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The structural dynamics of the interactions between defensins or lysozymes and various saccharide chains that are covalently linked to lipids or proteins were analyzed in relation to the sub-molecular architecture of the carbohydrate binding sites of lectins. Using tissue materials from rare and endangered domestic animals as well as from dogs it was possible to compare these results with data obtained from a human glioblastoma tissue. The binding mechanisms were analyzed on a cellular and a sub-molecular size level using biophysical techniques (e.g. NMR, AFM, MS) which are supported by molecular modeling tools. This leads to characteristic structural patterns being helpful to understand glyco-biochemical pathways in which galectins, defensins or lysozymes are involved. Carbohydrate chains have a distinct impact on cell differentiation, cell migration and immunological processes. The absence or the presence of sialic acids and the conformational dynamics in glycans are often correlated with zoonoses such as influenza- and coronavirus-infections. Receptor-sensitive glycomimetics could be a solution. The new findings concerning the function of galectin-3 in the nucleus in relation to differentiation processes can be understood when the binding specificity of neuroleptic molecules as well as the interactions between proteins and nucleic acids are describable on a sub-molecular size level.
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Short peptide-based supramolecular hydrogels hold enormous potential for a wide range of applications. However, the gelation of these systems is very challenging to control. Minor changes in the peptide sequence can significantly influence the self-assembly mechanism and thereby the gelation propensity. The involvement of SARS CoV E protein in the assembly and release of the virus suggests that it may have inherent self-assembling properties that can contribute to the development of hydrogels. Here, three pentapeptide sequences derived from C-terminal of SARS CoV E protein are explored with same amino acid residues but different sequence distributions and discovered a drastic difference in the gelation propensity. By combining spectroscopic and microscopic techniques, the relationship between peptide sequence arrangement and molecular assembly structure are demonstrated, and how these influence the mechanical properties of the hydrogel. The present study expands the variety of secondary structures for generating supramolecular hydrogels by introducing the 310-helix as the primary building block for gelation, facilitated by a water-mediated structural transition into ß-sheet conformation. Moreover, these Fmoc-modified pentapeptide hydrogels/supramolecular assemblies with tunable morphology and mechanical properties are suitable for tissue engineering, injectable delivery, and 3D bio-printing applications.
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Amyloid proteins and peptides play a pivotal role in the etiology of various neurodegenerative diseases, including Alzheimer's disease (AD). Synthetically designed small molecules/ peptides/ peptidomimetics show promise towards inhibition of various kinds of amyloidosis. However, exploration of compounds isolated from natural extracts having such potential is lacking. Herein, we have investigated the repurposing of a traditional Indian medicine Lasunadya Ghrita (LG) in AD. LG is traditionally used to treat gut dysregulation and mental illnesses. Various extracts of LG were obtained, characterized, and analyzed for inhibition of Aß aggregation. Biophysical studies show that the water extract of LG (LGWE) is more potent in inhibiting Aß peptide aggregation and defibrillation of Aß40/Aß42 aggregates. NMR studies showed that LGWE binds to the central hydrophobic area and C-terminal residues of Aß40/Aß42, thereby modulating the aggregation, and reducing cell membrane damage. Additionally, LGWE rescues Aß toxicity in neuronal SH-SY5Y cells evident from decreases in ROS generation, membrane leakage, cellular apoptosis, and calcium dyshomeostasis. Notably, LGWE is non-toxic to neuronal cells and mouse models. Our study thus delves into the mechanistic insights of a repurposed drug LGWE with the potential to ameliorate Aß induced neuroinflammation.
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Peptídeos beta-Amiloides , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/antagonistas & inibidores , Humanos , Animais , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Agregados Proteicos/efeitos dos fármacosRESUMO
Phase-separated protein accumulation through the formation of several aggregate species is linked to the pathology of several human disorders and diseases. Our current investigation envisaged detailed Raman signature and structural intricacy of bovine insulin in its various forms of aggregates produced in situ at an elevated temperature (60 °C). The amide I band in the Raman spectrum of the protein in its native-like conformation appeared at 1655 cm-1 and indicated the presence of a high content of α-helical structure as prepared freshly in acidic pH. The disorder content (turn and coils) also was predominately present in both the monomeric and oligomeric states and was confirmed by the presence shoulder amide I maker band at â¼1680 cm-1. However, the band shifted to â¼1671 cm-1 upon the transformation of the protein solution into fibrillar aggregates as produced for a longer time of incubation. The protein, however, maintained most of its helical conformation in the oligomeric phase; the low-frequency backbone α-helical conformation signal at â¼935 cm-1 was similar to that of freshly prepared aqueous protein solution enriched in helical conformation. The peak intensity was significantly weak in the fibrillar aggregates, and it appeared as a good Raman signature to follow the phase separation and the aggregation behavior of insulin and similar other proteins. Tyrosine phenoxy moieties in the protein may maintained its H-bond donor-acceptor integrity throughout the course of fibril formation; however, it entered in more hydrophobic environment in its journey of fibril formation. In addition, it was noticed that oligomeric bovine insulin maintained the orientation/conformation of the disulfide bonds. However, in the fibrillar state, the disulfide linkages became more strained and preferred to maintain a single conformation state.
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Fungal keratitis is the foremost cause of corneal infections worldwide, of which Fusariumspp. is the common etiological agent that causes loss of vision and warrants surgical intervention. An increase in resistance to the available drugs along with severe side effects of the existing antifungals demands for new effective antimycotics. Here, we demonstrate that antimicrobial peptide S100A12 directly binds to the phospholipids of the fungal membrane, disrupts the structural integrity, and induces generation of reactive oxygen species in fungus. In addition, it inhibits biofilm formation by Fusariumspp. and exhibits antifungal property against Fusariumspp. both in vitro and in vivo. Taken together, our results delve into specific effect of S100A12 against Fusariumspp. with an aim to investigate new antifungal compounds to combat fungal keratitis.
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Antifúngicos , Biofilmes , Membrana Celular , Fusarium , Proteína S100A12 , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Infecções Oculares Fúngicas/microbiologia , Fusarium/efeitos dos fármacos , Ceratite/microbiologia , Proteína S100A12/metabolismo , Proteína S100A12/farmacologia , Humanos , Membrana Celular/efeitos dos fármacos , Fosfolipídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The pursuit of a novel structural motif that can shed light on the key functional attributes is a primary focus in the study of protein folding disorders. Decades of research on Alzheimer's disease (AD) have centered on the Amyloid ß (Aß) pathway, highlighting its significance in understanding the disorder. The diversity in the Aß pathway and the possible silent tracks which are yet to discover, makes it exceedingly intimidating to the interdisciplinary scientific community. Over the course of AD research, Aß has consistently been at the forefront of scientific inquiry and discussion. In this review, we epitomize the role of a potential structural motif (GxxxG motif) that may provide a new horizon to the Aß conflict. We emphasize on how comprehensive understanding of this motif from a structure-function perspective may pave the way for designing novel therapeutics intervention in AD and related diseases.
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The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.
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Proteínas 14-3-3 , 3',5'-AMP Cíclico Fosfodiesterases , Proteínas Quinases Dependentes de AMP Cíclico , Sistema de Sinalização das MAP Quinases , Humanos , Proteínas 14-3-3/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Fosfosserina/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismoRESUMO
Amyloidogenesis, with its multifaceted nature spanning from peptide self-assembly to membrane-mediated structural transitions, presents a significant challenge for the interdisciplinary scientific community. Here, we emphasize on how Singular Value Decomposition (SVD) can be employed to reveal hidden patterns and dominant modes of interaction that govern the complex process of amyloidogenesis. We first utilize SVD analysis on Circular Dichroism (CD) spectral datasets to identify the intermediate structural species emerging during peptide-membrane interactions and to determine binding constants more precisely than conventional methods. We investigate the monomer loss kinetics associated with peptide self-assembly using Nuclear Magnetic Resonance (NMR) dataset and determine the global kinetic parameters through SVD. Furthermore, we explore the seeded growth of amyloid fibrils by analyzing a time-dependent NMR dataset, shedding light on the kinetic intricacies of this process. Our analysis uncovers two distinct states in the aggregation of Aß40 and pinpoints key residues responsible for this seeded growth. To strengthen our findings and enhance their robustness, we validate those using simulated data, thereby highlighting the physical interpretations derived from SVD. Overall, SVD analysis offers a model-free, global kinetic perspective, enabling the selection of optimal kinetic models. This study not only contributes valuable insights into the dynamics but also highlights the versatility of SVD in unravelling complex processes of amyloidogenesis.
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Amiloide , Peptídeos , Amiloide/química , Dicroísmo Circular , CinéticaRESUMO
Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the α-helical native human insulin conformers, disrupting their aggregation. Furthermore, it also increases the insulin secretion. This multipolar effect coupled with its non-toxic nature could be useful for developing highly bioactive, targeted and biostable therapeutic insulin.
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Insulinas , Corantes de Rosanilina , Humanos , Chaperonas MolecularesRESUMO
The global outbreak of SARS-CoV-2/COVID-19 provided the stage to accumulate an enormous biomedical data set and an opportunity as well as a challenge to test new concepts and strategies to combat the pandemic. New research and molecular medical protocols may be deployed in different scientific fields, e.g., glycobiology, nanopharmacology, or nanomedicine. We correlated clinical biomedical data derived from patients in intensive care units with structural biology and biophysical data from NMR and/or CAMM (computer-aided molecular modeling). Consequently, new diagnostic and therapeutic approaches against SARS-CoV-2 were evaluated. Specifically, we tested the suitability of incretin mimetics with one or two pH-sensitive amino acid residues as potential drugs to prevent or cure long-COVID symptoms. Blood pH values in correlation with temperature alterations in patient bodies were of clinical importance. The effects of biophysical parameters such as temperature and pH value variation in relation to physical-chemical membrane properties (e.g., glycosylation state, affinity of certain amino acid sequences to sialic acids as well as other carbohydrate residues and lipid structures) provided helpful hints in identifying a potential Achilles heel against long COVID. In silico CAMM methods and in vitro NMR experiments (including 31P NMR measurements) were applied to analyze the structural behavior of incretin mimetics and SARS-CoV fusion peptides interacting with dodecylphosphocholine (DPC) micelles. These supramolecular complexes were analyzed under physiological conditions by 1H and 31P NMR techniques. We were able to observe characteristic interaction states of incretin mimetics, SARS-CoV fusion peptides and DPC membranes. Novel interaction profiles (indicated, e.g., by 31P NMR signal splitting) were detected. Furthermore, we evaluated GM1 gangliosides and sialic acid-coated silica nanoparticles in complex with DPC micelles in order to create a simple virus host cell membrane model. This is a first step in exploring the structure-function relationship between the SARS-CoV-2 spike protein and incretin mimetics with conserved pH-sensitive histidine residues in their carbohydrate recognition domains as found in galectins. The applied methods were effective in identifying peptide sequences as well as certain carbohydrate moieties with the potential to protect the blood-brain barrier (BBB). These clinically relevant observations on low blood pH values in fatal COVID-19 cases open routes for new therapeutic approaches, especially against long-COVID symptoms.
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Aggregation of the human islets amyloid polypeptide, or hIAPP, is linked to ß-cell death in type II diabetes mellitus (T2DM). Different pancreatic ß-cell environmental variables such as pH, insulin and metal ions play a key role in controlling the hIAPP aggregation. Since insulin and hIAPP are co-secreted, it is known from numerous studies that insulin suppresses hIAPP fibrillation by preventing the initial dimerization process. On the other hand, zinc and copper each have an inhibitory impact on hIAPP fibrillation, but copper promotes the production of toxic oligomers. Interestingly, the insulin oligomeric equilibrium is controlled by the concentration of zinc ions when the effect of insulin and zinc has been tested together. Lower zinc concentrations cause the equilibrium to shift towards the monomer and dimer states of insulin, which bind to monomeric hIAPP and stop it from developing into a fibril. On the other hand, the combined effects of copper and insulin have not yet been studied. In this study, we have demonstrated how the presence of copper affects hIAPP aggregation and the toxicity of the resultant conformers with or without insulin. For this purpose, we have used a set of biophysical techniques, including NMR, fluorescence, CD etc., in combination with AFM and cell cytotoxicity assay. In the presence and/or absence of insulin, copper induces hIAPP to form structurally distinct stable toxic oligomers, deterring the fibrillation process. More specifically, the oligomers generated in the presence of insulin have slightly higher toxicity than those formed in the absence of insulin. This research will increase our understanding of the combined modulatory effect of two ß-cell environmental factors on hIAPP aggregation.
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Diabetes Mellitus Tipo 2 , Insulina , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cobre/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Zinco/farmacologia , Zinco/química , Amiloide/químicaRESUMO
Antimicrobial peptides (AMPs) with cell membrane lysing capability are considered potential candidates for the development of the next generation of antibiotics. Designing novel AMPs requires an in-depth understanding of the mechanism of action of the peptides. In this work, we used various biophysical techniques including 31P solid-state NMR to examine the interaction of model membranes with amphipathic de novo-designed peptides. Two such peptides, MSI-78 and VG16KRKP, were designed with different hydrophobicity and positive charges. The model lipid membranes were constituted by mixing lipids of varying degrees of 'area per lipid' (APL), which directly affected the packing properties of the membrane. The observed emergence of the isotropic peak in 31P NMR spectra as a function of time is a consequence of the fragmentation of the membrane mediated by the peptide interaction. The factors such as the charges, overall hydrophilicity of the AMPs, as well as lipid membrane packing, contributed to the kinetics of membrane fragmentation. Furthermore, we anticipate the designed AMPs follow the carpet and toroidal pore mechanisms when lysing the cell membrane. This study highlights the significance of the effect of the overall charges and the hydrophobicity of the novel AMPs designed for antimicrobial activity.
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Peptídeos Catiônicos Antimicrobianos , Peptídeos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Lipídeos , Bicamadas Lipídicas/químicaRESUMO
Aß (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα-H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix. However, conflicting data are reported in the literature. Some authors have suggested that hydrogen bonding does not have a stabilizing function. Here we synthesized pentapeptides having a GXXXG motif to explore its role in pore stability. We used molecular dynamics simulations, quantum mechanics, and experimental biophysical techniques to determine whether hydrogen bonding was formed and had a stabilizing function in ion-channel-like structures. Starting from our previous molecular dynamics data, molecular quantum mechanics simulations, and ATR data showed that a stable ion-channel-like pore formed and a band centered at 2910 cm-1 was attributed to the interaction between Gly 7 of an alpha-helix and Asp 23 of a vicinal alpha-helix.
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Aminoácidos , Canais Iônicos , Glicina/química , Ligação de Hidrogênio , Conformação Molecular , Simulação de Dinâmica Molecular , Peptídeos beta-Amiloides/químicaRESUMO
Targeting amyloidosis requires high-resolution insight into the underlying mechanisms of amyloid aggregation. The sequence-specific intrinsic properties of a peptide or protein largely govern the amyloidogenic propensity. Thus, it is essential to delineate the structural motifs that define the subsequent downstream amyloidogenic cascade of events. Additionally, it is important to understand the role played by extrinsic factors, such as temperature or sample agitation, in modulating the overall energy barrier that prompts divergent nucleation events. Consequently, these changes can affect the fibrillation kinetics, resulting in structurally and functionally distinct amyloidogenic conformers associated with disease pathogenesis. Here, we have focused on human Islet Polypeptide (hIAPP) amyloidogenesis for the full-length peptide along with its N- and C-terminal fragments, under different temperatures and sample agitation conditions. This helped us to gain a comprehensive understanding of the intrinsic role of specific functional epitopes in the primary structure of the peptide that regulates amyloidogenesis and subsequent cytotoxicity. Intriguingly, our study involving an array of biophysical experiments and ex vivo data suggests a direct influence of external changes on the C-terminal fibrillating sequence. Furthermore, the observations indicate a possible collaborative role of this segment in nucleating hIAPP amyloidogenesis in a physiological scenario, thus making it a potential target for future therapeutic interventions.
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Amiloidose , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Amiloide/química , Proteínas Amiloidogênicas , Epitopos , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/químicaRESUMO
Antimicrobial peptides (AMPs) have been a topic of substantial research as the next-generation antibiotics. They have been extensively studied for the selectivity and action against microbial membrane lipids in imparting their targeted functioning. To determine the effectivity of the peptides against the Gram-negative pathogens, it is imperative to elucidate their role in interacting with the lipopolysaccharide moieties. Lipopolysaccharide is a major component of the outer membrane of the Gram-negative bacteria. It serves to protect the bacteria as well as govern the functionality of several antibacterial agents. It can prevent the access of the agents into the inner membrane of the bacteria, thus rendering them inactive. Several techniques have been employed to study the interaction for better designing of peptides; NMR spectroscopy is one of the most widely used techniques in determining the interactive properties of peptides with LPS as it can provide the details in atomistic level. NMR spectroscopy provides information about the structural and conformational changes as well as the dynamics of the interactions.
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Peptídeos Catiônicos Antimicrobianos , Lipopolissacarídeos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos , Membrana Celular/química , Lipopolissacarídeos/química , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/análise , Testes de Sensibilidade MicrobianaRESUMO
The aggregation of proteins into ordered fibrillar structures called amyloids, and their disintegration represent major unsolved problems that limit the therapeutic applications of several proteins. For example, insulin, commonly used for the treatment of diabetes, is susceptible to amyloid formation upon exposure to non-physiological conditions, resulting in a loss of its biological activity. Here, we report a novel amphiphilic molecule called PAD-S, which acts as a chemical chaperone and completely inhibits fibrillation of insulin and its biosimilars. Mechanistic investigations and molecular docking lead to the conclusion that PAD-S binds to key hydrophobic regions of native insulin, thereby preventing its self-assembly. PAD-S treated insulin was biologically active as indicated by its ability to phosphorylate Akt, a protein in the insulin signalling pathway. PAD-S is non-toxic and protects cells from insulin amyloid induced cytotoxicity. The high aqueous solubility and easy synthetic accessibility of PAD-S facilitates its potential use in commercial insulin formulations. Notably, PAD-S successfully disintegrated preformed insulin fibrils to non-toxic smaller fragments. Since the structural and mechanistic features of amyloids are common to several human pathologies, the understanding of the amyloid disaggregation activity of PAD-S will inform the development of small molecule disaggregators for other amyloids.
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Medicamentos Biossimilares , Insulina , Amiloide/química , Proteínas Amiloidogênicas , Humanos , Insulina/química , Simulação de Acoplamento MolecularRESUMO
The treatment of invasive drug-resistant and potentially life-threatening fungal infections is limited to few therapeutic options that are usually associated with severe side effects. The development of new effective antimycotics with a more tolerable side effect profile is therefore of utmost clinical importance. Here, we used a combination of complementary in vitro assays and structural analytical methods to analyze the interaction of the de novo antimicrobial peptide VG16KRKP with the sterol moieties of biological cell membranes. We demonstrate that VG16KRKP disturbs the structural integrity of fungal membranes both invitro and in model membrane system containing ergosterol along with phosphatidylethanolamine lipid and exhibits broad-spectrum antifungal activity. As revealed by systematic structure-function analysis of mutated VG16KRKP analogs, a specific pattern of basic and hydrophobic amino acid side chains in the primary peptide sequence determines the selectivity of VG16KRKP for fungal specific membranes.
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Antifúngicos , Ergosterol , Antifúngicos/química , Antifúngicos/farmacologia , Membrana Celular/metabolismo , Ergosterol/química , Peptídeos/química , Peptídeos/farmacologia , Esteróis/metabolismoRESUMO
Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.