Dynamic Turnover of PI4P and PI(4,5)P2 under Hypoxia Controls Electrostatic Plasma Membrane Targeting

Phosphatidylinositol (PtdIns) 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PI(4,5)P2 or PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, the complex mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. Here we report that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays we showed that PI4KIIIa, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIIIa to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. Our results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins.

Sulfonylurea-insensitive permanent neonatal diabetes caused by a severe gain-of-function Tyr330His substitution in Kir6.2

Background/Aims: Mutations in KCNJ11, the gene encoding the Kir6.2 subunit of pancreatic and neuronal KATP channels, are associated with a spectrum of neonatal diabetes diseases. Methods: Variant screening was used to identify cause of neonatal diabetes, and continuous glucose monitoring used to assess effectiveness of sulfonylurea treatment. Electrophysiological analysis of variant KATP channel function was used to determine molecular basis. Results: We identified a previously uncharacterized KCNJ11 mutation, c.988T>C [pTyr330His], in an Italian child diagnosed with sulfonylurea-resistant permanent neonatal diabetes and developmental delay (iDEND). Functional analysis of recombinant KATP channels reveals that this mutation causes a drastic gain-of-function, due to a reduction in ATP-inhibition. Further, we demonstrate that the Tyr330His substitution causes a significant decrease in sensitivity to the sulfonylurea, glibenclamide. Conclusions: In this subject, the KCNJ11(c.988T>C) mutation provoked neonatal diabetes, with mild developmental delay, which was insensitive to correction by sulfonylurea therapy. This is explained by the molecular loss of sulfonylurea sensitivity conferred by the Tyr330His substitution, and highlights the need for molecular analysis of such mutations.

Structural insights into the mechanism of nucleotide regulation of pancreatic KATP channel

ATP-sensitive potassium channels (KATP) are metabolic sensors that convert the intracellular ATP/ADP ratio to the excitability of cells. They are involved in many physiological processes and implicated in several human diseases. Here we present the cryo-EM structures of the pancreatic KATP channel in both the closed state and the pre-open state, resolved in the same sample. The nucleotides bind at the inhibitory sites of the Kir6.2 channel in the closed state but not in the pre-open state. Structural comparisons reveal the mechanism for ATP inhibition and Mg-ADP activation, two fundamental properties of KATP channels. Moreover, the structure also uncovers the activation mechanism of diazoxide-type KATP openers.

Cholate Disrupts Regulatory Functions of Cytochrome c Oxidase

Cytochrome c oxidase (CytOx), the oxygen-accepting and rate-limiting enzyme of mitochondrial respiration, binds with 10 molecules of ADP, 7 of which are exchanged by ATP at high ATP/ADP-ratios. These bound ATP and ADP can be exchanged by cholate, which is generally used for the purification of CytOx. Many crystal structures of isolated CytOx were performed with the enzyme isolated from mitochondria using sodium cholate as a detergent. Cholate, however, dimerizes the enzyme isolated in non-ionic detergents and induces a structural change as evident from a spectral change. Consequently, it turns off the “allosteric ATP-inhibition of CytOx”, which is reversibly switched on under relaxed conditions via cAMP-dependent phosphorylation and keeps the membrane potential and ROS formation in mitochondria at low levels. This cholate effect gives an insight into the structural-functional relationship of the enzyme with respect to ATP inhibition and its role in mitochondrial respiration and energy production.

The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel

ATP-sensitive potassium (KATP) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we use molecular dynamics (MD) simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, K39 shows a greater preference to co-ordinate with PIP2 than ATP. A neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2, reducing ATP inhibition. We also find direct effects on nucleotide binding from mutating E179, a residue proposed to interact with PIP2. Our work suggests PIP2 and ATP interact allosterically to regulate KATP channel activity.

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.

Multiple Mechanisms Regulate Eukaryotic Cytochrome C Oxidase

Cytochrome c oxidase (COX), the rate-limiting enzyme of mitochondrial respiration, is regulated by various mechanisms. Its regulation by ATP (adenosine triphosphate) appears of particular importance, since it evolved early during evolution and is still found in cyanobacteria, but not in other bacteria. Therefore the “allosteric ATP inhibition of COX” is described here in more detail. Most regulatory properties of COX are related to “supernumerary” subunits, which are largely absent in bacterial COX. The “allosteric ATP inhibition of COX” was also recently described in intact isolated rat heart mitochondria.

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.

Purinergic P2Y1 and P2Y2/4 receptors elicit distinct Ca2+ signaling patterns in hepatocytes via differential feedback regulation by Protein Kinase C

Extracellular nucleotides are key regulators of liver physiology. In primary rat hepatocytes, P2Y1 receptor (P2Y1R) activation by ADP generates cytosolic calcium ([Ca2+]c) oscillations with narrow spikes, whereas P2Y2/4R activation by UTP led to more complex broad [Ca2+]c oscillations. Both [Ca2+]c oscillation signatures were observed with the common agonist ATP. Inhibition of Gαq signaling with YM-254890 abolished ATP-induced [Ca2+]c oscillations, indicating that they depend on inositol 1,4,5-trisphosphate (IP3), and are not mediated by P2X receptors. The narrow P2Y1-linked [Ca2+]c spikes and the broad P2Y2/4-linked [Ca2+]c spikes are shaped by differential and complex PKC-mediated feedback mechanisms. Downregulation of PKC broadened both ADP- and UTP-induced [Ca2+]c oscillations, with a more pronounced effect on the former. PKC downregulation also selectively elicited a more robust response to ADP stimulation, enhancing oscillatory and sustained [Ca2+]c responses. Acute PKC modulation confirmed the importance of the negative PKC feedback regulation of P2Y1R-linked [Ca2+]c signals; such that PKC activation decreased [Ca2+]c oscillation frequency and PKC inhibition increased [Ca2+]c spike width. However, both PKC activation and inhibition decreased the spike width of P2Y2/4R-induced [Ca2+]c oscillations, suggesting that multiple opposing PKC feedback mechanisms shape P2Y2/4R responses. Significantly, plasma membrane Ca2+ entry was required for negative PKC feedback on P2Y1R-linked [Ca2+]c oscillations, whereas P2Y2/4R-linked [Ca2+]c oscillations were less sensitive to negative regulation by PKC and independent of Ca2+ influx. Thus, differential feedback regulation by PKC gives rise to receptor-specific [Ca2+]c oscillation profiles, which can encode the diverse physiological and pathophysiological responses to distinct agonists that all act through the IP3 signaling cascade.