Frameshift Variant in AMPD2 in Cirneco dell'Etna Dogs with Retinopathy and Tremors.

While the manifestations of many inherited retinal disorders are limited to loss of vision, others are part of a syndrome that affects multiple tissues, particularly the nervous system. Most syndromic retinal disorders are thought to be recessively inherited. Two dogs out of a litter of Cirneco dell' Etna dogs, both males, showed signs of retinal degeneration, along with tremors and signs described as either atypical seizures or paroxysmal dyskinesias, while the other two male littermates were normal. We named this oculo-neurological syndrome CONS (Cirneco oculo-neurological syndrome), and undertook homozygosity mapping and whole-genome sequencing to determine its potential genetic etiology. Notably, we detected a 1-bp deletion in chromosome 6 that was predicted to cause a frameshift and premature stop codon within the canine AMPD2 gene, which encodes adenosine monophosphate deaminase, an enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP). Genotyping of the available Cirneco population suggested perfect segregation between cases and controls for the variant. Moreover, this variant was absent in canine genomic databases comprised of thousands of unaffected dogs. The AMPD2 genetic variant we identified in dogs presents with retinal manifestations, adding to the spectrum of neurological manifestations associated with AMPD2 variants in humans.

AMP deaminase: A crucial regulator in nitrogen stress and lipid metabolism in Mucor circinelloides.

Lipid biosynthesis is a significant metabolic response to nitrogen starvation in oleaginous fungi. The oleaginous fungus Mucor circinelloides copes with nitrogen stress by degrading AMP through AMP deaminase (AMPD). However, the mechanism of AMPD in regulating lipogenesis remains largely unclear. To elucidate the mechanism of AMPD in lipid synthesis in this M. circinelloides, we identified two genes (ampd1 and ampd2) encoding AMPD and constructed an ampd double knockout mutant. The engineered M. circinelloides strain elevated cell growth and lipid accumulation, as well as the content of oleic acid (OA) and gamma-linolenic acid (GLA). In addition to the expected increase in transcription levels of genes associated with lipid and TAG synthesis, we observed suppression of lipid degradation and reduced amino acid biosynthesis. This suggested that the deletion of AMPD genes induces the redirection of carbon towards lipid synthesis pathways. Moreover, the pathways related to nitrogen metabolism, including nitrogen assimilation and purine metabolism (especially energy level), were also affected in order to maintain homeostasis. Further analysis discovered that the transcription factors (TFs) related to lipid accumulation were also regulated. This study provides new insights into lipid biosynthesis in M. circinelloides, indicating that the trigger for lipid accumulation is not entirely AMPD-dependent and suggest that there may be additional mechanisms involved in the initiation of lipogenesis.

Role of AMP Deaminase in Mucor circinelloides: Implications for Nitrogen Utilization and Lipid Biosynthesis.

Lipid accumulation in oleaginous organisms is initiated by AMP deaminase (AMPD) after nitrogen depletion because it mediates the concentration of intracellular adenosine monophosphate (AMP). However, the role of AMPD in lipogenesis in the oleaginous fungus Mucor circinelloides is largely unknown. Therefore, we identified the genes (ampd1 and ampd2) encoding AMPD and investigated the role of AMPD in lipid synthesis in this fungus by overexpressing and deleting ampd genes. Deletion of ampd1 and ampd2 caused 21 and 28% increments in lipid contents under N-limited conditions, respectively. These increases were correlated with the activation of enzymes involved in lipogenesis and the alteration of energy balance. Unexpectedly, overexpression of ampd genes affected nitrogen consumption in both N-limited and N-excess media, which resulted in an increase in cell growth and lipid accumulation compared with the control strain when nitrogen was available. Furthermore, the increased lipid accumulation in the ampd-overexpressing mutants in N-excess media was accompanied by enhanced activities of lipid biosynthetic enzymes. These data suggested that nitrogen metabolism and energy metabolism are affected by AMPD, and overexpression of ampd genes induced lipid accumulation under nitrogen-rich conditions by mimicking the nitrogen limitation response. This highlights an intriguing function of AMPD in M. circinelloides.

Erythrocyte ENT1-AMPD3 Axis is an Essential Purinergic Hypoxia Sensor and Energy Regulator Combating CKD in a Mouse Model.

SIGNIFICANCE STATEMENT: Hypoxia drives kidney damage and progression of CKD. Although erythrocytes respond rapidly to hypoxia, their role and the specific molecules sensing and responding to hypoxia in CKD remain unclear. In this study, we demonstrated in a mouse model that erythrocyte ENT1-AMPD3 is a master energy regulator of the intracellular purinergic hypoxic compensatory response that promotes rapid energy supply from extracellular adenosine, eAMPK-dependent metabolic reprogramming, and O 2 delivery, which combat renal hypoxia and progression of CKD. ENT1-AMPD3-AMPK-BPGM comprise a group of circulating erythroid-specific biomarkers, providing early diagnostic and novel therapeutic targets for CKD. BACKGROUND: Hypoxia drives kidney damage and progression of CKD. Although erythrocytes respond rapidly to hypoxia, their role and the specific molecules sensing and responding to hypoxia in CKD remain unclear. METHODS: Mice with an erythrocyte-specific deficiency in equilibrative nucleoside transporter 1 ( eEnt1-/- ) and a global deficiency in AMP deaminase 3 ( Ampd3-/- ) were generated to define their function in two independent CKD models, including angiotensin II (Ang II) infusion and unilateral ureteral obstruction (UUO). Unbiased metabolomics, isotopic adenosine flux, and various biochemical and cell culture analyses coupled with genetic studies were performed. Translational studies in patients with CKD and cultured human erythrocytes examined the role of ENT1 and AMPD3 in erythrocyte function and metabolism. RESULTS: eEnt1-/- mice display severe renal hypoxia, kidney damage, and fibrosis in both CKD models. The loss of eENT1-mediated adenosine uptake reduces intracellular AMP and thus abolishes the activation of AMPK α and bisphosphoglycerate mutase (BPGM). This results in reduced 2,3-bisphosphoglycerate and glutathione, leading to overwhelming oxidative stress in eEnt1-/- mice. Excess reactive oxygen species (ROS) activates AMPD3, resulting in metabolic reprogramming and reduced O 2 delivery, leading to severe renal hypoxia in eEnt1-/- mice. By contrast, genetic ablation of AMPD3 preserves the erythrocyte adenine nucleotide pool, inducing AMPK-BPGM activation, O 2 delivery, and antioxidative stress capacity, which protect against Ang II-induced renal hypoxia, damage, and CKD progression. Translational studies recapitulated the findings in mice. CONCLUSION: eENT1-AMPD3, two highly enriched erythrocyte purinergic components that sense hypoxia, promote eAMPK-BPGM-dependent metabolic reprogramming, O 2 delivery, energy supply, and antioxidative stress capacity, which mitigates renal hypoxia and CKD progression.

AMPD2 plays important roles in regulating hepatic glucose and lipid metabolism.

Dysregulation of hepatic glucose and lipid metabolism can instigate the onset of various metabolic disorders including obesity, dyslipidemia, insulin resistance, type 2 diabetes, and fatty liver disease. Adenosine monophosphate (AMP) deaminase (AMPD), which converts AMP to inosine monophosphate, plays a key role in maintaining adenylate energy charge. AMPD2 is the major isoform present in the liver. However, the mechanistic link between AMPD2 and hepatic glucose and lipid metabolism remains elusive. In this study, we probed into the hepatic glucose and lipid metabolism in AMPD2-deficient (A2-/-) mice. These mice exhibited reduced body weight, fat accumulation, and blood glucose levels, coupled with enhanced insulin sensitivity while maintaining consistent calorie intake and spontaneous motor activity compared with wild type mice. Furthermore, A2-/- mice showed mitigated obesity and hyper-insulinemia induced by high-fat diet (HFD) but elevated levels of the serum triglyceride and cholesterol. The hepatic mRNA levels of several fatty acid and cholesterol metabolism-related genes were altered in A2-/- mice. RNA sequencing unveiled multiple alterations in lipid metabolic pathways due to AMPD2 deficiency. These mice were also more susceptible to fasting or HFD-induced hepatic lipid accumulation. The liver exhibited elevated AMP levels but unaltered AMP/ATP ratio. In addition, AMPD2 deficiency is not associated with the adenosine production. In summary, this study established a link between purine metabolism and hepatic glucose and lipid metabolism via AMPD2, providing novel insights into these metabolic pathways.

The AMP deaminase of the mollusk Helix pomatia is an unexpected member of the adenosine deaminase-related growth factor (ADGF) family.

We report here the first occurrence of an adenosine deaminase-related growth factor (ADGF) that deaminates adenosine 5' monophosphate (AMP) in preference to adenosine. The ADGFs are a group of secreted deaminases found throughout the animal kingdom that affect the extracellular concentration of adenosine by converting it to inosine. The AMP deaminase studied here was first isolated and biochemically characterized from the roman snail Helix pomatia in 1983. Determination of the amino acid sequence of the AMP deaminase enabled sequence comparisons to protein databases and revealed it as a member of the ADGF family. Cloning and expression of its cDNA in Pichia pastoris allowed the comparison of the biochemical characteristics of the native and recombinant forms of the enzyme and confirmed they correspond to the previously reported activity. Uncharacteristically, the H. pomatia AMP deaminase was determined to be dissimilar to the AMP deaminase family by sequence comparison while demonstrating similarity to the ADGFs despite having AMP as its preferred substrate rather than adenosine.

Downregulation of extramitochondrial BCKDH and its uncoupling from AMP deaminase in type 2 diabetic OLETF rat hearts.

Systemic branched-chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long-Evans-Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched-chain α-keto acid dehydrogenase (BCKDH), a rate-limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long-Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH-E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3-E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3-BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3-BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.

The discovery of 3,3-dimethyl-1,2,3,4-tetrahydroquinoxaline-1-carboxamides as AMPD2 inhibitors with a novel mechanism of action.

AMP deaminase 2 (AMPD2) has been thought to play an important role in energy homeostasis and immuno-oncology, while selective AMPD2 inhibitors are highly demanded to clarify the physiological function of AMPD2. In this report, we describe selective AMPD2 inhibitors inducing allosteric modulation. Based on hypothesis that compounds that exhibit increased inhibition by preincubation would cause conformational change of the enzyme, starting from HTS hit compound 4, we discovered compound 8 through the SAR study. From X-ray structural information of 8, this chemical series has a novel mechanism of action that changes the substrate pocket to prevent AMP from binding. Further elaboration of compound 8 led to the tool compound 21 which exhibited potent inhibitory activity of AMPD2 in ex vivo evaluation of mouse liver.

Delineating the phenotype and genetic basis of AMPD2-related pontocerebellar hypoplasia.

Pontocerebellar hypoplasia is a group of disorders with a wide range of presentations. We describe here the genetic and phenotypic features of PCH type 9 due to mutations in AMPD2. All patients have severe intellectual disability, and the vast majority manifest abnormal tone, cortical blindness, and microcephaly. Almost all have agenesis of the corpus callosum and severe cerebellar hypoplasia. The course is not progressive, however, few die in the first decade of life. Mutations are spread throughout the gene, and no hot spot can be identified. One of the mutations we report here is the most distal truncating variant known in this gene and is predicted to result in a truncated protein. The phenotype is severe in all cases; thus, no clear genotype-phenotype correlation can be established.

Mechanism of Polyhexamethylene Biguanide Resistance in Purpureocillium lilacinum Strains.

Purpureocillium lilacinum has been recently found to contaminate a 20% (200,000 µg/mL) aqueous solution of polyhexamethylene biguanide hydrochloride (PHMB) . We aimed to elucidate the mechanism underlying the resistance of P. lilacinum to PHMB. First, we induced the PHMB-resistant (IR) strains IFM 67050 (IR) and IFM 65838 (IR) from the type strain P. lilacinum CBS 284.36T via cultivation in a medium containing high concentrations of PHMB. We then analyzed the DNA sequences via Illumina sequencing to evaluate the presence of genetic mutations in IFM 65838 (IR) . Further, we established an IFM 65838 (IR) uridine/uracil auxotrophic strain, and using the orotidine-5'-decarboxylase gene, pyrG as a selection marker, we tried to knockout a mutant gene in IFM 65838 (IR) using the CRISPR-Cas9 genome-editing technique. The growth rates of IFM 67050 (IR) and IFM 65838 (IR) in medium containing PHMB increased, and the minimum inhibitory concentrations (MICs) against PHMB also increased. Based on the DNA sequence analysis, we found a nonsynonymous point mutation in the gene PLI-008146 (G779A) in IFM 67050 (IR) and IFM 65838 (IR) . This point mutation leads to site combinations of splicing changes that cause partial sequences deletion (p.Y251_G281del) in the ΔPLI-008146 locus of IFM 65838 (IR) , and deletion sequences include partial adenosine/AMP deaminase motif (PF00962) orthologous to adenosine deaminase (ADA) (GeneBank: OAQ82383.1) . Furthermore, the mutant gene ΔPLI-008146 was successfully knocked out from the resistanceinduced strain using a novel CRISPR-Cas9 gene transformation method. A considerable reduction in growth rate and MIC against PHMB was observed in the absence of the mutant gene. Therefore, ADA may represent an important resistance factor in PHMB-resistant P. lilacinum.

Skeletal muscle contraction kinetics and AMPK responses are modulated by the adenine nucleotide degrading enzyme AMPD1.

AMP deaminase 1 (AMPD1; AMP → IMP + NH3) deficiency in skeletal muscle results in an inordinate accumulation of AMP during strenuous exercise, with some but not all studies reporting premature fatigue and reduced work capacity. To further explore these inconsistencies, we investigated the extent to which AMPD1 deficiency impacts skeletal muscle contractile function of different muscles and the [AMP]/AMPK responses to different intensities of fatiguing contractions. To reduce AMPD1 protein, we electroporated either an inhibitory AMPD1-specific miRNA encoding plasmid or a control plasmid, into contralateral EDL and SOL muscles of C57BL/6J mice (n = 48 males, 24 females). After 10 days, isolated muscles were assessed for isometric twitch, tetanic, and repeated fatiguing contraction characteristics using one of four (None, LOW, MOD, and HIGH) duty cycles. AMPD1 knockdown (∼35%) had no effect on twitch force or twitch contraction/relaxation kinetics. However, during maximal tetanic contractions, AMPD1 knockdown impaired both time-to-peak tension (TPT) and half-relaxation time (½ RT) in EDL, but not SOL muscle. In addition, AMPD1 knockdown in EDL exaggerated the AMP response to contractions at LOW (+100%) and MOD (+54%) duty cycles, but not at HIGH duty cycle. This accumulation of AMP was accompanied by increased AMPK phosphorylation (Thr-172; LOW +25%, MOD +34%) and downstream substrate phosphorylation (LOW +15%, MOD +17%). These responses to AMPD1 knockdown were not different between males and females. Our findings demonstrate that AMPD1 plays a role in maintaining skeletal muscle contractile function and regulating the energetic responses associated with repeated contractions in a muscle- but not sex-specific manner.NEW & NOTEWORTHY AMP deaminase 1 (AMPD1) deficiency has been associated with premature muscle fatigue and reduced work capacity, but this finding has been inconsistent. Herein, we report that although AMPD1 knockdown in mouse skeletal muscle does not change maximal isometric force, it negatively impacts muscle function by slowing contraction and relaxation kinetics in EDL muscle but not SOL muscle. Furthermore, AMPD1 knockdown differentially affects the [AMP]/AMPK responses to fatiguing contractions in an intensity-dependent manner in EDL muscle.

Down-regulation of AMPD3 Is Associated With Poor Survival in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Adenosine monophosphate deaminase 3 (AMPD3) is an isoenzyme involved in the regulation of the energetic metabolism of mammalian cells. Cancer cells have a high demand for their energy supply. This experimental study aimed to illustrate the role of AMPD3 in human head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: Real-time quantitative reverse transcription-polymerase chain reaction was used to investigate the expression of the AMPD3 gene in human HNSCC tissues to assess the changes in cancerous and noncancerous parts and the correlation with different tumor behavior. The functions of AMPD3 were investigated using wound-healing and migration assays. RESULTS: AMPD3 was significantly down-regulated in cancerous tissues of HNSCC (p=0.001) and this was correlated with more advanced tumor and clinical stages. Patients with high expression had better 5-year survival. AMPD3 knock-down in SCC-4 and SCC-25 cells demonstrated reduction of proliferation but increased migration and invasion. CONCLUSION: To our knowledge, this is the first report evidencing the expression pattern of AMPD3 in HNSCC and demonstrated that high AMPD3 expression might represent a good prognostic biomarker. AMPD3 may have an antiproliferative potential but its down-regulation may not contribute to reducing the migration and invasion of HNSCC cells.

Rare variants in previously identified linkage regions associated with carotid plaque in Dominican Republic families.

Carotid plaque is a subclinical measure of atherosclerosis. We have previously shown measures of carotid plaque to be heritable in a sample of 100 Dominican families and found evidence for linkage and association of common variants (CVs) on 7q36, 11p15, 14q32 and 15q23 with plaque presence. Our current study aimed to refine these regions further and identify rare variants (RVs) influencing plaque presence. Therefore, we performed targeted sequencing of the one LOD unit down region on 7q36, 11p15, 14q32 and 15q23 in 12 Dominican families with evidence for linkage to plaque presence. Gene-based RV analyses were performed using the Sequence Association Test for familial data (F-SKAT) under two filtering algorithms; 1. all exonic RVs and 2. non-synonymous RVs. Replication analyses were performed using a sample of 22 Dominican families and 556 unrelated Dominicans with Exome Array data. To identify additional non-synonymous RVs influencing plaque, we looked for co-segregation of RVs with plaque in each of the sequenced families. Our most strongly associated gene with evidence for replication was AMPD3 which showed suggestive association with plaque presence in the sequenced families (exonic RV p = 0.003, nonsynonymous RV p = 0.005) and replication families (exonic RV p = 0.04, nonsynonymous RV p = 0.02). Examination of the sequenced family pedigrees revealed two missense variants on chromosome 11 which co-segregated with plaque presence in one of our families; rs61751342 (located in DENND2B), and rs61760882 (located in RNF141). The rs61751342 missense variant is an eQTL for SCUBE2 in the atrial appendage. Notably, SCUBE2 encodes a protein which interacts with vascular endothelial growth factor (VEGF) receptor 2 to regulate VEGF-induced angiogenesis, thus providing biologic plausibility for this gene in atherosclerosis. In conclusion, using targeted sequencing of previously-identified linkage regions, we have identified suggestive evidence for the role of RVs in carotid plaque pathogenesis.

Regulation of skeletal muscle AMP deaminase. Carbethoxylation of His-51 belonging to the zinc coordination sphere of the rabbit enzyme promotes its desensitization towards the inhibition by ATP.

BACKGROUND: Skeletal muscle AMP deaminase (AMPD1) regulates the concentration of adenine nucleotides during muscle contraction. We previously provided evidence that rabbit AMPD1 is composed by two HPRG 73 kDa subunits and two 85 kDa catalytic subunits with a dinuclear zinc site with an average of two histidine residues at each metal site. AMPD1 is mainly expressed in fast twitching fibers and is inhibited by ATP. The limited trypsinization of the 95-residue N-terminal domain of rabbit AMPD1 desensitizes the enzyme towards ATP inhibition at the optimal pH 6.5, but not at pH 7.1. METHODS: The modified residues of rabbit AMPD1 after incubation with radioactive diethyl pyrocarbonate ([14C]DEP) causing the desensitization to inhibition by ATP at pH 7.1 have been identified by sequence analysis and MS analysis of the radioactive peptides liberated from the carbethoxylated enzyme by limited proteolysis with trypsin. RESULTS: The study confirms the presence of a dinuclear zinc site in rabbit AMPD1 and shows that carbethoxylation of His-51 at the N-terminus of the catalytic subunit removes the inhibition of the enzyme by ATP at pH 7.1. CONCLUSIONS: The desensitization to ATP is due to the modification of His-51 of the Zn2 coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized. GENERAL SIGNIFICANCE: The progress in the study of the complex regulation of rabbit AMPD1 that shares an identical amino acid sequence with the human enzyme is important in relation to the role of the enzyme during mammalian evolution.

AMP deamination is sufficient to replicate an atrophy-like metabolic phenotype in skeletal muscle.

BACKGROUND: Skeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. METHODS: AMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. RESULTS: TA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (-25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (-16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (-24%), phosphorylation of AMPK substrates (-14%), and PGC-1α protein (-22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (-55%), basal ATP synthase-dependent (-13%), and maximal uncoupled oxygen consumption (-15%). CONCLUSIONS: Increased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting.

Purine Nucleotides Metabolism and Signaling in Huntington's Disease: Search for a Target for Novel Therapies.

Huntington's disease (HD) is a multi-system disorder that is caused by expanded CAG repeats within the exon-1 of the huntingtin (HTT) gene that translate to the polyglutamine stretch in the HTT protein. HTT interacts with the proteins involved in gene transcription, endocytosis, and metabolism. HTT may also directly or indirectly affect purine metabolism and signaling. We aimed to review existing data and discuss the modulation of the purinergic system as a new therapeutic target in HD. Impaired intracellular nucleotide metabolism in the HD affected system (CNS, skeletal muscle and heart) may lead to extracellular accumulation of purine metabolites, its unusual catabolism, and modulation of purinergic signaling. The mechanisms of observed changes might be different in affected systems. Based on collected findings, compounds leading to purine and ATP pool reconstruction as well as purinergic receptor activity modulators, i.e., P2X7 receptor antagonists, may be applied for HD treatment.

Surface AMP deaminase 2 as a novel regulator modifying extracellular adenine nucleotide metabolism.

Adenine nucleotides represent crucial immunomodulators in the extracellular environment. The ectonucleotidases CD39 and CD73 are responsible for the sequential catabolism of ATP to adenosine via AMP, thus promoting an anti-inflammatory milieu induced by the "adenosine halo". AMPD2 intracellularly mediates AMP deamination to IMP, thereby both enhancing the degradation of inflammatory ATP and reducing the formation of anti-inflammatory adenosine. Here, we show that this enzyme is expressed on the surface of human immune cells and its predominance may modify inflammatory states by altering the extracellular milieu. Surface AMPD2 (eAMPD2) expression on monocytes was verified by immunoblot, surface biotinylation, mass spectrometry, and immunofluorescence microscopy. Flow cytometry revealed enhanced monocytic eAMPD2 expression after TLR stimulation. PBMCs from patients with rheumatoid arthritis displayed significantly higher levels of eAMPD2 expression compared with healthy controls. Furthermore, the product of AMPD2-IMP-exerted anti-inflammatory effects, while the levels of extracellular adenosine were not impaired by an increased eAMPD2 expression. In summary, our study identifies eAMPD2 as a novel regulator of the extracellular ATP-adenosine balance adding to the immunomodulatory CD39-CD73 system.

Mechanism and structure based design of inhibitors of AMP and adenosine deaminase.

Inhibitors of the enzyme adenosine monophosphate deaminase (AMPD) show interesting levels of herbicidal activity. An enzyme mechanism-based approach has been used to design new inhibitors of AMPD starting from nebularine (6) and resulting in the synthesis of 2-deoxy isonebularine (16). This compound is a potent inhibitor of the related enzyme adenosine deaminase (ADA; IC50 16 nM), binding over 5000 times more strongly than nebularine. It is proposed that the herbicidal activity of compound 16 is due to 5́-phosphorylation in planta to give an inhibitor of AMPD. Subsequently, an enzyme structure-based approach was used to design new non-ribosyl AMPD inhibitors. The initial lead structure was discovered by in silico screening of a virtual library against plant AMPD. In a second step, binding to AMPD was further optimised via more detailed molecular modeling leading to 2-(benzyloxy)-5-(imidazo[2,1-f][1,2,4]triazin-7-yl)benzoic acid (36) (IC50 300 nM). This compound does not inhibit ADA and shows excellent selectivity for plant over human AMPD.