RESUMO
Monitoring exocellular adenosine-5'-triphosphate (ATP) is a demanding task but the biosensor development is limited by the low concentration and rapid degradation of ATP. Herein, we developed a simple yet effective biosensor based on ZIF-67 loaded with bi-enzymes of glucose (GOx) and hexokinase (HEX) for effective detection of ATP. In the confined space of the porous matrix, the bi-enzymes competed for the glucose substrate in the presence of ATP, facilitating the biosensor to detect low ATP concentrations down to the micromole level (3.75 µM) at working potential of 0.55 V (vs. Ag/AgCl). Furthermore, ZIF-67 with cobalt served as a porous matrix to specifically adsorb ATP molecules, allowing it to differentiate isomers with sensitivity of 0.53 nA/µM, RSD of 5.4%, and recovery rate of 93.3%. We successfully applied the fabricated biosensor to measure ATP secreted from rat PC12 cells in the pericellular space thus realizing time-resolving measurement. This work paved the path for real-time monitoring of ATP released by cells, which will aid in understanding tumor cell glycolysis and immune responses.
Assuntos
Enzimas Imobilizadas , Glucose Oxidase , Animais , Ratos , Enzimas Imobilizadas/química , Glucose Oxidase/química , Hexoquinase/química , Hexoquinase/metabolismo , Trifosfato de Adenosina/química , GlucoseRESUMO
Most of the polymeric membranes synthesized for decentralization of polluted water use fossil-based components. Thus, there is an urgent need to create robust and tunable nano/micro materials for confidently designing efficient and selective polymeric water filters with guaranteed sustainability. We have chosen a robust high-grade microfibrillated cellulose (MFC) as the functional material and selectively tuned it via enzymatic catalysis, which led to the attachment of phosphate group at the C6 position, followed by esterification (fatty acid attachment at C2 and C3 carbon), which led to the increase in its antifouling properties. We have demonstrated the robustness of the functionalization by measuring the separation of various metal ions, and the antifouling properties by adding foulants, such as Bovine Serum Albumin (BSA) and cancerous cells to the test solutions. These prototype affinity MFC membranes represent the most promising type of next-generation high-performance filtration devices for a more sustainable society.
Assuntos
Incrustação Biológica/prevenção & controle , Celulose/imunologia , Celulose/farmacologia , Cobalto/isolamento & purificação , Hexoquinase/metabolismo , Níquel/isolamento & purificação , Celulose/química , Celulose/metabolismo , Cobalto/química , Hexoquinase/química , Simulação de Dinâmica Molecular , Níquel/química , Soroalbumina Bovina/químicaRESUMO
Hexokinase II (HK2), a glycolytic enzyme is commonly overexpressed in most cancer types. The overexpression of HK2 is reported to promote the survival of cancer cells by facilitating the constant ATP generation and protecting the cancer cell against apoptotic cell death. Hence, HK2 is considered as potential target of many mitochondria targeting anticancerous agents (referred to as mitocans). Most of the existing mitocans are synthetic and hence such compounds are observed to exhibit adverse effects, witnessed through many experimental outcomes. These limitations necessitates hunting for an alternative source of mitocans with minimum/no side effects. The need for an alternative therapy points towards the ethnomedicinal herbs, known for their minimal side effects and effectiveness. Henceforth recent studies have put forth the effort to utilize anticancer herbs in formulating naturally derived mitocans as an add-on to improve cancer therapeutics. So, our study aims to explore the HK2 targeting potential of phytocompounds from the selected anticancerous herbs Andrographis paniculata (AP) and Centella asiatica (CA). 60 phytocompounds collectively from CA and AP were docked against HK2 and drug-likeness prediction of the selected phytocompounds was performed to screen the best possible ligand for HK2. Furthermore, the docked complexes were subjected to molecular dynamics simulations (MDS) to analyse the molecular mechanism of protein-ligand interactions. The results of the study suggest that the natural compounds asiatic acid and bayogenin (from CA) and andrographolide (from AP) can bepotential natural mitocans by targeting HK2. Further experimental studies (in-vitro and in-vivo) are required to validate the results.
Assuntos
Andrographis paniculata/química , Antineoplásicos/farmacologia , Centella/química , Hexoquinase/antagonistas & inibidores , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/farmacologia , Antineoplásicos/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hexoquinase/química , Hexoquinase/genética , Hexoquinase/metabolismo , Mitocôndrias/efeitos dos fármacos , Modelos Moleculares , Compostos Fitoquímicos/química , Fitoterapia , Conformação ProteicaRESUMO
It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.
Assuntos
Fosfatase Alcalina/química , Glucose Oxidase/química , Hexoquinase/química , Fosfatase Alcalina/metabolismo , Biocatálise , Glucose Oxidase/metabolismo , Hexoquinase/metabolismo , Modelos Moleculares , Estrutura Molecular , Agregados ProteicosRESUMO
Complex formation between hexokinase-II (HKII) and the mitochondrial VDAC1 is crucial to cell growth and survival. We hypothesize that HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we devised a hybrid approach. First, we describe membrane binding of HKII with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion capturing membrane insertion of its H-anchor. The insertion depth of the H-anchor was then used to derive positional restraints in subsequent millisecond-scale Brownian dynamics (BD) simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models for the complex were further refined and their structural stability probed with additional MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1's permeation pathway, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. We also show how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and has implications in mitochondria-mediated cell death.
Assuntos
Hexoquinase/metabolismo , Proteínas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Hexoquinase/química , Hexoquinase/genética , Humanos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Ligação Proteica , Domínios Proteicos , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Hexokinases (HXKs) and fructokinases (FRKs) are the only two families of enzymes in plants that have been identified as able to phosphorylate Glucose (Glc) and Fructose (Fru). Glc can only be phosphorylated in plants by HXKs, while Fru can be phosphorylated by either HXKs or FRKs. The various subcellular localizations of HXKs in plants indicate that they are involved in diverse functions, including anther dehiscence and pollen germination, stomatal closure in response to sugar levels, stomatal aperture and reducing transpiration. Its association with modulating programmed cell death, and responses to oxidative stress and pathogen infection (abiotic and biotic stresses) also have been reported. To extend our understanding about the function of HXK-like genes in the response of Prunus rootstocks to abiotic stress, we performed a detailed bioinformatic and functional analysis of hexokinase 3-like genes (HXK3s) from two Prunus rootstock genotypes, 'M.2624' (Prunus cerasifera Ehrh × P. munsoniana W.Wight & Hedrick) and 'M.F12/1' (P. avium L.), which are tolerant and sensitive to hypoxia stress, respectively. A previous large-scale transcriptome sequencing of roots of these rootstocks, showed that this HXK3-like gene that was highly induced in the tolerant genotype under hypoxia conditions. In silico analysis of gene promoters from M.2624 and M.F12/1 genotypes revealed regulatory elements that could explain differential transcriptional profiles of HXK3 genes. Subcellular localization was determinates by both bioinformatic prediction and expression of their protein fused to the green fluorescent protein (GFP) in protoplasts and transgenic plants of Arabidopsis. Both approaches showed that they are expressed in plastids. Metabolomics analysis of Arabidopsis plants ectopically expressing Prunus HXK3 genes revealed that content of several metabolites including phosphorylated sugars (G6P), starch and some metabolites associated with the TCA cycle were affected. These transgenic Arabidopsis plants showed improved tolerance to salt and drought stress under growth chamber conditions. Our results suggest that Prunus HXK3 is a potential candidate for enhancing tolerance to salt and drought stresses in stone fruit trees and other plants.
Assuntos
Arabidopsis/fisiologia , Hexoquinase/genética , Prunus/genética , Tolerância ao Sal/genética , Sequência de Aminoácidos , Arabidopsis/genética , Hexoquinase/química , Hipóxia/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Homologia de Sequência de AminoácidosRESUMO
The oleaginous yeast Yarrowia lipolytica is a potent cell factory as it is able to use a wide variety of carbon sources to convert waste materials into value-added products. Nonetheless, there are still gaps in our understanding of its central carbon metabolism. Here we present an in-depth study of Y. lipolytica hexokinase (YlHxk1), a structurally unique protein. The greatest peculiarity of YlHxk1 is a 37-amino acid loop region, a structure not found in any other known hexokinases. By combining bioinformatic and experimental methods we showed that the loop in YlHxk1 is essential for activity of this protein and through that on growth of Y. lipolytica on glucose and fructose. We further proved that the loop in YlHxk1 hinders binding with trehalose 6-phosphate (T6P), a glycolysis inhibitor, as hexokinase with partial deletion of this region is 4.7-fold less sensitive to this molecule. We also found that YlHxk1 devoid of the loop causes strong repressive effect on lipase-encoding genes LIP2 and LIP8 and that the hexokinase overexpression in Y. lipolytica changes glycerol over glucose preference when cultivated in media containing both substrates.
Assuntos
Expressão Gênica , Hexoquinase/química , Hexoquinase/metabolismo , Yarrowia/enzimologia , Yarrowia/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Biologia Computacional/métodos , Meios de Cultura/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Frutose/metabolismo , Proteínas Fúngicas/genética , Glucose/metabolismo , Glicerol/metabolismo , Glicólise/efeitos dos fármacos , Hexoquinase/antagonistas & inibidores , Hexoquinase/genética , Cinética , Lipase/genética , Organismos Geneticamente Modificados , Plasmídeos/genética , Fosfatos Açúcares/metabolismo , Fosfatos Açúcares/farmacologia , Trealose/análogos & derivados , Trealose/metabolismo , Trealose/farmacologia , Yarrowia/crescimento & desenvolvimentoRESUMO
Glucokinase (GLK) and Hexokinase (HK) have been characterized as essential targets in Trypanosoma cruzi (Tc)-mediated infection. A recent study reported the propensity of the concomitant inhibition of TcGLK and TcHK by compounds GLK2-003 and GLK2-004, thereby presenting an efficient approach in Chagas disease treatment. We investigated this possibility using atomic and molecular scaling methods. Sequence alignment of TcGLK and TcHK revealed that both proteins shared approximately 33.3 % homology in their glucose/inhibitor binding sites. The total binding free energies of GLK2-003 and GLK2-004 were favorable in both proteins. PRO92 and THR185 were pivotal to the binding and stabilization of the ligands in TcGLK, likewise their conserved counterparts, PRO163 and THR237 in TcHK. Both compounds also induced a similar pattern of perturbations in both TcGLK and TcHK secondary structure. Findings from this study therefore provide insights into the underlying mechanisms of dual inhibition exhibited by the compounds. These results can pave way to discover and optimize novel dual Tc inhibitors with favorable pharmacokinetics properties eventuating in the mitigation of Chagas disease.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucoquinase/antagonistas & inibidores , Hexoquinase/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Inibidores Enzimáticos/química , Glucoquinase/química , Glucoquinase/metabolismo , Hexoquinase/química , Hexoquinase/metabolismo , Humanos , Modelos Moleculares , Termodinâmica , Tripanossomicidas/química , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Hexokinase (HK) catalyzes the first step in glucose metabolism, making it an exciting target for the inhibition of tumor initiation and progression due to their elevated glucose metabolism. The upregulation of hexokinase-2 (HK2) in many cancers and its limited expression in normal tissues make it a particularly attractive target for the selective inhibition of cancer growth and the eradication of tumors with limited side effects. The design of such safe and effective anticancer therapeutics requires the development of HK2-specific inhibitors that will not interfere with other HK isozymes. As HK2 is unique among HKs in having a catalytically active N-terminal domain (NTD), we have focused our attention on this region. We previously found that NTD activity is affected by the size of the linker helix-α13 that connects the N- and C-terminal domains of HK2. Three nonactive site residues (D447, S449, and K451) at the beginning of the linker helix-α13 have been found to regulate the NTD activity of HK2. Mutation of these residues led to increased dynamics, as shown via hydrogen deuterium exchange analysis and molecular dynamic simulations. D447A contributed the most to the enhanced dynamics of the NTD, with reduced calorimetric enthalpy of HK2. Similar residues exist in the C-terminal domain (CTD) but are unnecessary for HK1 and HK2 activity. Thus, we postulate these residues serve as a regulatory site for HK2 and may provide new directions for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of HK2.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Hexoquinase/metabolismo , Catálise , Linhagem Celular Tumoral , Estabilidade Enzimática , Glicólise , Hexoquinase/antagonistas & inibidores , Hexoquinase/química , Humanos , Cinética , Domínios Proteicos , Especificidade por SubstratoRESUMO
Hexokinase (HXK) is the first key enzyme in the glycolytic pathway and plays an extremely important role in energy metabolism. By searching the microsporidian database, we found a sequence (NBO_27g0008) of Nosema bombycis Nägali, 1857 with high similarity to hexokinase-2, and named it as NbHXK2. The NbHXK2 gene has 894 bp and encodes 297 amino acids with 34.241 kD molecular weight and 5.26 isoelectric point. NbHXK2 contains 31 phosphorylation sites and 4 potential N-glycosylation sites with signal peptides and no transmembrane domain. Multiple sequence alignment showed that NbHXK2 shares more than 40% amino acid identity with that of other microsporidia, and the homology with hexokinase-2 of Nosema tyriae Canning, Curry, Cheney, Lafranchi-Tristem, Kawakami, Hatakeyama, Iwano et Ishihara, 1999, Nosema pyrausta (Paillot, 1927) and Nosema ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 was 89.17%, 87.82% and 69.86%, respectively. Phylogenetic analysis based on the amino acid sequence of hexokinase showed that all microsporidia cluster together in the same clade, and are far away from animals, plants and fungi, and that N. bombycis is closely related to N. tyriae; N. pyrausta; N. ceranae and Nosema apis Zander, 1909. Immunolocalisation with the prepared polyclonal antibody showed that NbHXK2 was mainly distributed in the cytoplasm and plasmalemma in proliferative, sporulation stage and mature spore of N. bombycis. qRT-PCR assay showed that the NbHXK2 expressed at higher level during spore germination and at early stage of proliferation. These results indicate that N. bombycis may use its own glycolytic pathways to supply energy for infection and development, especially germination and in the early stage of proliferation, and acquire energy from the host through certain ways as well.
Assuntos
Proteínas Fúngicas/genética , Hexoquinase/genética , Nosema/genética , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hexoquinase/química , Hexoquinase/metabolismo , Nosema/metabolismo , Filogenia , Alinhamento de SequênciaRESUMO
Sugars are the key regulatory molecules that impact diverse biological processes in plants. Hexokinase, the key rate-limiting enzyme in hexose metabolism, takes part in the first step of glycolytic pathway. Acting as a sensor that mediates sugar regulation, hexokinase has been proved to play significant roles in regulating plant growth and development. Here, we isolated a hexokinase gene SlHXK1 from tomato. Its transcript levels were higher in flowers and leaves than in other organs and decreased during leaf and petiole development. SlHXK1-RNAi lines displayed advanced leaf senescence and stunted plant growth. Physiological features including plant height, leaf length, thickness and size, the contents of chlorophyll, starch and MDA, and hexokinase activity were dramatically altered in SlHXK1-RNAi plants. Dark-induced leaf senescence were advanced and the transcripts of senescence-related genes after darkness treatment were markedly increased in SlHXK1-RNAi plants. RNA-seq and qRT-PCR analyses showed that the transcripts of genes related to plant hormones, photosynthesis, chloroplast development, chlorophyll synthesis and metabolism, cellular process, starch and sucrose metabolism, and senescence were significantly altered in SlHXK1-RNAi plants. Taken together, our data demonstrate that SlHXK1 is a significant gene involved in leaf senescence and plant growth and development in tomato through affecting starch turnover.
Assuntos
Hexoquinase/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Solanum lycopersicum/fisiologia , Sequência de Aminoácidos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hexoquinase/química , Hexoquinase/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Transcrição GênicaRESUMO
Cancer cells are often characterized by elevated levels of mitochondrion-bound hexokinase II (HKII), which facilitates their survival, proliferation, and metastasis. Here, we have designed a cancer-selective cell-penetrating peptide (CPP) by covalently coupling a short penetration-accelerating sequence (PAS) to the mitochondrial membrane-binding N-terminal 15 amino acids of HKII (pHK). PAS-pHK mediates efficient cellular uptake and cytosolic delivery of a synthetic mimic of miR-126, a tumor suppressor miRNA downregulated in many malignancies. Following uptake by breast cancer MCF-7 cells, the CPP-miRNA conjugate is distributed throughout the cytosol and shows strong colocalization with mitochondria, where PAS-pHK induces depolarization of mitochondrial membrane potential, inhibition of metabolic activities, depletion of intracellular ATP levels, release of cytochrome c, and, finally, apoptosis. Concomitantly, the miR-126 cargo synergistically enhances the anticancer effects of PAS-pHK. Importantly, the PAS-pHK-miR-126 conjugate is not toxic to noncancerous MCF-10A and HEK-93 cells. Our results demonstrate the potential of PAS-pHK-mediated delivery of miRNA mimics as a novel cancer-selective therapeutic strategy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Peptídeos Penetradores de Células , Sistemas de Liberação de Medicamentos , Hexoquinase/química , MicroRNAs , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Feminino , Células HEK293 , Humanos , Células MCF-7 , MicroRNAs/química , MicroRNAs/farmacologia , Proteínas de Neoplasias/metabolismoRESUMO
We describe an approach for the development of fluorescent sensors of metabolite binding in which a genetically encoded fluorescent non-canonical amino acid (fNCAA) containing a 7-hydroxycoumarin moiety (7-HCAA) forms a FRET pair with native tryptophan residues. Although previous studies demonstrated the potential for using 7-HCAA as an acceptor for tryptophan, this approach has not yet been explored within a single protein containing multiple tryptophan residues. A structure-based analysis of a hexokinase enzyme with multiple native tryptophan residues identified glutamate 50 as a potential site of 7-HCAA incorporation; Glu50 moves closer to the native tryptophans upon substrate binding. Substitution of 7-HCAA at residue 50 led to an increase in FRET efficiency in the presence of the substrate; this effect was not observed in a control protein where no change in distance between 7-HCAA and the native tryptophans occurs on substrate binding. This system was then used to directly observe differences in binding affinity of the hexokinase that occur at a number of pH values. Our approach builds on previous research in that it eliminates the need for the incorporation of multiple fNCAAs or fluorescent labels within a target protein and can be used to study substrate binding with native ligands. As such, it serves to expand the versatility of FRET-based techniques.
Assuntos
Aminoácidos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Hexoquinase/química , Umbeliferonas/química , Corantes Fluorescentes/síntese química , Hexoquinase/metabolismo , Ligantes , Modelos Moleculares , Conformação MolecularRESUMO
It have been significantly demonstrated that Hexokinase (HXK), Granule-bound starch synthase (GBSS) and ADP-glucose pyrophosphorylase (AGPase) are three critical enzymes in the starch biosynthetic pathway and are related to starch (amylose, amylopectin and total starch) content in lotus. It is important to develop functional markers in marker-assisted selection of lotus breeding. So far there have been few reports about lotus functional markers. In this study, based on insertion-deletions (INDELs) and single-nucleotide polymorphisms (SNPs), we developed three functional markers, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1. FMHXK-E1 was developed based on polymorphisms of two haplotypes of NnHXK. 26 lotus cultivars that the 320-bp fragment presented in NnHXK had a lower content of amylose and a higher content of amylopectin. FMGBSS-I8 was developed based on polymorphisms of two haplotypes of NnGBSS. The group containing 32 lotus cultivars with the 210-bp fragment had less amylose content and more amylopectin content. FMAGPL-I1 was developed based on polymorphisms of two haplotypes of NnAGPL (ADP-glucose pyrophosphorylase large subunit gene). The group containing 40 lotus cultivars with the 362-bp fragment had less amylopectin, total starch content and more amylose content. According to the study, FMHXK-E1, FMGBSS-I8 and FMAGPL-I1 are closely related to lotus starch content. It could be provided research basis for molecular assisted selection of lotus starch content improve breeding efficiency.
Assuntos
Lotus/genética , Locos de Características Quantitativas , Característica Quantitativa Herdável , Amido , Sequência de Bases , Hexoquinase/química , Hexoquinase/genética , Hexoquinase/metabolismo , Mutação INDEL , Lotus/metabolismo , Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único , Amido/biossíntese , Sintase do Amido/química , Sintase do Amido/genética , Sintase do Amido/metabolismoRESUMO
Extranodal nasal-type natural killer/T-cell lymphoma (ENKTL) is an Epstein-Barr virus (EBV) associated lymphoma that progresses rapidly and relapses frequently. Advanced ENKTL is multidrug chemoresistant and has a poor prognosis. In this study, we aim to develop a novel hexokinase domain component 1 (HKDC1)-based antitumor target for ENKTL that is involved with the antimetabolic signaling pathway, EBV replication, and P-glycoprotein (P-gp) expression. We showed that HKDC1 is highly upregulated in ENKTL cells and HKDC1 knockdown significantly suppresses ENKTL tumor growth. In addition, HKDC1 is highly identical with four other hexokinase isoforms, with the only difference being in the last eight amino acids (aa) at the C-terminal. Further investigation showed that peptide delivery of the last eight aa of HKDC1 at the C-terminal (HKC8) with D-configuration using transferrin (Tf) receptor internalization sequence (Tf-D-HKC8) inhibits HKDC1 association with vascular endothelial growth factor 1 (VDAC1), resulting in mitochondrial dysfunction and reactive oxygen species (ROS) overgeneration and subsequently suppressing EBV replication and P-gp expression, making it very effective in killing EBV-positive ENKTL cells. Further in vivo experiments showed that local injection of Tf-D-HKC8 peptide significantly suppresses ENKTL tumor growth and EBV replication in ENKTL xenograft mouse models. We conclude that HKDC1 C-terminal-based peptides inhibit ENKTL by modulation of mitochondrial function and EBV suppression.
Assuntos
Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4/efeitos dos fármacos , Hexoquinase/metabolismo , Linfoma Extranodal de Células T-NK/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Dano ao DNA , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Infecções por Vírus Epstein-Barr/virologia , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Hexoquinase/química , Humanos , Linfoma Extranodal de Células T-NK/etiologia , Linfoma Extranodal de Células T-NK/patologia , Camundongos , Peptídeos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.
Assuntos
Actinas/química , Adenosina Trifosfatases/química , Glucoquinase/química , Hexoquinase/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Glucoquinase/genética , Hexoquinase/genética , Polimerização , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Adenosine triphosphate (ATP) plays a crucial role in energy metabolism and extracellular purinergic signaling. A 3D bimetallic Au/Pt nanoflowers decorated ATP microelectrode biosensor prepared by facile and effective template-free electrodeposition was firstly reported, realizing local detection of cellular ATP secretion. The ATP biosensor was developed by co-immobilization of glucose oxidase and hexokinase, exhibiting long-term stability (79.39 ± 9.15% of its initial value remained after 14 days at 4 °C) and high selectivity with a limit of detection down to 2.5 µM (S/N = 3). The resulting ATP biosensor was then used for direct in situ monitoring of ATP secreted from living cells (PC12) with the stimulation of high K+ solutions. The obtained current was about 21.6 ± 3.4 nA (N = 6), corresponding to 12.2 ± 2.8 µM ATP released from cells, right in the micromolar range and consistent with the suggested levels. The 3D bimetallic Au/Pt nanoflowers possess excellent catalytic activity and large electroactive surface area, contributing to enzymatic activity preservation and long-term stability. This work provides a promising platform for long-time monitoring of other neurotransmitters and secretions in cellular glycolysis and apoptosis processes in the future.
Assuntos
Trifosfato de Adenosina/análise , Ligas/química , Ouro/química , Nanopartículas Metálicas/química , Platina/química , Animais , Técnicas Biossensoriais , Catálise , Galvanoplastia , Enzimas Imobilizadas/química , Glucose Oxidase/química , Hexoquinase/química , Peróxido de Hidrogênio/química , Limite de Detecção , Microeletrodos , Células PC12 , Ratos , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
Aerobic glycolysis promotes metastasis and correlates with poorer clinical outcomes in hepatocellular carcinoma (HCC), but the controllers and mechanisms of abnormally activated glycolysis remain unclear. Herein, we demonstrated that the fifth component of the constitutive photomorphogenic 9 (COP9) signalosome complex (COPS5/CSN5) was a controller of glycolysis. For the first time, we found that CSN5 could influence the expression of glycolytic metabolism-associated proteins, especially hexokinase 2 (HK2), a glycolytic rate-limiting enzyme. In addition, we found that CSN5 was associated with HK2 overexpression in HCC tissues. Silencing CSN5 expression caused a decrease in the level of the HK2 protein, glucose uptake, glycolysis capacity and the production of glycolytic intermediates in HCC cells. Re-expression of HK2 rescued the decreased glycolytic flux induced by CSN5 knockdown, whereas inhibition of HK2 alleviated CSN5-enhanced glycolysis. Functionally, CSN5 regulated HCC cell invasion and metastasis via HK2-mediated glycolysis. Mechanistically, we demonstrated that CSN5 attenuated the ubiquitin-proteasome system-mediated degradation of HK2 through its deubiquitinase function. Inhibition of CSN5 kinase activity by curcumin decreased HK2 protein expression and glycolysis, repressed the metastasis of HCC cells in vitro and in vivo, and prolonged the survival time of tumor-bearing nude mice. Overall, our study identified CSN5 as a controller of glycolysis, and it may be a potential treatment target for HCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Complexo do Signalossomo COP9/metabolismo , Carcinoma Hepatocelular/secundário , Glicólise , Hexoquinase/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/patologia , Peptídeo Hidrolases/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Complexo do Signalossomo COP9/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Nus , Peptídeo Hidrolases/genética , Prognóstico , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study identifies and validates hexokinase type 4 (HK4), an isozyme of hexokinase in the liver and pancreas, as an important target of C2-ß-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (ßdGT), a xanthone glucoside suggested to have antidiabetic property. In the study, we applied the computational pipeline of molecular docking followed by the molecular dynamics simulations to shortlist potential ßdGT protein targets. The analysis of protein dynamics and the binding free energy (ΔG) led us to the identification of HK4 as a key ßdGT target, whereby the binding mode and domain dynamics suggested the activator function of ßdGT. ßdGT bound to the allosteric site of the isozyme â¼13 Å away from the substrate (glucose)-binding site. The binding free energy of the ligand-protein complex was energetically feasible (ΔG, -41.61 kcal/mol) and the cleft angle deviation between the two (small and large) domains of HK4 revealed differential HK4 dynamics in response to ßdGT binding. 3D structure analysis of the isozyme-ligand complex highlighted the role of Arg63, Glu67 and Lys458 in ligand stabilization and hydrophobic interactions mediated by Tyr214 and Met235. Experimental validation of the results of computational analysis confirmed the activator function of ßdGT on HK4. The study has implication in diabetes as ßdGT may be used to lower the blood glucose level by activating hepatic and pancreatic hexokinase without the risk of hypoglycemia.Communicated by Ramaswamy H. Sarma.
Assuntos
Hexoquinase , Fígado , Pâncreas , Xantonas/química , Hexoquinase/química , Fígado/enzimologia , Simulação de Acoplamento Molecular , Pâncreas/enzimologia , Ligação ProteicaRESUMO
The substrate kinetic parameters of hexokinase (HK) and phosphofructokinase (PFK)-the key irreversible enzymes of glycolysis-in erythrocytes from type 2 diabetic subjects were examined in comparison with control subjects. It was observed that the kinetic parameters such as Km , Vmax , Apparent Kcat , Kcat /Km , and substrate (ATP) inhibition kinetic and substrate binding characteristics are significantly altered in the diabetic group. The observed changes are suggestive of compositional changes in the subunit makeup of HK and PFK. The implication of these findings in relation to energy status of the diabetic erythrocyte and its interrelationship with loss of cell deformability are discussed here.