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Contains fulltext : 229376.pdf (publisher's version ) (Open Access), Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45(-/-) mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Contains fulltext : 51968.pdf (Publisher’s version ) (Open Access), Matching blood flow to myocardial energy demand is vital for heart performance and recovery following ischemia. The molecular mechanisms responsible for transduction of myocardial energetic signals into reactive vasodilatation are, however, elusive. Adenylate kinase, associated with AMP signaling, is a sensitive reporter of the cellular energy state, yet the contribution of this phosphotransfer system in coupling myocardial metabolism with coronary flow has not been explored. Here, knock out of the major adenylate kinase isoform, AK1, disrupted the synchrony between inorganic phosphate P(i) turnover at ATP-consuming sites and gamma-ATP exchange at ATP synthesis sites, as revealed by (18)O-assisted (31)P NMR. This reduced energetic signal communication in the post-ischemic heart. AK1 gene deletion blunted vascular adenylate kinase phosphotransfer, compromised the contractility-coronary flow relationship, and precipitated inadequate coronary reflow following ischemia-reperfusion. Deficit in adenylate kinase activity abrogated AMP signal generation and reduced the vascular adenylate kinase/creatine kinase activity ratio essential for the response of metabolic sensors. The sarcolemma-associated splice variant AK1beta facilitated adenosine production, a function lost in the absence of adenylate kinase activity. Adenosine treatment bypassed AK1 deficiency and restored post-ischemic flow to wild-type levels, achieving phenotype rescue. AK1 phosphotransfer thus transduces stress signals into adequate vascular response, providing linkage between cell bioenergetics and coronary flow.

Item does not contain fulltext, To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant ATPase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.

Contains fulltext : 186282.pdf (Publisher’s version ) (Open Access), Previously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency.

Contains fulltext : 186091.pdf (Publisher’s version ) (Open Access), Genetic ablation of adenylate kinase 1 (AK1), a member of the AK family of phosphotransfer enzymes, disturbs muscle energetic economy and decreases tolerance to metabolic stress, despite rearrangements in alternative high energy phosphoryl transfer pathways. To define the mechanisms of this adaptive response, soleus and gastrocnemius muscles from AK1(-/-) mice were characterized by cDNA array profiling, Western blot and ultrastructural analysis. We demonstrate that AK1 deficiency induces fiber-type specific variation in groups of transcripts involved in glycolysis and mitochondrial metabolism and in gene products defining structural and myogenic events. This was associated with increased phosphotransfer capacities of the glycolytic enzymes pyruvate kinase and 3-phosphoglycerate kinase. Moreover, in AK1(-/-) mice, fast-twitch gastrocnemius, but not slow-twitch soleus, had an increase in adenine nucleotide translocator (ANT) and mitochondrial creatine kinase protein, along with a doubling of the intermyofibrillar mitochondrial volume. These results provide molecular evidence for wide-scale remodeling in AK1-deficient muscles aimed at preservation of efficient energetic communication between ATP producing and utilizing cellular sites.

Item does not contain fulltext, Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-P(i) and creatine phosphate-to-P(i) ratios. Postischemic AK1 knockout hearts maintained approximately 40% of beta-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.

Item does not contain fulltext, Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (K(ATP)) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K(ATP) channel-dependent membrane excitability remains elusive. Here, we identify that the response of K(ATP) channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the K(ATP) channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K(ATP) channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K(ATP) channels and associated functions.

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Contains fulltext : 185900.pdf (Publisher’s version ) (Open Access)