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1.
J Phys Chem B ; 125(40): 11099-11111, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34473498

RESUMO

Loss of function and aggregation of the neuronal protein α-Synuclein (A-Syn) underlies the pathogenesis of Parkinson's disease (PD), and both the function and aggregation of this protein happen to be mediated via its binding to the synaptic vesicles (SVs) at the presynaptic termini. An essential constituent of SV membranes is cholesterol, with which A-Syn directly interacts while binding to membranes. Thus, cholesterol content in SV membranes is likely to affect the binding of A-Syn to these vesicles and consequently its functional and pathogenic behaviors. Interestingly, the dyshomeostasis of cholesterol has often been associated with PD, with reports linking both high and low cholesterol levels to an increased risk of neurodegeneration. Herein, using SV-mimicking liposomes containing increasing percentages of membrane cholesterol, we show (with mathematical interpretation) that the binding of A-Syn to synaptic-like vesicles is strongest in the presence of an optimum cholesterol content, which correlates to its maximum function and minimum aggregation. This implicates a minimum risk of neurodegeneration at optimum cholesterol levels and rationalizes the existing controversial relationship between cholesterol levels and PD. Increased membrane cholesterol was, however, found to protect against damage caused by aggregated A-Syn, complementing previous reports and portraying one advantage of high cholesterol over low.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Colesterol , Humanos , Neurônios , Vesículas Sinápticas
2.
J Phys Chem B ; 125(33): 9402-9416, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34384214

RESUMO

ß-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent Ki,Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.


Assuntos
Agrobacterium tumefaciens , beta-Glucosidase , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Sítios de Ligação , Glucose , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , Especificidade por Substrato
3.
RSC Chem Biol ; 2(2): 592-605, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-34458802

RESUMO

Understanding of prion aggregation in a membrane environment may help to ameliorate neurodegenerative complications caused by the amyloid forms of prions. Here, we investigated the membrane binding-induced aggregation of yeast prion protein Sup35. Using the combination of fluorescence correlation spectroscopy (FCS) at single molecule resolution and other biophysical studies, we establish that lipid composition and lipid/protein ratio are key modulators of the aggregation kinetics of Sup35. In the presence of a zwitterionic membrane (DMPC), Sup35 exhibited novel biphasic aggregation kinetics at lipid/protein ratios ranging between 20 : 1 and 70 : 1 (termed here as the optimum lipid concentration, OLC). In ratios below (low lipid concentration, LLC) and above (ELC, excess lipid concentration) that range, the aggregation was found to be monophasic. In contrast, in the presence of negatively charged membranes, we did not observe any bi-phasic aggregation kinetics in the entire range of protein to lipid ratios. Our results provide a mechanistic description of the role that membrane concentration/composition-modulated aggregation may play in neurodegenerative diseases.

4.
Commun Biol ; 4(1): 518, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941845

RESUMO

The fibrillation pathway of alpha-Synuclein, the causative protein of Parkinson's disease, encompasses transient, heterogeneous oligomeric forms whose structural understanding and link to toxicity are not yet understood. We report that the addition of the physiologically-available small molecule heme at a sub-stoichiometric ratio to either monomeric or aggregated α-Syn, targets a His50 residue critical for fibril-formation and stabilizes the structurally-heterogeneous populations of aggregates into a minimally-toxic oligomeric state. Cryo-EM 3D reconstruction revealed a 'mace'-shaped structure of this monodisperse population of oligomers, which is comparable to a solid-state NMR Greek key-like motif (where the core residues are arranged in parallel in-register sheets with a Greek key topology at the C terminus) that forms the fundamental unit/kernel of protofilaments. Further structural analyses suggest that heme binding induces a distortion in the Greek key-like architecture of the mace oligomers, which impairs their further appending into protofilaments and fibrils. Additionally, our study reports a novel mechanism of prevention as well as reclamation of amyloid fibril formation by blocking an inter-protofilament His50 residue using a small molecule.


Assuntos
Amiloide/química , Heme/metabolismo , Neuroblastoma/patologia , Multimerização Proteica , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Heme/química , Humanos , Neuroblastoma/metabolismo , Conformação Proteica , Células Tumorais Cultivadas
5.
Elife ; 102021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33825682

RESUMO

Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, amyotrophic lateral sclerosis (ALS). Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid-induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association-mediated toxic aggregation and survival time scale after ALS diagnosis.


Assuntos
Esclerose Amiotrófica Lateral/enzimologia , Membrana Celular/enzimologia , Neurônios/enzimologia , Superóxido Dismutase-1/metabolismo , Zinco/metabolismo , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/patologia , Cobre/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Neurônios/patologia , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Superóxido Dismutase-1/genética
7.
ACS Chem Neurosci ; 11(20): 3442-3454, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33044818

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disorder, the pathogenesis of which is closely linked to the misfolding and aggregation of the neuronal protein α-Synuclein (A-Syn). Numerous molecules that inhibit/modulate the pathogenic aggregation of A-Syn in an effort to tackle PD pathogenesis have been reported, but none so far have been successful in treating the disease at the clinic. One major reason for this is the poor blood-brain barrier (BBB) permeability of most of the molecules being used. Therefore, using BBB-permeable (and biocompatible) nanomaterials as fibrillation modulators is gaining importance. In the present work, we show how nontoxic and ultrasmall gold nanoclusters (AuNCs) can systematically modulate the pathogenic fibrillation of A-Syn in vitro, based on the chemical nature of their capping agents, using two reported easily synthesizable AuNCs as models. In addition, we detect the BBB permeability in mice of one of these AuNCs solely by making use of its intrinsic fluorescence. Thus, our work exemplifies how AuNCs can be potential therapeutics against PD; while also acting as fluorescent probes for their own BBB permeability.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Barreira Hematoencefálica/metabolismo , Ouro , Camundongos , Neurônios/metabolismo , alfa-Sinucleína/metabolismo
8.
ACS Chem Neurosci ; 11(18): 2789-2792, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32880441

RESUMO

The recent outbreak of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) from Wuhan, China, was caused by a single-stranded RNA virus which has kept the entire world stranded. The outbreak was first diagnosed with respiratory illness, but recent findings of acute necrotizing hemorrhage of brain, brain encephalopathy, and the presence of the virus in the cerebrospinal fluid (CSF) have unveiled its neuroinvasivness. Various clinical features related to the central nervous system (CNS) and peripheral nervous system (PNS) due to COVID-19 infection are now identified. We demonstrate here an apparent similarity in neurological disorders of COVID-19 with CNS tuberculosis, which suggests that some anti-tubercular drugs may be used as therapeutic agents against COVID-19 infection.


Assuntos
Doenças do Sistema Nervoso Central/virologia , Infecções por Coronavirus/complicações , Pneumonia Viral/complicações , Tuberculose do Sistema Nervoso Central , Animais , Betacoronavirus , Encéfalo/virologia , COVID-19 , Humanos , Pandemias , SARS-CoV-2 , Tuberculose do Sistema Nervoso Central/imunologia , Tuberculose do Sistema Nervoso Central/patologia , Tuberculose do Sistema Nervoso Central/fisiopatologia
9.
Commun Biol ; 3(1): 382, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669705

RESUMO

It is now established that a protein can switch between multiple conformations to enable altered functions. Several pathogens including SARS COV2 utilize context-dependent conformational switches of particular proteins to invade host membrane to establish infections. In this perspective, we first discuss the understanding of the conformational switch of a protein towards the productive infections as a dark side of nature. Next, the unexplored binary combination of the sequences of SARS COV2 spike protein and the similarity with diverse pathogen derived proteins have been discussed to obtain novel molecular insights into the process of infection.


Assuntos
COVID-19/virologia , SARS-CoV-2/fisiologia , Proteínas Virais/metabolismo , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Membrana Celular/metabolismo , Membrana Celular/virologia , Desenho de Fármacos , Humanos , Infecções , Conformação Proteica , Proteínas Virais/química
10.
ACS Omega ; 5(27): 16395-16405, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32685802

RESUMO

We report here the preparation of an aminoxy amide-based pseudopeptide-derived building block using furanoid sugar molecules. Through the cyclo-oligomerization reaction, we generate a hybrid triazole/aminoxy amide macrocycle using the as-prepared building block. The novel conformation of the macrocycle has been characterized using NMR and molecular modeling studies, which show a strong resemblance of our synthesized compound to d-,l-α-aminoxy acid-based cyclic peptides that contain uniform backbone chirality. We observe that the macrocycle can efficiently and selectively bind Cl- ion and transport Cl- ion across a lipid bilayer. 1H NMR anion binding studies suggest a coherent relationship between the acidity of aminoxy amide N-H and triazole C-H proton binding strength. Using time-based fluorescence assay, we show that the macrocycle acts as a mobile transporter and follows an antiport mechanism. Our synthesized macrocycle imposes cancer cell death by disrupting ionic homeostasis through Cl- ion transport. The macrocycle induced cytochrome c leakage and changes in mitochondrial membrane potential along with activation of family of caspases, suggesting that the cellular apoptosis occurs through a caspase-dependent intrinsic pathway. The present results suggest the possibility of using the macrocycle as a biological tool of high therapeutic value.

11.
ACS Chem Neurosci ; 11(11): 1610-1619, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32407096

RESUMO

The accumulation of an inherently disordered protein α-synuclein (α-syn) aggregates in brain tissue play a pivotal role in the pathology and etiology of Parkinson's disease. Aggregation of α-syn has been found to be complex and heterogeneous, occurring through multitudes of early- and late-stage intermediates. Because of the inherent complexity and large dynamic range (between a few microseconds to several days under in vitro measurement conditions), it is difficult for the conventional biophysical and biochemical techniques to sample the entire time window of α-syn aggregation. Here, for the first time, we introduced the Z-scan technique as a novel tool to investigate different conformations formed in the early and late stage of temperature and mechanical stress-induced α-syn aggregation, in which different species showed its characteristic nonlinear characteristics. A power-dependent study was also performed to observe the changes in the protein nonlinearity. The perceived nonlinearity was accredited to the thermal-lensing effect. A switch in the sign of the refractive nonlinearity was observed for the first time as a signature of the late oligomeric conformation, a prime suspect that triggers cell death associated with neurodegeneration. We validate Z-scan results using a combination of different techniques, like thioflavin-T fluorescence assay, fluorescence correlation spectroscopy, Fourier-transform infrared spectroscopy, and atomic force microscopy. We believe that this simple, inexpensive, and sensitive method can have potential future applications in detecting/monitoring conformations in other essential peptides/proteins related to different neurodegenerative and other human diseases.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Encéfalo/metabolismo , Humanos , alfa-Sinucleína/metabolismo
12.
Langmuir ; 36(13): 3522-3530, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32160748

RESUMO

Kinetoplastid membrane protein-11 (KMP-11), expressed in all stages of leishmanial life cycle, is considered a potential candidate for leishmaniasis vaccine. KMP-11 is found on the membrane surface of the parasite. Although the biological function of KMP-11 is unknown, we hypothesize from its sequence analysis that it may interact with the macrophage membrane and may influence the entry process of the parasite into the host cell. To validate this hypothesis, we have investigated the interaction of KMP-11 with unilamellar anionic phospholipid vesicles and explored its pore-forming activity. The decrease in negative ζ-potential of the vesicles and reduction in the fluorescence intensity of membrane-bound dye DiI C-18 suggest a strong association of KMP-11 with the membrane. The fluorescence leakage experiment as well as phase contrast microscopy shows direct evidence of KMP-11-induced pore formation in an anionic membrane. Incorporation of cholesterol into the membrane has been found to inhibit pore formation induced by KMP-11, suggesting an important role of cholesterol in leishmaniasis. Interestingly, vesicles containing only neutral phospholipid do not exhibit any tendency toward pore formation.


Assuntos
Colesterol , Proteínas de Membrana , Fosfolipídeos , Leishmania , Membranas , Proteínas de Protozoários
13.
EMBO Mol Med ; 12(3): e11011, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32031337

RESUMO

HuR is a miRNA derepressor protein that can act as miRNA sponge for specific miRNAs to negate their action on target mRNAs. Here we have identified how HuR, by inducing extracellular vesicles-mediated export of miRNAs, ensures robust derepression of miRNA-repressed cytokines essential for strong pro-inflammatory response in activated mammalian macrophages. Leishmania donovani, the causative agent of visceral leishmaniasis, on the contrary alters immune response of the host macrophage by a variety of complex mechanisms to promote anti-inflammatory response essential for the survival of the parasite. We have found that during Leishmania infection, the pathogen targets HuR to promote onset of anti-inflammatory response in mammalian macrophages. In infected macrophages, Leishmania also upregulate protein phosphatase 2A that acts on Ago2 protein to keep it in dephosphorylated and miRNA-associated form. This causes robust repression of the miRNA-targeted pro-inflammatory cytokines to establish an anti-inflammatory response in infected macrophages. HuR has an inhibitory effect on protein phosphatase 2A expression, and mathematical modelling of macrophage activation process supports antagonistic miRNA-modulatory roles of HuR and protein phosphatase 2A which mutually balances immune response in macrophage by targeting miRNA function. Supporting this model, ectopic expression of the protein HuR and simultaneous inhibition of protein phosphatase 2A induce strong pro-inflammatory response in the host macrophage to prevent the virulent antimonial drug-sensitive or drug-resistant form of L. donovani infection. Thus, HuR can act as a balancing factor of immune responses to curtail the macrophage infection process by the protozoan parasite.


Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Leishmania donovani , Ativação de Macrófagos , Macrófagos/parasitologia , MicroRNAs , Animais , Leishmaniose Visceral
14.
Biomolecules ; 9(12)2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31817166

RESUMO

Superoxide dismutase (SOD) is the primary enzyme of the cellular antioxidant defense cascade. Misfolding, concomitant oligomerization, and higher order aggregation of human cytosolic SOD are linked to amyotrophic lateral sclerosis (ALS). Although, with two metal ion cofactors SOD1 is extremely robust, the de-metallated apo form is intrinsically disordered. Since the rise of oxygen-based metabolism and antioxidant defense systems are evolutionary coupled, SOD is an interesting protein with a deep evolutionary history. We deployed statistical analysis of sequence space to decode evolutionarily co-varying residues in this protein. These were validated by applying graph theoretical modelling to understand the impact of the presence of metal ion co-factors in dictating the disordered (apo) to hidden disordered (wild-type SOD1) transition. Contact maps were generated for different variants, and the selected significant residues were mapped on separate structure networks. Sequence space analysis coupled with structure networks helped us to map the evolutionarily coupled co-varying patches in the SOD1 and its metal-depleted variants. In addition, using structure network analysis, the residues with a major impact on the internal dynamics of the protein structure were investigated. Our results reveal that the bulk of these evolutionarily co-varying residues are localized in the loop regions and positioned differentially depending upon the metal residence and concomitant steric restrictions of the loops.


Assuntos
Análise de Sequência de Proteína/métodos , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética , Evolução Molecular , Humanos , Cadeias de Markov , Modelos Moleculares , Mutação , Conformação Proteica , Dobramento de Proteína
15.
PLoS One ; 14(9): e0222723, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31536559

RESUMO

Aggregation of the prion protein has strong implications in the human prion disease. Sup35p is a yeast prion, and has been used as a model protein to study the disease mechanism. We have studied the pattern of Sup35p aggregation inside live yeast cells under stress, by using confocal microscopy, fluorescence activated cell sorting and western blotting. Heat shock proteins are a family of proteins that are produced by yeast cells in response to exposure to stressful conditions. Many of the proteins behave as chaperones to combat stress-induced protein misfolding and aggregation. In spite of this, yeast also produce small molecules called osmolytes during stress. In our work, we tried to find the reason as to why yeast produce osmolytes and showed that the osmolytes are paramount to ameliorate the long-term effects of lethal stress in Saccharomyces cerevisiae, either in the presence or absence of Hsp104p.


Assuntos
Proteínas de Choque Térmico/metabolismo , Osmose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/genética , Microscopia Confocal , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico/fisiologia
16.
Biophys Rev ; 11(6): 851-872, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31444739

RESUMO

In order to maintain cellular function, biomolecules like protein, DNA, and RNAs have to diffuse to the target spaces within the cell. Changes in the cytosolic microenvironment or in the nucleus during the fulfillment of these cellular processes affect their mobility, folding, and stability thereby impacting the transient or stable interactions with their adjacent neighbors in the organized and dynamic cellular interior. Using classical Brownian motion to elucidate the diffusion behavior of these biomolecules is hard considering their complex nature. The understanding of biomolecular diffusion inside cells still remains elusive due to the lack of a proper model that can be extrapolated to these cases. In this review, we have comprehensively addressed the progresses in this field, laying emphasis on the different aspects of anomalous diffusion in the different biochemical reactions in cell interior. These experiment-based models help to explain the diffusion behavior of biomolecules in the cytosolic and nuclear microenvironment. Moreover, since understanding of biochemical reactions within living cellular system is our main focus, we coupled the experimental observations with the concept of sub-diffusion from in vitro to in vivo condition. We believe that the pairing between the understanding of complex behavior and structure-function paradigm of biological molecules would take us forward by one step in order to solve the puzzle around diseases caused by cellular dysfunction.

17.
Chem Commun (Camb) ; 55(74): 11052-11055, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31453599

RESUMO

Parkinson's disease (PD), closely associated with the misfolding and aggregation of the neuronal protein α-synuclein (A-Syn), is a neurodegenerative disorder with no cure to date. Here, we show that the commercially available, inexpensive, aminoglycoside antibiotic kanamycin effectively inhibits both lipid-induced and solution-phase aggregation of A-Syn in vitro, pointing towards the prospective repurposing of kanamycin as a potential anti-PD drug.


Assuntos
Antibacterianos/farmacologia , Canamicina/farmacologia , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Linhagem Celular Tumoral , Humanos , Canamicina/química , Canamicina/metabolismo , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
18.
ACS Chem Biol ; 14(7): 1601-1610, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31241303

RESUMO

Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (Mϕ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environment-sensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTB-secreted immunogenic MPT63 protein undergoes a switch from ß-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and Mϕ membranes, while the native ß-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing Mϕ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and Mϕ cell death.


Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/metabolismo , Proteínas de Bactérias/química , Morte Celular , Membrana Celular/microbiologia , Membrana Celular/patologia , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/patologia , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Tuberculose/microbiologia , Tuberculose/patologia
19.
Cell Mol Life Sci ; 76(20): 4145-4154, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31011770

RESUMO

A crucial contribution to the heterogeneity of the conformational landscape of a protein comes from the way an intermediate relates to another intermediate state in its journey from the unfolded to folded or misfolded form. Unfortunately, it is extremely hard to decode this relatedness in a quantifiable manner. Here, we developed an application of statistical cluster analyses to explore the conformational heterogeneity of a metalloenzyme, human cytosolic copper-zinc superoxide dismutase (SOD1), using the inputs from infrared spectroscopy. This study provides a quantifiable picture of how conformational information at one particular site (for example, the copper-binding pocket) is related to the information at the second site (for example, the zinc-binding pocket), and how this relatedness is transferred to the global conformational information of the protein. The distance outputs were used to quantitatively generate a network capturing the folding sub-stages of SOD1.


Assuntos
Cobre/química , Agregados Proteicos/genética , Superóxido Dismutase-1/química , Zinco/química , Sítios de Ligação , Clonagem Molecular , Análise por Conglomerados , Cobre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Zinco/metabolismo
20.
Mol Neurobiol ; 56(9): 6551-6565, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30868446

RESUMO

The amyloid cascade hypothesis dealing with the senile plaques is until date thought to be one of the causative pathways leading to the pathophysiology of Alzheimer's disease (AD). Though many aggregation inhibitors of misfolded amyloid beta (Aß42) peptide have failed in clinical trials, there are some positive aspects of the designed therapeutic peptides for diseases involving proteinaceous aggregation. Here, we evaluated a smart design of side chain tripeptide (Leu-Val-Phe)-based polymeric inhibitor addressing the fundamental hydrophobic amino acid stretch "Lys-Leu-Val-Phe-Phe-Ala" (KLVFFA) of the Aß42 peptide. The in vitro analyses performed through the thioflavin T (ThT) fluorescence assay, infrared spectroscopy, isothermal calorimetry, cytotoxicity experiments, and so on evinced a promising path towards the development of new age AD therapeutics targeting the inhibition of misfolded Aß42 peptide fibrillization. The in silico simulations done contoured the mechanism of drug action of the present block copolymer as the competitive inhibition of aggregate-prone hydrophobic stretch of Aß42. Graphical abstract The production of misfolded Aß42 peptide from amyloid precursor protein initiates amyloidosis pathway which ends with the deposition of fibrils via the oligomerization and aggregation of Aß42 monomers. The side chain tripeptide-based PEGylated polymer targets these Aß42 monomers and oligomers inhibiting their aggregation. This block copolymer also binds and helps degrading the preformed fibrils of Aß42.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Polietilenoglicóis/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Ligantes , Simulação de Dinâmica Molecular , Polietilenoglicóis/síntese química , Eletricidade Estática
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