Mitochondrial Adenylate Kinase Research Articles

Failure of metabolic checkpoint control during late-stage granulopoiesis drives neutropenia in reticular dysgenesis

AbstractCellular metabolism is highly dynamic during hematopoiesis, yet the regulatory networks that maintain metabolic homeostasis during differentiation are incompletely understood. Herein, we have studied the grave immunodeficiency syndrome reticular dysgenesis caused by loss of mitochondrial adenylate kinase 2 (AK2) function. By coupling single-cell transcriptomics in samples from patients with reticular dysgenesis with a CRISPR model of this disorder in primary human hematopoietic stem cells, we found that the consequences of AK2 deficiency for the hematopoietic system are contingent on the effective engagement of metabolic checkpoints. In hematopoietic stem and progenitor cells, including early granulocyte precursors, AK2 deficiency reduced mechanistic target of rapamycin (mTOR) signaling and anabolic pathway activation. This conserved nutrient homeostasis and maintained cell survival and proliferation. In contrast, during late-stage granulopoiesis, metabolic checkpoints were ineffective, leading to a paradoxical upregulation of mTOR activity and energy-consuming anabolic pathways such as ribonucleoprotein synthesis in AK2-deficient cells. This caused nucleotide imbalance, including highly elevated adenosine monophosphate and inosine monophosphate levels, the depletion of essential substrates such as NAD+ and aspartate, and ultimately resulted in proliferation arrest and demise of the granulocyte lineage. Our findings suggest that even severe metabolic defects can be tolerated with the help of metabolic checkpoints but that the failure of such checkpoints in differentiated cells results in a catastrophic loss of homeostasis.

Colorectal polyps increase the glycolytic activity.

In colorectal cancer (CRC) energy metabolism research, the precancerous stage of polyp has remained rather unexplored. By now, it has been shown that CRC has not fully obtained the glycolytic phenotype proposed by O. Warburg and rather depends on mitochondrial respiration. However, the pattern of metabolic adaptations during tumorigenesis is still unknown. Understanding the interplay between genetic and metabolic changes that initiate tumor development could provide biomarkers for diagnosing cancer early and targets for new cancer therapeutics. We used human CRC and polyp tissue material and performed high-resolution respirometry and qRT-PCR to detect changes on molecular and functional level with the goal of generally describing metabolic reprogramming during CRC development. Colon polyps were found to have a more glycolytic bioenergetic phenotype than tumors and normal tissues. This was supported by a greater GLUT1, HK, LDHA, and MCT expression. Despite the increased glycolytic activity, cells in polyps were still able to maintain a highly functional OXPHOS system. The mechanisms of OXPHOS regulation and the preferred substrates are currently unclear and would require further investigation. During polyp formation, intracellular energy transfer pathways become rearranged mainly by increasing the expression of mitochondrial adenylate kinase (AK) and creatine kinase (CK) isoforms. Decreased glycolysis and maintenance of OXPHOS activity, together with the downregulation of the CK system and the most common AK isoforms (AK1 and AK2), seem to play a relevant role in CRC development.

The palmitoyltransferase ZDHHC21 regulates oxidative phosphorylation to induce differentiation block and stemness in AML

AbstractAcute myeloid leukemia (AML) is an aggressive hematological malignancy. Nearly 50% of patients who receive the most intensive treatment inevitably experience disease relapse, likely resulting from the persistence of drug-resistant leukemia stem cells (LSCs). AML cells, especially LSCs, are highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for survival, but the mechanism involved in OXPHOS hyperactivity is unclear, and a noncytotoxic strategy to inhibit OXPHOS is lacking. To our knowledge, this study is the first to demonstrate that ZDHHC21 palmitoyltransferase serves as a key regulator of OXPHOS hyperactivity in AML cells. The depletion/inhibition of ZDHHC21 effectively induced myeloid differentiation and weakened stemness potential by inhibiting OXPHOS in AML cells. Interestingly, FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD)–mutated AML cells expressed significantly higher levels of ZDHHC21 and exhibited better sensitivity to ZDHHC21 inhibition. Mechanistically, ZDHHC21 specifically catalyzed the palmitoylation of mitochondrial adenylate kinase 2 (AK2) and further activated OXPHOS in leukemic blasts. Inhibition of ZDHHC21 arrested the in vivo growth of AML cells and extended the survival of mice inoculated with AML cell lines and patient derived xenograft AML blasts. Moreover, targeting ZDHHC21 to suppress OXPHOS markedly eradicated AML blasts and enhanced chemotherapy efficacy in relapsed/refractory leukemia. Together, these findings not only uncover a new biological function of palmitoyltransferase ZDHHC21 in regulating AML OXPHOS but also indicate that ZDHHC21 inhibition is a promising therapeutic regimen for patients with AML, especially relapsed/refractory leukemia.

Whole-exome sequencing in 415,422 individuals identifies rare variants associated with mitochondrial DNA copy number.

SummaryInter-individual variation in the number of copies of the mitochondrial genome, called mitochondrial DNA copy number (mtDNA-CN), reflects mitochondrial function and has been associated with various aging-related diseases. We examined 415,422 exomes of self-reported White ancestry individuals from the UK Biobank and tested the impact of rare variants, at the level of single variants and through aggregate variant-set tests, on mtDNA-CN. A survey across nine variant sets tested enrichment of putatively causal variants and identified 14 genes at experiment-wide significance and three genes at marginal significance. These included associations at known mtDNA depletion syndrome genes (mtDNA helicase TWNK, p = 1.1 × 10−30; mitochondrial transcription factor TFAM, p = 4.3 × 10−15; mtDNA maintenance exonuclease MGME1, p = 2.0 × 10−6) and the V617F dominant gain-of-function mutation in the tyrosine kinase JAK2 (p = 2.7 × 10−17), associated with myeloproliferative disease. Novel genes included the ATP-dependent protease CLPX (p = 8.4 × 10−9), involved in mitochondrial proteome quality, and the mitochondrial adenylate kinase AK2 (p = 4.7 × 10−8), involved in hematopoiesis. The most significant association was a missense variant in SAMHD1 (p = 4.2 × 10−28), found on a rare, 1.2-Mb shared ancestral haplotype on chromosome 20. SAMHD1 encodes a cytoplasmic host restriction factor involved in viral defense response and the mitochondrial nucleotide salvage pathway, and is associated with Aicardi-Goutières syndrome 5, a childhood encephalopathy and chronic inflammatory response disorder. Rare variants were enriched in Mendelian mtDNA depletion syndrome loci, and these variants implicated core processes in mtDNA replication, nucleoid structure formation, and maintenance. These data indicate that strong-effect mutations from the nuclear genome contribute to the genetic architecture of mtDNA-CN.

ADP and ATP Promote Mitochondrial Calcium Capacity Through Distinct Roles

The ratio of ADP and ATP is a natural indicator of cellular bioenergetic state, as such these analytes have become prominent factors in metabolism research. Beyond mitochondrial oxidative phosphorylation and ATPase activities, ADP and ATP act as steric regulators of enzymes, e.g. cytochrome C oxidase, and are major factors in mitochondrial calcium storage potential. Consideration of all routes of adenylate conversion is critical to understanding the role of each individual adenylate in the realm of mitochondrial function. Mitochondrial Adenylate Kinase activity dismutates ADP into ATP and AMP via a reversible reaction. Through the use of an Adenylate Kinase inhibitor we have noted that this enzymatic activity drives stark differences in the adenylate profile of isolated mitochondria when measured by 31-P magnetic resonance spectroscopy. These differences are further noted when the Adenylate Kinase reverse reaction is blocked by calcium. Additionally, by utilizing endogenous mitochondrial Adenylate Kinase activity, we identified distinct roles for ADP and ATP in promoting mitochondrial calcium capacity. These roles are such that ADP provides a defense against permeability transition, while ATP contributes to calcium storage through the formation of dense calcium phosphate granules. Our findings showcase how adenylate kinases elicit considerable impact on the outcome of a variety of mitochondrial assays through their drastic manipulation of the adenylate profile. Cytochrome c oxidase activity, P/O ratio, and mitochondrial calcium dynamics were all affected by Adenylate Kinase activity. Of particular note, it was revealed that ATP is required for a particularly dense form of mitochondrial amorphous calcium phosphate, which in the future may further entangle mitochondria with physiological calcium events.

Mitochondrial adenylate kinase 4 regulates dual AMPK and mTORC1 activation

In order to maintain cellular homeostasis, mitochondria integrate bioenergetic, metabolic, and redox cues. Signals both converge on and emanate from mitochondria to respond to varied cellular stressors. Therefore, mitochondrial proteins must play critical roles in maintaining cellular homeostasis. In a search for mitochondrial proteins that impact cellular bioenergetics, we identified the adenylate kinase, AK4, as the most potent regulator of ATP levels out of >1,000 mitochondrial proteins. We also found AK4 expression to significantly impact AMKP and mTORC1 activation status as well as cells' ability to respond to energetic stressors. Suppressing AK4 expression increases steady‐state cellular ATP levels by up to 30%, alters cellular respiration metabolite pools, and results in concomitant activation of both AMPK and mTORC1 signaling. Together, these effects of suppressed AK4 expression allow cells to thrive in the context of nutrient deprivation‐induced stress. The profound effects of AK4 expression on bioenergetics, survival under stress, and dual activation of the AMPK and mTORC1 energy sensing pathways position AK4 as a potential master regulator of mitochondria‐dependent cellular homeostasis.Support or Funding InformationNIH 1R15GM123382 CSUPERB New Investigator GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The influence of AICAR - direct activator of AMP-activated protein kinase (AMPK) - on liver proteome in apoE-knockout mice

There is a growing body of evidence that altered functioning of apoE may aggravate cellular energy homeostasis and stress response, leading to oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress and inflammation, leading to hypercholesterolemia, dyslipidemia, liver steatosis and neurodegeneration. One of the key cellular responses to mitochondria and ER-stress related processes and cellular energy imbalance is AMP-activated protein kinase (AMPK), considered as a cellular master energy sensor and critical regulator of mitochondrial homeostasis.The aim of our study was to use differential proteomics and transcriptomics approach to elucidate the effect of direct AMPK activator AICAR on liver proteome in apoE−/− mice – experimental model of atherosclerosis and moderate nonalcoholic steatosis. We applied Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling and two-dimensional chromatography coupled with mass spectrometry (2DLC-MS/MS) MudPIT strategy, as well as RT-PCR to investigate the changes in mitochondrial and cytosolic proteins and transcripts expression in 6-month old AICAR-treated apoE−/−.AICAR elicited induction of proteins related to mitochondrial β-oxidation, protein degradation and energy producing pathways (i.a. tricarboxylic acid cycle members and mitochondrial adenylate kinase 2). On the other hand, AICAR repressed inflammatory and pro-apoptotic markers in the apoE−/− mice liver, alongside reduction in several peroxisomal proteins, possibly suggesting induction of anti-oxidative pexophagy.

Linking Oxidative Stress to Cell Fate-Ipsc-Based Disease Modeling Identifies New Therapeutic Target in Reticular Dysgenesis

Reticular Dysgenesis (RD) is one of the most serious forms of severe combined immune deficiency (SCID). It is characterized by complete absence of circulating lymphocytes and neutrophils. In addition, patients suffer from sensorineural hearing loss. Before newborn screening for SCID was implemented, the majority of patients succumbed to infection long before hematopoietic cell transplantation (HCT) could be attempted. To this date, the prognosis for RD remains grim. RD is caused by mutations in the mitochondrial ADP-generator Adenylate Kinase 2 (AK2). AK1 is a cytosolic protein that may compensate in various tissues for the lack of AK2. However, AK1 is not expressed in leukocytes and the stria vascularis of the inner ear [1]. While this observation may explain where AK2 defects manifest, the molecular mechanisms how AK2 defects take effect, remain largely obscure. Significant obstacles to elucidating disease pathology have been the lack of a suitable animal models and the unavailability of patient specimens. Using skin fibroblasts from an RD-patient we have recently identified at Boston Children’s Hospital [2], we have generated induced pluripotent stem cells (iPSC) with homozygous loss of function mutation in AK2. In-vitro myeloid differentiation of AK2-mutated iPSCs recapitulates the characteristic maturation arrest at the promyelocyte stage observed in-vivo in patients with this condition. AK2 is expressed in the intermitochondrial space and serves as primary mitochondrial ADP generator by promoting the reversible reaction AMP + ATP = 2 ADP. Maintenance of adequate levels of ADP is critical to support ATP synthase activity. Using Mass Spectrometry, we have shown that decreased AK2 activity leads to an increase in the AMP/ADP ratio in iPS-derived myeloid cells, indicating that AK2 is indispensable in maintaining ADP supply in the myeloid lineage. We have also performed transcriptome analysis of AK2- mutated myeloid cells compared to control and found a significant down regulation of ATP-dependant transporters. Based on this data, we hypothesized that in patients with RD, ADP-depletion in myeloid progenitors leads to stage 4 respiration, a well defined state in mitochondrial biology, in which the ATP-synthase lacks substrate and decreases its activity. This causes a reduction in proton flux from the intermitochondrial space back into the matrix, transient rise in membrane potential, and an escalation in the formation of reactive oxygen species (ROS). The cell responds by activating “inducible uncoupling”, the opening of alternative proton pores, which allows proton flux back into the matrix, bypassing the ATP-synthase and foregoing the use of energy stored in the proton gradient. While this represents a cellular rescue mechanism in response to acute oxidative stress, extended oxidative-stress-induced uncoupling eventually leads to a decline in proton gradient and membrane potential and ultimately in demise of the cell. To test this hypothesis, we have added Glutathione, the primary endogenous cellular antioxidant, to the culture conditions. We also tested G-CSF and all-trans-retinoic acid (ATRA), agents known to promote promyelocyte differentiation to mature neutrophils in other conditions. While G-CSF had no, and ATRA clearly deleterious effects on myeloid maturation in AK2-mutated cells, Glutathione led to a significant improvement in differentiation, allowing development of mature neutrophils in-vitro. Our results suggest that cell fate in RD is linked to oxidative stress and identify antioxidants as a possible therapeutic approach that may help reduce early mortality due to severe infections in patients with RD. Disclosures No relevant conflicts of interest to declare.

A Mitochondrial RNAi Screen Defines Cellular Bioenergetic Determinants and Identifies an Adenylate Kinase as a Key Regulator of ATP Levels

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

Abstract 157: Metabolomic Profiling of AMP-Signaling Stress: Effects of Metformin on Wild Type and Adenylate Kinase AK1-/- Transgenic Mice

Metabolic drugs frequently target mitochondrial functions and AMP signaling. Metformin, one of the most prescribed anti-diabetic medications, exerts its glucose lowering effect through mild interference with mitochondrial energy metabolism triggering homeostatic adenylate kinase (AK)-catalyzed AMP and downstream AMPK signaling. However, metabolomic signature of drug-induced “therapeutic mitochondrial toxicity" and energetic imbalance-induced AMP signaling are poorly understood. This work was aimed to determine metabolomic representation of metformin-induced mitochondrial and AMP signaling stress by comparing wild type and AK1-deficient mice with compromised AMP-signaling. Metabolomic profiling of plasma and urine samples from wild type mice treated with metformin revealed distinct metabolic signature suggesting altered energy, Krebs cycle, lipid and ketone body metabolism, with higher levels of creatinine, malate, α-ketoglutarate, FFA, glycerol, glycerol-3-phosphate, 3-hydroxybutyrate and hypoxanthine. Contrary to expectations glucose lowering effect of metformin was greater in energetically and AMP-signaling compromised AK1-/- mice. Metformin action on AK1 deficient mice was characterized by increased levels of creatinine, lactate, alanine, FFA, phosphoserine, acetoacetate, 3-hydroxybutyrate, malate and α-ketoglutarate indicating broader alterations in energy, glycolytic, Krebs cycle, ketone body, lipid and amino acid metabolism. At baseline, livers from AK1-/- mice had higher AMP/ADP and pAMPKα/AMPKα ratios compared to wild type. There were no significant sustained changes in global nucleotide ratios after metformin treatment. Overall metabolomic profile of AK1-/- mice indicate larger disturbances in energetic and substrate metabolic networks after metformin treatment compared to wild type. Thus, this study revealed specific metabolomic signatures of metformin therapeutic action and mitochondrial toxicity indicating that genetic and metabolic background modulate individual response to metformin treatment.

Role of voltage-dependent anion channels of the mitochondrial outer membrane in regulation of cell metabolism

The role of voltage-dependent anion channels (VDAC/porins) of the mitochondrial outer membrane in the regulation of cell metabolism is assessed using an experimental model of ethanol toxicity in cultured hepatocytes. It is demonstrated that ethanol inhibits the phosphorylating and the uncoupled mitochondrial respiration, decreases the accessibility of mitochondrial adenylate kinase in the intermembrane space, and suppresses ureagenic respiration in the cells. Treatment with digitonin at high concentrations (>80 μM)—which creates pores in the mitochondrial outer membrane, allowing bypass of closed VDAC—restores all the processes suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial function because of closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space. Our studies also reveal the role of VDAC in the regulation of liver-specific intracellular processes such as ureagenesis. The data obtained can be used in development of pharmaceuticals that would prevent VDAC closure in mitochondria of ethanol-oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.

Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.

A Controlled SCID: Bringing Reticular Dysgenesis to a Halt

Reticular dysgenesis (aleukocytosis) is caused by mutations in the gene encoding mitochondrial adenylate kinase 2 Pannicke et al. (2009) Nature Genetics 41: 101–105 Human adenylate kinase 2 deficiency causes a profound hematopoietic defect associated with sensorineural deafness Lagresle-Peyrou et al. (2009) Nature Genetics 41: 106-111

Distinct behaviors of adenylate kinase and cytochrome c observed following induction of mitochondrial permeability transition by Ca2+ in the absence of respiratory substrate

For induction of the mitochondrial permeability transition (PT) by Ca(2+), the addition of a respiratory substrate such as succinate is required. However, earlier studies indicated the possible induction of the mitochondrial PT by Ca(2+) in the absence of a respiratory substrate (Hunter, D.R., and Haworth, R.A. (1979) Arch. Biochem. Biophys. 195, 453-459). In the present study, we obtained clear evidence showing that the mitochondrial PT could be induced by Ca(2+) even in the absence of respiratory substrate. We next examined the protein release from mitochondria that accompanied the induction of PT in the absence of a respiratory substrate. Interestingly, distinct from the ordinary mitochondrial PT induced by Ca(2+) in the presence of a respiratory substrate, which is associated with the release of mitochondrial cytochome c and adenylate kinase, the mitochondrial PT occurring in the absence of a respiratory substrate was associated with release of mitochondrial adenylate kinase but not with that of mitochondrial cytochrome c. This experimental system should be quite useful for understanding the mechanisms of protein release from mitochondria.

Reticular dysgenesis (aleukocytosis) is caused by mutations in the gene encoding mitochondrial adenylate kinase 2

Human severe combined immunodeficiencies (SCID) are phenotypically and genotypically heterogeneous diseases. Reticular dysgenesis is the most severe form of inborn SCID. It is characterized by absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood, hypoplasia of the thymus and secondary lymphoid organs, and lack of innate and adaptive humoral and cellular immune functions, leading to fatal septicemia within days after birth. In bone marrow of individuals with reticular dysgenesis, myeloid differentiation is blocked at the promyelocytic stage, whereas erythro- and megakaryocytic maturation is generally normal. These features exclude a defect in hematopoietic stem cells but point to a unique aberration of the myelo-lymphoid lineages. The dramatic clinical course of reticular dysgenesis and its unique hematological phenotype have spurred interest in the unknown genetic basis of this syndrome. Here we show that the gene encoding the mitochondrial energy metabolism enzyme adenylate kinase 2 (AK2) is mutated in individuals with reticular dysgenesis. Knockdown of zebrafish ak2 also leads to aberrant leukocyte development, stressing the evolutionarily conserved role of AK2. Our results provide in vivo evidence for AK2 selectivity in leukocyte differentiation. These observations suggest that reticular dysgenesis is the first example of a human immunodeficiency syndrome that is causally linked to energy metabolism and that can therefore be classified as a mitochondriopathy.

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.

The activities of nucleoside diphosphate kinase and adenylate kinase are influenced by their interaction

The enzyme nucleoside diphosphate kinase (NDPK) is responsible for maintaining the nucleotide balance in the cell, but is also involved in regulatory functions that often are mediated via protein-protein interactions. Here we present evidence of an interaction between the plant mitochondrial NDPK and a plant mitochondrial adenylate kinase. This is the most highly expressed adenylate kinase gene present in Arabidopsis thaliana. The adenylate kinase cDNA was amplified from A. thaliana and cloned into an expression vector. The recombinant His-tagged protein was produced in Escherichia coli and purified. In vitro experiments using the recombinant enzymes showed that NDPK and adenylate kinase affected the activity of each other. Adenylate kinase stimulated NDPK activity, whereas NDPK inhibited the activity of adenylate kinase, when the total ADP production of the enzymes was measured. The net effect could be an unchanged ADP production, albeit with different rates of substrate consumption. Possible implications in respiration and programmed cell death are discussed.

AK2 activates a novel apoptotic pathway through formation of a complex with FADD and caspase-10

Mitochondrial proteins function as essential regulators in apoptosis. Here, we show that mitochondrial adenylate kinase 2 (AK2) mediates mitochondrial apoptosis through the formation of an AK2-FADD-caspase-10 (AFAC10) complex. Downregulation of AK2 attenuates etoposide- or staurosporine-induced apoptosis in human cells, but not that induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL). During intrinsic apoptosis, AK2 translocates to the cytoplasm, whereas this event is diminished in Apaf-1 knockdown cells and prevented by Bcl-2 or Bcl-X(L). Addition of purified AK2 protein to cell extracts first induces activation of caspase-10 via FADD and subsequently caspase-3 activation, but does not affect caspase-8. AFAC10 complexes are detected in cells undergoing intrinsic cell death and AK2 promotes the association of caspase-10 with FADD. In contrast, AFAC10 complexes are not detected in several etoposide-resistant human tumour cell lines. Taken together, these results suggest that, acting in concert with FADD and caspase-10, AK2 mediates a novel intrinsic apoptotic pathway that may be involved in tumorigenesis.

Calcium and energy transfer

A recent article in The Journal of Physiology by Gueguen et al. (2005) shows that an efficient metabolic coupling between mitochondrial creatine kinase (mtCK) or adenylate kinase (AK2), and ADP rephosphorylation via mitochondrial respiration is tissue specific: it is functioning in skinned muscle of oxidative type I and type IIa but not in IIx type muscles, and this coupling can be modified by calcium. We would like to add some critical considerations which may contribute to solving some of discrepancies in this paper, and point out some methodological difficulties in studies of the functional coupling phenomenon. Calcium, ADP, AMP and creatine are known to act as important regulators of energy flux in muscle cells. High energy phosphates may be channelled out of mitochondria via direct ATP flux or by mtCK or AK2 via a phosphoryl-creatine (PCr) shuttle (Wallimann et al. 1992; Dzeja & Terzic, 2003; Saks et al. 2004). While all types of isolated mitochondria studied before show apparent Km (ADP) of 5–17 μm, this parameter is tissue specific in permeabilized cells in situ: Gueguen et al. (2005) showed that it is about the same in fast twitch muscle fibres of IIx type, but very high (212 μm) in slow twitch oxidative fibres of the type I and intermediate in the mixed types of fibres IIax and IIb where two apparent Km are observed. These results are in good agreement with earlier data (Veksler et al. 1995; Kuznetsov et al. 1996; Ventura-Clapier et al. 1998; Saks et al. 2004). Since the fibre diameter is usually bigger in fast glycolytic muscles, these data show very clearly the differences in the intracellular organization between different muscle types, and the very high apparent Km values are caused by specific local restrictions of the diffusion of ADP inside the type I oxidative fibres (Abraham et al. 2002; Vendelin et al. 2004). In their studies Gueguen et al. (2005) have made another remarkable observation: when the calcium concentration in the medium was elevated from 0.1 to 0.4 μm, the affinity of respiration for exogenous ADP was increased due to activation of actomyosin MgATPase. The increased affinity for exogenous ADP increased activation of respiration at submaximal ADP concentrations to the extent usually seen in the presence of creatine. Furthermore, calcium-induced activation of actomyosin ATPase also prevented the stimulation of respiration by creatine and AMP. The authors concluded from these results that mtCK and AK2 are no longer functionally coupled with mitochondrial respiration. This is a very surprising conclusion since under high work load, that is under elevated energy demand, the flux through the mtCK reaction was shown in vivo to increase significantly, e.g. during sea urchin sperm activation (Dorsten et al. 1997) and in muscle during increase of contraction frequency when directly assessed by the in vivo18O labelling technique (Pucar et al. 2001; Janssen et al. 2003), or alternatively, to decrease when demand was lowered (Joubert et al. 2002). In addition, calcium has been shown to strengthen the interaction between mtCK and voltage-dependent anion channels (VDAC) (Schlattner et al. 2001), which also would favour the formation of a VDAC–mtCK–adenine nucleotide translocase (ANT) multienzyme channelling complex, and thus facilitate the export of PCr from mitochondria. The experimental conditions described in the paper by Gueguen et al. (2005) are far from physiological conditions in the sense that the activating steady state level of calcium was continuously maintained at a high level and thus no relaxation was possible. Under these conditions, production of ATP by the activated mitochondrial respiration evidently results in hypercontraction of fibres, complete disturbance of myofibrillar architecture. This will also change the diffusion restrictions for ADP and results in a strong decrease of apparent Km for exogenous ADP (Andrienko et al. 2003). The rather artifactual system may explain the observations made under calcium addition. The fact that in the presence of vanadate, where the fibres are prevented from hypercontraction, calcium no longer ablates creatine-stimulated respiration (see Fig. 7) is direct proof that the observed phenomenon is related to supercontraction of the fibres, much more than to calcium itself. Under these conditions, exogenous ADP added already maximally activates respiration, and there will be no way for creatine to increase respiration further even if functional coupling is intact. For exactly the same reason, the very high apparent affinity of type II fibres for ADP makes it impossible to see the functional coupling between mtCK or AK2 and respiration from measurements at the fixed and relatively high ADP concentration of 0.1 mm (Fig. 5). To study functional coupling under these conditions, more sophisticated approach should be used (Moreadith & Jacobus, 1982). Clearly, metabolic microcompartments depend on the structural organization and integrity of the cells, and these considerations can no longer be ignored in studies of metabolic regulation of the muscle cell respiration (Schlattner & Wallimann, 2004).

Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix

The previously isolated cDNA encoding human adenylate kinase (AK) isozyme 3 was recently renamed AK4. Consequently, human AK3 cDNA remains to be identified and we have little information about the functional relationship between human AK3 and AK4. In pursuit of the physiological roles of both the AK3 and AK4 proteins, we first isolated an authentic human AK3 cDNA and compared their expression. Nucleotide sequencing revealed that the cDNA encoded a 227-amino-acid protein, with a deduced molecular mass of 25.6kDa, that shares greater homology with the AK3 cDNAs isolated from bovine and rat than that from human. We named the isolated cDNA AK3. Northern-blot analysis revealed that AK3 mRNA was present in all tissues examined, and was highly expressed in heart, skeletal muscle and liver, moderately expressed in pancreas and kidney, and weakly expressed in placenta, brain and lung. On the other hand, we found that human AK4 mRNA was highly expressed in kidney, moderately expressed in heart and liver and weakly expressed in brain. Western-blot analysis demonstrated expression profiles of AK3 and AK4 that were similar to their mRNA expression patterns in each tissue. Over expression of AK3, but not AK4, in both Escherichia coli CV2, a temperature-sensitive AK mutant, and a human embryonic kidney-derived cell line, HEK-293, not only produced significant GTP:AMP phosphotransferase (AK3) activity, but also complemented the CV2 cells at 42°C. Subcellular and submitochondrial fractionation analysis demonstrated that both AK3 and AK4 are localized in the mitochondrial matrix.