Adenylate kinase 9 is essential for sperm function and male fertility in mammals.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

Facile Method for High-throughput Identification of Stabilizing Mutations.

Characterizing the effects of mutations on stability is critical for understanding the function and evolution of proteins and improving their biophysical properties. High throughput folding and abundance assays have been successfully used to characterize missense mutations associated with reduced stability. However, screening for increased thermodynamic stability is more challenging since such mutations are rarer and their impact on assay readout is more subtle. Here, a multiplex assay for high throughput screening of protein folding was developed by combining deep mutational scanning, fluorescence-activated cell sorting, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of the method correlates with stability and that mutants with up to 13 °C increase in thermal melting temperature could be identified with low false positive rate. The discovery of many stabilizing mutations also enabled the analysis of general substitution patterns associated with increased stability in Adenylate kinase. This high throughput method to identify stabilizing mutations can be combined with functional screens to identify mutations that improve both stability and activity.

Aberrant DNA N6 -methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase-dependent 2'-deoxynucleotide pool sanitation.

Intracellular decay of N6 -methyladenine (m6A)-containing RNA potentially induces aberrant N6 -methyl-2'-adenine (6mdA) misincorporation into DNA. Biophysically, misincorporated 6mdA may destabilize the DNA duplex in a manner similar to bona fide methylated 6mdA DNA, thereby affecting DNA replication and transcription. Utilizing heavy stable isotope labeling and ultrasensitive UHPLC-MS/MS assay, we demonstrate that intracellular m6A-RNA decay does not generate free 6mdA species, nor lead to any misincorporated DNA 6mdA in most mammalian cell lines tested, unveiling the existence of a sanitation mechanism that prevents 6mdA misincorporation. Depletion of deaminase ADAL increases the levels of free 6mdA species, concomitant with the presence of DNA-misincorporated 6mdA resulting from intracellular RNA m6A decay, suggesting that ADAL catabolizes 6mdAMP in vivo. Furthermore, we show that the overexpression of adenylate kinase 1 (AK1) promotes 6mdA misincorporation, while AK1 knockdown diminishes 6mdA incorporation, in ADAL-deficient cells. We conclude that ADAL together with other factors (such as MTH1) contributes to 2'-deoxynucleotide pool sanitation in most cells but compromised sanitation (e.g., in NIH3T3 cells) and increased AK1 expression may facilitate aberrant 6mdA incorporation. This sanitation mechanism may provide a framework for the maintenance of the epigenetic 6mdA landscape.

Magnesium and cell energetics: At the junction of metabolism of adenylate and non-adenylate nucleotides.

Free magnesium (Mg2+) represents a powerful signal arising from interconversions of adenylates (ATP, ADP and AMP). This is a consequence of the involvement of adenylate kinase (AK) which equilibrates adenylates and uses defined species of Mg-complexed and Mg-free adenylates in both directions of its reaction. However, cells contain also other reversible Mg2+-dependent enzymes that equilibrate non-adenylate nucleotides (uridylates, cytidylates and guanylates), i.e. nucleoside monophosphate kinases (NMPKs) and nucleoside diphosphate kinase (NDPK). Here, we propose that AK activity is tightly coupled to activities of NMPK and NDPK, linking adenylate equilibrium to equilibria of other nucleotides, and with [Mg2+] controlling the ratios of Mg-chelated and Mg-free nucleotides. This coupling establishes main hubs for adenylate-driven equilibration of non-adenylate nucleotides, with [Mg2+] acting as signal arising from all nucleotides rather than adenylates only. Further consequences involve an overall adenylate control of UTP-, GTP- and CTP-dependent pathways and the availability of substrates for RNA and DNA synthesis.

The Effect of Heterozygous Mutation of Adenylate Kinase 2 Gene on Neutrophil Differentiation.

Mitochondrial ATP production plays an important role in most cellular activities, including growth and differentiation. Previously we reported that Adenylate kinase 2 (AK2) is the main ADP supplier in the mitochondrial intermembrane space in hematopoietic cells, especially in the bone marrow. AK2 is crucial for the production of neutrophils and T cells, and its deficiency causes reticular dysgenesis. However, the relationship between ADP supply by AK2 and neutrophil differentiation remains unclear. In this study, we used CRISPR/Cas9 technology to establish two heterozygous AK2 knock-out HL-60 clones as models for reticular dysgenesis. Their AK2 activities were about half that in the wild-type (WT). Furthermore, neutrophil differentiation was impaired in one of the clones. In silico analysis predicted that the obtained mutations might cause a structural change in AK2. Time course microarray analysis of the WT and mutants revealed that similar gene clusters responded to all-trans retinoic acid treatment, but their expression was lower in the mutants than in WT. Application of fructose partially restored neutrophil differentiation in the heterozygous knock-out HL-60 clone after all-trans retinoic acid treatment. Collectively, our study suggests that the mutation of N-terminal region in AK2 might play a role in AK2-dependent neutrophil differentiation and fructose could be used to treat AK2 deficiency.

Red cell adenylate kinase deficiency in China: molecular study of 2 new mutations (413G > A, 223dupA).

BACKGROUND: Adenylate kinase (AK) is a monomolecular enzyme widely found in a variety of organisms. It mainly catalyses the reversible transfer of adenosine nucleotide phosphate groups and plays an important role in maintaining energy metabolism. AK deficiency is a rare genetic disorder that is related to haemolytic anaemia. Chronic haemolytic anaemia associated with AK deficiency is a rare condition, and only 14 unrelated families have been reported thus far. Moreover, only 11 mutations have been identified in the AK1 gene, with only 3 cases of psychomotor impairment. CASE PRESENTATION: The patient was a 3-year-old boy with severe haemolytic anaemia and psychomotor retardation. A molecular study of the patient's AK gene revealed 2 different mutations: a heterozygous missense mutation in exon 6 (c.413G > A) and a heterozygous frameshift mutation in exon 5 (c.223dupA). Molecular modelling analyses indicated that AK gene inactivation resulted in a lack of AK activity. The patient recovered after regular blood transfusion therapy. CONCLUSIONS: AK1 deficiency was diagnosed on the basis of low enzymatic activity and the identification of a mutation in the AK1 gene located on chromosome 9q. Here, we report the first case of moderate red cell AK1 deficiency associated with chronic nonspherocytic haemolytic anaemia (CNSHA) in China. The genetic mutations were confirmed by Sanger sequencing. The variants were classified as pathogenic by bioinformatics tools, such as ACMG/AMP guidelines, Mutation Taster, SIFT, MACP, REVEL and PolyPhen2.2. Based on our evidence and previous literature reports, we speculate that the site of the AK1 gene c.413G > A (p.Arg138His) mutation may be a high-frequency mutation site and the other mutation (c.223dupA) might be related to the neuropathogenicity caused by AK1 deficiency. NGS should be a part of newborn to early childhood screening to diagnose rare and poorly diagnosed genetic diseases as early as possible.

Structural Dynamics Support Electrostatic Interactions in the Active Site of Adenylate Kinase.

Electrostatic preorganization as well as structural and dynamic heterogeneity are often used to rationalize the remarkable catalytic efficiency of enzymes. However, they are often presented as incompatible because the generation of permanent electrostatic effects implies that the protein structure remains rigid. Here, we use a metric, electric fields, that can treat electrostatic contributions and dynamics effects on equal footing, for a unique perspective on enzymatic catalysis. We find that the residues that contribute the most to electrostatic interactions with the substrate in the active site of Adenylate Kinase (our working example) are also the most flexible residues. Further, entropy-tuning mutations raise flexibility at the picosecond timescale where more conformations can be visited on short time periods, thereby softening the sharp heterogeneity normally visible at the microsecond timescale.

Adenylate kinase 1 overexpression increases locomotor activity in medaka fish.

Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

Differential effect of canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice.

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

Arjunolic acid from Cyclocarya paliurus ameliorates diabetic retinopathy through AMPK/mTOR/HO-1 regulated autophagy pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyclocarya paliurus (CP) is a traditional Chinese herb and possesses a variety of biological activities including anti-hyperglycemia, anti-hyperlipidemia, antioxidant and anti-inflammation. Arjunolic acid (AA) is an abundant and bioactive ingredient in CP that shows significant protection against many metabolic diseases such as diabetic complication. Diabetic retinopathy (DR) is a serious complication of diabetes and may lead to vision loss. However, the protective effects and underlying mechanisms of AA against DR is not still understood. AIM OF THE STUDY: We aimed to investigate whether AA activates AMPK/mTOR/HO-1 regulated autophagy pathway to alleviate DR. MATERIALS AND METHODS: In the study, the STZ-induced diabetic model of rats was established, and AA with 10 and 30 mg/kg dosages was given orally for ten weeks to investigate their effect on retinal injury of DR. H2O2-induced ARPE-19 cells were applied to evaluate anti-apoptosis and anti-oxidant effect of AA. RESULTS: The results revealed that AA could prevent STZ-induced weight loss and increase the retinal thickness and nuclei counts. The level of HO-1 protein was upregulated both in vivo and in vitro. In addition, AA prevented retinal damage and cell apoptosis through the AMPK-mTOR-regulated autophagy pathway. Furthermore, anti-apoptosis capacity, as well as the expression of HO-1 and LC3 protein, were effectively locked by AMPK inhibitor dorsomorphin dihydrochloride (compound C). CONCLUSIONS: This finding implies that AA may be a promising candidate drug by protecting retinal cells from STZ-induced oxidative stress and inflammation through the AMPK/mTOR/HO-1 regulated autophagy pathway.

A novel homozygous missense mutation in AK7 causes multiple morphological anomalies of the flagella and oligoasthenoteratozoospermia.

PURPOSE: To identify the genetic causes of multiple morphological anomalies of the flagella (MMAF) and oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing (WES) was performed on the proband to identify pathogenic mutation for infertility. Western blotting and immunofluorescence analysis detected the expression level and localization of adenylate kinase 7 (AK7). RESULTS: We identified a novel homozygous missense mutation (NM_152327: c.1846G > A; p.E616K) in AK7 in two brothers with MMAF and OAT from a consanguineous family by WES. Western blotting and immunofluorescence experiments determined that the expression level of AK7 decreased in the sperm from the proband. The proband and his wife underwent two cycles of intracytoplasmic sperm injection (ICSI) treatment but got unfavorable outcomes. CONCLUSION: This study could provide precise genetic diagnosis for the patient and expand the spectrum of AK7 mutations.

Conditional disruption of AMP kinase in dopaminergic neurons promotes Parkinson's disease-associated phenotypes in vivo.

Emerging studies implicate energy dysregulation as an underlying trigger for Parkinson's disease (PD), suggesting that a better understanding of the molecular pathways governing energy homeostasis could help elucidate therapeutic targets for the disease. A critical cellular energy regulator is AMP kinase (AMPK), which we have previously shown to be protective in PD models. However, precisely how AMPK function impacts on dopaminergic neuronal survival and disease pathogenesis remains elusive. Here, we showed that Drosophila deficient in AMPK function exhibits PD-like features, including dopaminergic neuronal loss and climbing impairment that progress with age. We also created a tissue-specific AMPK-knockout mouse model where the catalytic subunits of AMPK are ablated in nigral dopaminergic neurons. Using this model, we demonstrated that loss of AMPK function promotes dopaminergic neurodegeneration and associated locomotor aberrations. Accompanying this is an apparent reduction in the number of mitochondria in the surviving AMPK-deficient nigral dopaminergic neurons, suggesting that an impairment in mitochondrial biogenesis may underlie the observed PD-associated phenotypes. Importantly, the loss of AMPK function enhances the susceptibility of nigral dopaminergic neurons in these mice to 6-hydroxydopamine-induced toxicity. Notably, we also found that AMPK activation is reduced in post-mortem PD brain samples. Taken together, these findings highlight the importance of neuronal energy homeostasis by AMPK in PD and position AMPK pathway as an attractive target for future therapeutic exploitation.

Reticular dysgenesis exacerbated by hemophagocytic lymphohistiocytosis and the presence of unusual histiocyte-like cells in bone marrow.

We report a rare case of agranulocytosis and lymphopenia complicated with hemophagocytic lymphohistiocytosis. Diagnosis of reticular dysgenesis was made by detection of a pathogenic stop gain variant in the AK2 gene on targeted next generation sequencing and confirmed by Sanger sequencing. Parents were found to be carriers for this variant. Bone marrow aspirate and biopsy was also performed with a clinical diagnosis of severe combined immunodeficiency with HLH. However, no hemophagocytosis was noted in the bone marrow aspirate or trephine biopsy. Instead, it showed aggregates of large histiocyte-like cells, scattered erythroid precursors and megakaryocytes. These cells were confused to be some form of storage cells, but did not resemble storage cells seen in Gaucher's disease or Niemann Pick disease. Myeloid precursors were very few in number. Reticular dysgenesis was not suspected during admission due to a lack of awareness of this entity. Testing for sensorineural deafness in neonates with severe agranulocytosis and lymphopenia would facilitate an early diagnosis of reticular dysgenesis. To the best of our knowledge, hemophagocytic lymphohistiocytosis has not been previously reported in association with reticular dysgenesis.

The protective effect of sulforaphane against oxidative stress in granulosa cells of patients with polycystic ovary syndrome (PCOS) through activation of AMPK/AKT/NRF2 signaling pathway.

Increased production of reactive oxygen species (ROS) in granulosa cells (GCs) causes oxidative stress (OS) and plays a role in pathogenesis of polycystic ovary syndrome (PCOS). Sulforaphane (SFN) has received a great deal of attention as potent antioxidant because of its ability to induce expression of antioxidant enzymes through nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway. Therefore, the present study was done to investigate the protective effect of SFN against OS in granulosa-lutein cells (GLCs) of patients with PCOS through activation of AMP-activated protein kinase (AMPK)/AKT/NRF2 signaling pathway. GLCs were isolated from patients with PCOS and healthy fertile women, as control group, during egg retrieval procedure. Level of intracellular ROS and apoptosis was determined in the isolated cells. For investigating the protective effect of SFN against ROS production and apoptosis in GLCs, the cells were cultured for 24 h in the presence or absence of SFN. Finally, expression of AMPK, AKT, and NRF2 proteins and genes was evaluated by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The results indicated the increased ROS and apoptosis levels in GLCs isolated from patients with PCOS compared to the control group. Addition of SFN to culture medium of GLCs of patients with PCOS reduced intracellular ROS and apoptosis levels, and increased expression of AMPK, AKT, and NRF2 proteins and genes. Our findings demonstrated the protective effect of SFN against OS by lowering level of ROS and apoptosis possibly through activation of AMPK, AKT, and NRF2 proteins and genes expression.

Co-modification with MSC membrane and PDA prevents Fe3O4-induced pulmonary toxicity in mice via AMPK-ULK1 axis.

Fe3O4 nanoparticles are widely used in the diagnosis and treatment of diseases due to their superparamagnetism, but their toxicity in vivo, which can result in apoptosis or autophagy, cannot be ignored. It has been reported that polydopamine (PDA) modification can reduce the toxicity of Fe3O4 and increase its biocompatibility. However, more research is warranted to further improve the modification method. We therefore developed a new method to coat Fe3O4@PDA nanoparticles with the mesenchymal stem cell membrane (MSCM) and evaluated the toxicity of the modified particles in the lungs of mice. We found that the MSCM modification significantly reduced lung injury induced by Fe3O4 particles in mice. Compared with Fe3O4@PDA nanoparticles, co-modification with MSCM and PDA significantly reduced autophagy and apoptosis in mouse lung tissue, and reduced activation of autophagy mediated by the AMPK-ULK1 pathway axis. Thus, co-modification with MSCM and PDA prevents Fe3O4-induced pulmonary toxicity in mice by inhibiting autophagy, apoptosis, and oxidative stress.

Hypoglycemic effect of astragaloside IV via modulating gut microbiota and regulating AMPK/SIRT1 and PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Astragali, the dried root of Astragalus mongholicus Bunge, has long been used in traditional Chinese Medicine to treat diabetes. Astragaloside IV (AS-IV), one of the most active ingredients in the root, has been shown to have anti-diabetes ability; however, its underlying mechanism is still unclear. MATERIALS AND METHODS: In this study, we evaluated the hypoglycemic effect and possible mechanisms of AS-IV in diabetic mice and insulin resistance-HepG2 cells. The components of the intestinal microflora in mice with type 2 diabetes mellitus (T2DM) were determined using high-throughput 16S rRNA gene sequencing. Moreover, the molecular mechanisms of specific members of insulin signaling pathways were analyzed. RESULTS: AS-IV significantly reversed the abnormalities in blood lipids, glucose, insulin resistance, as well as oxidative stress levels in T2DM mice. Histological finding showed that AS-IV could protect the cellular architecture of the liver and pancreas. AS-IV also regulated the abundance and diversity of intestinal flora of T2DM mice in a positive direction and increased butyric acid levels. The active role of AS-IV as an anti-diabetic compound by regulating the AMPK/SIRT1 and PI3K/AKT signaling pathways was revealed using a T2DM model and verified through the intervention of inhibitors using insulin-resistance HepG2 cells. CONCLUSION: Our results suggested that AS-IV may be used as an anti-diabetic drug candidate owing to its effects of regulating gut microbiota and AMPK/SIRT1 and PI3K/AKT signaling pathways.

Evaluation of the immunomodulatory effects of C9-13-CPs in macrophages.

Short-chain chlorinated paraffins (SCCPs) have been listed as a new class of persistent organic pollutants by the Stockholm Convention. SCCPs exhibit carcinogenic-, endocrine-, and metabolism-disrupting effects. However, the knowledge of the immunomodulatory effects of SCCPs and their underlying mechanisms, especially in specific immune cells, remains limited. In addition to SCCPs, C9-13-CPs have also been detected in humans. In this study, murine RAW264.7 macrophages were exposed to C9-13-CPs at environmentally relevant concentrations to investigate whether or how C9-13-CPs exhibit immunomodulatory effects. The results showed that the exposure of RAW264.7 cells to C9-13-CPs increased cell viability, as assayed by MTT analysis at 490 nm, and also promoted cell proliferation, as indicated by EdU uptake assay, which was measured at excitation and emission wavelengths of 488 and 512 nm, respectively. In addition, exposure to C9-13-CPs not only led to elevated ATP level and intracellular Ca2+ level but also caused AMPK signaling activation and NF-κB signaling inhibition. Moreover, molecular docking showed that the ß2-AR receptor could bind to C9-13-CPs. Taken together, these results suggest that the immune dysfunction of RAW264.7 cells caused by C9-13-CPs is closely related to the ß2-AR/AMPK/NF-κB signaling axis.

Impacts of mutations on dynamic allostery of adenylate kinase.

Escherichia coli adenylate kinase (AK) is composed of CORE domain and two branch domains: LID and AMP-binding domain (AMPbd). AK exhibits considerable allostery in a reversible phosphoryl transfer reaction, which is largely attributed to the relative motion of LID and AMPbd with respect to CORE. Such an allosteric conformational change is also evident in the absence of ligands. Recent studies showed that the mutations in branch domains can adjust dynamic allostery and alter the substrate affinity and enzyme activity. In this work, we use all-atom molecular dynamics simulation to study the impacts of mutations in branch domains on AK's dynamic allostery by comparing two double mutants, i.e., LID mutant (Val135Gly, Val142Gly) and AMPbd mutant (Ala37Gly, Ala55Gly), with wild-type. Two mutants undergo considerable conformational fluctuation and exhibit deviation from the initially extended apo state to more compact structures. The LID domain in the LID mutant adjusts its relative position and firmly adheres to CORE. More strikingly, AMPbd mutations affect the relative positions of both the AMPbd domain and remote LID domain. Both domains undergo considerable movement, especially the inherent hinge swing motion of the flexible LID domain. In both mutants, the mutations can enhance the inter-domain interaction. The results about the conformation change of AK in both mutants are in line with the experiment of AK's affinity and activity. As revealed by our findings, the flexibility of branch domains and their inherent motions, especially LID domain, is highly relevant to dynamic allostery in the AK system.

Genome-Wide Identification and Expression Analysis of Tomato ADK Gene Family during Development and Stress.

Adenylate kinase (ADK) is widely distributed in organisms and plays an important role in cellular energy homeostasis. In plants, ADK has important functions in plant growth and development regulation as well as in adaptation to the environment. However, little information is available about the ADK genes in tomato (Solanum lycopersicum), an important economic crop. To investigate the characteristics and functions of ADK genes in tomato, a total of 11 ADK genes were identified and named according to their chromosomal locations. The ADK family in Arabidopsis, tomato, potato, and rice was divided into six groups, and motif analysis revealed that each SlADK protein contained five to eight conserved motifs. A total of 4 to 19 exons were identified in tomato ADK gene family members, and interestingly, most members possessed 4 exons. Several stress response elements were identified in the promoter regions of SlADKs. The 11 SlADKs were randomly distributed on 9 of the 12 tomato chromosomes. Three duplication events were observed between tomato chromosomes, and a high degree of conservation of synteny was demonstrated between tomato and potato. The online TomExpress platform prediction revealed that SlADKs were expressed in various tissues and organs, basically consistent with the data obtained from real-time quantitative PCR (qPCR). The qPCR verification was also performed to determine the expression level of SlADKs and demonstrated that the genes responded to multiple abiotic stresses, such as drought, salt, and cold. Besides, the qPCR results showed that SlADK transcription was responsive to most of the applied hormone treatment. For correlation network analysis under 44 global conditions, the results showed that the number of 17, 3, 4, and 6 coexpressed genes matched with SlADK5, 8, 9, and 11, respectively. For specific gene function analysis, expression of SlADK10 was inhibited using virus-induced gene silencing (VIGS). Compared to wild-type plants, plants with silenced SlADK10 gene had poor drought resistance, indicating SlADK10 regulated drought tolerance of tomato positively. In summary, the information provided in the present study will be helpful to understand the evolutionary relationship and their roles of tomato ADK gene family in further research.