Encoding Diacylglycerol Kinase Research Articles

Diacylglycerol kinase epsilon protects against renal ischemia/reperfusion injury in mice through Krüppel-like factor 15/klotho pathway

Although recent studies have indicated that mutations in the gene encoding diacylglycerol kinase epsilon (DGKE) result in some proteinuria related hereditary kidney diseases, the DGKE expression pattern in the kidney and its contribution to acute kidney injury (AKI) remain unknown. Therefore, the present study was designed to detect the role of DGKE in mice with AKI. DGKE expression was time-dependently altered in the kidneys of mice with renal ischemia/reperfusion injury (IRI). Compared with wild-type (WT) mice, DGKE- overexpressing mice (Rosa26-Dgke+/+ ) exhibited protective effects against renal IRI, including reduced serum creatinine, blood urea concentration, tubular cell death and inflammatory responses as well as improved morphological injuries. Consistently, in vitro, DGKE overexpression in human renal proximal tubule (HK-2) cells also protected against oxygen-glucose deprivation (OGD)/reoxygenation-induced cell death. Mechanistically, DGKE regulated Klotho expression, at least partly via the transcription factor Krüppel-like factor (KLF) 15. Moreover, a significant reduction in DGKE was also found in kidneys from patients with ischemia-associated acute tubular necrosis (ATN). Collectively, our studies demonstrate that DGKE protects against AKI in mice at least partly through KLF15/Klotho signaling pathway, indicating that DGKE may present an innovative therapeutic strategy for treating patients with AKI.

Molecular mapping and identification of a candidate gene for new locus Hg2 conferring hairy glume in wheat

Glume hairiness or pubescence that occurs in hexaploid common wheat and its relatives at different ploidy levels is a distinct morphological marker. Current knowledge about the genetic control of wheat glume hairiness is based on study of Hg1 (formerly Hg) on chromosome 1AS. Here, we report characterization of a new locus for hairy glume Hg2 in synthetic hexaploid wheat line CIGM86.944. Hg2 was inherited a dominant allele. Bulked segregant analysis and RNA-sequencing (BSR-Seq) was performed on an F2:3 population from cross CIGM86.944 × Shannong 29 (glabrous glume), which localized Hg2 in a 2.02 cM genetic interval corresponding to ∼1.08 Mb (754,001,564–755,082,433 Mb) on chromosome 2BL in the Chinese Spring reference genome. Gene annotation and expression identified TraesCS2B02 G562300.1 encoding diacylglycerol kinase 5 protein and TraesCS2B02 G561400.1 encoding a wound-responsive family protein as possible candidate genes regulating development of glume hairiness. The identification of Hg2 provides new insights into the genetic control of glume hairiness in wheat.

The Lactococcal dgkB (yecE) and dxsA Genes for Lipid Metabolism Are Involved in the Resistance to Cell Envelope-Acting Antimicrobials.

The emergence of antibiotic-resistant bacteria led to an urgent need for next-generation antimicrobial agents with novel mechanisms of action. The use of positively charged antimicrobial peptides that target cytoplasmic membrane is an especially promising strategy since essential functions and the conserved structure of the membrane hinder the development of bacterial resistance. Aureocin A53- and enterocin L50-like bacteriocins are highly cationic, membrane-targeting antimicrobial peptides that have potential as next-generation antibiotics. However, the mechanisms of resistance to these bacteriocins and cross-resistance against antibiotics must be examined before application to ensure their safe use. Here, in the model bacterium Lactococcus lactis, we studied the development of resistance to selected aureocin A53- and enterocin L50-like bacteriocins and its correlation with antibiotics. First, to generate spontaneous resistant mutants, L. lactis was exposed to bacteriocin BHT-B. Sequencing of their genomes revealed single nucleotide polymorphisms (SNPs) in the dgkB (yecE) and dxsA genes encoding diacylglycerol kinase and 1-deoxy-D-xylulose 5-phosphate synthase, respectively. Then, selected mutants underwent susceptibility tests with a wide array of bacteriocins and antibiotics. The highest alterations in the sensitivity of studied mutants were seen in the presence of cytoplasmic membrane targeting bacteriocins (K411, Ent7, EntL50, WelM, SalC, nisin) and antibiotics (daptomycin and gramicidin) as well as lipid II cycle-blocking bacteriocins (nisin and Lcn972) and antibiotics (bacitracin). Interestingly, decreased via the SNPs accumulation sensitivity to membrane-active bacteriocins and antibiotics resulted in the concurrently increased vulnerability to bacitracin, carbenicillin, or chlortetracycline. It is suspected that SNPs may result in alterations to the efficiency of the nascent enzymes rather than a total loss of their function as neither deletion nor overexpression of dxsA restored the phenotype observed in spontaneous mutants.

The Phenotypic Spectrum of Nephropathies Associated with Mutations in Diacylglycerol Kinase ε.

The recent discovery of mutations in the gene encoding diacylglycerol kinase ε (DGKE) identified a novel pathophysiologic mechanism leading to HUS and/or MPGN. We report ten new patients from eight unrelated kindreds with DGKE nephropathy. We combined these cases with all previously published cases to characterize the phenotypic spectrum and outcomes of this new disease entity. Most patients presented with HUS accompanied by proteinuria, whereas a subset of patients exhibited clinical and histologic patterns of MPGN without TMA. We also report the first two patients with clinical and histologic HUS/MPGN overlap. DGKE-HUS typically manifested in the first year of life but was not exclusively limited to infancy, and viral triggers frequently preceded HUS episodes. We observed signs of complement activation in some patients with DGKE-HUS, but the role of complement activation remains unclear. Most patients developed a slowly progressive proteinuric nephropathy: 80% of patients did not have ESRD within 10 years of diagnosis. Many patients experienced HUS remission without specific treatment, and a few patients experienced HUS recurrence despite complete suppression of the complement pathway. Five patients received renal allografts, with no post-transplant recurrence reported. In conclusion, we did not observe a clear genotype-phenotype correlation in patients with DGKE nephropathy, suggesting additional factors mediating phenotypic heterogeneity. Furthermore, the benefits of anti-complement therapy are questionable but renal transplant may be a feasible option in the treatment of patients with this condition.

DGKE and atypical HUS

A new study reports that recessive loss-of-function mutations in the gene encoding diacylglycerol kinase ε result in atypical hemolytic-uremic syndrome. Notably, mutations in DGKE are not associated with activation of the complement pathway, the only other identified cause of this disorder so far, and have important implications for patient management.

Recessive mutations in DGKE cause atypical hemolytic-uremic syndrome

Pathologic thrombosis is a major cause of mortality. Hemolytic-uremic syndrome (HUS) features episodes of small vessel thrombosis resulting in microangiopathic hemolytic anemia, thrombocytopenia and renal failure1. Atypical HUS (aHUS) can result from genetic or autoimmune factors2 that lead to pathologic complement cascade activation3. By exome sequencing we identify recessive mutations in DGKE (diacylglycerol kinase epsilon) that co-segregate with aHUS in 9 unrelated kindreds, defining a distinctive Mendelian disease. Affected patients present with aHUS before age 1, have persistent hypertension, hematuria and proteinuria (sometimes nephrotic range), and develop chronic kidney disease with age. DGKE is found in endothelium, platelets, and podocytes. Arachidonic acid-containing diacylglycerols (DAG) activate protein kinase C, which promotes thrombosis. DGKE normally inactivates DAG signaling. We infer that loss of DGKE function results in a pro-thrombotic state. These findings identify a new mechanism of pathologic thrombosis and kidney failure and have immediate implications for treatment of aHUS patients.

Common variants in DGKK are strongly associated with risk of hypospadias

Hypospadias is a common congenital malformation of the male external genitalia. We performed a genome-wide association study using pooled DNA from 436 individuals with hypospadias (cases) and 494 controls of European descent and selected the highest ranked SNPs for individual genotyping in the discovery sample, an additional Dutch sample of 133 cases and their parents, and a Swedish series of 266 cases and 402 controls. Individual genotyping of two SNPs (rs1934179 and rs7063116) in DGKK, encoding diacylglycerol kinase κ, produced compelling evidence for association with hypospadias in the discovery sample (allele-specific odds ratio (OR) = 2.5, P = 2.5 × 10⁻¹¹ and OR = 2.3, P = 2.9 × 10⁻⁹, respectively) and in the Dutch (OR = 3.9, P = 2.4 × 10⁻⁵ and OR = 3.8, P = 3.4 × 10⁻⁵) and Swedish (OR = 2.5, P = 2.6 × 10⁻⁸ and OR = 2.2, P = 2.7 × 10⁻⁶) replication samples. Expression studies showed expression of DGKK in preputial tissue of cases and controls, which was lower in carriers of the risk allele of rs1934179 (P = 0.047). We propose DGKK as a major risk gene for hypospadias.

Diacylglycerol Kinase in T Cell Anergy

Defective Ras signaling contributes to T cell anergy, which occurs when T cells are presented with antigens in the absence of costimulatory signals and leads to T cells’ unresponsiveness to subsequent stimulation. T cell anergy is a key mechanism by which inappropriate immune responses are prevented. Previous work had shown that diacylglycerol (DAG)-mediated signaling was defective in anergic T cells and that abundance of the transcript encoding diacylglycerol kinase α (DGK-α) was decreased in activated T cells and increased in anergic T cells. Two groups (Zha et al . and Olenchock et al .) extend these observations and show that DGK-α, an enzyme that converts DAG to phosphatic acid, contributes to T cell anergy. Zha et al . showed that introduction of constitutively active Ras, one of the components downstream of DAG, restored T cell responsiveness [interleukin-2 (IL-2) production and mitogen-activated protein kinase (MAPK) phosphorylation] to cells previously rendered anergic. Expression of the Dgka gene was increased in anergic T cells in the system used by Zha et al ., and overexpression of DGK-α in resting T cells diminished recruitment of the Ras guanine nucleotide exchange factor RasGRP1 to the plasma membrane, decreased IL-2 production, and decreased MAPK activation in response to stimulation. Olenchock et al . used cells from Dgka −/− mice to show that T cells from these animals failed to undergo anergy in response to T cell receptor activation in the absence of costimulatory signals. Together, these two reports demonstrate a key role in DAG metabolism in promoting T cell anergy (see Mueller). What remains to be determined is how DGK activity is regulated to prevent excessive activity in cells that receive an activating signal and to promote activity in cells that receive a signal that produces anergy. Y. Zha, R. Marks, A. W. Ho, A. C. Peterson, S. Janardhan, I. Brown, K. Praveen, S. Stang, J. C. Stone, T. F. Gajewski, T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-α. Nat. Immunol. 7 , 1166-1173 (2006). [PubMed] B. A. Olenchock, R. Guo, J. H. Carpenter, M. Jordan, M. K. Topham, G. A. Koretzky, X.-P. Zhong, Disruption of diacylglycerol metabolism impairs the induction of T cell anergy. Nat. Immunol. 7 , 1174-1181 (2006). [PubMed] D. L. Mueller, Linking diacylglycerol kinase to T cell anergy. Nat. Immunol. 7 , 1132-1134 (2006). [PubMed]

Molecular Basis of Amplification in Drosophila Phototransduction : Roles for G Protein, Phospholipase C, and Diacylglycerol Kinase

In Drosophila photoreceptors, the amplification responsible for generating quantum bumps in response to photoisomerization of single rhodopsin molecules has been thought to be mediated downstream of phospholipase C (PLC), since bump amplitudes were reportedly unaffected in mutants with greatly reduced levels of either G protein or PLC. We now find that quantum bumps in such mutants are reduced ∼3- to 5-fold but are restored to near wild-type values by mutations in the rdgA gene encoding diacylglycerol kinase (DGK) and also by depleting intracellular ATP. The results demonstrate that amplification requires activation of multiple G protein and PLC molecules, identify DGK as a key enzyme regulating amplification, and implicate diacylglycerol as a messenger of excitation in Drosophila phototransduction.

Genes expressed in Pseudomonas putida during colonization of a plant-pathogenic fungus.

In vivo expression technology (IVET) was employed to study colonization of Phytophthora parasitica by a biological control bacterium, Pseudomonas putida 06909, based on a new selection marker. The pyrB gene, which encodes aspartate transcarbamoylase, an enzyme used for pyrimidine biosynthesis, was cloned from P. putida 06909. A pyrB-disrupted mutant did not grow in pyrimidine-deficient media unless it was complemented with pyrBC' behind an active promoter. Thirty clones obtained from P. putida 06909 that were expressed on fungal hyphae but not on culture media were isolated by IVET based on the promoterless transcriptional fusion between pyrBC' and lacZ. Nineteen of these clones were induced during late-stage bacterial growth in vitro, while 11 of the clones were expressed only when they were inoculated onto fungal hyphae. Restriction analysis of these 11 clones revealed that there were five unique clones. Sequence analyses of three of the five unique clones showed that the 3' ends of the clones fused to pyrB were similar to genes encoding diacylglycerol kinase (DAGK), bacterial ABC transporters, and outer membrane porins. The sequences of the two other clones were not similar to the sequences of any of the genes in the database used. A LuxR family response regulator was found upstream of DAGK, and a LysR family response regulator was found upstream of the ABC transporter. The location of the inducible promoter of two clones suggested that DAGK and the ABC transporter are induced and may play a role in colonization of the fungus P. parasitica by P. putida 06909.

Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana.

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 CDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.

The nucleotide sequence of a 2·1 kb fragment from chromosome VI of Saccharomyces cerevisiae identifies a tRNAGly gene, part of a delta element and a palindromic sequence

The nucleotide sequence was determined of a 2.1 kb DNA fragment located at approximately 35 kb to the right of the centromere of chromosome VI from Saccharomyces cerevisiae. Analysis revealed the presence of a tRNA(GLy) gene, part of a delta element and a remarkable palindromic sequence. The longest open reading frame found encodes a putative protein of 195 amino acids. Although the fragment was isolated by hybridization to a human diacylglycerol kinase cDNA, no evidence was obtained for the presence of a gene encoding diacylglycerol kinase.