An essential Noc3p dimerization cycle mediates ORC double-hexamer formation in replication licensing.

Replication licensing, a prerequisite of DNA replication, helps to ensure once-per-cell-cycle genome duplication. Some DNA replication-initiation proteins are sequentially loaded onto replication origins to form pre-replicative complexes (pre-RCs). ORC and Noc3p bind replication origins throughout the cell cycle, providing a platform for pre-RC assembly. We previously reported that cell cycle-dependent ORC dimerization is essential for the chromatin loading of the symmetric MCM double-hexamers. Here, we used Saccharomyces cerevisiae separation-of-function NOC3 mutants to confirm the separable roles of Noc3p in DNA replication and ribosome biogenesis. We also show that an essential and cell cycle-dependent Noc3p dimerization cycle regulates the ORC dimerization cycle. Noc3p dimerizes at the M-to-G1 transition and de-dimerizes in S-phase. The Noc3p dimerization cycle coupled with the ORC dimerization cycle enables replication licensing, protects nascent sister replication origins after replication initiation, and prevents re-replication. This study has revealed a new mechanism of replication licensing and elucidated the molecular mechanism of Noc3p as a mediator of ORC dimerization in pre-RC formation.

ANP32 Family as Diagnostic, Prognostic, and Therapeutic Biomarker Related to Immune Infiltrates in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, with high incidence and mortality rate. There is an urgent need to identify effective diagnostic and prognostic biomarkers for HCC. Members of the acidic leucine-rich nucleophosphoprotein 32 (ANP32) family, which mainly includes ANP32A, ANP32B, and ANP32E, are abnormally expressed and have prognostic value in certain cancers. However, the diagnostic, prognostic, and therapeutic value of ANP32 family members in HCC has not yet been fully studied. In this study, we identified the diagnostic and prognostic value of ANP32 family members in HCC. Transcriptome data from public databases, such as the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, suggested that ANP32A, ANP32B, and ANP32E were upregulated in HCC tissues, and high expression of ANP32 family members was associated with advanced pathologic stage and histologic grade. Our immunohistochemistry and western blot results further verified the differential expression of ANP32 family members. ANP32A, ANP32B, and ANP32E had an outstanding diagnostic potential. Survival analysis of HCC patients in TCGA databases demonstrated that ANP32A, ANP32B, and ANP32E were associated with poor overall survival (OS) and disease-specific survival (DSS). Univariate and multivariate Cox analyses suggested the capability of ANP32B and ANP32E to independently predict the OS and DSS of HCC patients. Gene set enrichment analysis (GSEA) showed that ANP32 family members were associated with immune response, epidermal cell differentiation, and stem cell proliferation. Expression of ANP32 family members was associated with immune cell infiltration and immune status in the tumor microenvironment of HCC, and patients with high ANP32 family expression had poor sensitivity to immunotherapy. Finally, we identified potential chemotherapy drugs for HCC patients with high ANP32 family expression by CellMiner database. This study suggested the diagnostic, prognostic, and therapeutic roles of the ANP32 family in HCC patients, providing potential therapeutic targets for HCC.

Epithelial-mesenchymal transition, regulated by ß-catenin and Twist, leads to esophageal wall remodeling in pediatric eosinophilic esophagitis.

Eosinophilic Esophagitis (EoE) is an antigen-triggered inflammatory condition of the esophageal lining characterized by eosinophilic infiltration. EoE is associated with significant remodeling, and although this remodeling is reversed by current treatment regimens, symptoms of EoE and associated remodeling reappear upon cessation of therapies. We hypothesized that structural remodeling of cell-cell adhesion is a key factor in the pathogenesis of EoE and that epithelial to mesenchymal transition (EMT) was a viable molecular process to lead to this remodeling. Endoscopically obtained biopsy samples from 18 EoE and 18 control pediatric patients were evaluated by transmission electron microscopy to measure intercellular spaces (IS) between cells. Biopsy samples from all groups were analyzed for cellular levels of cell-cell adhesion proteins: E-cadherin, zonula occludens associated protein-1 (ZO-1), and N-cadherin. We also analyzed for cellular levels and localization two of transcription factors, Twist1 and ß-catenin, that are associated with promoting EMT. The IS was significantly increased in the EoE group compared to the control. We observed a significant decrease in E-cadherin and ZO-1 levels and a concomitant increase in N-cadherin levels in EoE samples compared to control. Further, while there was no significant change in cellular levels of ß-catenin, we observed an altered localization of the protein from the cell membrane in control tissue to a nuclear/perinuclear localization in EoE. We observed higher levels of the transcription factor Twist1 in the EoE group compared to normal which was localized mainly at the nucleus. Our results suggest that the integrity of normally sealed esophageal epithelia is compromised in the EoE patients compared to control subjects, and this is due to alterations in the expression of cell adhesion molecules at the esophageal epithelium. Our data also suggest that EMT, potentially regulated by transcription factors ß-catenin and Twist1, may be responsible for the molecular alteration which leads to the remodeling of esophageal epithelia in EoE.

Spop ameliorates diabetic nephropathy through restraining NLRP3 inflammasome.

Diabetic nephropathy (DN) is one of the most common causes for end-stage renal disease without effective therapies available. NLR family, pyrin domain-containing 3 (NLRP3) inflammasome possesses a fundamental effect to facilitate the pathogenesis of DN. Unfortunately, how NLRP3 inflammasome is mediated still remains largely unclear. In the present study, an E3 ubiquitin ligase Speckle-type BTB-POZ protein (Spop) was identified as a suppressor of NLRP3 inflammasome. We first showed that Spop expression was extensively down-regulated in kidney of DN patients, which was confirmed in kidney of streptozotocin (STZ)-challenged mice and in high glucose (HG)-stimulated podocytes. Intriguingly, we showed that conditional knockout (cKO) of Spop in podocytes considerably accelerated renal dysfunction and pathological changes in the glomerulus of STZ-induced mice with DN, along with severe podocyte injury. Furthermore, Spop specific ablation in podocytes dramatically facilitated inflammatory response in glomeruli of DN mice via enhancing NLRP3 inflammasome and nuclear factor κB (NF-κB) signaling pathways, which were confirmed in HG-cultured podocytes. Notably, our findings indicated that Spop directly interacted with NLRP3. More importantly, Spop promoted NLRP3 degradation via elevating K48-linked polyubiquitination of NLRP3. Collectively, our findings disclosed a mechanisms through which Spop limited NLRP3 inflammasome under HG condition, and illustrated that Spop may be a novel therapeutic target to suppress NLRP3 inflammasome, contributing to the DN management.

Regulator of Cell Cycle Protein (RGCC/RGC-32) Protects against Pulmonary Fibrosis.

Some previous studies in tissue fibrosis have suggested a profibrotic contribution from elevated expression of a protein termed either RGCC (regulator of cell cycle) or RGC-32 (response gene to complement 32 protein). Our analysis of public gene expression datasets, by contrast, revealed a consistent decrease in RGCC mRNA levels in association with pulmonary fibrosis. Consistent with this observation, we found that stimulating primary adult human lung fibroblasts with transforming growth factor (TGF)-ß in cell cultures elevated collagen expression and simultaneously attenuated RGCC mRNA and protein levels. Moreover, overexpression of RGCC in cultured lung fibroblasts attenuated the stimulating effect of TGF-ß on collagen levels. Similar to humans with pulmonary fibrosis, the levels of RGCC were also decreased in vivo in lung tissues of wild-type mice challenged with bleomycin in both acute and chronic models. Mice with constitutive RGCC gene deletion accumulated more collagen in their lungs in response to chronic bleomycin challenge than did wild-type mice. RNA-Seq analyses of lung fibroblasts revealed that RGCC overexpression alone had a modest transcriptomic effect, but in combination with TGF-ß stimulation, induced notable transcriptomic changes that negated the effects of TGF-ß, including on extracellular matrix-related genes. At the level of intracellular signaling, RGCC overexpression delayed early TGF-ß-induced Smad2/3 phosphorylation, elevated the expression of total and phosphorylated antifibrotic mediator STAT1, and attenuated the expression of a profibrotic mediator STAT3. We conclude that RGCC plays a protective role in pulmonary fibrosis and that its decline permits collagen accumulation. Restoration of RGCC expression may have therapeutic potential in pulmonary fibrosis.

The role of Siah2 in tumorigenesis and cancer therapy.

Seven in absentia homolog 2 (Siah2), an RING E3 ubiquitin ligases, has been characterized to play the vital role in tumorigenesis and cancer progression. Numerous studies have determined that Siah2 promotes tumorigenesis in a variety of human malignancies such as prostate, lung, gastric, and liver cancers. However, several studies revealed that Siah2 exhibited tumor suppressor function by promoting the proteasome-mediated degradation of several oncoproteins, suggesting that Siah2 could exert its biological function according to different stages of tumor development. Moreover, Siah2 is subject to complex regulation, especially the phosphorylation of Siah2 by a variety of protein kinases to regulate its stability and activity. In this review, we describe the structure and regulation of Siah2 in human cancer. Moreover, we highlight the critical role of Siah2 in tumorigenesis. Furthermore, we note that the potential clinical applications of targeting Siah2 in cancer therapy.

The Intrinsically Disordered Proteins MLLT3 (AF9) and MLLT1 (ENL) - Multimodal Transcriptional Switches With Roles in Normal Hematopoiesis, MLL Fusion Leukemia, and Kidney Cancer.

AF9 (MLLT3) and ENL (MLLT1) are members of the YEATS family (named after the five proteins first shown to contain this domain: Yaf9, ENL, AF9, Taf14, Sas5) defined by the presence of a YEATS domain. The YEATS domain is an epigenetic reader that binds to acetylated and crotonylated lysines, unlike the bromodomain which can only bind to acetylated lysines. All members of this family have been shown to be components of various complexes with roles in chromatin remodeling, histone modification, histone variant deposition, and transcriptional regulation. MLLT3 is a critical regulator of hematopoiesis with a role in maintaining the hematopoietic stem or progenitor cell (HSPC) population. Approximately 10% of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) patients harbor a translocation involving MLL (mixed lineage leukemia). In the context of MLL fusion patients with AML and ALL, MLL-AF9 and MLL-ENL fusions are observed in 34 and 31% of the patients, respectively. The intrinsically disordered C-terminal domain of MLLT3 (AHD, ANC1 homology domain) undergoes coupled binding and folding upon interaction with partner proteins AF4, DOT1L, BCOR, and CBX8. Backbone dynamics studies of the complexes suggest a role for dynamics in function. Inhibitors of the interaction of the intrinsically disordered AHD with partner proteins have been described, highlighting the feasibility of targeting intrinsically disordered regions. MLLT1 undergoes phase separation to enhance recruitment of the super elongation complex (SEC) and drive transcription. Mutations in MLLT1 observed in Wilms tumor patients enhance phase separation and transcription to drive an aberrant gene expression program.

miR-190-5p Alleviates Myocardial Ischemia-Reperfusion Injury by Targeting PHLPP1.

OBJECTIVE: Myocardial ischemia-reperfusion (I/R) injury (MIRI) refers to the more serious myocardial injury after blood flow recovery, which seriously affects the prognosis of patients with ischemic cardiomyopathy. This study explored the new targets for MIRI treatment by investigating the effects of miR-190-5p and its downstream target on the structure and function of myocardial cells. METHODS: We injected agomir miR-190-5p into the tail vein of rats to increase the expression of miR-190-5p in rat myocardial cells and made an I/R rat model by coronary artery occlusion. We used 2,3,5-triphenyl tetrazolium chloride staining, lactate dehydrogenase (LDH) detection, echocardiography, and hematoxylin-eosin (HE) staining to determine the degree of myocardial injury in I/R rats. In addition, we detected the expression of inflammatory factors and apoptosis-related molecules in rat serum and myocardial tissue to determine the level of inflammation and apoptosis in rat myocardium. Finally, we determined the downstream target of miR-190-5p by Targetscan system and dual luciferase reporter assay. RESULTS: The expression of miR-190-5p in an I/R rat myocardium was significantly lower than that in normal rats. After treatment of I/R rats with agomir miR-190-5p, the ischemic area of rat myocardium and the concentration of LDH decreased. The results of echocardiography and HE staining also found that overexpression of miR-190-5p improved the structure and function of rat myocardium. miR-190-5p was also found to improve the viability of H9c2 cells in vitro and reduce the level of apoptosis of H9c2 cells. The results of Targetscan system and dual luciferase reporter assay found that miR-190-5p targeted to inhibit pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1). In addition, inhibition of PHLPP1 was found to improve the viability of H9c2 cells. CONCLUSION: Therefore, miR-190-5p can reduce the inflammation and apoptosis of myocardium by targeting PHLPP1, thereby alleviating MIRI.

Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance.

BACKGROUND: Eya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown. METHODS: We engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo. RESULTS: Eya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death-inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor-specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor-specific expression in response to Six2 activity. CONCLUSIONS: Our results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

Nitrosative stress induces downregulation of ribosomal protein genes via MYCT1 in vascular smooth muscle cells.

OBJECTIVE: In our previous genomic studies in human intracranial aneurysms, we observed downregulations in the expression of a number of ribosomal protein genes and the c-Myc-related gene MYC target 1 (MYCT1). So far there is no information about the roles of MYCT1 in vascular cells. Our study aims to investigate the functional roles of MYCT1 in vascular smooth muscle cells (SMCs). MATERIALS AND METHODS: Primary SMCs were isolated from rat thoracic aorta and cultured in vitro. The mRNA and protein expressions were determined by real-time PCR and western blot respectively. Apoptosis was detected by measuring caspase 3/7 activity. Collagen production was determined with ELISA. RESULTS: Using PCR, we validated our previous genomic data showing that the expressions of MYCT1 and ribosomal protein genes were decreased in human aneurysm tissues. In vascular SMCs, we showed that nitrosative stress downregulated the expression of both MYCT1 and ribosomal proteins. Knockdown of MYCT1 mimicked the effects of nitrosative stress on ribosomal protein expressions, whereas overexpression of MYCT1 blunted the effects of nitrosative stress. MYCT1-dependent downregulation of ribosomal proteins compromised the protein translational capacity of the cells for collagen production. Moreover, the endogenously expressed MYCT1 in vascular SMCs was involved in maintaining normal cellular functions including survival, proliferation and migration. CONCLUSIONS: MYCT1-dependent gene regulation may, at least partly, explain the downregulated expressions of ribosomal proteins observed in human intracranial aneurysms. It is suggested that MYCT1 may represent a novel molecular target for counteracting the decreased activity of aneurysmal SMCs for tissue repairmen/regeneration.

ZNF545 loss promotes ribosome biogenesis and protein translation to initiate colorectal tumorigenesis in mice.

Ribosome biogenesis plays a pivotal role in tumorigenesis by supporting robust protein translation. We investigate the functional and molecular mechanism of Zinc finger protein 545 (ZNF545), a transcriptional repressor for ribosomal RNA (rRNA), in colorectal cancer (CRC). ZNF545 was silenced in CRC compared to adjacent normal tissues (P < 0.0001), implying a tumor-suppressive role. Colon-specific Znf545 knockout in mice accelerated CRC in ApcMin/+ and azoxymethane/dextran sulfate sodium-induced CRC. Mechanistically, we demonstrated that ZNF545 uses its two zinc finger clusters to bind to minimal rDNA promoter, where it assembled transcriptional repressor complex by interacting with KAP1. Znf545 deletion in mouse embryonic fibroblasts not only increased rRNA transcription rate and the nucleolar size and number but also altered the nucleolar composition and architecture with an increased number of fibrillar centers surrounded by net-like dense fibrillar components. Consequently, Znf545 deletion promoted the gene expression of translation machinery, protein translation, and cell growth. Consistent with its tumor-suppressive role, ZNF545 overexpression in CRC cells induced growth arrest and apoptosis. Finally, administration of rRNA synthesis inhibitor, CX-5461, inhibited CRC development in Znf545Δ/ΔApcMin/+ mice. In conclusion, ZNF545 suppresses CRC through repressing rRNA transcription and protein translation. Targeting rRNA biosynthesis in ZNF545-silenced tumors is a potential therapeutic strategy for CRC.

The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks.

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.

Let-7a targets Rsf-1 to modulate radiotherapy response of non-small cell lung cancer cells through Ras-MAPK pathway.

PURPOSE: Radiotherapy is the most commonly selective medical treatment for non-small cell lung cancer (NSCLC) and the multiple underlying mechanisms are considered as the effectively theoretical foundation. Herein, we investigated the effects of let-7a targets Rsf-1 on modulating the radiotherapy response in NSCLC cells by Ras-MAPK pathway. METHODS: A549 cells were divided into different groups to investigate the role of let-7a and Rsf-1 on the radiotherapy response. The expression of let-7a and Rsf-1 were detected by RT-PCR. Bioinformatic analysis indicated that Rsf-1 is the target of let-7a. The binding site of let-7a in the Rsf-1 3'UTR was detected based on double luciferase reporter assay and Western blot. The cell variability and proliferation were assessed by MTT and colony formation assay. The expression levels of Ras-MARK signaling pathway related proteins were assessed by RT-PCR. RESULTS: RT-PCR results showed that radiotherapy could up-regulate the expression of let-7a, thereby reducing the expression of Rsf-1, and the correlation between the two factors was negatively correlated. At the same time, let-7a overexpression and Rsf-1 silencing could further reduce the activity of A549 cells after radiotherapy, have an inhibitory effect on cell proliferation and inhibit the expression of related proteins in the Ras-MAPK pathway. CONCLUSIONS: Rsf-1 is the target of Let-7a. The present study provides evidence that let-7a targeting Rsf-1 can modulate radiotherapy response in NSCLC cells through Ras-MAPK pathway.

Photoactivation of mitochondrial reactive oxygen species-mediated Src and protein kinase C pathway enhances MHC class II-restricted T cell immunity to tumours.

High fluence low-level laser (HF-LLL), a mitochondria-targeted tumour phototherapy, results in oxidative damage and apoptosis of tumour cells, as well as damage to normal tissue. To circumvent this, the therapeutic effect of low fluence LLL (LFL), a non-invasive and drug-free therapeutic strategy, was identified for tumours and the underlying molecular mechanisms were investigated. We observed that LFL enhanced antigen-specific immune response of macrophages and dendritic cells by upregulating MHC class II, which was induced by mitochondrial reactive oxygen species (ROS)-activated signalling, suppressing tumour growth in both CD11c-DTR and C57BL/6 mice. Mechanistically, LFL upregulated MHC class II in an MHC class II transactivator (CIITA)-dependent manner. LFL-activated protein kinase C (PKC) promoted the nuclear translocation of CIITA, as inhibition of PKC attenuated the DNA-binding efficiency of CIITA to MHC class II promoter. CIITA mRNA and protein expression also improved after LFL treatment, characterised by direct binding of Src and STAT1, and subsequent activation of STAT1. Notably, scavenging of ROS downregulated LFL-induced Src and PKC activation and antagonised the effects of LFL treatment. Thus, LFL treatment altered the adaptive immune response via the mitochondrial ROS-activated signalling pathway to control the progress of neoplastic disease.

Upregulation of lncRNA NIFK-AS1 in hepatocellular carcinoma by m6A methylation promotes disease progression and sorafenib resistance.

Long non-coding RNAs (LncRNAs) have recently emerged as vital regulators in the development and progression of hepatocellular carcinoma (HCC), providing new opportunities as novel therapeutic targets. Here we identified the lncRNA NIFK-AS1 as being highly expressed in HCC tissues and cells and showed this up-regulation resulted from METTL3-dependent m6A methylation. Functionally, knockdown of NIFK-AS1 inhibited the proliferation, colony formation, migration, and invasion of HCC cells. Moreover, these effects were elicited though AKT1 and we uncovered a ceRNA network involving an NIFK-AS1/miR-637/AKT1 axis with downstream effects on HCC progression involving regulation of MMP-7 and MMP-9 expression. From the clinical perspective, we showed that knockdown of NIFK-AS1 sensitized HCC cells to sorafenib through the up-regulation of the drug transporters OATP1B1 and OATP1B3. Clinical investigations showed HCC patients with low NIFK-AS1 expression benefited from sorafenib therapy and this phenomenon was reproduced in patient-derived tumor xenograft models (PDX) comparing HCC with low and high expression of NIFK-AS1. Taken together, these results suggest an essential role for NIFK-AS1 in HCC progression and promote NIFK-AS1 as a new therapeutic target and predictor of sorafenib benefit in HCC patients.

Lipin1 Alleviates Autophagy Disorder in Sciatic Nerve and Improves Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is a chronic complication of diabetes, and its neural mechanisms underlying the pathogenesis remain unclear. Autophagy plays an important role in neurodegenerative diseases and nerve tissue injury. Lipin1 is a phosphatidic acid phosphatase enzyme that converts phosphatidic acid (PA) into diacylglycerol (DAG), a precursor of triacylglycerol and phospholipids which plays an important role in maintaining normal peripheral nerve conduction function. However, whether Lipin1 involved in the pathogenesis of DPN via regulation of autophagy is not elucidated. Here, we show that the Lipin1 expression was downregulated in streptozotocin (STZ)-induced DPN rat model. Interestingly, STZ prevented DAG synthesis, and resulted in autophagic hyperactivity, effects which may increase the apoptosis of Schwann cells and lead to demyelination in sciatic nerve in DPN rats. More importantly, upregulation of lipin1 in the DPN rats ameliorated autophagy disorders and pathological changes of the sciatic nerve, which associated with the increase of the motor nerve conductive velocity (MNCV) in DPN rats. In contrast, knockdown of lipin1 exacerbates neuronal abnormalities and facilitates the genesis of DPN phenotypes in rats. In addition, overexpression of lipin1 in RSC96 cells also significantly decreased the autophagic hyperactivity and apoptosis induced by hyperglycemia. These results suggest that lipin1 may exert neuroprotection within the sciatic nerve anomalies and may serve as a potential therapeutic target for the treatment of DPN.

DeSUMOylation of Apoptosis Inhibitor 5 by Avibirnavirus VP3 Supports Virus Replication.

SUMOylation is a reversible posttranslational modification involved in the regulation of diverse biological processes. Growing evidence suggests that virus infection can interfere with the SUMOylation system. In the present study, we discovered that apoptosis inhibitor 5 (API5) is a SUMOylated protein. Amino acid substitution further identified that Lys404 of API5 was the critical residue for SUMO3 conjugation. Moreover, we found that Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. In addition, our results further revealed that viral protein VP3 inhibited the SUMOylation of API5 by targeting API5 and promoting UBC9 proteasome-dependent degradation through binding to the ubiquitin E3 ligase TRAF3. Furthermore, we revealed that wild-type but not K404R mutant API5 inhibited IBDV replication by enhancing MDA5-dependent IFN-ß production. Taken together, our data demonstrate that API5 is a UBC9-dependent SUMOylated protein and deSUMOylation of API5 by viral protein VP3 aids in viral replication. IMPORTANCE Apoptosis inhibitor 5 (API5) is a nuclear protein initially identified for its antiapoptotic function. However, so far, posttranslational modification of API5 is unclear. In this study, we first identified that API5 K404 can be conjugated by SUMO3, and Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. Mechanically, viral protein VP3 directly interacts with API5 and inhibits SUMOylation of API5. Additionally, the cellular E3 ligase TNF receptor-associated factor 3 (TRAF3) is employed by VP3 to facilitate UBC9 proteasome-dependent degradation, leading to the reduction of API5 SUMOylation. Moreover, our data reveal that SUMOylation of API5 K404 promotes MDA5-dependent beta interferon (IFN-ß) induction, and its deSUMOylation contributes to IBDV replication. This work highlights a critical role of conversion between SUMOylation and deSUMOylation of API5 in regulating viral replication.

RGC-32 Acts as a Hub to Regulate the Transcriptomic Changes Associated With Astrocyte Development and Reactive Astrocytosis.

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-ß signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32's ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-ß. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-ß-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.

Coilin enhances phosphorylation and stability of DGCR8 and promotes miRNA biogenesis.

MicroRNAs (miRNAs) are ∼22 nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The biogenesis of miRNAs involves a series of processing steps beginning with cropping of the primary miRNA transcript by the Microprocessor complex, which is composed of Drosha and DGCR8. Here we report a novel regulatory interaction between the Microprocessor components and coilin, the Cajal body (CB) marker protein. Coilin knockdown causes alterations in the level of primary and mature miRNAs, let-7a and miR-34a, and their miRNA targets, HMGA2 and Notch1, respectively. We also found that coilin knockdown affects the levels of DGCR8 and Drosha in cells with (HeLa) and without (WI-38) CBs. To further explore the role of coilin in miRNA biogenesis, we conducted a series of coimmunoprecipitation experiments using coilin and DGCR8 constructs, which revealed that coilin and DGCR8 can form a complex. Additionally, our results indicate that phosphorylation of DGCR8, which has been shown to increase protein stability, is impacted by coilin knockdown. Collectively, our results implicate coilin as a member of the regulatory network governing miRNA biogenesis.