Subunit Isoforms Research Articles

POU2F2 activates the Akt/mTOR signalling pathway and enhances B lymphocyte function during diabetic kidney disease by promoting PIK3CD transcription.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) is the predominant isoform of the catalytic subunit of PI3K in lymphocytes. Based on comprehensive bioinformatics, this study explores the functions of PIK3CD in diabetic kidney disease (DKD) progression in mice and B lymphocyte activity. Non-obese diabetic (NOD) mice that spontaneously develop DKD were applied as animal models. Lentiviral vector-mediated PIK3CD silencing was introduced in mice, followed by histological staining and biomarker assessments to evaluate DKD-associated symptoms. The activity of the Akt/mTOR signalling pathway was determined by western blot analysis. Following bioinformatics analyses, the interaction between POU class 2 homeobox 2 (POU2F2) and PIK3CD in B lymphocytes was determined by chromatin immunoprecipitation and luciferase reporter assays. Their functions in B cell function were identified by MTT, flow cytometry and IgG deposition assessment. PIK3CD was found to be highly expressed in the kidney of DKD mice. Knockdown of PIK3CD inactivated the Akt/mTOR signalling, thus ameliorating tissue injury and fibrosis, enhancing the expression of AQP1 and decreasing urinary NGAL contents, UACR, BUN and β-NAG/creatinine ratio. These effects were negated by the Akt-specific activator SC79. POU2F2 was found to promote PIK3CD transcription by binding to its promoter, thus activating the Akt/mTOR pathway. Knockdown of POU2F2 suppressed B cell proliferation in vitro and decreased B cell population and IgG deposition in the mouse kidney. However, these trends were reversed upon additional PIK3CD overexpression. This study demonstrates that POU2F2 mediates PIK3CD-dependent Akt/mTOR signalling activation, thus enhancing B lymphocyte function and promoting DKD progression.

Role of Na+-K+ ATPase Alterations in the Development of Heart Failure.

Na+-K+ ATPase is an integral component of cardiac sarcolemma and consists of three major subunits, namely the α-subunit with three isoforms (α1, α2, and α3), β-subunit with two isoforms (β1 and β2) and γ-subunit (phospholemman). This enzyme has been demonstrated to transport three Na and two K ions to generate a trans-membrane gradient, maintain cation homeostasis in cardiomyocytes and participate in regulating contractile force development. Na+-K+ ATPase serves as a receptor for both exogenous and endogenous cardiotonic glycosides and steroids, and a signal transducer for modifying myocardial metabolism as well as cellular survival and death. In addition, Na+-K+ ATPase is regulated by different hormones through the phosphorylation/dephosphorylation of phospholemman, which is tightly bound to this enzyme. The activity of Na+-K+ ATPase has been reported to be increased, unaltered and depressed in failing hearts depending upon the type and stage of heart failure as well as the association/disassociation of phospholemman and binding with endogenous cardiotonic steroids, namely endogenous ouabain and marinobufagenin. Increased Na+-K+ ATPase activity in association with a depressed level of intracellular Na+ in failing hearts is considered to decrease intracellular Ca2+ and serve as an adaptive mechanism for maintaining cardiac function. The slight to moderate depression of Na+-K+ ATPase by cardiac glycosides in association with an increased level of Na+ in cardiomyocytes is known to produce beneficial effects in failing hearts. On the other hand, markedly reduced Na+-K+ ATPase activity associated with an increased level of intracellular Na+ in failing hearts has been demonstrated to result in an intracellular Ca2+ overload, the occurrence of cardiac arrhythmias and depression in cardiac function during the development of heart failure. Furthermore, the status of Na+-K+ ATPase activity in heart failure is determined by changes in isoform subunits of the enzyme, the development of oxidative stress, intracellular Ca2+-overload, protease activation, the activity of inflammatory cytokines and sarcolemmal lipid composition. Evidence has been presented to show that marked alterations in myocardial cations cannot be explained exclusively on the basis of sarcolemma alterations, as other Ca2+ channels, cation transporters and exchangers may be involved in this event. A marked reduction in Na+-K+ ATPase activity due to a shift in its isoform subunits in association with intracellular Ca2+-overload, cardiac energy depletion, increased membrane permeability, Ca2+-handling abnormalities and damage to myocardial ultrastructure appear to be involved in the progression of heart failure.

The pro-drug C13 activates AMPK by two distinct mechanisms.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is expressed in almost all eukaryotic cells. In the canonical activation mechanism, it is activated by increases in AMP:ATP and ADP:ATP ratios that signify declining cellular energy status. Once activated, AMPK phosphorylates numerous targets that promote catabolic pathways generating ATP, while inhibiting anabolic and other processes that consume ATP, thus acting to restore energy homeostasis. Pharmacological agents that activate AMPK have been useful in identifying downstream targets and have potential as drugs for treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. One such agent is C13, a pro-drug with a phosphonate bis(isobutyryloxymethyl) ester moiety, with the isobutyryloxymethyl groups increasing membrane permeability. Following cellular uptake, C13 is cleaved to release C2, an AMP analogue and potent AMPK activator that is specific for complexes containing the α1 (but not the α2) catalytic subunit isoform. This has previously been assumed to be the sole mechanism by which C13 activates AMPK, with potential roles for the isobutyryloxymethyl groups being ignored. We now report that, following cleavage from C13, these protective groups are metabolized to formaldehyde, an agent that inhibits mitochondrial function and increases cellular AMP:ATP ratios, thus providing additional AMPK activation by the canonical mechanism.

Novel secreted regulators of glucose and lipid metabolism in the development of metabolic diseases.

The tight regulation of glucose and lipid metabolism is crucial for maintaining metabolic health. Dysregulation of these processes can lead to the development of metabolic diseases. Secreted factors, or hormones, play an essential role in the regulation of glucose and lipid metabolism, thus also playing an important role in the development of metabolic diseases such as type 2 diabetes and obesity. Given the important roles of secreted factors, there has been significant interest in identifying new secreted factors and new functions for existing secreted factors that control glucose and lipid metabolism. In this review, we evaluate novel secreted factors or novel functions of existing factors that regulate glucose and lipid metabolism discovered in the last decade, including secreted isoform of endoplasmic reticulum membrane complex subunit 10, vimentin, cartilage intermediate layer protein 2, isthmin-1, lipocalin-2, neuregulin-1 and neuregulin-4. We discuss their discovery, tissues of origin, mechanisms of action and sex differences, emphasising their potential to regulate metabolic processes central to diabetes. Additionally, we discuss the translational barriers, particularly the absence of identified receptors, that hamper their functional characterisation and further therapeutic development. Ultimately, the identification of new secreted factors may give insights into previously unidentified pathways of disease progression and mechanisms of glucose and lipid homeostasis.

Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation

Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.

Exploring the anti-inflammatory and immunomodulatory potential of licochalcone B against psoralidin-induced liver injury

Ethnopharmacological relevanceHerb-induced liver injury (HILI) represents an exacerbated inflammatory response, with Psoraleae fructus (PF) and its preparations recently associated with hepatotoxicity. Licorice, historically recognized as a detoxifying herbal remedy, is considered to possess hepatoprotective properties. Our previous research identified bavachin, bakuchiol, and psoralidin (PSO) as potential toxic constituents in PF, while licochalcone B (LCB) and echinatin were identified as bioactive components in licorice. However, evidence regarding the interactions of active compounds in herbs and their underlying mechanisms remains limited. Aim of the studyThe objective of this study is to assess the potential mechanisms through which LCB modulates immunological and anti-inflammatory responses to treat PSO-induced liver injury by using human hepatocyte cells (L02) and LPS-primed mice. MethodsThe ameliorative effects of LCB and echinatin on bavachin, bakuchiol, and PSO-induced liver injury were demonstrated in L02 cells. Subsequently, the efficacy of LCB on PSO-induced idiosyncratic liver injury was further validated in C57BL/6 mice under moderate inflammatory stress induced by LPS priming. The mechanisms were preliminarily explored with an integrated strategy of molecular docking, RT-PCR verification, and untargeted metabolomics. ResultsThe study shows that LCB significantly reduced cell injury induced by the three chemicals in PF and provided substantial protection against PSO-induced hepatic damage, as indicated by the levels of ALT, AST, and LDH. LCB normalized liver function and remarkedly alleviated hepatic lesions and inflammation caused by PSO in mice under moderate inflammatory stress. The mRNA profiles of both L02 cells and mice liver tissue revealed that LCB mitigated PSO-induced hepatotoxicity by regulating the gene expression of pro-inflammatory cytokines IL1B and TNF, as well as immunoinflammatory genes PIK3CA, AKT1, NFKB1, and NLRP3. Furthermore, untargeted metabolomics of liver tissue indicated that LCB could reverse the abnormal expression of 11 discriminatory metabolites, with the interrelationship between differential metabolites and target genes primarily clustering in glycerophospholipid metabolism, arachidonic acid metabolism, and phosphatidylinositol signaling system. ConclusionLCB demonstrated a superior anti-inflammatory and immunomodulatory effect on PSO-induced hepatotoxicity by modulating the inflammatory response and metabolic signaling system. Key interactive targets included phosphatidylcholine, phosphatidic acid, and subunit isoforms of PI3K.

High expression of eukaryotic elongation factor 1-alpha-2 in lung adenocarcinoma is associated with poor prognosis.

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) encodes an isoform of the alpha subunit of the elongation factor 1 complex and is responsible for the enzymatic delivery of aminoacyl tRNA to the ribosome. Our proteomic analysis has identified eEF1A2 as one of the proteins expressed during malignant progression from adenocarcinoma in situ (AIS) to early invasive lung adenocarcinoma. The expression level of eEF1A2 in 175 lung adenocarcinomas was examined by immunohistochemical staining in relation to patient prognosis and clinicopathological factors. Quantitative PCR analysis and fluorescence in situ hybridization (FISH) were performed to evaluate the amplification of the eEF1A2 gene. Relatively high expression of eEF1A2 was observed in invasive adenocarcinoma (39/144 cases) relative to minimally invasive adenocarcinoma (1/10 cases) or AIS (0/21 cases). Among invasive adenocarcinomas, solid-type adenocarcinoma (15/32 cases, 47%) showed higher expression than other histological subtypes (23/92, 25%). Patients with eEF1A2-positive tumors had a significantly poorer prognosis than those with eEF1A2-negative tumors. Of the five tumors that were eEF1A2-positive, two cases showed amplified genomic eEF1A2 DNA, which was confirmed by both qPCR and FISH. These findings indicate that eEF1A2 overexpression occurs in the course of malignant transformation of lung adenocarcinomas and is partly due to eEF1A2 gene amplification.

Progressive substitution of fish oil with Schizochytrium-derived algal oil in the diet of Atlantic salmon (Salmo salar) parr subjected to winter signal period

The aim of this study was to investigate the impact of progressive replacement of fish oil with Schizochytrium-derived oil from Veramaris® (VMO) in the diets of Atlantic salmon, focusing on its effect during the pre-adaptation to smoltification phase of parr, with an emphasis on the winter signal period. Substitution of fish oil with VMO during this critical developmental phase of salmon (pre-adaptation to smoltification phase) can contribute to the goal of reducing dependence on wild fisheries as a primary source of essential fatty acids in aquafeeds. Three isoproteic, isolipidic and isoenergetic diets were formulated; a control diet containing 5% fish oil and 12% plant oil (0VMO) and two other diets, in which 50% and 100% of the EPA+DHA from fish oil were replaced with VMO, henceforth referred to as 50VMO and 100VMO, respectively. The fish were exposed to a 12 L:12D photoperiod over a 49-day window with samples collected at five time points, including a 48-h salt water challenge to evaluate the effects on common smoltification-related biomarkers including the expression of two genes coding for the two protein isoforms of the NKA alpha catalytic subunit (nkaa1a, nkaa1b), the gene coding for NA+/K+Cl⁻ co-transporter (nkcc1) in the gills as well as osmolality, Cl-, Mg2+, Ca2+, cortisol, growth hormone, and insulin-like growth factor-1 in plasma. The results showed non-significant differences in growth performance and only minor differences in smoltification-related parameters between the dietary treatment groups. Overall, the unconventional source of EPA+DHA was not detrimental to growth, hypoosmoregulatory ability or general welfare, suggesting that the progressive substitution of fish oil with Schizochytrium-derived oil in diets for Atlantic salmon parr is a viable option for adoption by the aquaculture industry.

Calcineurin mediates the inhibitory effects of TNF on NKCC2 phosphorylation

We previously showed that TNF inhibits NKCC2 activity and phosphorylation along the TAL, however, the mechanism has not yet been defined. As calcineurin (CN) activity in synoviocytes was increased by proinflammatory cytokines such as IL-1 and TNF, we tested the hypothesis that induction of CN activity and expression of CN isoforms is part of a pathway by which TNF inhibits NKCC2 phosphorylation. In this study, we found that CN activity increased by approximately 2-fold in primary cultures of medullary thick ascending limb (mTAL) cells that were maintained at 37oC/5%CO2 then challenged with mouse recombinant TNF (1 nM) for 60 min. Similarly, TNF induced a 3-fold increase in CN activity when mTAL cells were exposed to hypertonic media (400 mOsm/Kg H2O), an effect that was prevented by cyclosporine A (CsA). In contrast, silencing TNF production in mTAL cells using lentivirus (U6-TNF-ex4) significantly reduced CN activity by approximately 56% in cells exposed to 400 mOsm/Kg H2O for 1 h. CN activity also was inhibited in cells pretreated with CsA for 30 min followed by incubation in 400 mOsm/Kg H2O. Our data indicate that NKCC2 phosphorylation was significantly decreased in cultured mTAL cells challenged with TNF (1 nM) for 4 hr while inhibition of CN activity with CsA increased pNKCC2 expression. Analysis of qRT-PCR experiments showed that mTAL cells express the calcineurin A subunit (CNA) isoforms a (Sequence ID: NM_001293622.1) and b (sequence ID: NM_001310426.1). However, only the mRNA abundance of the CNAb isoform increased when mTAL cells were incubated with TNF (1 nM) for 4 hr under NS or HS (400 mOsm/Kg H2O) conditions as TNF had no effect on the a isoform of CNA. In vivo, both TNF and CNAb expression increased in outer medulla (OM) from mice given 1% NaCl in the drinking water (HS) for 7 days while intrarenal silencing of TNF with the U6-TNF-ex4 lentivirus reduced expression of CNAb under NS or HS conditions. Similarly, intrarenal injection of a lentivirus construct that specifically silenced CNAb in the kidney (U6-CNAb-ex6) reduced expression of CNAb in mice ingesting HS for 7 days. Interestingly, co-administration of U6-CNAb-ex6 with U6-TNF-ex4 increased pNKCC2 expression by approximately 63% in the OM from mice compared with silencing of TNF alone. Collectively, these data are the first to demonstrate that TNF increases CN activity as well as specific isoform expression in the kidney. Since NKCC2 has been identified as a target of the CNAb isoform, which is increased by TNF, these data demonstrate that a calcineurin-dependent signaling pathway is part of the mechanism by which TNF inhibits the phosphorylation of NKCC2. NIH grants R01 HL133077 and HL153525. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Maackia amurensis seed lectin (MASL) and soluble human podoplanin (shPDPN) sequence analysis and effects on human oral squamous cell carcinoma (OSCC) cell migration and viability

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent.

GABPB1 plays a cancer-promoting role in non-small cell lung cancer

BackgroundGABPB1, the gene that encodes two isoforms of the beta subunit of GABP, has been identified as an oncogene in multiple malignant tumors. However, the role and mode of action of GABPB1 in malignant tumors, especially in lung cancer, are not well understood and need further research.MethodsOur research focused on examining the biological function of GABPB1 in NSCLC (Non-Small Cell Lung Cancer). We analysed tumor data from public databases to assess the expression of GABPB1 in NSCLC and its correlation with patient prognosis and investigated GABPB1 expression and methylation patterns in relation to the tumor microenvironment. In parallel, experiments were conducted using short hairpin RNA (shRNA) to suppress the GABPB1 gene in human lung cancer cells to evaluate the effects on cell proliferation, viability, and apoptosis.ResultsGABPB1 was widely expressed in various tissues of the human body. Compared to that in normal tissues, the expression of this gene was different in multiple tumor tissues. GABPB1 was highly expressed in lung cancer tissues and cell lines. Its expression was associated with molecular subtype and cellular signalling pathways, and a high level of GABPB1 expression was related to a poor prognosis in lung adenocarcinoma patients. The expression and methylation of GABPB1 affect the tumor microenvironment. After suppressing the expression of GABPB1 in both A549 and H1299 cells, we found a decrease in cell growth and expression, the formation of clones and an increase in the apoptosis rate.ConclusionsOur research verified that GABPB1 promotes the tumorigenesis of NSCLC and has an inhibitory effect on tumor immunity. The specific role of GABPB1 may vary among different pathological types of NSCLC. This molecule can serve as a prognostic indicator for lung adenocarcinoma, and its methylation may represent a potential breakthrough in treatment by altering the tumor immune microenvironment in lung squamous cell carcinoma. The role and mechanism of action of GABPB1 in NSCLC should be further explored.

CUL4B mutations impair human cortical neurogenesis through PP2A-dependent inhibition of AKT and ERK

Mutation in CUL4B gene is one of the most common causes for X-linked intellectual disability (XLID). CUL4B is the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complex. While the roles of CUL4B in cancer progression and some developmental processes like adipogenesis, osteogenesis, and spermatogenesis have been studied, the mechanisms underlying the neurological disorders in patients with CUL4B mutations are poorly understood. Here, using 2D neuronal culture and cerebral organoids generated from the patient-derived induced pluripotent stem cells and their isogenic controls, we demonstrate that CUL4B is required to prevent premature cell cycle exit and precocious neuronal differentiation of neural progenitor cells. Moreover, loss-of-function mutations of CUL4B lead to increased synapse formation and enhanced neuronal excitability. Mechanistically, CRL4B complex represses transcription of PPP2R2B and PPP2R2C genes, which encode two isoforms of the regulatory subunit of protein phosphatase 2 A (PP2A) complex, through catalyzing monoubiquitination of H2AK119 in their promoter regions. CUL4B mutations result in upregulated PP2A activity, which causes inhibition of AKT and ERK, leading to premature cell cycle exit. Activation of AKT and ERK or inhibition of PP2A activity in CUL4B mutant organoids rescues the neurogenesis defect. Our work unveils an essential role of CUL4B in human cortical development.

Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function.

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, β, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2-deficient mice displayed visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor-knockout mice. These findings highlight a therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a potentially previously uncharacterized mechanism affecting IMPDH activity.

Sensational site: the sodium pump ouabain-binding site and its ligands.

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and β subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology.

BAY-3827 and SBI-0206965: Potent AMPK Inhibitors That Paradoxically Increase Thr172 Phosphorylation.

AMP-activated protein kinase (AMPK) is the central component of a signalling pathway that senses energy stress and triggers a metabolic switch away from anabolic processes and towards catabolic processes. There has been a prolonged focus in the pharmaceutical industry on the development of AMPK-activating drugs for the treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. However, recent findings suggest that AMPK inhibitors might be efficacious for treating certain cancers, especially lung adenocarcinomas, in which the PRKAA1 gene (encoding the α1 catalytic subunit isoform of AMPK) is often amplified. Here, we study two potent AMPK inhibitors, BAY-3827 and SBI-0206965. Despite not being closely related structurally, the treatment of cells with either drug unexpectedly caused increases in AMPK phosphorylation at the activating site, Thr172, even though the phosphorylation of several downstream targets in different subcellular compartments was completely inhibited. Surprisingly, the two inhibitors appear to promote Thr172 phosphorylation by different mechanisms: BAY-3827 primarily protects against Thr172 dephosphorylation, while SBI-0206965 also promotes phosphorylation by LKB1 at low concentrations, while increasing cellular AMP:ATP ratios at higher concentrations. Due to its greater potency and fewer off-target effects, BAY-3827 is now the inhibitor of choice for cell studies, although its low bioavailability may limit its use in vivo.

Identification, classification, and characterization of alpha and beta subunits of LVP1 protein from the venom gland of four Iranian scorpion species

Scorpion venoms contain bioactive peptides and proteins. Some, can be used for pharmaceutical purposes. So, identification of venom proteins matters because, in addition to determining the function of the toxins can also be an excellent guide to developing new drugs. Here, we got transcriptome of venom glands from four Iranian scorpion species, including Hemsicorpius lepturus, Mesobuthus eupeus, Andructunus crassicuada, and Hottentotta saulcyi using cDNA library synthesis and high-throughput transcriptomic analysis of the venom glands. In a comparative way, we identified the cDNA encoding isoforms of subunits (alpha and beta) of BotLVP1/BmLVP1-like protein in the venom gland of three species except for H. lepturus. Characterization and structure determination of the LVP1_like proteins combined with gene map analysis provided evidence of the existence of some isoforms of LVP1_like proteins, encoded by genes with two exons and one intron, which can be classified in CSαβ superfamily in the venom gland of three Iranian scorpion species. According to the high similarity with BotLVP1 and BmLVP1, these proteins could also be potent to mediate cholesterol homeostasis. However, further research is needed to prove it, and this study just may lay the foundation lead to light up this way.

Identification of Melittin-Like Proteins with a Molecular Weight of 67 kDa that Interact with Na+/K+-ATPase

Melittin, a peptide from bee venom, was found to be able to interact with many proteins, including calmodulin target proteins and ion-transporting P-type ATPases. It is assumed that melittin mimics a protein module involved in protein-protein interactions within cells. Previously, a Na+/K+-ATPase containing the α1 isoform of the catalytic subunit was found to co-precipitate with a protein with a molecular weight of about 70 kDa that interacts with antibodies against melittin by cross immunoprecipitation. In the presence of a specific Na+/K+-ATPase inhibitor (ouabain), the amount of protein with a molecular weight of 70 kDa interacting with Na+/K+-ATPase increases. In order to identify melittin-like protein from murine kidney homogenate, a fraction of melittin-like proteins with a molecular weight of approximately 70 kDa was obtained using affinity chromatography with immobilized antibodies specific to melittin. By mass spectrometry analysis, the obtained protein fraction was found to contain three molecular chaperones of Hsp70 superfamily: mitochondrial mtHsp70 (mortalin), Hsp73, Grp78 (BiP) of endoplasmic reticulum. These data suggest that chaperones from the HSP-70 superfamily contain a melittin-like module.

Frequent loss-of-function mutations in the AMPK-α2 catalytic subunit suggest a tumour suppressor role in human skin cancers.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status activated by increases in AMP or ADP relative to ATP. Once activated, it phosphorylates targets that promote ATP-generating catabolic pathways or inhibit ATP-consuming anabolic pathways, helping to restore cellular energy balance. Analysis of human cancer genome studies reveals that the PRKAA2 gene (encoding the α2 isoform of the catalytic subunit) is often subject to mis-sense mutations in cancer, particularly in melanoma and non-melanoma skin cancers, where up to 70 mis-sense mutations have been documented, often accompanied by loss of the tumour suppressor NF1. Recently it has been reported that knockout of PRKAA2 in NF1-deficient melanoma cells promoted anchorage-independent growth in vitro, as well as growth as xenografts in immunodeficient mice in vivo, suggesting that AMPK-α2 can act as a tumour suppressor in that context. However, very few of the mis-sense mutations in PRKAA2 that occur in human skin cancer and melanoma have been tested to see whether they cause loss-of-function. We have addressed this by making most of the reported mutations and testing their activity when expressed in AMPK knockout cells. Of 55 different mis-sense mutations (representing 75 cases), 9 (12%) appeared to cause a total loss of activity, 18 (24%) a partial loss, 11 (15%) an increase in phenformin-stimulated kinase activity, while just 37 (49%) had no clear effect on kinase activity. This supports the idea that AMPK-α2 acts as a tumour suppressor in the context of human skin cancer.

Existence and importance of Na+K+-ATPase in the plasma membrane of boar spermatozoa.

Sodium-potassium-ATPase (Na+K+-ATPase), a target to treat congestive heart failure, is the only known receptor for cardiac glycosides implicated in intracellular signaling and additionally functions enzymatically in ion transport. Spermatozoa need transmembrane ion transport and signaling to fertilize, and Na+K+-ATPase is identified here for the first time in boar spermatozoa. Head plasma membrane (HPM) isolated from boar spermatozoa was confirmed pure by marker enzymes acid and alkaline phosphatase (218±23% and 245±38% enrichment, respectively, versus whole spermatozoa). Western immunoblotting detected α and β subunits (isoforms α1, α3, β1, β2, and β3) in different concentrations in whole spermatozoa and HPM. Immunofluorescence of intact sperm only detected α3 on the post-equatorial exterior membrane; methanol-permeabilized sperm also had α3 post-equatorially and other isoforms on the acrosomal ridge and cap. Mass spectrometry confirmed the presence of all isoforms in HPM. Incubating boar sperm in capacitating media to induce the physiological changes preceding fertilization significantly increased the percentage of capacitated sperm compared to 0h control (33.0±2.6%vs. 19.2±2.6% capacitated sperm, respectively; p=0.014) and altered the β2 immunofluorescence pattern. These results demonstrate the presence of Na+K+-ATPase in boar sperm HPM and that it changes during capacitation.

The B1 H + -ATPase ( Atp6v1b1 ) Subunit in Non-Type A Intercalated Cells is Required for Driving Pendrin Activity and the Renal Defense Against Alkalosis.

In the kidney, the B1 H + -ATPase subunit is mostly expressed in intercalated cells (IC). Its importance in acid-secreting type A ICs is evident in patients with inborn distal renal tubular acidosis and ATP6V1B1 mutations. However, the protein is also highly expressed in alkali-secreting non-type A ICs where its function is incompletely understood. We demonstrate in Atp6v1b1 knock out mice that the B1 subunit is critical for the renal response to defend against alkalosis during an alkali load or chronic furosemide treatment. These findings highlight the importance of non-type A ICs in maintaining acid-base balance in response to metabolic challenges or commonly used diuretics. Non-type A ICs in the collecting duct system express the luminal Cl - /HCO 3- exchanger pendrin and apical and/or basolateral H + -ATPases containing the B1 subunit isoform. Non-type A ICs excrete bicarbonate during metabolic alkalosis. Mutations in the B1 subunit (ATP6V1B1) cause distal renal tubular acidosis due to its role in acid secretory type A ICs. The function of B1 in non-type A ICs has remained elusive. We examined the responses of Atp6v1b1-/- and Atp6v1b1+/+ mice to an alkali load and to chronic treatment with furosemide. An alkali load or 1 week of furosemide resulted in a more pronounced hypokalemic alkalosis in male ATP6v1b1-/- versus Atp6v1b1+/+ mice that could not be compensated by respiration. Total pendrin expression and activity in non-type A ICs of ex vivo microperfused cortical collecting ducts were reduced, and β2 -adrenergic stimulation of pendrin activity was blunted in ATP6v1b1-/- mice. Basolateral H + -ATPase activity was strongly reduced, although the basolateral expression of the B2 isoform was increased. Ligation assays for H + -ATPase subunits indicated impaired assembly of V 0 and V 1 H + -ATPase domains. During chronic furosemide treatment, ATP6v1b1-/- mice also showed polyuria and hyperchloremia versus Atp6v1b1+/+ . The expression of pendrin, the water channel AQP2, and subunits of the epithelial sodium channel ENaC were reduced. Our data demonstrate a critical role of H + -ATPases in non-type A ICs function protecting against alkalosis and reveal a hitherto unrecognized need of basolateral B1 isoform for a proper H + -ATPase complexes assembly and ability to be stimulated.