RESUMO
Human islet amyloid polypeptide (amylin or hIAPP) is a 37 residue hormone co-secreted with insulin from ß cells of the pancreas. In patients suffering from type-2 diabetes, amylin self-assembles into amyloid fibrils, ultimately leading to the death of the pancreatic cells. However, a research gap exists in preventing and treating such amyloidosis. Plumbagin, a natural compound, has previously been demonstrated to have inhibitory potential against insulin amyloidosis. Our investigation unveils collapsible regions within hIAPP that, upon collapse, facilitates hydrophobic and pi-pi interactions, ultimately leading to aggregation. Intriguingly plumbagin exhibits the ability to bind these specific collapsible regions, thereby impeding the aforementioned interactions that would otherwise drive hIAPP aggregation. We have used atomistic molecular dynamics approach to determine secondary structural changes. MSM shows metastable states forming native like hIAPP structure in presence of PGN. Our in silico results concur with in vitro results. The ThT assay revealed a striking 50% decrease in fluorescence intensity at a 1:1 ratio of hIAPP to Plumbagin. This finding suggests a significant inhibition of amyloid fibril formation by plumbagin, as ThT fluorescence directly correlates with the presence of these fibrils. Further TEM images revealed disappearance of hIAPP fibrils in plumbagin pre-treated hIAPP samples. Also, we have shown that plumbagin disrupts the intermolecular hydrogen bonding in hIAPP fibrils leading to an increase in the average beta strand spacing, thereby causing disaggregation of pre-formed fibrils demonstrating overall disruption of the aggregation machinery of hIAPP. Our work is the first to report a detailed atomistic simulation of 22 µs for hIAPP. Overall, our studies put plumbagin as a potential candidate for both preventive and therapeutic candidate for hIAPP amyloidosis.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas , Simulação de Dinâmica Molecular , Naftoquinonas , Naftoquinonas/química , Naftoquinonas/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Humanos , Cadeias de Markov , Ligação Proteica , Interações Hidrofóbicas e Hidrofílicas , Agregados Proteicos/efeitos dos fármacos , Ligação de HidrogênioRESUMO
Polyphenols has attained pronounced attention due to their beneficial values of health and found to prevent several chronic diseases. Here, we elucidated binding mechanism between frequently consumed polyphenol "tea catechin" and milk protein bovine beta-lactoglobulin (ß-Lg). We investigated the conformational changes of ß-Lg due to interaction with catechin using spectroscopic and in silico studies. Fluorescence quenching data (Stern-Volmer quenching constant) revealed that ß-Lg interacted with catechin via dynamic quenching. Thermodynamic data revealed that the interaction between ß-Lg and catechin is endothermic and spontaneously interacted mainly through hydrophobic interactions. The UV-Vis absorption and far-UV circular dichroism (CD) spectroscopy exhibited that the tertiary as well as secondary structure of ß-Lg distorted after interaction with catechin. Molecular docking and simulation studies also confirm that catechin binds at the central cavity of ß-Lg with high affinity (~105 M-1) and hydrophobic interactions play significant role in the formation of a stable ß-Lg-catechin complex.
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Dengue and chikungunya fevers are transmitted by the common mosquito vector Aedes and malaria by Anopheles. Concurrent infections are reported due to co-circulation of these pathogens, especially in endemic regions. We report a rare case of triple infection with 3 arthropod-borne pathogens (Plasmodium vivax and the dengue and chikungunya viruses) in a 3-year-old child from New Delhi, India, in August 2016. The viruses were identified by RT-PCR and the parasite by microscopy and antigen detection. The dengue virus serotype 3 sequence was clustered in the genotype III by the phylogenetic analysis. Mixed infection with multiple pathogens is a challenge for accurate diagnosis due to the overlapping clinical symptoms. The accurate and timely diagnosis of multiple pathogens in such cases is important for rapid and effective patient management.
Assuntos
Febre de Chikungunya/complicações , Coinfecção , Dengue/complicações , Malária Vivax/complicações , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Vírus Chikungunya/isolamento & purificação , Pré-Escolar , Dengue/diagnóstico , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/isolamento & purificação , Diagnóstico Diferencial , Humanos , Índia , Malária Vivax/diagnóstico , Malária Vivax/parasitologia , Masculino , Filogenia , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , SorogrupoRESUMO
Numerous pathophysiological conditions known as amyloidosis, have been connected to protein misfolding leading to aggregation of proteins. Inhibition of cytotoxic aggregates or disaggregation of the preformed fibrils is thus one of the important strategies in the prevention of such diseases. Growing interest and exploration of identification of small molecules mainly natural compounds can prevent or delay amyloid fibril formation. We examined the mechanism of interaction and inhibition of human lysozyme (HL) aggregates with luteolin (LT). Biophysical and computational approaches have been employed to study the effect of LT on HL amyloid aggregation. Transmission Electronic Microscopy, Thioflavin T fluorescence, UV-vis spectroscopy, and RLS demonstrates that LT inhibit HL fibril formation. ANS fluorescence and hemolytic assay was also employed to examine the effect of the LT on toxicity of HL aggregation. Docking and molecular dynamics results showed that LT interacted with HL via hydrophobic and hydrogen interactions, thus reducing fibrillation levels. These findings highlight the benefit of polyphenols as safe therapy for preventing amyloid related diseases.
Assuntos
Amiloidose , Luteolina , Humanos , Luteolina/farmacologia , Muramidase/química , Amiloide/química , Proteínas AmiloidogênicasRESUMO
The increasing frequency of Dengue is a cause of severe epidemics and therefore demands strategies for effective prevention, diagnosis, and treatment. DENV-protease is being investigated as a potential therapeutic target. However, due to the flat and highly charged active site of the DENV-protease, designing orthosteric medicines is very difficult. In this study, we have done a thorough analysis of pH-dependent conformational changes in recombinantly expressed DENV protease using various spectroscopic techniques. Our spectroscopic study of DENV protease (NS2B-NS3pro) at different pH conditions gives important insights into the dynamicity of structural conformation. At physiological pH, the DENV-protease exists in a random-coiled state. Lowering the pH promotes the formation of alpha-helical and beta-sheet structures i.e. gain of secondary structure as shown by Far-UV CD. The light scattering and Thioflavin T (ThT)-binding assay proved the aggregation-prone tendency of DENV-protease at pH 4.0. Further, the confocal microscopy image intensity showed the amorphous aggregate formation of DENV protease at pH 4.0. Thus, the DENV protease acquires different conformations with changes in pH conditions. Together, these results have the potential to facilitate the design of a conformation destabilizer-based therapeutic strategy for dengue fever.
Assuntos
Vírus da Dengue , Serina Endopeptidases , Serina Endopeptidases/química , Proteínas não Estruturais Virais/química , Domínio Catalítico , Concentração de Íons de Hidrogênio , Inibidores de Proteases/farmacologiaRESUMO
Protein misfolding and related formation of amyloid fibrils are associated with several conformational diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases, and Diabetes mellitus, Type 2 (DM-II). Several molecules including antibiotics, polyphenols, flavonoids, anthraquinones, and other small molecules are implicated to modulate amyloid assembly. The stabilization of the native forms of the polypeptides and prevention of their misfolding and aggregation are of clinical and biotechnological importance. Among the natural flavonoids, luteolin is of great importance because of its therapeutic role against neuroinflammation. Herein, we have explored the inhibitory effect of luteolin (LUT) on aggregation of a model protein, human insulin (HI). To understand the molecular mechanism of the inhibition of aggregation of HI by LUT, we employed molecular simulation, UV-Vis, fluorescence, and circular dichroism (CD) spectroscopies along with the dynamic light scattering (DLS). The analysis of the tuning of the HI aggregation process by luteolin revealed that interaction of HI with LUT resulted in the decrease in binding of the various fluorescent dyes, such as thioflavin T (ThT) and 8-anilinonaphthalene-1-sulfonic acid (ANS) to this protein. Retention of the native-like CD spectra and resistance to the aggregation in the presence of LUT has confirmed the aggregation inhibitory potential of LUT. The maximum inhibitory effect was found at the protein-to-drug ratio of 1:12, and no significant change was observed beyond this concentration.
Assuntos
Proteínas Amiloidogênicas , Luteolina , Humanos , Amiloide/química , Insulina/química , PeptídeosRESUMO
Geminiviruses consist of a single-stranded DNA genome that replicates by a rolling circle (RCR) and recombination-dependent (RDR) modes of replication. The AC1 or Rep is the indispensable viral protein required for the RCR mode of replication. Since these viruses encode only a few proteins, they depend on several host factors for replication, transcription, and other physiological processes. To get insights into the repertoire of host factors influencing the replication of geminiviruses, we performed phage display experiments which led to the identification of putative mungbean yellow mosaic India virus (MYMIV) Rep interacting host proteins. These proteins might directly or indirectly participate in geminivirus biology. MCM3 was one of the Rep-interacting partners obtained in the phage display results. Using bimolecular fluorescence complementation (BiFC), the interaction of the MYMIV Rep with Arabidopsis thaliana MCM3 (AtMCM3) was confirmed. We report the involvement of AtMCM3 in the replication of MYMIV DNA through an ex vivo system. The physiological relevance of the interaction between AtMCM3 and MYMIV Rep is reflected by yeast replication assay.Communicated by Ramaswamy H. Sarma.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Geminiviridae , Componente 3 do Complexo de Manutenção de Minicromossomo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Viral/genética , DNA Viral/metabolismo , Geminiviridae/fisiologia , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Replicação Viral , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
Since the first appearance of a novel coronavirus pneumonia (NCP) caused by a novel human coronavirus, and especially after the infection started its rapid spread over the world causing the COVID-19 (coronavirus disease 2019) pandemics, a very substantial part of the scientific community is engaged in the intensive research dedicated to finding of the potential therapeutics to cure this disease. As repurposing of existing drugs represents the only instant solution for those infected with the virus, we have been working on utilization of the structure-based virtual screening method to find some potential medications. In this study, we screened a library of 646 FDA approved drugs against the receptor-binding domain of the SARS-CoV-2 spike (S) protein and the main protease of this virus. Scoring functions revealed that some of the anticancer drugs (such as Pazopanib, Irinotecan, and Imatinib), antipsychotic drug (Risperidone), and antiviral drug (Raltegravir) have a potential to interact with both targets with high efficiency. Further we performed molecular dynamics simulations to understand the evolution in protein upon interaction with drug. Also, we have performed a phylogenetic analysis of 43 different coronavirus strains infecting 12 different mammalian species.Communicated by Ramaswamy H. Sarma.
Assuntos
COVID-19 , Animais , Humanos , SARS-CoV-2 , Filogenia , Reposicionamento de Medicamentos/métodos , Simulação de Acoplamento Molecular , Antivirais/farmacologia , Antivirais/química , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , MamíferosRESUMO
The re-emergence of Chikungunya virus (CHIKV) infection in humans with no approved antiviral therapies or vaccines is one of the major problems with global significance. In the present investigation, we screened 80 in-house quinoline derivatives for their anti-CHIKV activity by computational techniques and found 4-hydroxy-1-methyl-3-(3-morpholinopropanoyl)quinoline-2(1H)-one (QVIR) to have potential binding affinities with CHIKV nsP2 and E2 glycoproteins. QVIR was evaluated in vitro for its anti-CHIKV potential. QVIR showed strong inhibition of CHIKV infection with an EC50 (50% effective concentration) value of 2.2 ± 0.49 µM without significant cytotoxicity (CC50 > 200 µM) and was chosen for further elucidation of its antiviral mechanism. The infectious viral particle formation was abolished by approximately 72% at a QVIR concentration of 20 µM during infection in the BHK-21 cell line, and the CHIKV RNA synthesis was diminished by 84% for nsP2 as well as 74% for E2, whereas the levels of viral proteins were decreased by 69.9% for nsP2 and 53.9% for E2. Flow cytometry analysis confirmed a huge decline in the expression of viral nsP2 and E2 proteins by 71.84 and 67.7%, respectively. Time of addition experiments indicated that QVIR inhibited viral infection at early and late stages of viral replication cycle, and the optimal inhibition was observed at 16 h post infection. The present study advocates for the first time that QVIR acts as a substantial and potent inhibitor against CHIKV and might be as an auspicious novel drug candidate for the development of therapeutic agents against CHIKV infections.
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Cataract is one of the major causes of blindness worldwide. Several factors including post-translational modification, thermal and solar radiations promote cataractogenesis. The camel lens proteins survive very harsh desert conditions and resist cataractogenesis. The folding and aggregation mechanism of camel lens proteins are poorly characterized. The camel lens contains three ubiquitous crystallins (α-, ß-, and γ-crystallin) and a novel protein (ζ-crystallin) in large amounts. In this study, a sequence similarity search of camel α-crystallin with that of other organisms showed that the camel αB-crystallin consists of an extended N-terminal domain. Our results indicate that camel α-crystallin efficiently prevented aggregation of ζ-crystallin, with or without an obligate cofactor up to 89 °C. It performed a quick and efficient holdase function irrespective of the unfolding stage or aggregation. Camel α-crystallin exhibits approximately 20% chaperone activity between 30 and 40 °C and is completely activated above 40 °C. Camel α-crystallin underwent a single reversible thermal transition without loss of ß-sheet secondary structure. Intrinsic tryptophan fluorescence and ANS binding experiments revealed two transitions which corresponded to activation of its chaperone function. In contrast to earlier studies, camel α-crystallin completely protected lens proteins during thermal stress.
Assuntos
Estresse Fisiológico , Temperatura , alfa-Cristalinas/química , zeta-Cristalinas/química , Animais , Camelus , Catarata , Fluorometria/métodos , Insulina/química , Cinética , Cristalino , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Filogenia , Agregados Proteicos , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes , Análise Espectral , alfa-Cristalinas/isolamento & purificação , zeta-Cristalinas/isolamento & purificaçãoRESUMO
Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host-pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host-pathogen interaction.
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BACKGROUND: Dengue is a rapidly emerging arthropod borne viral infection affecting tropical and sub-tropical regions of the world. Dengue is an acute febrile illness but sometimes causes more fatal complications like dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Delhi, the capital of India has become hyper endemic for dengue virus because all the four serotypes are circulating here. METHODS: The present study describes the identification of dengue virus from clinical samples collected from the suspected dengue patients from New Delhi, India during 2016. The CprM region of Dengue virus genome was analyzed for phylogenetic, selection pressure and Shannon entropy analyses. RESULTS: The present study reports circulation of a single serotype (DENV-3) in New Delhi, during 2016. The phylogenetic analysis revealed that Indian subcontinent (genotype III) of DENV-3 was circulating in Delhi during this period. Neutral selection pressure in the analyzed region revealed relatively conserved nature of this part of the Dengue virus genome. Amino acid at 31 was positively selected and had high entropy value suggesting probability of variation at this position. CONCLUSIONS: The changing trend in circulation of dengue virus serotypes necessitates the continuous epidemiological surveillance for the dengue outbreaks in this region.
Assuntos
Vírus da Dengue/genética , Dengue/epidemiologia , Filogenia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise por Conglomerados , Dengue/sangue , Vírus da Dengue/classificação , Surtos de Doenças , Entropia , Feminino , Variação Genética , Genoma Viral , Genótipo , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , RNA Viral/genética , Seleção Genética , Sorogrupo , Adulto JovemRESUMO
Arthropod-borne infections like malaria, dengue and chikungunya fever are transmitted by mosquitoes. The present report describes an unusual case of mixed infection with malaria, dengue and chikungunya viruses in a 21 year old male patient from New Delhi, India during monsoon season of 2016. The malarial fever was diagnosed by thin slide microscopy and antigen test. Chikungunya virus IgM was detected in the sample by the card test. Dengue and chikungunya viruses were further confirmed by RT-PCR for CprM and E1 gene respectively. Phylogenetic analysis clustered the study dengue virus serotype 3 sequence in the genotype III. Thus, the mono infections can not be differentiated from concurrent infections on the basis of clinical symptoms, the appropriate laboratory diagnosis is essential for the accurate pathogen confirmation. Precise and appropriate identification of the multiple pathogens in such clinical cases will assist in the effective management of these arthropod mediated infections.