PLD4

Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5'-to-3' manner and are blocked by 5'-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a 'link-and-release' two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3'- phosphistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.

Animal models of diabetes, such as db/db mice, are a useful tool for deciphering the genetic background of molecular changes at the initial stages of disease development. Our goal was to find early transcriptomic changes in three tissues involved in metabolism regulation in db/db mice: adipose tissue, muscle tissue and liver tissue. Nine animals (three per time point) were studied. Tissues were collected at 8, 12 and 16 weeks of age. Transcriptome-wide analysis was performed using mRNA-seq. Libraries were sequenced on NextSeq (Illumina). Differential expression (DE) analysis was performed with edgeR. The analysis of the gene expression profile shared by all three tissues revealed eight upregulated genes (Irf7, Sp100, Neb, Stat2, Oas2, Rtp4, H2-T24 and Oasl2) as early as between 8 and 12 weeks of age. The most pronounced differences were found in liver tissue: nine DE genes between 8 and 12 weeks of age (Irf7, Ly6a, Ly6g6d, H2-Dma, Pld4, Ly86, Fcer1g, Ly6e and Idi1) and five between 12 and 16 weeks of age (Irf7, Plac8, Ifi44, Xaf1 and Ly6a) (adj. p-value < 0.05). The mitochondrial transcriptomic profile also changed with time: we found two downregulated genes in mice between 8 and 12 weeks old (Ckmt2 and Cox6a2) and five DE genes between 12 and 16 weeks of age (Mavs, Tomm40L, Mtfp1, Ckmt2 and Cox6a2). The KEGG pathway analysis showed significant enrichment in pathways related to the autoimmune response and cytosolic DNA sensing. Our results suggest an important involvement of the immunological response, mainly cytosolic nucleic acid sensing, and mitochondrial signalling in the early stages of diabetes and obesity.

Geese evolved from migratory birds, and when they consume excessive high-energy feed, glucose is converted into triglycerides. A large amount of triglyceride deposition can induce incomplete oxidation of fatty acids, leading to lipid accumulation in the liver and the subsequent formation of fatty liver. In the Chaoshan region of Guangdong, China, Shitou geese develop a unique form of fatty liver through 24 h overfeeding of brown rice. To investigate the mechanisms underlying the formation of fatty liver in Shitou geese, we collected liver samples from normally fed and overfed geese. The results showed that the liver size in the treatment group was significantly larger, weighing 3.5 times more than that in the control group. Extensive infiltration of lipid droplets was observed in the liver upon staining of tissue sections. Biochemical analysis revealed that compared to the control group, the treatment group showed significantly elevated levels of total cholesterol (T-CHO), triglycerides (TG), and glycogen in the liver. However, no significant differences were observed in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are common indicators of liver damage. Furthermore, we performed a combined transcriptomic and lipidomic analysis of the liver samples and identified 1,510 differentially expressed genes (DEGs) and 1,559 significantly differentially abundant metabolites (SDMs). The enrichment analysis of the DEGs revealed their enrichment in metabolic pathways, cellular process-related signaling pathways, and specific lipid metabolism pathways. We also conducted KEGG enrichment analysis of the SDMs and compared them with the enriched signaling pathways obtained from the DEGs. In this study, we identified 3 key signaling pathways involved in the formation of fatty liver in Shitou geese, namely, the biosynthesis of unsaturated fatty acids, glycerol lipid metabolism, and glycerophospholipid metabolism. In these pathways, genes such as glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), diacylglycerol O-acyltransferase 2 (DGAT2), lipase, endothelial (LIPG), lipoprotein lipase (LPL), phospholipase D family member 4 (PLD4), and phospholipase A2 group IVF (PLA2G4F) may regulate the synthesis of metabolites, including triacylglycerol (TG), phosphatidate (PA), 1,2-diglyceride (DG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). These genes and metabolites may play a predominant role in the development of fatty liver, ultimately promoting the accumulation of TG in the liver and leading to the progression of fatty liver.