ABSTRACT
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-phosphohistidine 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.
Subject(s)
Catalytic Domain , Models, Molecular , Phospholipase D , Phospholipase D/metabolism , Phospholipase D/chemistry , Phospholipase D/genetics , Humans , Substrate Specificity , Crystallography, X-Ray , Mutation , Lysosomes/metabolism , Lysosomes/enzymology , Phosphorylation , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/chemistry , Protein Multimerization , Protein Binding , ExodeoxyribonucleasesABSTRACT
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.
ABSTRACT
Rationale: Psychological stress has been linked to cancer development and resistance to therapy by many epidemiological and clinical studies. Stress-induced immunosuppressive microenvironment by stress hormones, in particular glucocorticoids, has been extensively studied. However, the impacts of other stress-related neurotransmitters, such as serotonin (5-hydroxytryptamine, 5-HT), on cancer development just start to be revealed. Here, we aimed to identify novel neurotransmitters involved in stress-induced growth and dissemination of ovarian cancer (OC) and reveal the major underlying signaling pathway and the therapeutic significance. Methods: Through a genome-wide CRISPR/Cas9 knockout screen in the murine orthotopic model of ovarian carcinoma (OC), we identified candidate genes regulating the peritoneal dissemination of OC. Among them, we picked out HTR1E, one member of 5-HT receptor family specifically expressed in the ovary and endometrium in addition to brain. The correlation of HTR1E expression with OC progression was analyzed in OC patient specimen by quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC). Gain-of-function and loss-of-function analyses were performed to explore the functions of 5-HT/HTR1E signaling in OC growth and dissemination in vitro and in vivo. In addition, we investigated the therapeutic values of HTR1E specific agonist and small molecular inhibitors against HTR1E downstream factor SRC in a stressed murine OC xenograft model. Results: In OC patients, the HTR1E expression is dramatically decreased in peritoneal disseminated OC cells, which correlates with poor clinical outcome. Silence of HTR1E in OC cells greatly promotes cell proliferation and epithelial mesenchymal transition (EMT) by the activation of SRC-mediated downstream signaling pathways. Furthermore, chronic stress results in significantly decreased serotonin in the ovary and the enhanced OC growth and peritoneal dissemination in mice, which can be strongly inhibited by specific HTR1E agonist or the SRC inhibitor. Conclusions: We discovered the essential role of serotonin/HTR1E signaling in preventing the chronic psychological stress-promoted progression of OC, suggesting the potential therapeutic value of the HTR1E specific agonist and the SRC inhibitor for OC patients who are suffering from psychological stress.
Subject(s)
Adenocarcinoma/metabolism , Ovarian Neoplasms/metabolism , Receptors, Serotonin, 5-HT1/metabolism , Receptors, Serotonin/metabolism , Serotonin/pharmacology , Stress, Physiological , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Behavior Rating Scale , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/ethics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Disease Progression , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Genetic Testing , Humans , Immunohistochemistry , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplasm Invasiveness/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , RNA-Seq , Real-Time Polymerase Chain Reaction , Receptors, Serotonin/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Stress, Physiological/drug effects , Stress, Physiological/genetics , Xenograft Model Antitumor AssaysABSTRACT
Glioma is one of the most common types of malignant primary central nervous system tumor, and prognosis for this disease is poor. As autophagic drugs have been reported to induce glioma cell death, we investigated the potential prognostic role of autophagy-associated long non-coding RNA (lncRNA) in glioma patients. In this study, we obtained 879 lncRNAs and 216 autophagy genes from the Chinese Glioma Genome Atlas microarray, and found that 402 lncRNAs are correlated with the autophagy genes. Subsequently, 10 autophagy-associated lncRNAs with prognostic value (PCBP1-AS1, TP53TG1, DHRS4-AS1, ZNF674-AS1, GABPB1-AS1, DDX11-AS1, SBF2-AS1, MIR4453HG, MAPKAPK5-AS1 and COX10-AS1) were identified in glioma patients using multivariate Cox regression analyses. A prognostic signature was then established based on these prognostic lncRNAs, dividing patients into low-risk and high-risk groups. The overall survival time was shorter in the high-risk group than that in the low-risk group [hazard ratio (HR) = 5.307, 95% CI: 4.195-8.305; P < 0.0001]. Gene set enrichment analysis revealed that the gene sets were significantly enriched in cancer-related pathways, including interleukin (IL) 6/Janus kinase/signal transducer and activator of transcription (STAT) 3 signaling, tumor necrosis factor α signaling via nuclear factor κB, IL2/STAT5 signaling, the p53 pathway and the KRAS signaling pathway. The Cancer Genome Atlas dataset was used to validate that high-risk patients have worse survival outcomes than low-risk patients (HR = 1.544, 95% CI: 1.110-2.231; P = 0.031). In summary, our signature of 10 autophagy-related lncRNAs has prognostic potential for glioma, and these autophagy-related lncRNAs may play a key role in glioma biology.
