RESUMO
Aggregation of the human islet amyloid polypeptide (hIAPP) contributes to the development and progression of Type 2 Diabetes (T2D). hIAPP aggregates within a few hours at few micromolar concentration in vitro but exists at millimolar concentrations in vivo. Natively occurring inhibitors of hIAPP aggregation might therefore provide a model for drug design against amyloid formation associated with T2D. Here, we describe the combined ability of low pH, zinc, and insulin to inhibit hIAPP fibrillation. Insulin dose-dependently slows hIAPP aggregation near neutral pH but had less effect on the aggregation kinetics at acidic pH. We determine that insulin alters hIAPP aggregation in two manners. First, insulin diverts the aggregation pathway to large nonfibrillar aggregates with ThT-positive molecular structure, rather than to amyloid fibrils. Second, soluble insulin suppresses hIAPP dimer formation, which is an important early aggregation event. Further, we observe that zinc significantly modulates the inhibition of hIAPP aggregation by insulin. We hypothesize that this effect arose from controlling the oligomeric state of insulin and show that hIAPP interacts more strongly with monomeric than oligomeric insulin.
Assuntos
Insulina , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Agregados Proteicos , Zinco , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Concentração de Íons de Hidrogênio , Humanos , Zinco/farmacologia , Zinco/metabolismo , Zinco/química , Insulina/metabolismo , Agregados Proteicos/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Cinética , Amiloide/metabolismo , Amiloide/química , Agregação Patológica de Proteínas/metabolismoRESUMO
Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention.
RESUMO
The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining atomic force microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains only in the fluid phase of membranes and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.
Assuntos
Bicamadas Lipídicas , Microscopia de Força Atômica , Staphylococcus aureus , Microscopia de Força Atômica/métodos , Bicamadas Lipídicas/química , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Amiloide/química , Amiloide/toxicidade , Interações Hidrofóbicas e HidrofílicasRESUMO
The aggregation of human islet amyloid polypeptide (hIAPP) is linked to the death of pancreatic ß-cells in type II diabetes. The process of fibril formation by hIAPP is thought to cause membrane damage, but the precise mechanisms are still unclear. Previously, we showed that the aggregation of hIAPP in the presence of membranes containing anionic lipids is dominated by secondary nucleation events, which occur at the interface between existing fibrils and the membrane surface. Here, we used vesicles with different lipid composition to explore the connection between hIAPP aggregation and vesicle leakage. We found that different anionic lipids promote hIAPP aggregation to the same extent, whereas remarkably stochastic behaviour is observed on purely zwitterionic membranes. Vesicle leakage induced by hIAPP consists of two distinct phases for any of the used membrane compositions: (i) an initial phase in which hIAPP binding causes a certain level of leakage that is strongly dependent on osmotic conditions, membrane composition and the used dye, and (ii) a main leakage event that we attribute to elongation of hIAPP fibrils, based on seeded experiments. Altogether, our results shed more light on the relationship between hIAPP fibril formation and membrane damage, and strongly suggest that oligomeric intermediates do not considerably contribute to vesicle leakage.
RESUMO
The synthetic peptide ERα17p (sequence: PLMIKRSKKNSLALSLT), which corresponds to the 295-311 region of the human estrogen receptor α (ERα), induces apoptosis in breast cancer cells. In mice and at low doses, it promotes not only the decrease of the size of xenografted triple-negative human breast tumors, but also anti-inflammatory and anti-nociceptive effects. Recently, we have shown that these effects were due to its interaction with the seven-transmembrane G protein-coupled estrogen receptor GPER. Following modeling studies, the C-terminus of this peptide (sequence: NSLALSLT) remains compacted at the entrance of the GPER ligand-binding pocket, whereas its N-terminus (sequence: PLMI) engulfs in the depth of the same pocket. Thus, we have hypothesized that the PLMI motif could support the pharmacological actions of ERα17p. Here, we show that the PLMI peptide is, indeed, responsible for the GPER-dependent antiproliferative and anti-nociceptive effects of ERα17p. By using different biophysical approaches, we demonstrate that the NSLALSLT part of ERα17p is responsible for aggregation. Overall, the tetrapeptide PLMI, which supports the action of the parent peptide ERα17p, should be considered as a hit for the synthesis of new GPER modulators with dual antiproliferative and anti-nociceptive actions. This study highlights also the interest to modulate GPER for the control of pain.
Assuntos
Receptor alfa de Estrogênio , Neoplasias de Mama Triplo Negativas , Animais , Humanos , Camundongos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios , Peptídeos , Receptores Acoplados a Proteínas G , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Homeoprotein transcription factors have the property of interacting with membranes through their DNA-binding homeodomain, which is involved in unconventional internalization and secretion. Both processes depend on membrane-translocating events but their detailed molecular mechanisms are still poorly understood. We have previously characterized the conformational properties of Engrailed 2 homeodomain (EnHD) in aqueous solution and in micelles as membrane-mimetic environments. In the present study, we used small isotropic lipid bicelles as a more relevant membrane-mimetic model to characterize the membrane-bound state of EnHD. We show that lipid bicelles, in contrast to micelles, adequately reproduce the requirement of anionic lipids in the membrane binding and conformational transition of EnHD. The fold-unfold transition of EnHD induced by anionic lipids was characterized by NMR using 1H, 13C, 15N chemical shifts, nuclear Overhauser effects, residual dipolar couplings, intramolecular and intermolecular paramagnetic relaxation enhancements induced by site-directed spin-label or paramagnetic lipid probe, respectively. A global unpacking of EnHD helices is observed leading to a loss of the native fold. However, near-native propensities of EnHD backbone conformation are maintained in membrane environment, including not only the three helices but also the turn connecting helices H2 and H3. NMR and coarse-grained molecular dynamics simulations reveal that the EnHD adopts a shallow insertion in the membrane, with the three helices oriented parallel to the membrane. EnHD explores extended conformations and closed U-shaped conformations, which are stabilized by anionic lipid recruitment.
Assuntos
Micelas , Simulação de Dinâmica Molecular , Proteínas de Homeodomínio/química , Lipídeos , Estrutura Secundária de ProteínaRESUMO
Loss of pancreatic ß-cell mass is deleterious for type 2 diabetes patients since it reduces insulin production, critical for glucose homeostasis. The main research axis developed over the last few years was to generate new pancreatic ß-cells or to transplant pancreatic islets as occurring for some specific type 1 diabetes patients. We evaluate here a new paradigm consisting in preservation of ß-cells by prevention of human islet amyloid polypeptide (hIAPP) oligomers and fibrils formation leading to pancreatic ß-cell death. We review the hIAPP physiology and the pathology that contributes to ß-cell destruction, deciphering the various cellular steps that could be involved. Recent progress in understanding other amyloidosis such as Aß, Tau, α-synuclein or prion, involved in neurodegenerative processes linked with inflammation, has opened new research lines of investigations to preserve neuronal cells. We evaluate and estimate their transposition to the pancreatic ß-cells preservation. Among them is the control of reactive oxygen species (ROS) production occurring with inflammation and the possible implication of the mitochondrial translocator protein as a diagnostic and therapeutic target. The present review also focuses on other amyloid forming proteins from molecular to physiological and physiopathological points of view that could help to better decipher hIAPP-induced ß-cell death mechanisms and to prevent hIAPP fibril formation.
Assuntos
Diabetes Mellitus Tipo 2 , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Amiloide/química , Morte Celular , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Inflamação , Polipeptídeo Amiloide das Ilhotas Pancreáticas/químicaRESUMO
Type II diabetes is characterized by the loss of pancreatic ß-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, that is, the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent ß-cell death in type II diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Amiloide/química , Catálise , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cinética , LipídeosRESUMO
The islet amyloid polypeptide (IAPP) is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The aggregation of IAPP is known to cause cell death, where the cell membrane plays a dual role: being a catalyst of IAPP aggregation and being the target of IAPP toxicity. Using ATR-FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Distinct IAPP-membrane interaction modes for the various IAPP variants are revealed. Membrane binding causes IAPP to fold into an amphipathic α-helix, which in the case of H18K-, and H18R-IAPP readily moves beyond the headgroup region. For all IAPP variants but H18E-IAPP, the membrane-bound helix is an intermediate on the way to amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel ß-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel ß-sheets as well as amorphous aggregates. Our results emphasize the decisive role of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good therapeutic target for destabilizing membrane-bound IAPP fibrils to inhibit their toxic actions.
RESUMO
Amyloid diseases are degenerative pathologies, highly prevalent today because they are closely related to aging, that have in common the erroneous folding of intrinsically disordered proteins (IDPs) which aggregate and lead to cell death. Type 2 Diabetes involves a peptide called human islet amyloid polypeptide (hIAPP), which undergoes a conformational change, triggering the aggregation process leading to amyloid aggregates and fibers rich in ß-sheets mainly found in the pancreas of all diabetic patients. Inhibiting the aggregation of amyloid proteins has emerged as a relevant therapeutic approach and we have recently developed the design of acyclic flexible hairpins based on peptidic recognition sequences of the amyloid ß peptide (Aß1-42) as a successful strategy to inhibit its aggregation involved in Alzheimer's disease. The present work reports the extension of our strategy to hIAPP aggregation inhibitors. The design, synthesis, conformational analyses, and biophysical evaluations of dynamic ß-hairpin like structures built on a piperidine-pyrrolidine ß-turn inducer are described. By linking to this ß-turn inducer three different arms (i) pentapeptide, (ii) tripeptide, and (iii) α/aza/aza/pseudotripeptide, we demonstrate that the careful selection of the peptide-based arms from the sequence of hIAPP allowed to selectively modulate its aggregation, while the peptide character can be decreased. Biophysical assays combining, Thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis, and mass spectrometry showed that the designed compounds inhibit both the oligomerization and the fibrillization of hIAPP. They are also capable to decrease the aggregation process in the presence of membrane models and to strongly delay the membrane-leakage induced by hIAPP. More generally, this work provides the proof of concept that our rational design is a versatile and relevant strategy for developing efficient and selective inhibitors of aggregation of amyloidogenic proteins.
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/químicaRESUMO
The peptide ERα17p, which corresponds to the 295-311 fragment of the hinge/AF2 domains of the human estrogen receptor α (ERα), exerts apoptosis in breast cancer cells through a mechanism involving the G protein-coupled estrogen-dependent receptor GPER. Besides this receptor-mediated mechanism, we have detected a direct interaction (Kd value in the micromolar range) of this peptide with lipid vesicles mimicking the plasma membrane of eukaryotes. The reversible and not reversible pools of interacting peptide may correspond to soluble and aggregated membrane-interacting peptide populations, respectively. By using circular dichroism (CD) spectroscopy, we have shown that the interaction of the peptide with this membrane model was associated with its folding into ß sheet. A slight leakage of the 5(6)-fluorescein was also observed, indicating lipid bilayer permeability. When the peptide was incubated with living breast cancer cells at the active concentration of 10 µM, aggregates were detected at the plasma membrane under the form of spheres. This insoluble pool of peptide, which seems to result from a fibrillation process, is internalized in micrometric vacuoles under the form of fibrils, without evidence of cytotoxicity, at least at the microscopic level. This study provides new information on the interaction of ERα17p with breast cancer cell membranes as well as on its mechanism of action, with respect to direct membrane effects.
Assuntos
Neoplasias da Mama/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Fenômenos Biofísicos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Bicamadas Lipídicas/química , Células MCF-7 , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic ß cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic ß-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death.
Assuntos
Amiloide/química , Morte Celular , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Neoplasias Pancreáticas/metabolismo , Vesículas Secretórias/metabolismo , Sequência de Aminoácidos , Agonistas dos Receptores da Amilina/farmacologia , Amiloide/efeitos dos fármacos , Animais , Células Cultivadas , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/tratamento farmacológico , Insulinoma/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Ratos , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/patologiaRESUMO
Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Multimerização Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Cinética , Agregados Proteicos , Ratos , TermodinâmicaRESUMO
Type 2 diabetes mellitus and Alzheimer's disease are characterized by the accumulation of fibrillar amyloid deposits consisting mainly of islet amyloid polypeptide (IAPP) and amyloid-ß (Aß), respectively. Fibril formation is a multi-step nucleation process that involves the transient build-up of oligomeric species that are thought to be the most toxic components. To gain more insight into the molecular mechanism of early IAPP aggregated species formation, we performed a combination of direct and indirect biophysical approaches on IAPP and also on Aß42 for the sake of comparison. Thioflavin T fluorescence kinetics measurements revealed a stronger autocatalytic behaviour of IAPP and a weaker concentration dependence of fibrillization half-time t1/2, as compared to Aß42. Our NMR experiments highlight the absence of micelle reservoir or supercritical regime in the studied concentration range, indicating that the low concentration dependence of IAPP fibril formation can be ascribed to saturable pathways. IAPP and Aß42 displayed marked differences in formation of oligomeric species, as observed by 1D 1H, pulsed-field gradient (PFG) diffusion and saturation transfer difference (STD) NMR experiments. A fast equilibrium between monomer and oligomeric species was detected in the case of Aß42 but not IAPP, with a significant build-up of aggregated species, as shown by the time dependence of diffusion coefficient and STD magnetization transfer efficiency during the aggregation process. Altogether our data show significant differences between IAPP and Aß42 regarding the microscopic events of amyloid species formation.
Assuntos
Peptídeos beta-Amiloides/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Agregados Proteicos/fisiologia , Sequência de Aminoácidos , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
Amyloid fibril formation has been implicated in a wide range of human diseases and the interactions of amyloidogenic proteins with cell membranes are considered to be important in the aetiology of these pathologies. In type 2 diabetes mellitus (T2DM), the human islet amyloid polypeptide (hIAPP) forms amyloid fibrils which impair the functionality and viability of pancreatic ß cells. The mechanisms of hIAPP cytotoxicity are linked to the ability of the peptide to self-aggregate and to interact with membranes. Previous studies have shown that the N-terminal part of hIAPP from residues 1 to 19 is the membrane binding domain. The non-amyloidogenic and nontoxic mouse IAPP differs from hIAPP by six residues out of 37, among which a single one, residue 18, lies in the membrane binding region. To gain more insight into hIAPP-membrane interactions we herein performed comprehensive biophysical studies on four analogues (H18R-IAPP, H18K-IAPP, H18E-IAPP and H18A-IAPP). Our data reveal that all peptides are able to insert efficiently in the membrane, indicating that residue 18 is not essential for hIAPP membrane binding and insertion. However, only wild-type hIAPP and H18K-IAPP are able to form fibrils at the membrane. Importantly, all peptides induce membrane damage; wild-type hIAPP and H18K-IAPP presumably cause membrane disruption mainly by fibril growth at the membrane, while for H18R-IAPP, H18E-IAPP and H18A-IAPP, membrane leakage is most likely due to high molecular weight oligomeric species. These results highlight the importance of the residue at position 18 in IAPP for modulating fibril formation at the membrane and the mechanisms of membrane leakage.
RESUMO
Type 2 diabetes mellitus is characterized histopathologically by the presence of fibrillary amyloid deposits in the pancreatic islets of Langerhans. Human islet amyloid polypeptide (hIAPP), the 37-residue pancreatic hormone, is the major constituent of these amyloid deposits. The propensity of IAPP to form amyloid fibrils is strongly dependent on its primary sequence. An intriguing example is His at residue 18. Although H18 is located outside the amyloidogenic region, it has been suggested that this residue and its charge state play an important role in the kinetics of conformational changes and fibril formation as well as in mediating cell toxicity. To gain more insight into the importance of this residue, we have synthesized four analogues (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and we performed a full biophysical study on the properties of these peptides. Kinetic experiments as monitored by thioflavin-T fluorescence, transmission electron microscopy, circular dichroism and cell toxicity assays revealed that all variants are less fibrillogenic and less toxic than native hIAPP both at neutral pH and at low pH. This demonstrates that the effect of H18 in native IAPP is not simply determined by its charge state, but rather that residue 18 is important for specific intra- and intermolecular interactions that occur during fibril formation and that may involve charge, size and hydrophobicity. Furthermore, our results indicate that H18R-IAPP has a strong inhibiting effect on native hIAPP fibril formation. Together these results highlight the large impact of modifying a single residue outside the amyloidogenic domain on fibril formation and cell toxicity induced by IAPP, opening up new avenues for design of inhibitors or modulators of IAPP aggregation.
Assuntos
Amiloide/química , Amiloide/toxicidade , Ilhotas Pancreáticas/metabolismo , Agregados Proteicos , Sequência de Aminoácidos , Amiloide/genética , Animais , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Mutação , RatosRESUMO
How anti-Alzheimer's drug candidates that reduce amyloid 1-42 peptide fibrillization interact with the most neurotoxic species is far from being understood. We report herein the capacity of sugar-based peptidomimetics to inhibit both Aß1-42 early oligomerization and fibrillization. A wide range of bio- and physicochemical techniques, such as a new capillary electrophoresis method, nuclear magnetic resonance, and surface plasmon resonance, were used to identify how these new molecules can delay the aggregation of Aß1-42. We demonstrate that these molecules interact with soluble oligomers in order to maintain the presence of nontoxic monomers and to prevent fibrillization. These compounds totally suppress the toxicity of Aß1-42 toward SH-SY5Y neuroblastoma cells, even at substoichiometric concentrations. Furthermore, demonstration that the best molecule combines hydrophobic moieties, hydrogen bond donors and acceptors, ammonium groups, and a hydrophilic ß-sheet breaker element provides valuable insight for the future structure-based design of inhibitors of Aß1-42 aggregation.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Desenho de Fármacos , Glicopeptídeos/farmacologia , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptidomiméticos , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glicopeptídeos/síntese química , Glicopeptídeos/química , Humanos , Estrutura Molecular , Neuroblastoma/patologia , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
Human islet amyloid polypeptide (hIAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Mature hIAPP, a 37-aa peptide, is natively unfolded in its monomeric state but forms islet amyloid in T2DM. In common with other misfolded and aggregated proteins, amyloid formation involves aggregation of monomers of hIAPP into oligomers, fibrils, and ultimately mature amyloid deposits. hIAPP is coproduced and stored with insulin by the pancreatic islet ß-cells and is released in response to the stimuli that lead to insulin secretion. Accumulating evidence suggests that hIAPP amyloid deposits that accompany T2DM are not just an insignificant phenomenon derived from the disease progression but that hIAPP aggregation induces processes that impair the functionality and the viability of ß-cells. In this review, we particularly focus on hIAPP structure, hIAPP aggregation, and hIAPP-membrane interactions. We will also discuss recent findings on the mechanism of hIAPP-membrane damage and on hIAPP-induced cell death. Finally, the development of successful antiamyloidogenic agents that prevent hIAPP fibril formation will be examined.