid in type 2 diabetes, a process which contributes to ß-cell death in the disease. The role of the N-terminal region of amylin in amyloid formation is relatively unexplored, although removal of the disulfide bridged loop between Cys-2 and Cys-7 accelerates amyloid formation. We examine the des Lys-1 variant of human amylin (h-amylin), a variant which is likely produced in vivo. Lys-1 is a region of high charge density in the h-amylin amyloid fiber. The des Lys-1 polypeptide forms amyloid on the same time scale as wild-type amylin in phosphate buffered saline, but does so more rapidly in Tris. The des Lys-1 variant is somewhat less toxic to cultured INS cells than wild type. The implications for the in vitro mechanism of amyloid formation and for comparative analysis of amyloidogenicity are discussed.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Agregados Proteicos , Sequência de Aminoácidos , Tampões (Química) , Linhagem Celular , Humanos , Cinética
9.
PLoS One ; 14(10): e0223456, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31600260

RESUMO

Expression of the Alzheimer's disease associated polypeptide Aß42 and the human polypeptide hormon islet amyloid polypeptide (hIAPP) and the prohormone precursor (hproIAPP) in neurons of Drosophila melanogaster leads to the formation of protein aggregates in the fat body tissue surrounding the brain. We determined the structure of these membrane-encircled protein aggregates using transmission electron microscopy (TEM) and observed the dissolution of protein aggregates after starvation. Electron tomography (ET) as an extension of transmission electron microscopy revealed that these aggregates were comprised of granular subunits having a diameter of 20 nm aligned into highly ordered structures in all three dimensions. The three dimensional (3D) lattice of hIAPP granules were constructed of two unit cells, a body centered tetragonal (BCT) and a triclinic unit cell. A 5-fold twinned structure was observed consisting of the cyclic twinning of the BCT and triclinic unit cells. The interaction between the two nearest hIAPP granules in both unit cells is not only governed by the van der Waals forces and the dipole-dipole interaction but potentially also by filament-like structures that can connect the nearest neighbors. Hence, our 3D structural analysis provides novel insight into the aggregation process of hIAPP in the fat body tissue of Drosophila melanogaster.


Assuntos
Drosophila melanogaster/metabolismo , Imageamento Tridimensional , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Animais , Cristalização , Drosophila melanogaster/ultraestrutura , Corpo Adiposo/metabolismo , Corpo Adiposo/ultraestrutura , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Agregados Proteicos , Subunidades Proteicas/química
10.
Phys Chem Chem Phys ; 21(36): 20239-20251, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31490518

RESUMO

The amyloid formation of human islet amyloid polypeptide (hIAPP)-an intrinsically disordered peptide, is associated with type II diabetes. Cellular membranes, especially those composed of negatively-charged lipids, accelerate the hIAPP amyloid fibrillation, and their integrity is disrupted during the aggregation process, leading to cell apoptosis. However, the underlying molecular mechanism is not well understood. Herein, we investigated the conformational dynamics during the interactions of hIAPP monomer with POPG membrane bilayer, by carrying out µs-long all-atom molecular dynamics simulations. Starting from the metastable coiled conformations in water, hIAPP monomers tend to adopt transient α-helical and ß-sheet structures when adsorbing to the membrane surface. The amphiphilic N-terminal region further inserts into the membrane interior and is located at the lipid head-tail interface, mainly in turn and α-helical structures. In contrast, the ß-hairpin structures reside on the membrane surface without insertion, and expand laterally with the hydrophobic residues exposed to the solvent. Moreover, the adsorption and insertion of hIAPP monomers induce two distinct local membrane deformations: (1) the hIAPP adsorption on the membrane surface mainly causes membrane bending; (2) the insertion of both turns and α-helices synchronizes with the formation of hydrophobic defects on the POPG membrane, leading to stronger membrane stretching and a longer coherence length of membrane thinning. Based on the structural and dynamical results, we propose that ß-hairpin structures may be a precursor for the fibrillation on the membrane surface due to the flat geometry and hydrophobic regions exposed to solvent, while N-terminal amphiphilic α-helices would facilitate hIAPP assembling into toxic oligomers inside the membrane.


Assuntos
Membrana Celular/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo
11.
Phys Chem Chem Phys ; 21(36): 20083-20094, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31482893

RESUMO

The formation of amyloid aggregates is the hallmark of many protein misfolding diseases, including Type-II diabetes mellitus, which is caused by the fibrillation of amylin protein. It is established that nano-sized ligands such as curcumin, resveratrol and graphene quantum dots can modify protein aggregation rates. In this article, we report a comparative study of these ligands to estimate their protein aggregation rates and fluorescence quenching using various experimental techniques. Through light scattering experiments, the RH of bare amylin was found to increase at a rate of 43% per day, whereas in the presence of the ligands in different molar ratios (A1C10, A1R10 and A1GQDs20), the sizes of the complexes were found to grow at rates of 7%, 8% and 13% per day, respectively. We observed fluorescence quenching using photoluminescence experiments for all three protein-ligand complexes. The protein aggregation rate and fluorescence quenching exhibited a concentration-dependent competitive role in the inhibition process. Interestingly, for graphene quantum dots, the protein aggregation rate is more affected at lower concentrations, while fluorescence quenching dominates at higher concentrations; this is in contrast to curcumin and resveratrol, where fluorescence quenching dominates at all concentrations of the ligands in the complex. The FTIR data showed appreciable conversion of ß-sheets into less aggregation-prone secondary structures for all three amylin-ligand ratios; however, the inhibition performance of curcumin overshadowed those of the other two inhibitors. The inhibition behavior of these three ligands was corroborated by analysis of analytical and high-resolution TEM images of the fibrils.


Assuntos
Modelos Químicos , Agregação Patológica de Proteínas , Fluorescência , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Microscopia Eletrônica de Transmissão
12.
J Mol Model ; 25(9): 263, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31428870

RESUMO

The USFDA has approved pramlintide, commercially named Symlin (sIAPP), as adjunctive therapy for type 2 diabetes (T2D). This analogue of the human amylin peptide (hIAPP) has triple proline substitutions typical of the rat isoform (rIAPP). Recently, it was proposed that pramlintide solubility and aggregation resistance might be improved by incorporating further mutations, as S20R, screened from the wild-type porcine isoform (pIAPP), which leads to the variant named sIAPP+. To better elucidate how such properties might be systematically induced in rationally designed analogues, we performed comparative assessments of rIAPP, sIAPP, and sIAPP+ using replica-exchange molecular dynamics (REMD) with an accurate combination of force field Charmm22* and explicit aqueous solvation TIP4P/Ew. Our thermo-structural analyses show that sIAPP exhibits a thermal conversion channel of helices[Formula: see text]-sheets resembling hIAPP. This channel is depleted in rIAPP and is absent in sIAPP+. As a consequence, sIAPP+ presents an overall decrease of ß-like secondary structures and an overstabilization of α-helices. Additionally, we observed in rIAPP and sIAPP+ an increase in the backbone RMSF of molecular terminals and the exposed area of key residues. These structural features of sIAPP+ suggest a nonamyloidogenic character, which is corroborated by our judicious estimate of the electrostatic component of the solvation free energy using a generalized Born model, and so it may constitute an alternative strategy to sIAPP as a peptide analogue of hIAPP. Furthermore, our findings confirm that different aggregation propensities of amylin and its analogues are synergistically modulated by long-range effects of key mutations. Graphical Abstract S20R-Pramlintide.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Simulação de Dinâmica Molecular , Mutação , Agregação Patológica de Proteínas , Animais , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Estrutura Secundária de Proteína , Ratos , Água/química
13.
PLoS One ; 14(8): e0219130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31404073

RESUMO

The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer's Aß peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aß also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aß amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aß amyloid formation and inhibit Aß induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured ß-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aß and h-amylin amyloid formation, illustrates the limitation of using Aß inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.


Assuntos
Agonistas dos Receptores da Amilina/farmacologia , Amiloide/antagonistas & inibidores , Bioensaio/normas , Desenho de Fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Corantes de Rosanilina/farmacologia , Compostos de Tritil/farmacologia , Amiloide/metabolismo , Naftalenossulfonato de Anilina/farmacologia , Benzotiazóis/farmacologia , Corantes Fluorescentes/farmacologia , Humanos , Indicadores e Reagentes/farmacologia
14.
Ann Neurol ; 86(4): 539-551, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31376172

RESUMO

OBJECTIVE: Alzheimer disease (AD) is the leading cause of dementia, and although its etiology remains unclear, it seems that type 2 diabetes mellitus (T2DM) and other prediabetic states of insulin resistance could contribute to the appearance of sporadic AD. As such, we have assessed whether tau and ß-amyloid (Aß) deposits might be present in pancreatic tissue of subjects with AD, and whether amylin, an amyloidogenic protein deposited in the pancreas of T2DM patients, might accumulate in the brain of AD patients. METHODS: We studied pancreatic and brain tissue from 48 individuals with no neuropathological alterations and from 87 subjects diagnosed with AD. We examined Aß and tau accumulation in the pancreas as well as that of amylin in the brain. Moreover, we performed proximity ligation assays to ascertain whether tau and/or Aß interact with amylin in either the pancreas or brain of these subjects. RESULTS: Cytoplasmic tau and Aß protein deposits were detected in pancreatic ß cells of subjects with AD as well as in subjects with a normal neuropathological examination but with a history of T2DM and in a small cohort of control subjects without T2DM. Furthermore, we found amylin deposits in the brain of these subjects, providing histological evidence that amylin can interact with Aß and tau in both the pancreas and hippocampus. INTERPRETATION: The presence of both tau and Aß inclusions in pancreatic ß cells, and of amylin deposits in the brain, provides new evidence of a potential overlap in the mechanisms underlying the pathogenesis of T2DM and AD. ANN NEUROL 2019;86:539-551.


Assuntos
Doença de Alzheimer/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pâncreas/metabolismo , Estudos Retrospectivos , Proteínas tau/metabolismo
15.
Nano Lett ; 19(9): 6535-6546, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31455083

RESUMO

Human amyloids and plaques uncovered post mortem are highly heterogeneous in structure and composition, yet literature concerning the heteroaggregation of amyloid proteins is extremely scarce. This knowledge deficiency is further exacerbated by the fact that peptide delivery is a major therapeutic strategy for targeting their full-length counterparts associated with the pathologies of a range of human diseases, including dementia and type 2 diabetes (T2D). Accordingly, here we examined the coaggregation of full-length human islet amyloid polypeptide (IAPP), a peptide associated with type 2 diabetes, with its primary and secondary amyloidogenic fragments 19-29 S20G and 8-20. Single-molecular aggregation dynamics was obtained by high-speed atomic force microscopy, augmented by transmission electron microscopy, X-ray diffraction, and super-resolution stimulated emission depletion microscopy. The coaggregation significantly prolonged the pause phase of fibril elongation, increasing its dwell time by 3-fold. Surprisingly, unidirectional elongation of mature fibrils, instead of protofilaments, was observed for the coaggregation, indicating a new form of tertiary protein aggregation unknown to existing theoretical models. Further in vivo zebrafish embryonic assay indicated improved survival and hatching, as well as decreased frequency and severity of developmental abnormalities for embryos treated with the heteroaggregates of IAPP with 19-29 S20G, but not with 8-20, compared to the control, indicating the therapeutic potential of 19-29 S20G against T2D.


Assuntos
Amiloidose/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Agregação Patológica de Proteínas/tratamento farmacológico , Amiloidose/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Agregação Patológica de Proteínas/metabolismo , Peixe-Zebra/metabolismo
16.
Biophys Chem ; 254: 106239, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31442763

RESUMO

The intrinsically disordered human islet amyloid polypeptide (hIAPP) is a 37 amino acid peptide hormone that is secreted by pancreatic beta cells along with glucagon and insulin. The glucose metabolism of humans is regulated by a balanced ratio of insulin and hIAPP. The disturbance of this balance can result in the development of type-2 diabetes mellitus (T2DM), whose pathogeny is associated by self-assembly induced aggregation and amyloid deposits of hIAPP into nanofibrils. Here, we report pressure- and temperature-induced changes of NMR chemical shifts of monomeric hIAPP in bulk solution to elucidate the contribution of conformational substates in a residue-specific manner in their role as molecular determinants for the initial self-assembly. The comparison with a similar peptide, the Alzheimer peptide Aß(1-40), which is leading to self-assembly induced aggregation and amyloid deposits as well, reveals that in both peptides highly homologous areas exist (Q10-|L16 and N21-L27 in hIAPP and Q15-A21 and S26-I32 in Aß). The N-terminal area of hIAPP around amino acid residues 3-20 displays large differences in pressure sensitivity compared to Aß, pinpointing to a different structural ensemble in this sequence element which is of helical origin in hIAPP. Knowledge of the structural nature of the highly amyloidogenic hIAPP and the differences with respect to the conformational ensemble of Aß(1-40) will help to identify molecular determinants of self-assembly as well as cross-seeded assembly initiated aggregation and help facilitate the rational design of drugs for therapeutic use.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Pressão , Conformação Proteica , Especificidade por Substrato , Temperatura
17.
J Phys Chem Lett ; 10(14): 3836-3842, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31246039

RESUMO

Immunosensors use antibodies to detect and quantify biomarkers of disease, though the sensors often lack structural information. We create a surface-sensitive two-dimensional infrared (2D IR) spectroscopic immunosensor for studying protein structures. We tether antibodies to a plasmonic surface, flow over a solution of amyloid proteins, and measure the 2D IR spectra. The 2D IR spectra provide a global assessment of antigen structure, and isotopically labeled proteins give residue-specific structural information. We report the 2D IR spectra of fibrils and monomers using a polyclonal antibody that targets human islet amyloid polypeptide (hIAPP). We observe two fibrillar polymorphs differing in their structure at the G24 residue, which supports the hypothesis that hIAPP polymorphs form from a common oligomeric intermediate. This work provides insight into the structure of hIAPP, establishes a new method for studying protein structures using 2D IR spectroscopy, and creates a spectroscopic immunoassay applicable for studying a wide range of biomarkers.


Assuntos
Amiloide/química , Técnicas Biossensoriais , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Humanos , Conformação Proteica , Espectrofotometria Infravermelho
18.
ACS Appl Mater Interfaces ; 11(26): 22973-22978, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31252497

RESUMO

Misfolding and abnormal assembly of proteins cause many intractable diseases. The modulation of the assembly process of these proteins could contribute to understanding and controlling amyloid protein aggregation. Previous works focused mainly on the inhibition of the assembly process. To broaden the interaction modality of modulators with proteins for developing new modulators, in this work, we designed and synthesized two reactive poly ( p-phenylene vinylene) polymers, respectively, functionalized with N-hydroxysuccinimide ester (PPV-NHS) and pentafluorophenol ester (PPV-PFP), which exhibited the prevention or co-assembly effect on the aggregation process of islet amyloid polypeptide (IAPP). Cell assays demonstrated that both of the two polymers could effectively eliminate the cytotoxicity of IAPP. Moreover, PPV-NHS also could irreversibly disrupt preformed IAPP fibrils. We envision that PPV-NHS and PPV-PFP might offer a new design method for the modulation of protein assembly.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polímeros/química , Agregação Patológica de Proteínas/tratamento farmacológico , Sequência de Aminoácidos/genética , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , Fluorbenzenos/síntese química , Fluorbenzenos/química , Fluorbenzenos/farmacologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Fenóis/síntese química , Fenóis/química , Fenóis/farmacologia , Polímeros/síntese química , Polímeros/farmacologia , Agregação Patológica de Proteínas/genética , Succinimidas/síntese química , Succinimidas/química , Succinimidas/farmacologia
19.
PLoS One ; 14(6): e0218561, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31206565

RESUMO

The biologically active pancreatic hormone peptide islet amyloid polypeptide (IAPP) regulates brain functions such as appetite and cognition. It also plays a role in clearance of amyloid beta (Aß), a peptide implicated in the dementia disorder Alzheimer's disease (AD). If IAPP becomes modified, it loses its biological activity and starts to aggregate. Such aggregations have been found in the AD brain and decreased plasma levels of the unmodified IAPP (uIAPP) have been shown in the same patients. In the current study, we analyze levels of uIAPP and total IAPP (unmodified and modified) in cerebrospinal fluid (CSF) to investigate its potential as a biomarker for AD. We found no differences in neither CSF nor plasma levels of uIAPP or total IAPP in AD patients compared to cognitive healthy individuals (NC). The levels of uIAPP in CSF of NC were positively correlated with uIAPP in plasma, Q-albumin and albumin levels in CSF, but negatively correlated with CSF levels of t-tau and p-tau. These findings were not seen in AD patients. Levels of total CSF IAPP correlated positively with total Q-albumin and albumin levels in CSF in both AD and NC. In addition, total plasma IAPP correlated positively with CSF t-tau and p-tau in NC and negatively with CSF Aß42 in AD patients. To conclude, our studies did not find evidence supporting the use of CSF IAPP as an AD biomarker. However, our findings, indicating a compromised translocation of uIAPP in and out of the brain in AD patients as well as the correlations between total plasma IAPP and AD biomarkers, encourage further research on the role for IAPP in AD.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/sangue , Polipeptídeo Amiloide das Ilhotas Pancreáticas/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas tau/líquido cefalorraquidiano
20.
Nat Commun ; 10(1): 2679, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213603

RESUMO

The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by ß-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, ß-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, ß-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of ß-cell function.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Estresse do Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Adulto , Animais , Animais Geneticamente Modificados , Apoptose , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Glicólise/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Masculino , Pessoa de Meia-Idade , Mitofagia/fisiologia , Fosforilação Oxidativa , Fosfofrutoquinase-2/metabolismo , Agregados Proteicos/fisiologia , Ratos
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