ATP Inhibition Research Articles

Nano β-tricalcium phosphate/hydrogel encapsulated scaffolds promote osteogenic differentiation of bone marrow stromal cells through ATP metabolism

An ideal scaffold should have good biocompatibility and osteogenic differentiation properties and provide interconnected porous networks for cell penetration and nutrient and waste transportation. To improve the biological activity of hydrogels, with nano-β-tricalcium phosphate (TCP) as a model, we successfully designed β-TCP nanoparticle/hydrogel encapsulated scaffolds, which improved the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) and focused on the osteogenic differentiation mechanism of the β-TCP nanoparticle-mediated microenvironment and phosphorus. Results showed that β-TCP nanoparticles affected phosphorylated ERK1/2 and BMP-2 signalling molecules in BMSCs through ATP metabolism. Addition of an ATP inhibitor (NEM) and SLC20a1 inhibitor (Foscarnet), significantly inhibited expression of phosphorylated ERK1/2 and BMP-2. This study also provides an understanding of the positive role of β-TCP nanoparticles and β-TCP nanoparticle/hydrogel-encapsulated scaffolds in the bone repair field.

Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Given evidence of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation and exerts its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. Metformin blocked LPS-induced ATP-dependent synthesis of the NLRP3 ligand mtDNA independently of AMP-activated protein kinase (AMPK) or NF-κB. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.

Another Form of Modified, Highly-Active 6-Phosphofructo-1-Kinase in Cancer Cells

Enhanced glycolytic flux is a hallmarks of cancer cells. Posttranslational modification of the key regulatory enzyme of glycolysis, 6-Phosphofructo-1- Kinase (Pfk1) might trigger metabolic flux deregulation. In the cancer cells the human 85 kDa muscle type nPfk-M enzyme can be proteolytically cleaved to form highly-active 47 kDa shorter fragments that retain activity but become resistant to feed-back inhibition. In several tumorigenic cell lines, no native 85 kDa liver type nPfk-L isoforms could be either found and only 70 kDa shorter fragments were detected by immune-blotting. To learn more about the cancer-specific modified sfPfk-L enzyme, the truncated human sfPfk-L gene encoding 70 kDa fragments was inserted into the pfk null yeast S.cerevisiae cell. The recombinant modified enzyme showed higher affinity toward the substrate fructose-6-phosphate, reduced sensitivity toward the citrate and ATP inhibition in respect to the recombinant native PFK-L enzyme. Partially purified cancer-specific sfPfk-L fragments lacking the C-portion of the enzyme showed some instability under the diluted conditions in the buffer in respect to the tetrameric native nPfk-L enzyme. Growth characteristics of the yeast transformant encoding short sfPfk-L enzymes were similar to those encoding shorter sfPfk-M enzymes. No growth of the transformant with the sfPfk-L gene was observed on glucose but it grew faster than the transformant with the native human nPfk-L enzyme in a narrow ecological niche with low maltose concentration and 10 mM of ethanol in the medium. Similar to modified 47 kDa sfPfk-M fragments, also the short 70 kDa nPfk- Lfragments might cause deregulation of the glycolytic flux in the yeast and in the cancer cells. In yeast, deregulated metabolic flux unbalances redox potential that results in reduced growth rate. However, the cancer cells beat the redox unbalance by rapid re-oxidation of redundant NADH that results in lactate formation while the growth rate remains high.

Targeting the HSP90-CDC37-kinase chaperone cycle: A promising therapeutic strategy for cancer.

Heat shock protein 90 (HSP90) is an indispensable molecular chaperone that facilitates the maturation of numerous oncoproteins in cancer cells, including protein kinases, ribonucleoproteins, steroid hormone receptors, and transcription factors. Although over 30 HSP90 inhibitors have steadily entered clinical trials, further clinical advancement has been restricted by their limited efficacy, inevitable heat shock response, and multiple side-effects, likely induced via an ATP inhibition mechanism. Since both ATP and various co-chaperones play essential roles in the HSP90 chaperone cycle to achieve integrated function, optimal therapeutics require an understanding of the dynamic interactions among HSP90, ATP, and cochaperones. To date, continuous research has promoted the exploration of the cochaperone cell division cycle 37 (CDC37) as a kinase-specific recognizer and has shown that the HSP90-CDC37-kinase complex is particularly relevant in cancers. Indeed, disrupting the HSP90-CDC37-kinase complex, rather than totally blocking the ATP function of HSP90, is emerging as an alternative way to avoid the limitations of current inhibitors. In this review, we first briefly introduce the HSP90-CDC37-kinase cycle and present the currently available approaches for inhibitor development targeting this cycle and provide insights into selective regulation of the kinase clients of HSP90 by more directional ways.

The KCNJ11-E23K Gene Variant Hastens Diabetes Progression by Impairing Glucose-Induced Insulin Secretion.

The ATP-sensitive K+ (KATP) channel controls blood glucose levels by coupling glucose metabolism to insulin secretion in pancreatic β-cells. E23K, a common polymorphism in the pore-forming KATP channel subunit (KCNJ11) gene, has been linked to increased risk of type 2 diabetes. Understanding the risk-allele-specific pathogenesis has the potential to improve personalized diabetes treatment, but the underlying mechanism has remained elusive. Using a genetically engineered mouse model, we now show that the K23 variant impairs glucose-induced insulin secretion and increases diabetes risk when combined with a high-fat diet (HFD) and obesity. KATP-channels in β-cells with two K23 risk alleles (KK) showed decreased ATP inhibition, and the threshold for glucose-stimulated insulin secretion from KK islets was increased. Consequently, the insulin response to glucose and glycemic control was impaired in KK mice fed a standard diet. On an HFD, the effects of the KK genotype were exacerbated, accelerating diet-induced diabetes progression and causing β-cell failure. We conclude that the K23 variant increases diabetes risk by impairing insulin secretion at threshold glucose levels, thus accelerating loss of β-cell function in the early stages of diabetes progression.

Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches.

6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.

New insights into KATP channel gene mutations and neonatal diabetes mellitus

To read the full abstract: Nat Rev Endocrinol 2020 May 6. doi: 10.1038/s41574-020-0351-y. [Epub ahead of print]. PMID:32376986

Antimicrobial Terpenoids as a Potential Substitute in Overcoming Antimicrobial Resistance.

There was a golden era where everyone thought that microbes can no longer establish threat to humans but the time has come where microbes are proposing strong resistance against the majority of antimicrobials. Over the years, the inappropriate use and easy availability of antimicrobials have made antimicrobial resistance (AMR) to emerge as the world's third leading cause of death. Microorganisms over the time span have acquired resistance through various mechanisms such as efflux pump, transfer through plasmids causing mutation, changing antimicrobial site of action, or modifying the antimicrobial which will lead to become AMR as the main cause of death worldwide by 2030. In order to overcome the emerging resistance against majority of antimicrobials, there is a need to uncover drugs from plants because they have proved to be effective antimicrobials due to the presence of secondary metabolites such as terpenoids. Terpenoids abundant in nature are produced in response to microbial attack have huge potential against various microorganisms through diverse mechanisms such as membrane disruption, anti-quorum sensing, inhibition of protein synthesis and ATP. New approaches like combination therapy of terpenoids and antimicrobials have increased the potency of treatment against various multidrug resistant microorganisms by showing synergism to each other.

Ethanol antagonizes P2X4 receptors in ventral tegmental area neurons

P2X4 receptors are found throughout the central nervous system, and studies have shown that these purinergic receptors are important regulators of alcohol intake. The ventral tegmental area (VTA) is an important region for the rewarding and reinforcing properties of alcohol, but the role of P2X4 receptors in this region is unknown. Using both immunohistochemical and electrophysiological methods, we examined the interaction between P2X4 receptors and alcohol on VTA neurons. Incubation of brain slices containing the VTA for 2 h with siRNA targeting P2X4 receptors resulted in about a 25% reduction in P2X4 immunoreactivity in tyrosine hydroxylase positive VTA neurons. In electrophysiological experiments, ATP (0.5-3 mM) produced a reduction in the spontaneous firing rate, and ethanol significantly reduced this inhibition. Exposure to siP2X4 for 2 h via the recording micropipette resulted in a suppression of the response of VTA neurons to ATP, but no significant reduction in the ethanol inhibition of the ATP response was observed after this P2X4 downregulation. These results support the idea that VTA neurons are inhibited by ATP, ethanol antagonizes this inhibition, and the ethanol-sensitive component of ATP inhibition is mediated by P2X4 receptors. This interaction of ethanol with P2X4 receptors may be an important regulator of the rewarding effects of ethanol, making P2X4 receptors an intriguing target for the development of agents to treat alcohol use disorders.

2070-P: KCNJ11 (Kir6.2) Gain-of-Function Mutation in Zebrafish Leads to Transient Neonatal Diabetes

The majority of neonatal diabetes (NDM), defined by early onset of hyperglycemia due to impaired β-cell function, is caused by gain-of-function (GOF) mutations in the ATP-sensitive potassium (KATP) channel genes (ABCC8 encoding SUR1 and KCNJ11 encoding Kir6.2). These GOF mutations lead to increased KATP channel activity, decreased β-cell excitability, and impaired insulin secretion. A zebrafish model of NDM can provide a high throughput platform for understanding mechanisms of NDM and testing potential therapeutic approaches. To create such a model, we used CRISPR mutagenesis to introduce the most common human Kir6.2 GOF mutation (R201H) into the equivalent zebrafish locus. Patch-clamp analysis demonstrates decreased ATP sensitivity in β-cells isolated from mutant fish (K1/2 for ATP inhibition = 17.21 ±1.66 mM in WT, 40.38 ± 7.6 mM in heterozygous R201H). Increasing [glucose] causes a robust increase of [Ca]i in WT islets expressing a GCaMP6s fluorescent reporter, but there is no glucose-dependent increase in R201H islets. R201H fish have smaller overall body size, indicative of failure to thrive, and β-cell density is reduced ∼50% in R201H compared to WT. Whole body glucose was measured from larval zebrafish using a fluorometric assay. Starting at 4 weeks of age, blood glucose was directly measured by glucometer from the transected cardiac cavity. While R201H animals are markedly hyperglycemic (BG > 250 mg/dl) at 4 weeks, blood sugar normalizes (BG <120 mg/dl) by ∼12 weeks of age. In conclusion, R201H mutants demonstrate the expected KATP GOF, loss of glucose-dependence of [Ca]i, and marked hyperglycemia early in life, proving a viable model of the molecular and cellular consequences of NDM. Intriguingly, early hyperglycemia is followed by normalization of blood sugar in older fish. This resolution of hyperglycemia is an unexpected finding, and may provide further insight into physiologic differences between transient and permanent NDM. Disclosure J. Ikle: None. S.S. Singareddy: None. R.C. Tryon: None. N. York: None. L. Yin: None. W. Chen: None. C. Nichols: None. Funding National Institutes of Health (DK109407); Pediatric Endocrine Society; Endocrine Fellows Foundation

New insights into KATP channel gene mutations and neonatal diabetes mellitus.

The ATP-sensitive potassium channel (KATP channel) couples blood levels of glucose to insulin secretion from pancreatic β-cells. KATP channel closure triggers a cascade of events that results in insulin release. Metabolically generated changes in the intracellular concentrations of adenosine nucleotides are integral to this regulation, with ATP and ADP closing the channel and MgATP and MgADP increasing channel activity. Activating mutations in the genes encoding either of the two types of KATP channel subunit (Kir6.2 and SUR1) result in neonatal diabetes mellitus, whereas loss-of-function mutations cause hyperinsulinaemic hypoglycaemia of infancy. Sulfonylurea and glinide drugs, which bind to SUR1, close the channel through a pathway independent of ATP and are now the primary therapy for neonatal diabetes mellitus caused by mutations in the genes encoding KATP channel subunits. Insight into the molecular details of drug and nucleotide regulation of channel activity has been illuminated by cryo-electron microscopy structures that reveal the atomic-level organization of the KATP channel complex. Here we review how these structures aid our understanding of how the various mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) lead to a reduction in ATP inhibition and thereby neonatal diabetes mellitus. We also provide an update on known mutations and sulfonylurea therapy in neonatal diabetes mellitus.

Studies on Structures and Functions of Kinases leading to Prostate Cancer and Their Inhibitors

Background:Cancer is the uncontrolled growth of abnormal cells in any part of the body. These abnormalities in the cells make them cancer cells, malignant cells, or tumour cells. These cells can infiltrate normal body tissues. Prostate Cancer begins when cells in the prostate gland start to grow out of control.Introduction:According to the National Cancer Institute, an estimated 20 percent of men experience Prostate Cancer in their lifetimes. Prostate Cancer can be divided into castration sensitive or hormone- sensitive Prostate Cancer (CSPC or HSPC) and castration-resistant Prostate Cancer (CRPC). Different therapies showed potential for the treatment of Prostate Cancer in that androgen receptor antagonist, cytochrome p17 inhibitors, radiation therapy, brachytherapy, surgical removal of the gland, androgen deprivation therapy and LnRH antagonists are some of the important ones. Despite various available treatment options in our understanding of the biological basis of Prostate Cancer, the management of the disease, especially in the castration-resistant phase, remains a significant challenge. Several Tyrosine kinase inhibitors (TKIs) have been evaluated in the preclinical setting in Advanced Prostate Cancer. TKIs are small drug molecules that work by competitive ATP inhibition at the catalytic binding site of tyrosine kinase. This results in complete inhibition of the catalytic activity of certain enzymes. If chosen correctly, TKIs can target and inhibit critical, mutated pathways important for the development, progression and metastasis of Prostate Cancer. The review focuses on various tyrosine kinase drug targets and their chemical structure to discuss the mechanism and pathways in the treatment of Prostate Cancer.Methods:The method adopted for the study was mainly based on the secondary search through a systematic literature review. Targets discussed in this review include the epidermal growth factor family (EGFR), vascular endothelial growth factor family (VEGF) receptor, c-Src family kinases (Proto-oncogene tyrosine-protein kinase) (c-Src), platelet-derived growth factor (PDGF) and cmesenchymal- epithelial transition factor (c-Met), which showed some promising results in various studies.Results:Even with the strong scientific rationale for many TKIs in the treatment of Prostate Cancer, the clinical trial experience showed some negative results in advanced phases. However, despite various challenges, the validation studies targeting kinases hold great potential in Prostate Cancer. Given the success of kinase inhibitors across multiple other cancer types, it is likely that this drug class will eventually improve outcomes in Prostate Cancer.Conclusion:Modifications in structures and certain other aspects of TKIs may make these agents promising for the treatment of Prostate Cancer.

Peptidomimetic Polo-Box-Targeted Inhibitors that Engage PLK1 in Tumor Cells and Are Selective against the PLK3 Tumor Suppressor.

The polo-box domain (PBD) of PLK1 determines mitotic substrate recognition and subcellular localization. Compounds that target PLK1 selectively are required due to the tumor-suppressor roles of PLK3. A structure-activity analysis of the PBD phosphopeptide binding motif has identified potent peptides that delineate the determinants required for mimicry by nonpeptidic inhibitors and provide insights into the structural basis for the selectivity of inhibitors for the PLK1 PBD. Fragment-ligated inhibitory peptides (FLIPs) obtained through REPLACE have been optimized to enhance in vitro binding and a systematic analysis of selectivity for PLK1 vs PLK3 has been carried out for peptides and peptidomimetics. Furthermore, these more drug-like non-ATP-competitive inhibitors had on-target engagement in a cellular context, as evidenced by stabilization of PLK1 in a thermal-shift assay and by inhibition of the phosphorylation of TCTP, a target of PLK1. Investigation in cells expressing a mutant PLK1 showed that these cells are sensitive to PBD inhibitors but dramatically resistant to clinically investigated ATP-competitive compounds. These results further validate targeting the PBD binding site in the move towards PLK1 inhibitors that are active against tumors resistant to ATP inhibitors.

Phosphoenolpyruvate carboxylase from the cyanobacterium Synechocystis sp. PCC 6803 is under global metabolic control by PII signaling.

Phosphoenolpyruvate carboxylase (PEPC) is the second major carbon-fixing enzyme in photoautotrophic organisms. PEPC is required for the synthesis of amino acids of the glutamate and aspartate family by replenishing the TCA cycle. Furthermore, in cyanobacteria, PEPC, together with malate dehydrogenase and malic enzyme, forms a metabolic shunt for the synthesis of pyruvate from PEP. During this process, CO2 is first fixed and later released again. Due to its central metabolic position, it is crucial to fully understand the regulation of PEPC. Here, we identify PEPC from the cyanobacterium Synechocystis sp. PCC 6803 (PEPC) as a novel interaction partner for the global signal transduction protein PII . In addition to an extensive characterization of PEPC, we demonstrate specific PII -PEPC complex formation and its enzymatic consequences. PEPC activity is tuned by the metabolite-sensing properties of PII : Whereas in the absence of PII, PEPC is subjected to ATP inhibition, it is activated beyond its basal activity in the presence of PII . Furthermore, PII -PEPC complex formation is inhibited by ADP and PEPC activation by PII -ATP is mitigated in the presence of 2-OG, linking PEPC regulation to the cell's global carbon/nitrogen status. Finally, physiological relevance of the in vitro measurements was proven by metabolomic analyses of Synechocystis wild-type and PII -deficient cells.

The acupuncture effect on mitochondria of experimental psoriasis.

Psoriasis is a chronic inflammation with alteration in keratinocyte proliferation, where the inflammatory condition is established through activation of dendritic cells, effectors T cells generated and attracted to skin tissue and, epithelial cells. The ancient science, acupuncture, one of key of Tradicional Chinese Medicine, acts especially in the skin. The importance of T lymphocytes in the inflammatory process is well established, such as the participation of mitochondrial dynamics in the activity of these cells. Therefore, we chose to evaluate mitofusin‐2 ( MTF2) and acetyl Coa carboxylase (ACC) proteins in this process.The aim of this study is to continue the evaluation of the effect of acupuncture on experimental psoriasis in mice, in this moment through the skin mitochondria.The study used males Balb/C mice (20 – 30 g), randomly sorted into 5 groups: control (2), psoriasis induced by IMQ (5), psoriasis induced by IMQ treated with acupuncture(10), treated with acupuncture (5), psoriasis induced by IMQ treated with sham acupuncture (5) . The psoriasis was induced by Imiquimod (IMQ), received, on the shaved back, a dose of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals)during 15 days. It was adopted acupuncture of Tung’s method, 88.17, 88.18 and 88.19 points or point Yintang as Sham during 15 consecutive days, starting on day 3 of IMQ at animals which received acupuncture treatment. It was collected fragments of the hurt tissue for histological study and spleen, adrenal glands, axial and inguinal lymph nodes were weighed, immunohistochemistry by mitochondrial biogenesis. The blades were treated with Leishman stain, mft‐2 and pACC antibodies. Statistic tests ANOVA one way.Treatment with IMQ caused a significant inflammatory process when compared to control, showing intense cellularity, mainly lymphocytes. The acupuncture application has reduced cell migration. In order to understand more deeply the influence of acupuncture on inflammatory skin disease, the pathway of mitochondrial biogenesis has been altered. The animals treated with acupuncture showed significative diference about mft‐2 presence when compared with group without treatment. In other hand, the acupuncture animals had very near results with only IMQ when ACC antibodies were .Mitochondrial dynamics act on metabolic balance and T cell response, in this context mitochondrial protein mitofusin‐2 plays an important role in controlling cell proliferation and apoptosis in vitro and in vivo. Thus, the cellular hyperproliferation observed in psoriasis may be a consequence of the destabilization of this protein. We observed that the treated animals suffered alteration in the expression of this protein when compared to the untreated ones.The enzyme acetyl‐CoA carboxylase participates in the AMP‐activated protein kinase (AMPK) cycle that regulates glucose metabolism. This leads to ATP generation and inhibition of ATP‐consuming events, such as fatty acid synthesis. Therefore, the presence of ACC may be a marker of cellular activity.The results observed in the acupuncture treatment demonstrated the influence in the mitochondrial biogenesis and in the immune response, altering the cellular environment.Support or Funding InformationLaboratory of Skin Pharmacology and PhysiopathologyComparing IMQ and ACP cellularityFigure 1

The structure of LRRK2 and the conformational changes that are associated with LRRK2 activation and Parkinson’s disease pathogenesis

Parkinson’s Disease (PD), a major neurodegenerative disorder, is characterized by chronic and progressive loss of dopaminergic neurons ultimately leading to death. Leucine‐rich repeat kinase 2 (LRRK2) is one of the greatest known genetic contributors to PD. LRRK2 is a large multi‐domain protein that contains a kinase domain as well as a Ras/GTPase (ROC) domain, followed by a COR domain. LRRK2 also acts as scaffold for the assembly of signaling complexes with its armadillo, ankyrin, leucine‐rich and WD40 domains at the N‐ and C‐termini. Studies have shown that most of the common familial mutations resulted in the formation of fibrous structures docked on microtubules in cells. However, how the fibrous structure is associated with LRRK2 activation is still unclear. Little is known about how LRRK2 activity is regulated, as well as the pathogenic mechanisms that drive PD. In this study, we use hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) to measure the solvent exposed regions of LRRK2. Purified truncated LRRK2 (RCKW), which includes ROC, COR, kinase and WD40 domain, was used to map conformational changes associated with ATP, GTP and kinase inhibitor binding. Mutations in the kinase domain, including two familial mutations (G2019S and I2020T), are characterized as well. This information will help us to understand the protein interfaces within and between LRRK2 oligomers involved in LRRK2 activity. Also, it will help to understand the conformation that drives the activation of LRRK2 and pathological docking onto microtubules.

Effects of different ATP contents on phosphorylation level of glycogen phosphorylase and its activity in lamb during incubation at 4 ℃ in vitro

AbstractThe aim of this study was to investigate the effects of ATP on glycogen phosphorylase activity in lamb during incubation at 4℃ in vitro. Sarcoplasmic proteins from lamb were extracted and treated with different contents of ATP to get three groups of glycogen phosphorylase with low, middle and high ATP content groups, the amount of ATP were 0.5, 2.0 and 2.5 μM per 100 μg protein, respectively. The control group was without ATP adding. The results showed that ATP promoted the phosphorylation of glycogen phosphorylase, and phosphorylation modification promoted its activity. But ATP inhibited the activity of glycogen phosphorylase and ATP preferentially participated in phosphorylation. When ATP concentration was 0.5 μM per 100 μg protein, the effect of phosphorylation modification on the activity of glycogen phosphorylase was equal to the inhibition of ATP. The effect of glycogen phosphorylase phosphorylation on its activity gradually became dominant as incubation time prolonged.

Locally-Induced CaMKII Translocation Requires Nucleotide Binding

Calcium-calmodulin-dependent protein kinase (CaMKII) is a molecule involved in several cell processes including plasticity related to learning and memory. Activation of NMDA-type glutamate receptors results in translocation of CaMKII to synapses. However, there are at least two distinct mechanisms by which glutamate-dependent CaMKII translocation occurs: one well-studied process resulting from whole-cell glutamate stimulation and one resulting from brief, local glutamate application. Unlike the relatively fast CaMKII translocation seen following whole-cell glutamate delivery (seconds), local application results in CaMKII translocation that occurs gradually within 6–10 min. This locally-induced translocation of CaMKII requires L-type Ca2+ channel co-activation but does not rely on GluN2B receptor subunit expression, unlike translocation following whole-cell application of glutamate. The current study examined if nucleotide binding is necessary for locally-induced CaMKII translocation, similar to CaMKII translocation resulting from whole-cell glutamate application. Three different mechanisms of inhibition were employed: staurosporine (ATP inhibitor), CaMKII(281–302) peptide inhibitor and expression of the K42M mutation. Locally-induced CaMKII translocation was moderately suppressed in the presence of either the broad-spectrum kinase inhibitor staurosporine (100 nm) or the CaMKII(281–302) peptide inhibitor. However, expression of the catalytically dead K42M mutation that prevents ATP-binding to CaMKII, significantly inhibited locally-induced translocation. Thus, CaMKII translocation following brief, local glutamate application requires nucleotide binding, providing support for future research into the molecular mechanisms of this distinct form of CaMKII translocation.

Regulation of TRPC5 currents by intracellular ATP: Single channel studies.

TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel levelusing patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels.

Virtual docking screening and QSAR studies to explore AKT and mTOR inhibitors acting on PI3K in cancers.

The phosphoinositide 3-kinase (PI3K) pathway is an important regulator of cell proliferation and metabolism. PI3K activation initiates a signal transduction cascade, of which the major effectors are the kinases AKT and mTOR. Aberrant activation of the PI3K/AKT/mTOR pathway is frequently observed in many human malignancies and the combination of compounds simultaneously targeting different related molecules in the PI3K/AKT/mTOR pathway leads to synergistic activity. To explore the competing common ATP inhibitors PI3K/AKT and PI3K/mTOR we developed a model PI3K-SAR 2D which made it possible to predict the bioactivity of inhibitors of AKT and mTOR towards PI3K; the interaction of the best inhibitors was evaluated by docking analysis and compared to that of dactolisib and pictilisib. A PI3K-SAR model with a correlation coefficient (R2) of 0.81706 and an RMSE of 0.16029 was obtained, which was validated and evaluated by a cross-validation method, LOO. The most predicted AKT and mTOR inhibitors present respectively pIC50 activities between 9.26–9.93 and 9.59–9.87.After docking and several comparisons, inhibitors with better predictions showed better affinity and interaction with PI3K compared to pictilisib and dactolisib, so we found that 4 inhibitors of AKT and 14 mTOR inhibitors met the criteria of Lipinski and Veber and could be future drugs.