Hexokinase gene OsHXK1 positively regulates leaf senescence in rice.

BACKGROUND: Leaf senescence is a highly complex and meticulous regulatory process, and the disruption of any factor involved in leaf senescence might lead to premature or delayed leaf senescence and thus result in reduced or increased crop yields. Despite sincere efforts by scientists, there remain many unsolved problems related to the regulatory factors and molecular mechanisms of leaf senescence. RESULTS: This study successfully revealed that OsHXK1 was highly expressed in senescent leaves of rice. The upregulation of OsHXK1 led to premature senescence of rice leaves, a decreased level of chlorophyll, and damage to the chloroplast structure. The overexpression of OsHXK1 resulted in increases in glucose and ROS levels and produced programmed cell death (PCD) signals earlier at the booting stage. Further analysis showed that expression level of the respiratory burst oxidase homolog (RBOH) genes and OsGLO1 were increased in OsHXK1-overexpressing plants at the booting stage. CONCLUSIONS: Overall, the outcomes of this study suggested that OsHXK1 could act as a positive regulator of rice leaf senescence by mediating glucose accumulation and inducing an increase in ROS.

HK2 Mediated Glycolytic Metabolism in Mouse Photoreceptors Is Not Required to Cause Late Stage Age-Related Macular Degeneration-Like Pathologies.

Age-related macular degeneration (AMD) is a multifactorial disease of unclear etiology. We previously proposed that metabolic adaptations in photoreceptors (PRs) play a role in disease progression. We mimicked these metabolic adaptations in mouse PRs through deletion of the tuberous sclerosis complex (TSC) protein TSC1. Here, we confirm our previous findings by deletion of the other complex protein, namely TSC2, in rod photoreceptors. Similar to deletion of Tsc1, mice with deletion of Tsc2 in rods develop AMD-like pathologies, including accumulation of apolipoproteins, migration of microglia, geographic atrophy, and neovascular pathologies. Subtle differences between the two mouse models, such as a significant increase in microglia activation with loss of Tsc2, were seen as well. To investigate the role of altered glucose metabolism in disease pathogenesis, we generated mice with simulation deletions of Tsc2 and hexokinase-2 (Hk2) in rods. Although retinal lactate levels returned to normal in mice with Tsc2-Hk2 deletion, AMD-like pathologies still developed. The data suggest that the metabolic adaptations in PRs that cause AMD-like pathologies are independent of HK2-mediated aerobic glycolysis.

Guard cells control hypocotyl elongation through HXK1, HY5, and PIF4.

The hypocotyls of germinating seedlings elongate in a search for light to enable autotrophic sugar production. Upon exposure to light, photoreceptors that are activated by blue and red light halt elongation by preventing the degradation of the hypocotyl-elongation inhibitor HY5 and by inhibiting the activity of the elongation-promoting transcription factors PIFs. The question of how sugar affects hypocotyl elongation and which cell types stimulate and stop that elongation remains unresolved. We found that overexpression of a sugar sensor, Arabidopsis hexokinase 1 (HXK1), in guard cells promotes hypocotyl elongation under white and blue light through PIF4. Furthermore, expression of PIF4 in guard cells is sufficient to promote hypocotyl elongation in the light, while expression of HY5 in guard cells is sufficient to inhibit the elongation of the hy5 mutant and the elongation stimulated by HXK1. HY5 exits the guard cells and inhibits hypocotyl elongation, but is degraded in the dark. We also show that the inhibition of hypocotyl elongation by guard cells' HY5 involves auto-activation of HY5 expression in other tissues. It appears that guard cells are capable of coordinating hypocotyl elongation and that sugar and HXK1 have the opposite effect of light on hypocotyl elongation, converging at PIF4.

Hexokinase 2 in Cancer: A Prima Donna Playing Multiple Characters.

Hexokinases are a family of ubiquitous exose-phosphorylating enzymes that prime glucose for intracellular utilization. Hexokinase 2 (HK2) is the most active isozyme of the family, mainly expressed in insulin-sensitive tissues. HK2 induction in most neoplastic cells contributes to their metabolic rewiring towards aerobic glycolysis, and its genetic ablation inhibits malignant growth in mouse models. HK2 can dock to mitochondria, where it performs additional functions in autophagy regulation and cell death inhibition that are independent of its enzymatic activity. The recent definition of HK2 localization to contact points between mitochondria and endoplasmic reticulum called Mitochondria Associated Membranes (MAMs) has unveiled a novel HK2 role in regulating intracellular Ca2+ fluxes. Here, we propose that HK2 localization in MAMs of tumor cells is key in sustaining neoplastic progression, as it acts as an intersection node between metabolic and survival pathways. Disrupting these functions by targeting HK2 subcellular localization can constitute a promising anti-tumor strategy.

Toxicity and metabolism of 3-bromopyruvate in Caenorhabditis elegans.

In this study, we aimed to evaluate the toxic effects, changes in life span, and expression of various metabolism-related genes in Caenorhabditis elegans, using RNA interference (RNAi) and mutant strains, after 3-bromopyruvate (3-BrPA) treatment. C. elegans was treated with various concentrations of 3-BrPA on nematode growth medium (NGM) plates, and their survival was monitored every 24 h. The expression of genes related to metabolism was measured by the real-time fluorescent quantitative polymerase chain reaction (qPCR). Nematode survival in the presence of 3-BrPA was also studied after silencing three hexokinase (HK) genes. The average life span of C. elegans cultured on NGM with 3-BrPA was shortened to 5.7 d compared with 7.7 d in the control group. hxk-1, hxk-2, and hxk-3 were overexpressed after the treatment with 3-BrPA. After successfully interfering hxk-1, hxk-2, and hxk-3, the 50% lethal concentration (LC50) of all mutant nematodes decreased with 3-BrPA treatment for 24 h compared with that of the control. All the cyp35 genes tested were overexpressed, except cyp-35B3. The induction of cyp-35A1 expression was most obvious. The LC50 values of the mutant strains cyp-35A1, cyp-35A2, cyp-35A4, cyp-35B3, and cyp-35C1 were lower than that of the control. Thus, the toxicity of 3-BrPA is closely related to its effect on hexokinase metabolism in nematodes, and the cyp-35 family plays a key role in the metabolism of 3-BrPA.

Toxicity and metabolism of 3-bromopyruvate in Caenorhabditis elegans / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In this study, we aimed to evaluate the toxic effects, changes in life span, and expression of various metabolism-related genes in Caenorhabditis elegans, using RNA interference (RNAi) and mutant strains, after 3-bromopyruvate (3-BrPA) treatment. C. elegans was treated with various concentrations of 3-BrPA on nematode growth medium (NGM) plates, and their survival was monitored every 24 h. The expression of genes related to metabolism was measured by the real-time fluorescent quantitative polymerase chain reaction (qPCR). Nematode survival in the presence of 3-BrPA was also studied after silencing three hexokinase (HK) genes. The average life span of C. elegans cultured on NGM with 3-BrPA was shortened to 5.7 d compared with 7.7 d in the control group. hxk-1, hxk-2, and hxk-3 were overexpressed after the treatment with 3-BrPA. After successfully interfering hxk-1, hxk-2, and hxk-3, the 50% lethal concentration (LC50) of all mutant nematodes decreased with 3-BrPA treatment for 24 h compared with that of the control. All the cyp35 genes tested were overexpressed, except cyp-35B3. The induction of cyp-35A1 expression was most obvious. The LC50 values of the mutant strains cyp-35A1, cyp-35A2, cyp-35A4, cyp-35B3, and cyp-35C1 were lower than that of the control. Thus, the toxicity of 3-BrPA is closely related to its effect on hexokinase metabolism in nematodes, and the cyp-35 family plays a key role in the metabolism of 3-BrPA.

Hepatic hexokinase domain containing 1 (HKDC1) improves whole body glucose tolerance and insulin sensitivity in pregnant mice.

Hexokinase domain containing 1, a recently discovered putative fifth hexokinase, is hypothesized to play key roles in glucose metabolism. Specifically, during pregnancy in a recent genome wide association study (GWAS), a strong correlation between HKDC1 and 2-h plasma glucose in pregnant women from different ethnic backgrounds was shown. Our earlier work also reported diminished glucose tolerance during pregnancy in our whole body HKDC1 heterozygous mice. Therefore, we hypothesized that HKDC1 plays important roles in gestational metabolism, and designed this study to assess the role of hepatic HKDC1 in whole body glucose utilization and insulin action during pregnancy. We overexpressed human HKDC1 in mouse liver by injecting a human HKDC1 adenoviral construct; whereas, for the liver-specific HKDC1 knockout model, we used AAV-Cre constructs in our HKDC1fl/fl mice. Both groups of mice were subjected to metabolic testing before and during pregnancy on gestation day 17-18. Our results indicate that hepatic HKDC1 overexpression during pregnancy leads to improved whole-body glucose tolerance and enhanced hepatic and peripheral insulin sensitivity while hepatic HKDC1 knockout results in diminished glucose tolerance. Further, we observed reduced gluconeogenesis with hepatic HKDC1 overexpression while HKDC1 knockout led to increased gluconeogenesis. These changes were associated with significantly enhanced ketone body production in HKDC1 overexpressing mice, indicating that these mice shift their metabolic needs from glucose reliance to greater fat oxidation and ketone utilization during fasting. Taken together, our results indicate that hepatic HKDC1 contributes to whole body glucose disposal, insulin sensitivity, and aspects of nutrient balance during pregnancy.

Genome-wide identification of hexokinase gene family in Brassica napus: structure, phylogenetic analysis, expression, and functional characterization.

MAIN CONCLUSION: Genome-wide identification, expression analysis, and functional characterization of previously uncharacterized hexokinase family of oil crop, Brassica napus, underscore the importance of this gene family in plant growth and development. In plants, the multi-gene family of dual-function hexokinases (HXKs) plays important roles in sugar metabolism and sensing that affect growth and development. Rapeseed (Brassica napus L.) is an important oil crop; however, little is known about the B. napus HXK gene family. We identified 19 putative HXKs in B. napus genome. B. rapa and B. oleracea, the two diploid progenitors of B. napus, contributed almost equally to the BnHXK genes. Phylogenetic analysis divided the 19 BnHXKs into four groups. The exon-intron structures of BnHXKs share high similarity to those of HXKs in Arabidopsis and rice. The group III and IV BnHXKs are highly expressed in roots, whereas group I members preferentially express in leaves. Analysis of seed transcriptomes at different developmental stages showed that most of group I and IV HXKs are highly expressed 2-weeks after pollination (2WAP), compared to 4WAP for group III. BnHKXs are differentially expressed in susceptible and tolerant B. napus cultivars after fungal infection, suggesting the possible involvement in defense response. We generated rapeseed RNAi lines for BnHXK9, a member of relatively less characterized group IV, by pollen-mediated gene transformation. The seedlings of BnHXK9-RNAi lines showed delayed growth compared to the wild type. The RNAi plants were dwarf with curly leaves, suggesting the involvement of BnHXK9 in plant development. Collectively, our findings provides a comprehensive account of BnHXK gene family in an important crop and a starting point for further elucidation of their roles in sugar metabolism and sensing, as well as plant growth and development.

Reciprocal role of SIRT6 and Hexokinase 2 in the regulation of autophagy driven monocyte differentiation.

Emerging evidences suggest the impact of autophagy on differentiation but the underlying molecular links between metabolic restructuring and autophagy during monocyte differentiation remain elusive. An increase in PPARγ, HK2 and SIRT6 expression was observed upon PMA induced monocyte differentiation. While PPARγ positively regulated HK2 and SIRT6 expression, the latter served as a negative regulator of HK2. Changes in expression of these metabolic modelers were accompanied by decreased glucose uptake and increase in Chibby, a potent antagonist of ß-catenin/Wnt pathway. Knockdown of Chibby abrogated PMA induced differentiation. While inhibition of HK2 either by Lonidamine or siRNA further elevated PMA induced Chibby, mitochondrial ROS, TIGAR and LC3II levels; siRNA mediated knock-down of SIRT6 exhibited contradictory effects as compared to HK2. Notably, inhibition of autophagy increased HK2, diminished Chibby level and CD33 expression. In addition, PMA induced expression of cytoskeletal architectural proteins, CXCR4, phagocytosis, acquisition of macrophage phenotypes and release of pro-inflammatory mediators was found to be HK2 dependent. Collectively, our findings highlight the previously unknown reciprocal influence of SIRT6 and HK2 in regulating autophagy driven monocyte differentiation.

[Interaction of DNAJB13 with HK1 in mouse].

OBJECTIVE: To investigate the presence of interactions between DNAJB13 and HK1. METHODS: The open reading frame of Dnajb13 gene was amplified from mouse testis cDNA by PCR. The PCR products were then inserted into pGEX-4T-1 vector after double digestion and identified by sequencing. The recombinant plasmids were transformated into competent DH5a cells, and the fusion protein was expressed with IPTG induction. SDS-PAGE Coomassie brilliant blue staining and Western blot analysis were used to detect the fusion protein expression. The protein precipitated by GST-DNAJB13 in GST pull down assay was detected by Western blotting. RESULTS: The recombinant plasmid pGEX-4T-1-Dnajb13 was successfully constructed and verified. E.coli transformed with the recombinant plasmid expressed abundant fusion protein. GST pull down assay showed interactions between DNAJB13 and HK1. CONCLUSION: DNAJB13 interacts with HK1 in mouse testis and probably participates in spermatogenesis and the regulation of sperm motility.

Involvement of Arabidopsis Hexokinase1 in Cell Death Mediated by Myo-Inositol Accumulation.

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants.

Role of hexokinase-1 in the survival of HIV-1-infected macrophages.

Viruses have developed various strategies to protect infected cells from apoptosis. HIV-1 infected macrophages are long-lived and considered reservoirs for HIV-1. One significant deciding factor between cell survival and cell death is glucose metabolism. We hypothesized that HIV-1 protects infected macrophages from apoptosis in part by modulating the host glycolytic pathway specifically by regulating hexokinase-1 (HK-1) an enzyme that converts glucose to glucose-6-phosphate. Therefore, we analyzed the regulation of HK-1 in HIV-1 infected PBMCs, and in a chronically HIV-1 infected monocyte-like cell line, U1. Our results demonstrate that HIV-1 induces a robust increase in HK-1 expression. Surprisingly, hexokinase enzymatic activity was significantly inhibited in HIV-1 infected PBMCs and in PMA differentiated U1 cells. Interestingly, we observed increased levels of mitochondria-bound HK-1 in PMA induced U1 cells and in the HIV-1 accessory protein, viral protein R (Vpr) transduced U937 cell derived macrophages. Dissociation of HK-1 from mitochondria in U1 cells using a pharmacological agent, clotrimazole (CTZ) induced mitochondrial membrane depolarization and caspase-3/7 mediated apoptosis. Dissociation of HK-1 from mitochondria in Vpr transduced U937 also activated caspase-3/7 activity. These observations indicate that HK-1 plays a non-metabolic role in HIV-1 infected macrophages by binding to mitochondria thereby maintaining mitochondrial integrity. These results suggest that targeting the interaction of HK-1 with the mitochondria to induce apoptosis in persistently infected macrophages may prove beneficial in purging the macrophage HIV reservoir.

VDAC electronics: 1. VDAC-hexo(gluco)kinase generator of the mitochondrial outer membrane potential.

The simplest mechanism of the generation of the mitochondrial outer membrane potential (OMP) by the VDAC (voltage-dependent anion channel)-hexokinase complex (VHC), suggested earlier, and by the VDAC-glucokinase complex (VGC), was computationally analyzed. Even at less than 4% of VDACs bound to hexokinase, the calculated OMP is high enough to trigger the electrical closure of VDACs beyond the complexes at threshold concentrations of glucose. These results confirmed our previous hypothesis that the Warburg effect is caused by the electrical closure of VDACs, leading to global restriction of the outer membrane permeability coupled to aerobic glycolysis. The model showed that the inhibition of the conductance and/or an increase in the voltage sensitivity of a relatively small fraction of VDACs by factors like tubulin potentiate the electrical closure of the remaining free VDACs. The extrusion of calcium ions from the mitochondrial intermembrane space by the generated OMP, positive inside, might increase cancer cell resistance to death. Within the VGC model, the known effect of induction of ATP release from mitochondria by accumulated glucose-6-phosphate in pancreatic beta cells might result not only of the known effect of GK dissociation from the VDAC-GK complex, but also of a decrease in the free energy of glucokinase reaction, leading to the OMP decrease and VDAC opening. We suggest that the VDAC-mediated electrical control of the mitochondrial outer membrane permeability, dependent on metabolic conditions, is a fundamental physiological mechanism of global regulation of mitochondrial functions and of cell death.

Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in heart.

Mitochondrially bound hexokinase II (mtHKII) has long been known to confer cancer cells with their resilience against cell death. More recently, mtHKII has emerged as a powerful protector against cardiac cell death. mtHKII protects against ischaemia-reperfusion (IR) injury in skeletal muscle and heart, attenuates cardiac hypertrophy and remodelling, and is one of the major end-effectors through which ischaemic preconditioning protects against myocardial IR injury. Mechanisms of mtHKII cardioprotection against reperfusion injury entail the maintenance of regulated outer mitochondrial membrane (OMM) permeability during ischaemia and reperfusion resulting in stabilization of mitochondrial membrane potential, the prevention of OMM breakage and cytochrome C release, and reduced reactive oxygen species production. Increasing mtHK may also have important metabolic consequences, such as improvement of glucose-induced insulin release, prevention of acidosis through enhanced coupling of glycolysis and glucose oxidation, and inhibition of fatty acid oxidation. Deficiencies in expression and distorted cellular signalling of HKII may contribute to the altered sensitivity of diabetes to cardiac ischaemic diseases. The interaction of HKII with the mitochondrion constitutes a powerful endogenous molecular mechanism to protect against cell death in almost all cell types examined (neurons, tumours, kidney, lung, skeletal muscle, heart). The challenge now is to harness mtHKII in the treatment of infarction, stroke, elective surgery and transplantation. Remote ischaemic preconditioning, metformin administration and miR-155/miR-144 manipulations are potential means of doing just that.

Hexokinase 2 overexpression promotes the proliferation and survival of laryngeal squamous cell carcinoma.

Proliferating cancer cells preferentially use anaerobic glycolysis rather than oxidative phosphorylation for energy production. Hexokinase 2 (HK2) is highly expressed in many malignant cells and is necessary for anaerobic glycolysis. The role of HK2 in laryngeal squamous cell carcinoma (LSCC) is unknown. In this study, the expression of HK2 in LSCC was investigated and the effect of inhibiting HK2 expression with small hairpin RNA (shRNA) on tumor growth was investigated. Using immunohistochemistry, HK2 expression was assessed in LSCC tissues. Human laryngeal carcinoma Hep-2 cells were stably transfected with a plasmid expressing HK2 shRNA (pGenesil-1.1-HK2) and were compared to control cells with respect to the cell cycle, cell viability, apoptosis, and their ability to form xenograft tumors. HK2 expression was significantly higher in LSCC than in papilloma or glottis polypus. Tumor samples of higher T, N, and TNM stage often had stronger HK2 staining. HK2 shRNA reduced HK2 mRNA, protein levels, and HK activity in Hep-2 cells. HK2 cells expressing shRNA demonstrated a higher G0-G1 ratio, increased apoptosis, and reduced viability. Xenograft tumors derived from cells expressing HK2 shRNA were smaller and had lower proliferation than those from untransfected or control-plasmid-transfected cells. In conclusion, depletion of HK2 expression resulted in reduced xenograft tumor development likely by reducing proliferation, altering the cell cycle, reducing cell viability and activating apoptosis. These data suggest that HK2 plays an important role in the development of LSCC and represents a potential therapeutic target for LSCC.

Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes.

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and -0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.

Hexokinase mediates stomatal closure.

Stomata, composed of two guard cells, are the gates whose controlled movement allows the plant to balance the demand for CO2 for photosynthesis with the loss of water through transpiration. Increased guard-cell osmolarity leads to the opening of the stomata and decreased osmolarity causes the stomata to close. The role of sugars in the regulation of stomata is not yet clear. In this study, we examined the role of hexokinase (HXK), a sugar-phosphorylating enzyme involved in sugar-sensing, in guard cells and its effect on stomatal aperture. We show here that increased expression of HXK in guard cells accelerates stomatal closure. We further show that this closure is induced by sugar and is mediated by abscisic acid. These findings support the existence of a feedback-inhibition mechanism that is mediated by a product of photosynthesis, namely sucrose. When the rate of sucrose production exceeds the rate at which sucrose is loaded into the phloem, the surplus sucrose is carried toward the stomata by the transpiration stream and stimulates stomatal closure via HXK, thereby preventing the loss of precious water.

A dual role of tobacco hexokinase 1 in primary metabolism and sugar sensing.

Hexokinase (HXK) is present in all virtually living organisms and is central to carbohydrate metabolism catalysing the ATP-dependent phosphorylation of hexoses. In plants, HXKs are supposed to act as sugar sensors and/or to interact with other enzymes directly supplying metabolic pathways such as glycolysis, the nucleotide phosphate monosaccharide (NDP-glucose) pathway and the pentose phosphate pathway. We identified nine members of the tobacco HXK gene family and observed that among RNAi lines of these nine NtHXKs, only RNAi lines of NtHXK1 showed an altered phenotype, namely stunted growth and leaf chlorosis. NtHXK1 was also the isoform with highest relative expression levels among all NtHXKs. GFP-tagging and immunolocalization indicated that NtHXK1 is associated with mitochondrial membranes. Overexpression of NtHXK1 resulted in elevated glucose phosphorylation activity in leaf extracts or chloroplasts. Moreover, NtHXK1 was able to complement the glucose-insensitive Arabidopsis mutant gin2-1 suggesting that NtHXK1 can take over glucose sensing functions. RNAi lines of NtHXK1 showed severely damaged leaf and chloroplast structure, coinciding with an excess accumulation of starch. We conclude that NtHXK1 is not only essential for maintaining glycolytic activity during respiration but also for regulating starch turnover, especially during the night.

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

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, (2) repressed by PML-RARA, and (3) functionally involved in neutrophil differentiation and cell viability of APL cells.

PABP interacting protein 2A (PAIP2A) regulates specific key proteins during spermiogenesis in the mouse.

During spermiogenesis, expression of the specific proteins needed for proper differentiation of male germ cells is under translational control. We have shown that PAIP2A is a major translational regulator involved in the maturation of male germ cells and male fertility. To identify the proteins controlled by PAIP2A during spermiogenesis, we characterized the proteomic profiles of elongated spermatids from wild-type (WT) mice and mice that were Paip2a/Paip2b double-null mutants (DKO). Elongated spermatid populations were obtained and proteins were extracted and separated on gradient polyacrylamide gels. The gels were digested with trypsin and peptides were identified by mass spectrometry. We identified 632 proteins with at least two unique peptides and a confidence level of 95%. Only 209 proteins were consistently detected in WT or DKO replicates with more than five spectra. Twenty-nine proteins were differentially expressed with at least a 1.5-fold change; 10 and 19 proteins were down- and up-regulated, respectively, in DKO compared to WT mice. We confirmed the significantly different expression levels of three proteins, EIF4G1, AKAP4, and HK1, by Western blot analysis. We have characterized novel proteins that have their expression controlled by PAIP2A; of these, 50% are involved in flagellar structure and sperm motility. Although several proteins affected by abrogation of Paip2a have established roles in reproduction, the roles of many others remain to be determined.