Effects of pH on Human Cardiac and Neuronal Sodium Channels

Voltage-gated sodium channels are large transmembrane proteins that selectively allow the passage of sodium ions across the cell membrane in response to membrane depolarizations (Yu and Catterall, 2003). The resulting influx of positive charge further depolarizes the membrane and is responsible for the upstroke of the action potential. Voltage-gated sodium channels are vital to the initiation and propagation of action potentials in excitable cells such as neurons and myocytes. In vertebrates, there are many different tissue-specific sodium channel isoforms; these include the neuronal (Nav1.2) and cardiac (Nav1.5) isoforms. Although very closely related in structure and functional characteristics, the different channel types have unique functional properties well suited to the physiological functions of their respective tissues

ATP Consumption Promotes Cancer Metabolism

Cancer cells metabolize glucose by aerobic glycolysis, a phenomenon known as the Warburg effect. Fang et al. (2010) show that the endoplasmic reticulum enzyme ENTPD5 promotes ATP consumption and favors aerobic glycolysis. The findings suggest that nutrient uptake in cancer cells is limited by ATP and satisfies energy requirements other than ATP production

2-Oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase

Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.National Human Genome Research Institute (U.S.) (Molecular Libraries Initiative of the NIH Roadmap for Medical Research

Cell-State-Specific Metabolic Dependency in Hematopoiesis and Leukemogenesis

The balance between oxidative and non-oxidative glucose metabolism is essential for a number of pathophysiological processes. By deleting enzymes that affect aerobic glycolysis with different potencies, we examine how modulating glucose metabolism specifically affects hematopoietic and leukemic cell populations. We find that deficiency in the M2 pyruvate kinase isoform (PKM2) reduces levels of metabolic intermediates important for biosynthesis and impairs progenitor function without perturbing hematopoietic stem cells (HSC), whereas lactate dehydrogenase-A (LDHA) deletion significantly inhibits the function of both HSC and progenitors during hematopoiesis. In contrast, leukemia initiation by transforming alleles putatively affecting either HSC or progenitors is inhibited in the absence of either PKM2 or LDHA, indicating that the cell state-specific responses to metabolic manipulation in hematopoiesis do not apply to the setting of leukemia. This finding suggests that fine-tuning the level of glycolysis may be therapeutically explored for treating leukemia while preserving HSC function.National Institutes of Health (U.S.) (Grants P30CA147882 and R01CA168653)Smith Family FoundationBurroughs Wellcome FundVirginia and D.K. Ludwig Fund for Cancer ResearchDamon Runyon Cancer Research Foundatio

Salt Uptake in Natural Channels Traversing Mancos Shales in the Price River Basin, Utah

Field and laboratory measurements of process rates for runoff and salt movement were used to develop and calibrate a hydrosalinity model of outlfows from the Price River Basin at Woodside, Utah. The field measurements were specifically used to formulate a model for estimating surface flow (both overland and from small ephemeral channels) in the Coal Creek Basin on the valley floor of the Price River Basin. The basin simulation assessment model (BSAM) was used to combine local flows and model total outflow from the Price River. The results must be regarded as a first generation model that, while giving ostensibly reasonable results, needs much additional refinement and validation by collecting additional field data. As to field data, observed salt loading rates reached 518 pounds per square mile daily, groundwater inflow declined steadily throughout the summer but maintained constant salt concentrations, channel efflorescence varied more than 100 fold with the largest concentrations occurring in saturated bed material, and turbulent mixing and cyclic drying added to salt dissolution rates. Extrapolation of the results with the Coal Creek model showed only a very small percentage of the salt loading from the valley floor to originate from natural lands. BSAM showed average annual salt leaving the Basin at Woodside to be 190,000 tons, 114,000 coming from the mountain area and 76,000 from the valley floor. Of the valley floor contribution, only 3,500 tons are produced by surface runoff from nonirrigated areas. Topics to be emphasized in further model development include salt contribution from percolation snowmelt on natural lands, groundwater movement, the formation and dissolution of efflorescence, and salt-sediment transport by the sharp hydrographs on small ephemeral streams

Germline loss of PKM2 promotes metabolic distress and hepatocellular carcinoma

Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase (PK), and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2-null mice (Pkm2[superscript −/−]). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2[superscript −/−] mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism.National Cancer Institute (U.S.) (Cancer Center Support Grant P30CA14051)Howard Hughes Medical InstituteBurroughs Wellcome FundSmith Family FoundationUnited States. Dept. of Health and Human Services (P01CA117969)United States. Dept. of Health and Human Services (R01CA168653)American Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipJane Coffin Childs Memorial Fund for Medical Research (Postdoctoral Fellowship

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.National Institutes of Health (U.S.) (NIH grant R01 GM56203)National Institutes of Health (U.S.) (grant NIH 5P01CA120964)Dana-Farber/Harvard Cancer Center (NIH 5P30CA006516)National Institutes of Health (U.S.) (NIH grant R03MH085679)National Human Genome Research Institute (U.S.) (Intramural Research Program)National Institutes of Health (U.S.) (Molecular Libraries Initiative of the NIH Roadmap for Medical Research

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism

The role of pyruvate kinase regulation in tumor growth and metabolism

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis. Vita.Includes bibliographical references.Cancer is a disease of inappropriate cell proliferation, and central carbon metabolism is highly regulated to support the unique anabolic needs of proliferating cells. Pyruvate kinase, the enzyme catalyzing the final step of glycolysis, is an important point of regulation. Mammals have four pyruvate kinase isoforms, and one in particular, the M2 isoform, is preferentially expressed in proliferative tissues, including cancers. We sought to determine the role and importance of PKM2 in cancer. The activity of pyruvate kinase M2 (PKM2) is down-regulated by pro-proliferative signaling in the cell, and reduced PK activity appears to be important for proliferative metabolism. Because oncogenic signaling reduces PKM2 activity, we hypothesized that artificially high intracellular pyruvate kinase activity would disrupt proliferative metabolism and hinder tumor growth. We found that increased PK activity due to expression of the constitutively-active PKM1 isoform or direct pharmacological activation of PKM2 perturbs metabolism and reduces tumor growth in a lung cancer xenograft model. These results suggested that PKM2 expression is selected for in cancers because PKM2 activity can be down-regulated in a controlled fashion. We next sought to determine if the PKM2 isoform is necessary for tumor proliferation. Deletion of PKM2 in a mouse model of BRCA1-deficient breast cancer demonstrated that PKM2 is not required for tumor formation or growth. PKM2-null tumors exhibited heterogeneous PKM1 expression, and tumor cell proliferation was associated with low PKM1 expression in the absence of PKM2. Analysis of human breast tumors revealed highly variable PKM2 protein expression, and heterozygous PKM2 mutations were found in many cancer types. These mutations cause truncations or amino acid changes in conserved regions of the enzyme. Determination of kinetic parameters of purified wild-type and mutant PKM2 showed that cancer-associated missense mutations reduce affinity of the enzyme for substrate, reduce maximum velocity, or disrupt response of the enzyme to activation by its allosteric activator FBP. These results suggest that cancer cells tolerate or select for reduced pyruvate kinase activity. We conclude that PKM2 is important because its activity is down-regulated to support proliferation, but the PKM2 isoform itself is not required for tumor formation or cancer cell proliferation.by William James Israelsen.Ph. D