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1.
Biochem Biophys Res Commun ; 725: 150253, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-38880080

RESUMO

Type1 Non-specific Lipid Transfer Protein (CsLTP1) from Citrus sinensis is a small cationic protein possessing a long tunnel-like hydrophobic cavity. CsLTP1 performing membrane trafficking of lipids is a promising candidate for developing a potent drug delivery system. The present work includes in-silico studies and the evaluation of drugs binding to CsLTP1 using biophysical techniques along with the investigation of CsLTP1's ability to enhance the efficacy of drugs employing cell-based bioassays. The in-silico investigations identified Panobinostat, Vorinostat, Cetylpyridinium Chloride, and Fulvestrant with higher affinities and stability of binding to the hydrophobic pocket of CsLTP1. SPR studies revealed strong binding affinities of anticancer drugs, Panobinostat (KD = 1.40 µM) and Vorinostat (KD = 2.17 µM) to CsLTP1 along with the binding and release kinetics. CD and fluorescent spectroscopy revealed drug-induced conformational changes in CsLTP1. CsLTP1-associated drug forms showed remarkably enhanced efficacy in MCF-7 cells, representing increased cell cytotoxicity, intracellular ROS, reduced mitochondrial membrane potential, and up-regulation of proapoptotic markers than the free drugs employing qRT-PCR and western blot analysis. The findings demonstrate that CsLTP1 binds strongly to hydrophobic drugs to facilitate their transport, hence improving their therapeutic efficacy revealed by the in-vitro investigations. This study establishes an excellent foundation for developing CsLTP1-based efficient drug delivery system.


Assuntos
Antineoplásicos , Proteínas de Transporte , Citrus sinensis , Humanos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Células MCF-7 , Citrus sinensis/química , Sistemas de Liberação de Medicamentos/métodos , Simulação de Acoplamento Molecular , Apoptose/efeitos dos fármacos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Ligação Proteica
2.
Arch Biochem Biophys ; 753: 109888, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38232797

RESUMO

The haloacid dehalogenase superfamily implicated in bacterial pathogenesis comprises different enzymes having roles in many metabolic pathways. Staphylococcus lugdunensis, a Gram-positive bacterium, is an opportunistic human pathogen causing infections in the central nervous system, urinary tract, bones, peritoneum, systemic conditions and cutaneous infection. The haloacid dehalogenase superfamily proteins play a significant role in the pathogenicity of certain bacteria, facilitating invasion, survival, and proliferation within host cells. The genome of S. lugdunensis encodes more than ten proteins belonging to this superfamily. However, none of them have been characterized. The present work reports the characterization of one of the haloacid dehalogenase superfamily proteins (SLHAD1) from Staphylococcus lugdunensis. The functional analysis revealed that SLHAD1 is a metal-dependent acid phosphatase, which catalyzes the dephosphorylation of phosphorylated metabolites of cellular pathways, including glycolysis, gluconeogenesis, nucleotides, and thiamine metabolism. Based on the substrate specificity and genomic analysis, the physiological function of SLHAD1 in thiamine metabolism has been tentatively assigned. The crystal structure of SLHAD1, lacking 49 residues at the C-terminal, was determined at 1.7 Å resolution with a homodimer in the asymmetric unit. It was observed that SLHAD1 exhibited time-dependent cleavage at a specific point, occurring through a self-initiated process. A combination of bioinformatics, biochemical, biophysical, and structural studies explored unique features of SLHAD1. Overall, the study revealed a detailed characterization of a critical enzyme of the human pathogen Staphylococcus lugdunensis, associated with several life-threatening infections.


Assuntos
Fosfatase Ácida , Staphylococcus lugdunensis , Humanos , Staphylococcus lugdunensis/metabolismo , Hidrolases/química , Bactérias , Tiamina
3.
Pestic Biochem Physiol ; 200: 105844, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38582571

RESUMO

Enzymes have attracted considerable scientific attention for their crucial role in detoxifying a wide range of harmful compounds. In today's global context, the extensive use of insecticides has emerged as a significant threat to the environment, sparking substantial concern. Insects, including economically important pests like Helicoverpa armigera, have developed resistance to conventional pest control methods through enzymes like carboxyl/cholinesterases. This study specifically focuses on a notable carboxyl/cholinesterase enzyme from Helicoverpa armigera (Ha006a), with the goal of harnessing its potential to combat environmental toxins. A total of six insecticides belonging to two different classes displayed varying inhibitory responses towards Ha006a, thereby rendering it effective in detoxifying a broader spectrum of insecticides. The significance of this research lies in discovering the bioremediation property of Ha006a, as it hydrolyzes synthetic pyrethroids (fenvalerate, λ-cyhalothrin and deltamethrin) and sequesters organophosphate (paraoxon ethyl, profenofos, and chlorpyrifos) insecticides. Additionally, the interaction studies between organophosphate insecticides and Ha006a helped in the fabrication of a novel electroanalytical sensor using a modified carbon paste electrode (MCPE). This sensor boasts impressive sensitivity, with detection limits of 0.019 µM, 0.15 µM, and 0.025 µM for paraoxon ethyl, profenofos, and chlorpyrifos, respectively. This study provides a comprehensive biochemical and biophysical characterization of the purified esterase Ha006a, showcasing its potential to remediate different classes of insecticides.


Assuntos
Clorpirifos , Inseticidas , Mariposas , Organotiofosfatos , Paraoxon/análogos & derivados , Piretrinas , Animais , Inseticidas/farmacologia , Inseticidas/metabolismo , Carboxilesterase/metabolismo , Helicoverpa armigera , Piretrinas/farmacologia , Piretrinas/metabolismo , Colinesterases , Resistência a Inseticidas
4.
Sci Rep ; 14(1): 19615, 2024 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-39179653

RESUMO

Electrospinning stands out as a flexible and viable method, presenting designed nanoscale materials with customized properties. This research demonstrates the immobilization of carboxylesterase protein Ha006a, reported for its adequacy in pesticide bioremediation by utilizing the electrospinning strategy. This strategy was utilized to create nanofibers by incorporating variable mixtures of biodegradable and cost-effective polyvinyl alcohol (PVA)-chitosan (CS) nanofiber solution (PVA100, PVA96, PVA94, PVA92 and PVA90). All the mixtures were electrospun at a reliable voltage of 21 kV, maintaining a gap of 12 cm from the nozzle. The Ha006a, sourced from Helicoverpa armigera, was consolidated into the optimized PVA90 polymer mixture. The electrospun nanofibers experienced comprehensive characterization utilizing distinctive microscopy and spectroscopy procedures counting FESEM, TGA, XRD and FTIR. The comparative investigation of the esterase property, ideal parameters and stability of the unbound and bound/immobilized Ha006a was scrutinized. The results uncovered an essential elevation in the ideal conditions of enzyme activity post-immobilization. The PVA-CS control nanofiber and Ha006a-PVA-CS showed a smooth structure, including an average breadth of around 170.5 ± 44.2 and 222.5 ± 66.5 nm, respectively. The enzyme-immobilized nanofibers displayed upgraded stability and comprehensive characterization of the nanofiber, which guaranteed genuineness and reproducibility, contributing to its potential as a potent device for bioremediation applications. This investigation opens the way for the manufacture of pesticide-resistant insect enzyme-based nanofibers, unlocking their potential for assorted applications, counting pesticide remediation and ensuring environmental sustainability.


Assuntos
Carboxilesterase , Quitosana , Estabilidade Enzimática , Enzimas Imobilizadas , Nanofibras , Álcool de Polivinil , Álcool de Polivinil/química , Nanofibras/química , Quitosana/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Carboxilesterase/metabolismo , Carboxilesterase/química , Animais , Concentração de Íons de Hidrogênio
5.
Protein J ; 2024 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-39306651

RESUMO

Cationic amino acid binding protein (CLasArgBP), one of the two amino acid binding receptor in Candidatus Liberibacter asiaticus (CLas), is predominately expressed in citrus psyllids as a part of ATP-binding cassette transport system. The present study describes characterization of CLasArgBP by various biophysical techniques and in silico study, to identify potential inhibitor molecules against CLasArgBP through virtual screening and MD simulations. Further, in planta study was carried out to assess the effect of selected inhibitors on Huanglongbing infected Mosambi plants. The results showed that CLasArgBP exhibits pronounced specificity for arginine, histidine and lysine. Surface plasmon resonance (SPR) study reports highest binding affinity for arginine (Kd, 0.14 µM), compared to histidine and lysine (Kd, 15 µΜ and 26 µΜ, respectively). Likewise, Differential Scanning Calorimetry (DSC) study showed higher stability of CLasArgBP for arginine, compared to histidine and lysine. N(omega)-nitro-L-arginine, Gamma-hydroxy-L-arginine and Gigartinine emerged as lead compounds through in silico study displaying higher binding energy and stability compared to arginine. SPR reports elevated binding affinities for N(omega)-nitro-L-arginine and Gamma-hydroxy-L-arginine (Kd, 0.038 µΜ and 0.061 µΜ, respectively) relative to arginine. DSC studies showed enhanced thermal stability for CLasArgBP in complex with selected inhibitors. Circular dichroism and fluorescence studies showed pronounced conformational changes in CLasArgBP with selected inhibitors than with arginine. In planta study demonstrated a substantial decrease in CLas titer in treated plants as compared to control plants. Overall, the study provides the first comprehensive characterization of cationic amino acid binding protein from CLas, as a potential drug target to manage HLB disease.

6.
Biochim Biophys Acta Proteins Proteom ; 1872(4): 141015, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38615986

RESUMO

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.


Assuntos
Aminoimidazol Carboxamida , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacologia , Fosforribosilaminoimidazolcarboxamida Formiltransferase/metabolismo , Fosforribosilaminoimidazolcarboxamida Formiltransferase/química , Simulação de Acoplamento Molecular , Ribonucleotídeos/metabolismo , Ribonucleotídeos/química , Cinética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/antagonistas & inibidores , Nucleotídeo Desaminases/metabolismo , Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/genética , Especificidade por Substrato , Proliferação de Células/efeitos dos fármacos , Hidroximetil e Formil Transferases/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/antagonistas & inibidores , Complexos Multienzimáticos
7.
Int J Biol Macromol ; 265(Pt 1): 130811, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38490399

RESUMO

Lipid Transfer Protein1 (LTP1) is a cationic, multifaceted protein belonging to the pathogenesis-related protein (PR14) family. Despite being involved in diverse physiological processes and defense mechanisms, the precise in-vivo role of LTP1 remains undiscovered. This work presents the characterization of recombinant Citrus sinensis LTP1 (CsLTP1) along with lipid binding studies through in-silico and in-vitro approaches. CsLTP1 demonstrated great thermal and pH stability with a huge biotechnological potential. It showed in-vitro binding capacity with jasmonic acid and lipids involved in regulating plant immune responses. Gene expression profiling indicated a significant upregulation of CsLTP1 in Candidatus-infected Citrus plants. CsLTP1 disrupted the cell membrane integrity of various pathogens, making it a potent antimicrobial agent. Further, in-vivo antimicrobial and insecticidal properties of CsLTP1 have been explored. The impact of exogenous CsLTP1 treatment on rice crop metabolism for managing blight disease has been studied using GC-MS. CsLTP1 triggered crucial metabolic pathways in rice plants while controlling the blight disease. CsLTP1 effectively inhibited Helicoverpa armigera larvae by impeding mid-gut α-amylase activity and obstructing its developmental stages. This study highlights the pivotal role of CsLTP1 in plant defense by offering insights for developing multi-target therapeutic agent or disease-resistant varieties to comprehensively tackle the challenges towards crop protection.


Assuntos
Anti-Infecciosos , Citrus sinensis , Citrus , Citrus sinensis/metabolismo , Proteínas de Transporte/metabolismo , Anti-Infecciosos/farmacologia , Anti-Infecciosos/metabolismo , Citrus/metabolismo
8.
Sci Rep ; 14(1): 21596, 2024 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-39285183

RESUMO

In the realm of disease vectors and agricultural pest management, insecticides play a crucial role in preserving global health and ensuring food security. The pervasive use, particularly of organophosphates (OPs), has given rise to a substantial challenge in the form of insecticide resistance. Carboxylesterases emerge as key contributors to OP resistance, owing to their ability to sequester or hydrolyze these chemicals. Consequently, carboxylesterase enzymes become attractive targets for the development of novel insecticides. Inhibiting these enzymes holds the potential to restore the efficacy of OPs against which resistance has developed. This study aimed to screen the FooDB library to identify potent inhibitory compounds targeting carboxylesterase, Ha006a from the agricultural pest Helicoverpa armigera. The ultimate objective is to develop effective interventions for pest control. The compounds with the highest scores underwent evaluation through docking studies and pharmacophore analysis. Among them, four phytochemicals-donepezil, protopine, 3',4',5,7-tetramethoxyflavone, and piperine-demonstrated favorable binding affinity. The Ha006a-ligand complexes were subsequently validated through molecular dynamics simulations. Biochemical analysis, encompassing determination of IC50 values, complemented by analysis of thermostability through Differential Scanning Calorimetry and interaction kinetics through Isothermal Titration Calorimetry was conducted. This study comprehensively characterizes Ha006a-ligand complexes through bioinformatics, biochemical, and biophysical methods. This investigation highlights 3',4',5,7-tetramethoxyflavone as the most effective inhibitor, suggesting its potential for synergistic testing with OPs. Consequently, these inhibitors offer a promising solution to OP resistance and address environmental concerns associated with excessive insecticide usage, enabling a significant reduction in their overuse.


Assuntos
Carboxilesterase , Inseticidas , Simulação de Acoplamento Molecular , Compostos Fitoquímicos , Animais , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Carboxilesterase/antagonistas & inibidores , Carboxilesterase/metabolismo , Carboxilesterase/química , Inseticidas/farmacologia , Inseticidas/química , Simulação de Dinâmica Molecular , Mariposas/enzimologia , Mariposas/efeitos dos fármacos , Controle de Pragas/métodos , Resistência a Inseticidas , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/química , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Helicoverpa armigera
9.
3 Biotech ; 13(6): 175, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37188291

RESUMO

Juvenile hormone (JH) plays pivotal roles in several critical developmental processes in insects, including metamorphosis and reproduction. JH-biosynthetic pathway enzymes are considered highly promising targets for discovering novel insecticides. The oxidation of farnesol to farnesal, catalysed by farnesol dehydrogenase (FDL), represents a rate-limiting step in JH biosynthesis. Here, we report farnesol dehydrogenase (HaFDL) from H. armigera as a promising insecticidal target. The inhibitory potential of natural substrate analogue geranylgeraniol (GGol) was tested in vitro, wherein it showed a high binding affinity (kd 595 µM) for HaFDL in isothermal titration calorimetry (ITC) and subsequently exhibited dose-dependent enzyme inhibition in GC-MS coupled qualitative enzyme inhibition assay. Moreover, the experimentally determined inhibitory activity of GGol was augmented by the in silico molecular docking simulation which showed that GGol formed a stable complex with HaFDL, occupied the active site pocket and interacted with key active site residues (Ser147 and Tyr162) as well as other residues that are crucial in determining the active site architecture. Further, the diet-incorporated oral feeding of GGol caused detrimental effects on larval growth and development, exhibiting a significantly reduced rate of larval weight gain (P < 0.01), aberrant pupal and adult morphogenesis, and a cumulative mortality of ~ 63%. To the best of our knowledge, the study presents the first report on evaluating GGol as a potential inhibitor for HaFDL. Overall, the findings revealed the suitability of HaFDL as a potential insecticidal target for the management H. armigera.

10.
J Biomol Struct Dyn ; 41(5): 1978-1987, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-35037838

RESUMO

Helicoverpa armigera (Ha), a polyphagous pest, causes significant damage to several crop plants, including cotton. The control of this cosmopolitan pest is largely challenging due to the development of resistance to existing management practices. The Juvenile Hormone (JH) plays a pivotal role in the life cycle of insects by regulating their morphogenetic and gonadotropic development. Hence, enzymes involved in JH biosynthesis are an attractive target for the development of selective insecticides. Farnesyl diphosphate synthase (FPPS), a member protein of (E)-prenyl-transferases, is one of the most crucial enzymes in the biosynthetic pathway of JHs. It catalyzes the condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), forming farnesyl diphosphate (FPP), a precursor of JH. The study was designed to identify an effective small inhibitory molecule that could inhibit the activity of Helicoverpa armigera - FPPS (HaFPPS) for an effective pest control intervention. Therefore, a 3D model of FPPS protein was generated using homology modeling. The FooDB database library of small molecules was selected for virtual screening, following which binding affinities were evaluated using docking studies. Three top-scored molecules were analyzed for various pharmacophore properties. Further, molecular dynamics (MD) simulation analysis showed that the identified molecules (mitraphylline-ZINC1607834, chlorogenic acid-ZINC2138728 and llagate-ZINC3872446) had a reasonably acceptable binding affinity for HaFPPS and resulted in the formation of a stable HaFPPS-inhibitor(s) complex. The identified phytochemical molecules may be used as potent inhibitors of HaFPPS thus, paving the way for further developing environment-friendly insect growth regulator(s). Communicated by Ramaswamy H. Sarma.


Assuntos
Geraniltranstransferase , Mariposas , Animais , Geraniltranstransferase/química , Geraniltranstransferase/metabolismo
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