Bi-enzyme competition based on ZIF-67 co-immobilization for real-time monitoring of exocellular ATP.

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.

Chemo-enzymatic functionalized sustainable cellulosic membranes: Impact of regional selectivity on ions capture and antifouling behavior.

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.

Molecular docking and simulation studies of phytocompounds derived from Centella asiatica and Andrographis paniculata against hexokinase II as mitocan agents.

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.

Linker residues regulate the activity and stability of hexokinase 2, a promising anticancer target.

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.

Hexokinase II-Derived Cell-Penetrating Peptide Mediates Delivery of MicroRNA Mimic for Cancer-Selective Cytotoxicity.

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.

HKDC1 C-terminal based peptides inhibit extranodal natural killer/T-cell lymphoma by modulation of mitochondrial function and EBV suppression.

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.

Polymerization in the actin ATPase clan regulates hexokinase activity in yeast.

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.

3D bimetallic Au/Pt nanoflowers decorated needle-type microelectrode for direct in situ monitoring of ATP secreted from living cells.

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.

CSN5 upregulates glycolysis to promote hepatocellular carcinoma metastasis via stabilizing the HK2 protein.

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.

Type 2 diabetes differentially affects the substrate saturation kinetic attributes of erythrocyte hexokinase and phosphofructokinase.

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.

KRAS4A directly regulates hexokinase 1.

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region1. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins-each capable of transforming cells-are encoded when KRAS is activated by mutation2. No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells3 in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen4 (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes3-5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation-depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.

Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays.

Adenosine triphosphate (ATP) is a crucial substrate and energy source commonly used in enzyme reactions. However, we demonstrated that the addition of this acidic compound to enzyme assay buffers can serve as a source of unnoticed pH changes. Even relatively low concentrations of ATP (up to 5 mM) shifted pH of reaction mixtures to acidic values. For example, Tris buffer lost buffering capacity at pH 7.46 by adding ATP at a concentration higher than 2 mM. In addition to the buffering capacity, the pH shifts differed with respect to the buffer concentration. High ATP concentrations are commonly used in hexokinase assays. We demonstrated how the presence of ATP affects pH of widely used enzyme assay buffers and inversely affected KM of human hexokinase 2 and S0.5 of human glucokinase. The pH optimum of human glucokinase was never reported before. We found that previously reported optimum of mammalian glucokinase was incorrect, affected by the ATP-induced pH shifts. The pH optimum of human glucokinase is at pH 8.5-8.7. Suggested is the full disclosure of reaction conditions, including the measurement of pH of the whole reaction mixtures instead of measuring pH prior to the addition of all the components.

[False paraprotein-induced hypoglycemia in the measurement of glucose by the hexokinase method]. / Fausse hypoglycémie induite par les paraprotéines lors de la mesure du glucose par la méthode de l'hexokinase.

A 67 years old woman with a Waldenström disease was admitted in the intensive care unit for dyspnea and fever. During hospitalization, episodes of undetectable glycemia were observed without any hypoglycemia symptoms. Plasma glucose was determined with the hexokinase method (recommended). From this observation, a literature review on PubMed was performed to investigate similar cases. In patients with protides in excess (e.g. immunoproliferative syndrome), absorption measurements could be disrupted by the precipitation of excess protein (IgM in most cases). Other parameters could be affected: bilirubin, phosphate, HDL cholesterol, GGT, CRP and calcemia. In our case, the main difficulty was to identify the cause of the interference and then correct it. Using a series of dilution, we prevented protide precipitation allowing correct glucose determination. Those interferences are rare, but present a real analytical difficulty. Biologists should be aware of those interferences because of dramatics consequences.

Identification of hexokinase family members in pear (Pyrus × bretschneideri) and functional exploration of PbHXK1 in modulating sugar content and plant growth.

Hexokinase (HXK) is a multifunctional protein that serves as a sugar sensor for glucose signaling and a catalyst for glycolysis. It has been well studied in many species, however, there is far less information about this family in pear. To investigate the roles of HXK in the growth and development of pear fruit, we performed a genome-wide analysis and identified the HXK gene family members in pear. In addition, we functionally characterized a glucose sensor gene, PbHXK1, in P. bretschneideri. In total, 10 HXK genes were identified in pear, and a multiple sequence alignment and phylogenetic analysis showed that PbHXK1 is a Type B HXK that contains four conserved domains, phosphate 1 and 2, sugar binding and adenosine, which are specific to plant HXKs and essential for enzymatic functions. A qRT-PCR analysis revealed that the relative expression levels of PbHXK1 were negatively correlated with sugar content but significantly positively correlated with HXK activity during pear fruit development. Furthermore, the overexpression of PbHXK1 in tomatoes significantly enhanced the HXK activity and decreased the sugar content. In addition, the growth of transgenic tomato plants overexpressing PbHXK1 was inhibited, leading to shortened internodes and smaller leaves. Thus, in pear, PbHXK1 encodes HXK, which regulated the sugar content in fruit and affected the growth and development of plants.

Crystal structures of rice hexokinase 6 with a series of substrates shed light on its enzymatic mechanism.

Hexokinases (HXKs) have determined to be multifaceted proteins, and they are the only ones able to phosphorylate glucose in plants. However, the binding mode for ATP to plant HXKs remains unclear. Here, we report the crystal structures of rice hexokinase 6 (OsHXK6) in four different forms: (i) apo-form, (ii) binary complex with D-Glc, (iii) quaternary complex with ADP, PO4 and Mg2+, and (iv) pentanary complex with D-Glc, ADP, PO4, and Mg2+. The apo form is in the open state conformation, and the three others are in the closed state, indicating that glucose and ADP-PO4 binding induces a large conformational change by domain rearrangement. The quaternary complex is a novel intermediate during the catalytic reaction we trapped for the first time, which provides a new evidence for the enzymatic mechanism of HXKs. In addition, the latter two complexes reveal the binding mode for ADP-PO4 to plant HXKs, which provide the structural explanation for the dual-function of OsHXK6. In addition, we identified that residues Gly112, Thr261, Gly262, and Gly450 are essential to the binding between ADP-PO4 and OsHXK6 by a series of single mutations and enzymatic assays. Our study provide structural basis for the other functional studies of OsHXK6 in rice.

Luteolin-7-O-ß-d-Glucoside Inhibits Cellular Energy Production Interacting with HEK2 in Keratinocytes.

Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems. Their functional phenolic groups are able to mediate antioxidant effects by scavenging free radicals. Molecules of this class have been found able to modulate the activity of kinases, phospholipase A2, cyclooxygenases, lipoxygenase, glutathione S-transferase, and many others. Recently, it has been demonstrated that luteolin, in the form of Luteolin-7-O-ß-d-glucoside (LUT-7G) is able to induce the keratinocyte differentiation process in vitro. This flavonoid is able to counteract the proliferative effects of IL-22/IL6 pathway by the inhibition of STAT3 activity also in vivo in a psoriatic mouse model. Observations on energy metabolism changes of differentiating cells led us to perform a complete metabolomics analysis using human primary keratinocytes treated with LUT-7G. Our results show that LUT-7G, is not only able to impair the nuclear translocation of STAT3, but it also blocks the energy metabolism pathway, depressing the glycolytic and Krebs pathway by the inhibition of hexokinase 2 activity. These data confirm that LUT-7G can be proposed as a potential candidate for the treatment of inflammatory and proliferative diseases, but its role as a hexokinase 2 (HEK2) inhibitor opens new perspectives in nutritional science, and especially in cancer therapy, in which the inhibition of the Warburg effect could be relevant.

Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics.

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelis⁻Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.

Substrate-driven chemotactic assembly in an enzyme cascade.

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.

The high proliferation rate of tumor cells demands high energy and metabolites that are sustained by a high glycolytic flux known as the 'Warburg effect'. The activation and further metabolism of glucose is initiated by hexokinase, a focal point of metabolic regulation. The human hexokinase 2 (HK2) is overexpressed in all aggressive tumors and predominantly found on the outer mitochondrial membrane, where interactions through its N-terminus initiates and maintains tumorigenesis. Here, we report the structure of HK2 in complex with glucose and glucose-6-phosphate (G6P). Structural and biochemical characterization of the mitochondrial conformation reveals higher conformational stability and slow protein unfolding rate (ku) compared with the cytosolic conformation. Despite the active site similarity of all human hexokinases, the N-domain of HK2 is catalytically active but not in hexokinase 1 and 3. Helix-α13 that protrudes out of the N-domain to link it to the C-domain of HK2 is found to be important in maintaining the catalytic activity of the N-half. In addition, the N-domain of HK2 regulates the stability of the whole enzyme in contrast with the C-domain. Glucose binding enhanced the stability of the wild-type (WT) enzyme and the single mutant D657A of the C-domain, but it did not increase the stability of the D209A mutant of the N-domain. The interaction of HK2 with the mitochondria through its N-half is proposed to facilitate higher stability on the mitochondria. The identification of structural and biochemical differences between HK2 and other human hexokinase isozymes could potentially be used in the development of new anticancer therapies.

Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria-transformed leukocytes.

One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKA is an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.