Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

Rijksen, Gert; Staal, Gerard E.J.; Beks, P.J.; Streefkerk, M.; Akkerman, Jan Willem N.

doi:10.1016/0304-4165(82)90230-6

Cited by 27 publications

(19 citation statements)

References 17 publications

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“…Confocal microscopic analysis confirmed that membrane-associated GLUT1 was similar between HD and GSD-Ib neutrophils ( Figure 2E). The expression of HK1 mRNA is downregulated and HK2 is unchanged, but the expression of HK3, the major HK isozyme in neutrophils, 35,36 BLOOD, 1 MAY 2014 x VOLUME 123, NUMBER 18 …”

Section: Resultsmentioning

confidence: 99%

Molecular mechanisms of neutrophil dysfunction in glycogen storage disease type Ib

Jun

Weinstein

Lee

et al. 2014

Blood

114

133

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• A deficiency in G6PT impairs neutrophil energy homeostasis characterized by reduced intracellular levels of G6P, ATP, lactate, and NADPH. • Impaired energy homeostasis and activation of the HIF-1a/ PPAR-g pathway underlie neutrophil dysfunction in GSD-Ib.Glycogen storage disease type Ib (GSD-Ib) is an autosomal-recessive syndrome characterized by neutropenia and impaired glucose homeostasis resulting from a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT). The underlying cause of GSD-Ib neutropenia is an enhanced neutrophil apoptosis, but patients also manifest neutrophil dysfunction of unknown etiology. Previously, we showed G6PT interacts with the enzyme glucose-6-phosphatase-b (G6Pase-b) to regulate the availability of G6P/ glucose in neutrophils. A deficiency in G6Pase-b activity in neutrophils impairs both their energy homeostasis and function. We now show that G6PT-deficient neutrophils from GSD-Ib patients are similarly impaired. Their energy impairment is characterized by decreased glucose uptake and reduced levels of intracellular G6P, lactate, adenosine triphosphate, and reduced NAD phosphate, whereas functional impairment is reflected in reduced neutrophil respiratory burst, chemotaxis, and calcium mobilization. We further show that the mechanism of neutrophil dysfunction in GSD-Ib arises from activation of the hypoxia-inducible factor-1a/peroxisome-proliferators-activated receptor-g pathway.

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Section: Resultsmentioning

confidence: 99%

Molecular mechanisms of neutrophil dysfunction in glycogen storage disease type Ib

Jun

Weinstein

Lee

et al. 2014

Blood

114

133

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“…An analogous situation exists for the enzyme hexokinase, which binds to a proteinaceous binding site in the membrane of mitochondria. This enzyme is more resistant to digitonin solubilization than adenylate kinase, an enzyme whose liberation depends only on the permeabilization of the mitochondrial membrane (20,21). (iii) The third compartment is the chromaffmi granule, which is relatively insensitive to digitonin permeabilization (16,17) and therefore retains intragranular components such as dopamine 3-hydroxylase and catecholamines.…”

Section: Resultsmentioning

confidence: 99%

“…The plasma membrane of isolated chromaffin cells becomes leaky to proteins and small molecules, while the chromaffin granule membrane is unaffected and the calcium-sensitive secretory machinery remains relatively intact (16,17). Digitonin has been used to investigate the subcellular distribution of many small molecules and proteins (18)(19)(20)(21)(22)(23) Lactate dehydrogenase was measured by monitoring the formation of NADH from NAD. Aliquots (100 ,ul of cell lysate or digitonin medium) were incubated with 60 mM lactate/100 mM Tris, pH 9.0/1 mM NAD in a final vol of 1.1 ml at room temperature for 30 min.…”

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confidence: 99%

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Kelner¹,

Morita²,

Rossen³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosie,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14. 16.2] and phenylethanolamine N-methyltransferase (PMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1. 1.28) are involved in catecholamine biosynthesis and are considered soluble proteins. However, they may actually be localized on the surface of the chromaffin granule. We have used the detergent digitonin to permeabilize the plasma membrane of cultured adrenal chromaffln cells to investigate the subcellular localization of TyrOHase and PMTase. A digitonin titration of the release of proteins and catecholamines revealed the existence of at least three subcellular compartments that are distinguished by their digitonin sensitivity: (i) soluble proteins, which were released upon treatment of the cells with low digitonin concentrations (S jM), (ii) a "digitonin-sensitive" cytoplasnic protein pool, which required higher concentrations of digitonin for release (10 ,uM) and included TyrOHase and PMTase, and (iii) the chromaffln granule, which was insensitive to digitonin. Analysis of the rates of release of all of these proteins revealed that the rate of TyrOHase and PMTase release was slower at 10 IAM than at 40 jaM digitonin, while the rates of release of the other proteins were similar at both concentrations and varied in proportion to their respective sizes. Treatment with cytoskeletal disrupting agents had no effect on TyrOHase or PMTase eftlux. These data suggest that TyrOHase and PMTase are in a detergent-labile association in the cell. This is consistent with the concept that TyrOHase and PMTase may be localized on the surface of the chromffin granule.In epinephrine-forming cells of the adrenal medulla, the catecholamine biosynthetic pathway is composed of four enzymes, tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], aromatic amino acid decarboxylase (aromatic L-amino acid carboxy-lyase, EC 4.1.1.28), dopamine ,B-hydroxylase [dopamine l3-monooxygenase; 3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (P-hydroxylating), EC 1.14.17.1] and phenylethanolamine N-methyltransferase (PMTase; Sadenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28). Dopamine o-hydroxylase is localized in the interior of the chromaffin granule and on the internal face of the granule membrane (1), while TyrOHase, aromatic amino acid decarboxylase, and PMTase are generally considered to be "soluble" cytoplasmic enzymes and ate synthesized on free ribosomes (2-4). However, morphological and subcellular fractionation studies suggest the possible particulate localization of PMTase and TyrOHase, perhaps on the surface of the chromaffmi granule. By immunocytochemistry, both PMTase and TyrOHase have been seen to be associated with chromaffin granules (5-9), and one report shows TyrOHase to be localized on microtubules (8). In purification procedur...

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“…14,15 In particular, HK3 is the major hexokinase member present in granulocytes (70%-80% of total activity), with the remaining 20% to 30% of the activity provided by HK1. 16 Here we describe that HK3 is significantly down-regulated in primary APL, and its expression is restored on ATRA therapy. We further identified HK3 as direct transcriptional target of PU.1 and PML-RARA.…”

Section: Introductionmentioning

confidence: 96%

PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells

Federzoni

Valk

Torbett

et al. 2012

Blood

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The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, IntroductionAcute promyelocytic leukemia (APL or AML-M3) accounts for 5% to 8% of acute myeloid leukemia (AML) subtypes. 1 APL is characterized by a balanced chromosomal translocation involving the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor␣ (RARA) on chromosome 17 that results in expression of the oncogenic fusion gene PML-RARA. 2 PML-RARA is a gain-of-function protein that promotes leukemic transformation by impairing the formation of functional PML nuclear bodies 3 and repressing RARA target genes in a dominantnegative manner. 4 This repression interferes with gene expression programs involved in differentiation, apoptosis, and self-renewal and leads to increased cell survival and inhibition of terminal differentiation, with an accumulation of promyelocytes. The block in differentiation that is observed in APL cells can be reverted with pharmacologic doses of ATRA by triggering PML-RARA degradation and thus restoring normal myeloid differentiation (reviewed by de Thé and Chen 5 ).The Ets-family member PU.1 is a master transcriptional regulator of myeloid differentiation, 6,7 as evidenced in PU.1 null mice that die at birth because of the lack of functional myeloid cells. 7 Inhibition of PU.1-induced myeloid differentiation may represent a critical step in APL leukemogenesis. On the one hand, PU.1 is inhibited at the transcriptional level by PML-RARA binding to its promoter region 8 ; on the other hand, a recent study identified PML-RARA as a binding partner of PU.1, 4 resulting in repression of PU.1-activated genes. A majority of PU.1 target genes are directly involved in myeloid differentiation and function, such as CD11b, CD45, the granulocyte/macrophage colonystimulating factor receptor (GM-CSFR), myeloperoxidase, lysozyme, and neutrophil elastase (reviewed...

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Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

Cited by 27 publications

References 17 publications

Molecular mechanisms of neutrophil dysfunction in glycogen storage disease type Ib

Molecular mechanisms of neutrophil dysfunction in glycogen storage disease type Ib

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells

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