CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

De novo variants in the PSMC3 proteasome AAA-ATPase subunit gene cause neurodevelopmental disorders associated with type I interferonopathies

A critical step in preserving protein homeostasis by the ubiquitin-proteasome system (UPS) is the recognition, binding, unfolding, and translocation of protein substrates by AAA-ATPase proteasome subunits for degradation by 26S proteasomes. Here, we identified fourteen different de novo missense variants in the PSMC3 gene encoding the AAA-ATPase proteasome subunit Rpt5 in twenty-two unrelated heterozygous subjects with an autosomal dominant form of neurodevelopmental delay and intellectual disability. Indeed, depletion of PSMC3 impaired reversal learning capabilities in a Drosophila model. The PSMC3 variants cause proteasome dysfunction in patient-derived cells by disruption of substrate translocation, proteotoxic stress and proteostatic imbalances, as well as alterations in proteins controlling developmental and innate immune programs. Molecular analysis confirmed the induction of cellular stress responses and dysregulated mitophagy along with an elevated type I interferon (IFN) signature. Our data define PSMC3 variants as the genetic cause of proteotoxic stress alerting the innate immune system to mount a type I IFN response and link neurodevelopmental syndromes to interferonopathies.

SMARCA4 Depletion Induces Cisplatin Resistance by Activating YAP1-Mediated Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer

The role of SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, in genomic organization is well studied in various cancer types. However, its oncogenic role and therapeutic implications are relatively unknown in triple-negative breast cancer (TNBC). We investigated the clinical implication and downstream regulation induced by SMARCA4 inactivation using large-scale genome and transcriptome profiles. Additionally, SMARCA4 was knocked out in MDA-MB-468 and MDA-MB-231 using CRISPR/Cas9 to identify gene regulation and a targetable pathway. First, we observed an increase in SMARCA4 mutations in cisplatin resistance and metastasis in TNBC patients. Its inactivation was associated with the mesenchymal-like (MSL) subtype. Gene expression analysis showed that the epithelial-to-mesenchymal transition (EMT) pathway was activated in SMARCA4-deficient patients. Next, the Hippo pathway was activated in the SMARCA4 inactivation group, as evidenced by the higher CTNNB1, TGF-β, and YAP1 oncogene signature scores. In SMARCA4 knockout cells, EMT was upregulated, and the cell line transcriptome changed from the SL to the MSL subtype. SMARCA4 knockout cells showed cisplatin resistance and Hippo-YAP/TAZ target gene activation. The YAP1 inhibitor verteporfin suppressed the expression of YAP1 target genes, and decreased cell viability and invasiveness on SMARCA4 knockout cells. SMARCA4 inactivation in TNBC endowed the resistance to cisplatin via EMT activation. The YAP1 inhibitor could become a novel strategy for patients with SMARCA4-inactivated TNBC.

First Analyses of Lysine Succinylation Proteome and Overlap between Succinylation and Acetylation in Solenopsis Invicta Buren (Hymenoptera: Formicidae)

Background: Lysine succinylation (Ksu) exists in both eukaryotes and prokaryotes, and influences a variety of metabolism processes. However, little attention has been paid to Ksu in insects, especially the notorious invasive pest Solenopsis invicta. Results: In this study, the first analyses of Ksu proteome and overlap between Ksu and lysine acetylation (Kac) in S. invicta were presented. 3,753 succinylated sites in 893 succinylated proteins were tested. The dihydrolipoyl dehydrogenase, V-type proton ATPase subunit G, and tubulin alpha chain all had evolutionary conservatism among diverse ant or bee species. Immunoblotting validation showed that there were many Ksu protein bands with a wide range of molecular mass. In addition, 1,230 sites in 439 proteins were highly overlapped between Ksu and Kac. 54.05% of Ksu proteins in cytoplasm were acetylated. The results demonstrated that Ksu may play a vital part in the allergization, redox metabolism, sugar, fat, and protein metabolism, energy production, immune response, and biosynthesis of various secondary metabolites.Conclusions: Ksu and Kac were two ubiquitous protein post-translational modifications participated in a variety of biological processes. Our results may supply rich resources and a starting point for the molecular basic research of regulation on metabolic pathways and other biological processes by succinylation and acetylation.

Knockdown of Vacuolar ATPase Subunit G Gene Affects Larval Survival and Impaired Pupation and Adult Emergence in Henosepilachna vigintioctopunctata

The vATPase holoenzyme consists of two functional subcomplexes, the cytoplasmic (peripheral) V1 and the membrane-embedded V0. Both V1 and V0 sectors contain eight subunits, with stoichiometry of A3B3CDE3FG3H in V1 and ac8c’c”def(Voa1p) in V0 respectively. However, the function of G subunit has not been characterized in any non-Drosophilid insect species. In the present paper, we uncovered that HvvATPaseG was actively transcribed from embryo to adult in a Coleopteran pest Henosepilachna vigintioctopunctata. Its mRNA levels peaked in larval hindgut and Malpighian tubules. RNA interference (RNAi)-mediated knockdown of HvvATPaseG significantly reduced larval feeding, affected chitin biosynthesis, destroyed midgut integrity, damaged midgut peritrophic membrane, and retarded larval growth. The function of Malpighian tubules was damaged, the contents of glucose, trehalose, lipid, total soluble amino acids and protein were lowered and the fat bodies were lessened in the HvvATPaseG RNAi larvae, compared with those in the PBS- and dsegfp-fed beetles. In contrast, the amount of glycogen was dramatically increased in the HvvATPaseG depletion ladybirds. As a result, the development was arrested, pupation was inhibited and adult emergence was impaired in the HvvATPaseG hypomorphs. Our results demonstrated that G subunit plays a critical role during larval development in H. vigintioctopunctata.

A Pan-Cancer Analysis of SMARCA4 Alterations in Human Cancers

BackgroundSMARCA4, the essential ATPase subunit of SWI/SNF chromatin remodeling complex, regulates transcription through the control of chromatin structure and is increasingly thought to play significant roles in human cancers. This study aims to explore the potential role of SMARCA4 with a view to providing insights on pathologic mechanisms implicated here.MethodsThe potential roles of SMARCA4 in different tumors were explored based on The Cancer Genome Atlas (TCGA), Genotype-tissue expression (GTEx), Tumor Immune Estimation Resource (TIMER), and Gene Set Enrichment Analysis (GSEA) datasets. The expression difference, mutation and phosphorylation status, survival, pathological stage, DNA methylation, tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MMR), tumor microenvironment (TME), and immune cell infiltration related to SMARCA4 were analyzed.ResultsHigh expression levels of SMARCA4 were observed in most cancer types. SMARCA4 expression in tumor samples correlates with poor overall survival in several cancers. Lung adenocarcinoma cases with altered SMARCA4 showed a poorer prognosis. Enhanced phosphorylation levels of S613, S695, S699, and S1417 were observed in several tumors, including breast cancer. SMARCA4 correlated with tumor immunity and associated with different immune cells and genes in different cancer types. TMB, MSI, MMR, and DNA methylation correlated with SMARCA4 dysregulation in cancers. SMARCA4 expression was negatively associated with CD8+ T-cell infiltration in several tumors. Furthermore, the SWI/SNF superfamily-type complex and ATPase complex may be involved in the functional mechanisms of SMARCA4, albeit these data require further confirmation.ConclusionsOur study offers a comprehensive understanding of the oncogenic roles of SMARCA4 across different tumors. SMARCA4 may correlate with tumor immunity.

Phenotypic characterization of rabbit model for aortic valve stenosis: a novel medial calcification paradigm

Introduction Calcific aortic valve stenosis (CAVS) is the result of subtle, chronic inflammation and osteoblastic differentiation. As we lack human specimens of the early stages, reliable and reproducible animal models are needed to facilitate research. We previously demonstrated the ability of a novel rabbit CAVS vitamin D2 toxicity protocol to produce calcification and valve stenosis (1). We sought to characterize the phenotype of the model at the final stage. Methods Twelve New Zealand Rabbits were randomized 1:1 to control (normal chaw) and experimental group (normal chaw+1% cholesterol+3.500 I.U.s Vitamin D2, in oil in a biscuit) for 7 weeks. Animals were sacrificed and aortic valve cusps were snap frozen or formalin-fixed paraffin embedded. Cusps were then mechanically homogenized in buffer optimized for protein extraction and total protein measured with Bradford method. Part of the extract was subjected to trypsinization, in-gel digestion and untargeted LC-MS/MS. The rest was used to quantitate BMP-2 with total protein-normalized sandwitch competitive ELISA. Thin tissue sections were stained with Masson's trichrome, Von Kossa and H&E. Osteopontin, Bone sialoprotein II (BSPII), tissue non-specific alkaline phosphatase (TNAP) and osteocalcin (OCN) were detected on tissue with immunohistochemistry. Femoral bones from the same animals served as positive controls. Results Aortic valve cusp demonstrate large areas of collagen degradation and calcification in the medial layer, almost sparing the intima. Osteopontin deposits were colocalized with the calcification area in the media, whereas BSPII, TNAP and OCN were not expressed in the lesion, although present in bones. Similarly, BMP-2 levels were not significantly different between groups (experimental = 43.45 vs controls = 62.75 pg/ml, Mann-Whitney U test p=0.496). Proteomic analysis revealed a set of 96 differentially expressed proteins between cases and controls, interestingly including sortilin, osteonectin, beta-crystallin A2, Matrix Gla protein, Na/H exchanger 3, V-type H ATPase subunit D, Y-box binding protein. Conclusion The novel rabbit vitamin D2 toxicity protocol leads to excessive medial calcification of the aortic valve, with overexpression of osteopontin but without other classic markers of CAVS. Proteomics analysis reveals novel pathways with pathophysiological implications for the model and medial calcification. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Hellenic Cardiology Society, Hellenic Heart Foundation