Co-immunoprecipitation Assays Research Articles

Abnormal function of EPHA2/p.R957P mutant in congenital cataract.

To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity. A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo. A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant's impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics. Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

LncRNA HOXC-AS3 accelerates malignant proliferation of cervical cancer cells via stabilizing KDM5B

BackgroundCervical cancer (CC) is a common malignancy amongst women globally. Ubiquitination plays a dual role in the occurrence and development of cancers. This study analyzed the mechanism of long noncoding RNA HOXC cluster antisense RNA 3 (lncRNA HOXC-AS3) in malignant proliferation of CC cells via mediating ubiquitination of lysine demethylase 5B (KDM5B/JARID1B).MethodsThe expression patterns of lncRNA HOXC-AS3 and KDM5B were measured by real-time quantitative polymerase chain reaction or Western blot analysis. After transfection with lncRNA HOXC-AS3 siRNA and pcDNA3.1-KDM5B, proliferation of CC cells was assessed by the cell counting kit-8, colony formation, and 5-Ethynyl-2’-deoxyuridine staining assays. The xenograft tumor model was established to confirm the impact of lncRNA HOXC-AS3 on CC cell proliferation in vivo by measuring tumor size and weight and the immunohistochemistry assay. The subcellular location of lncRNA HOXC-AS3 and the binding of lncRNA HOXC-AS3 to KDM5B were analyzed. After treatment of lncRNA HOXC-AS3 siRNA or MG132, the protein and ubiquitination levels of KDM5B were determined. Thereafter, the interaction and the subcellular co-location of tripartite motif-containing 37 (TRIM37) and KDM5B were analyzed by the co-immunoprecipitation and immunofluorescence assays.ResultsLncRNA HOXC-AS3 and KDM5B were upregulated in CC tissues and cells. Depletion of lncRNA HOXC-AS3 repressed CC cell proliferation and in vivo tumor growth. Mechanically, lncRNA HOXC-AS3 located in the nucleus directly bound to KDM5B, inhibited TRIM37-mediated ubiquitination of KDM5B, and upregulated the protein levels of KDM5B. KDM5B overexpression attenuated the inhibitory role of silencing lncRNA HOXC-AS3 in CC cell proliferation in vivo and in vitro.ConclusionNucleus-located lncRNA HOXC-AS3 facilitated malignant proliferation of CC cells via stabilization of KDM5B protein levels.

Astragaloside IV inhibits cell viability and glycolysis of hepatocellular carcinoma by regulating KAT2A-mediated succinylation of PGAM1

BackgroundAstragaloside IV (AS-IV) is one of the basic components of Astragali radix, that has been shown to have preventive effects against various diseases, including cancers. This study aimed to explore the role of AS-IV in hepatocellular carcinoma (HCC) and its underlying mechanism.MethodsThe cell viability, glucose consumption, lactate production, and extracellular acidification rate (ECAR) in SNU-182 and Huh7 cell lines were detected by specific commercial kits. Western blot was performed to analyze the succinylation level in SNU-182 and Huh7 cell lines. The interaction between lysine acetyltransferase (KAT) 2 A and phosphoglycerate mutase 1 (PGAM1) was evaluated by co-immunoprecipitation and immunofluorescence assays. The role of KAT2A in vivo was explored using a xenografted tumor model.ResultsThe results indicated that AS-IV treatment downregulated the protein levels of succinylation and KAT2A in SNU-182 and Huh7 cell lines. The cell viability, glucose consumption, lactate production, ECAR, and succinylation levels were decreased in AS-IV-treated SNU-182 and Huh7 cell lines, and the results were reversed after KAT2A overexpression. KAT2A interacted with PGAM1 to promote the succinylation of PGAM1 at K161 site. KAT2A overexpression promoted the viability and glycolysis of SNU-182 and Huh7 cell lines, which were partly blocked following PGAM1 inhibition. In tumor-bearing mice, AS-IV suppressed tumor growth though inhibiting KAT2A-mediated succinylation of PGAM1.ConclusionAS-IV inhibited cell viability and glycolysis in HCC by regulating KAT2A-mediated succinylation of PGAM1, suggesting that AS-IV might be a potential and suitable therapeutic agent for treating HCC.

Ginsenoside Rb2 Inhibits the Pyroptosis in Myocardial Ischemia Progression Through Regulating the SIRT1 Mediated Deacetylation of ASC.

Myocardial ischemic (MI) injury is a common cardiovascular disease, and the potential therapeutic effects of ginsenoside Rb2 (Rb2) have been lately the focus of interest. Therefore, this study aimed to investigate the effects of Rb2 on pyroptosis of cardiomyocytes in MI progression. An in vitro MI model was developed by subjecting rat's cardiomyocytes (H9c2) to hypoxia/reoxygenation (H/R). The cell viability was determined by CCK-8 assay, while cell death was analyzed by propidium iodide staining. Similarly, pyroptosis-related protein levels and acetylation levels of apoptosis-associated speck-like protein containing a CARD (ASC) were detected by western blotting, and the relationship between Sirtuin 1 (SIRT1) and ASC was confirmed by co-immunoprecipitation (Co-IP) assay. Moreover, hematoxylin-eosin (H&E) and triphenyl tetrazolium chloride staining were used to study pathological structure and infarct size. It was found that post-Rb2 treatment significantly increased the cell viability and decreased the cell death and lactic dehydrogenase release, while the increased gasdermin D-N, NOD-like receptor thermal protein domain-associated protein 3, ASC, and cleaved-caspase-1 protein levels were significantly decreased in H/R-stimulated H9c2 cells. Moreover, the acetylation levels of H92c cells were decreased post-Rb2 treatment via increasing SIRT1 levels, while knocking down SIRT1, translated into an increase in ASC acetylation levels, leading to the increase in ASC protein stability and expressions. Additionally, the Rb2 effects on pyroptosis in H/R-stimulated H92c cells were reversed by overexpressing ASC, while reduced myocardial tissue damage was observed in MI rats following in vivo Rb2 treatment. Rb2 treatment inhibited pyroptosis in MI progression by decreasing the ASC levels. Mechanistically, Rb2 treatment increased the SIRT1 levels, further increasing the acetylation levels of ASC and decreasing the protein stability of ASC.

UCHL3 induces radiation resistance and acquisition of mesenchymal phenotypes by deubiquitinating POLD4 in glioma stem cells

BackgroundThe high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive.MethodExpression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS.ResultWe determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model.ConclusionThese findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.

DYNLL1 accelerates cell cycle via ILF2/CDK4 axis to promote hepatocellular carcinoma development and palbociclib sensitivity.

Disorder of cell cycle represents as a major driver of hepatocarcinogenesis and constitutes an attractive therapeutic target. However, identifying key genes that respond to cell cycle-dependent treatments still facing critical challenges in hepatocellular carcinoma (HCC). Increasing evidence indicates that dynein light chain 1 (DYNLL1) is closely related to cell cycle progression and plays a critical role in tumorigenesis. In this study, we explored the role of DYNLL1 in the regulation of cell cycle progression in HCC. We analysed clinical specimens to assess the expression and predictive value of DYNLL1 in HCC. The oncogenic role of DYNLL1 was determined by gain or loss-of-function experiments in vitro, and xenograft tumour, liver orthotopic, and DEN/CCl4-induced mouse models in vivo. Mass spectrometry analysis, RNA sequencing, co-immunoprecipitation assays, and forward and reverse experiments were performed to clarify the mechanism by which DYNLL1 activates the interleukin-2 enhancer-binding factor 2 (ILF2)/CDK4 signalling axis. Finally, the sensitivity of HCC cells to palbociclib and sorafenib was assessed by apoptosis, cell counting kit-8, and colony formation assays in vitro, and xenograft tumour models and liver orthotopic models in vivo. DYNLL1 was significantly higher in HCC tissues than that in normal liver tissues and closely related to the clinicopathological features and prognosis of patients with HCC. Importantly, DYNLL1 was identified as a novel hepatocarcinogenesis gene from both in vitro and in vivo evidence. Mechanistically, DYNLL1 could interact with ILF2 and facilitate the expression of ILF2, then ILF2 could interact with CDK4 mRNA and delay its degradation, which in turn activates downstream G1/S cell cycle target genes CDK4. Furthermore, palbociclib, a selective CDK4/6 inhibitor, represents as a promising therapeutic strategy for DYNLL1-overexpressed HCC, alone or particularly in combination with sorafenib. Our work uncovers a novel function of DYNLL1 in orchestrating cell cycle to promote HCC development and suggests a potential synergy of CDK4/6 inhibitor and sorafenib for the treatment of HCC patients, especially those with increased DYNLL1.

METTL3 facilitates prostate cancer progression via inducing HOXC6 m6A modification and stabilizing its expression through IGF2BP2-dependent mechanisms.

HOXC6 (Homeobox C6) and methyltransferase-like 3 (METTL3) have been shown to be involved in the progression of prostate cancer (PCa). However, whether HOXC6 performs oncogenic effects in PCa via METTL3-mediated N6-methyladenosine (m6A) modification is not yet reported. The Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, scratch, sphere formation assays were applied for cell growth, invasion, migration and stemness analyses. Glycolysis was evaluated by measuring glucose consumption, lactate generation and ATP/ADP ratio. The N6-methyladenine (m6A) modification profile was determined by RNA immunoprecipitation (Me-RIP) assay. The proteins that interact with PGK1 (phosphoglycerate kinase 1) were confirmed by Co-immunoprecipitation assay. Tumor formation experiments in mice were conducted for in vivo assay. PCa tissues and cells showed highly expressed HOXC6 and METTL3. Functionally, the silencing of HOXC6 or METTL3 suppresses PCa cell proliferation, invasion, migration, stemness, and glycolysis. Moreover, METTL3-induced HOXC6 m6A modification to stabilize its expression. In addition, the m6A reader IGF2BP2 directly recognized and bound to HOXC6 mRNA, and maintained its stability, and was involved in the regulation of HOXC6 expression by METTL3. Furthermore, IGF2BP2 knockdown impaired PCa cell proliferation, invasion, migration, stemness, and glycolysis by regulating HOXC6. Besides that HOXC6 interacted with the glycoytic enzyme PGK1 in PCa cells. In vivo assays further showed that METTL3 silencing reduced the expression of HOXC6 and PGK1, and impeded PCa growth. METTL3 promoted PCa progression by maintaining HOXC6 expression in an m6A-IGF2BP2-dependent mechanism.

C/EBPβ-Lin28a positive feedback loop triggered by C/EBPβ hypomethylation enhances the proliferation and migration of vascular smooth muscle cells in restenosis.

The main cause of restenosis after percutaneous transluminal angioplasty (PTA) is the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Lin28a has been reported to play critical regulatory roles in this process. However, whether CCAAT/enhancer-binding proteins β (C/EBPβ) binds to the Lin28a promoter and drives the progression of restenosis has not been clarified. Therefore, in the present study, we aim to clarify the role of C/EBPβ-Lin28a axis in restenosis. Restenosis and atherosclerosis rat models of type 2 diabetes (n = 20, for each group) were established by subjecting to PTA. Subsequently, the difference in DNA methylation status and expression of C/EBPβ between the two groups were assessed. EdU, Transwell, and rescue assays were performed to assess the effect of C/EBPβ on the proliferation and migration of VSMCs. DNA methylation status was further assessed using Methyltarget sequencing. The interaction between Lin28a and ten-eleven translocation 1 (TET1) was analysed using co-immunoprecipitation (Co-IP) assay. Student's t-test and one-way analysis of variance were used for statistical analysis. C/EBPβ expression was upregulated and accompanied by hypomethylation of its promoter in restenosis when compared with atherosclerosis. In vitroC/EBPβ overexpression facilitated the proliferation and migration of VSMCs and was associated with increased Lin28a expression. Conversely, C/EBPβ knockdown resulted in the opposite effects. Chromatin immunoprecipitation assays further demonstrated that C/EBPβ could directly bind to Lin28a promoter. Increased C/EBPβ expression and enhanced proliferation and migration of VSMCs were observed after decitabine treatment. Further, mechanical stretch promoted C/EBPβ and Lin28a expression accompanied by C/EBPβ hypomethylation. Additionally, Lin28a overexpression reduced C/EBPβ methylation via recruiting TET1 and enhanced C/EBPβ-mediated proliferation and migration of VSMCs. The opposite was noted in Lin28a knockdown cells. Our findings suggest that the C/EBPβ-Lin28a axis is a driver of restenosis progression, and presents a promising therapeutic target for restenosis.

USP9X-mediated REV1 deubiquitination promotes lung cancer radioresistance via the action of REV1 as a Rad18 molecular scaffold for cystathionine γ-lyase.

Radioresistance is a key clinical constraint on the efficacy of radiotherapy in lung cancer patients. REV1 DNA directed polymerase (REV1) plays an important role in repairing DNA damage and maintaining genomic stability. However, its role in the resistance to radiotherapy in lung cancer is not clear. This study aims to clarify the role of REV1 in lung cancer radioresistance, identify the intrinsic mechanisms involved, and provide a theoretical basis for the clinical translation of this new target for lung cancer treatment. The effect of targeting REV1 on the radiosensitivity was verified by in vivo and in vitro experiments. RNA sequencing (RNA-seq) combined with nontargeted metabolomics analysis was used to explore the downstream targets of REV1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the content of specific amino acids. The coimmunoprecipitation (co-IP) and GST pull-down assays were used to validate the interaction between proteins. A ubiquitination library screening system was constructed to investigate the regulatory proteins upstream of REV1. Targeting REV1 could enhance the radiosensitivity in vivo, while this effect was not obvious in vitro. RNA sequencing combined with nontargeted metabolomics revealed that the difference result was related to metabolism, and that the expression of glycine, serine, and threonine (Gly/Ser/Thr) metabolism signaling pathways was downregulated following REV1 knockdown. LC-MS/MS demonstrated that REV1 knockdown results in reduced levels of these three amino acids and that cystathionine γ-lyase (CTH) was the key to its function. REV1 enhances the interaction of CTH with the E3 ubiquitin ligase Rad18 and promotes ubiquitination degradation of CTH by Rad18. Screening of the ubiquitination compound library revealed that the ubiquitin-specific peptidase 9 X-linked (USP9X) is the upstream regulatory protein of REV1 by the ubiquitin-proteasome system, which remodels the intracellular Gly/Ser/Thr metabolism. USP9X mediates the deubiquitination of REV1, and aberrantly expressed REV1 acts as a scaffolding protein to assist Rad18 in interacting with CTH, promoting the ubiquitination and degradation of CTH and inducing remodeling of the Gly/Ser/Thr metabolism, which leads to radioresistance. A novel inhibitor of REV1, JH-RE-06, was shown to enhance lung cancer cell radiosensitivity, with good prospects for clinical translation.

TRIM47 silencing inhibits the malignant biological behaviors of prostate cancer cells by regulating MDM2/p53 signaling.

Prostate cancer (PCa) is a prevalent male malignancy globally. Tripartite motif 47 (TRIM47) has been reported to be associated with PCa. However, how TRIM47 acts on PCa is still incompletely understood. Here, we explored the biological roles of TRIM47 in PCa cells and investigated its potential regulatory mechanism. TRIM47 expression in PCa cells was detected by qRT-PCR and western blot. After TRIM47 silencing, the viability of PCa cells was measured using CCK-8 method. Flow cytometry was employed to estimate cell cycle. Cell apoptotic level was subjected to appraisement with TUNEL assay. Additionally, wound healing- and transwell assays were adopted for evaluation of migration and invasion of PCa cells. Moreover, the Biogrid database and HDOCK SERVER predicated that TRIM47 could interact with mouse double minute 2 (MDM2), which was detected using the Co-immunoprecipitation (co-IP) assay and glutathione S-transferase (GST) pull-down assay. The expression of proteins in MDM2/p53 signaling was detected by western blot analysis. Results indicated that TRIM47 expression was highly expressed in PCa cells. TRIM47 knockdown inhibited PCa proliferation and cell cycle whereas promoted cell apoptosis. Besides, TRIM47 knockdown significantly inhibited the migration and invasion of PCa cells. In addition, TRIM47 was proved to bind to MDM2 and regulated MDM2/p53 expression. Importantly, MDM2 overexpression counteracted the impacts of TRIM47 knockdown on cell viability, cell cycle, apoptosis, migration and invasion by regulating the MDM2/p53 pathway. Collectively, our results suggested that TRIM47 silencing inhibits the malignant biological behaviors of prostate cancer cells by regulating MDM2/p53 signaling, which may provide a novel therapeutic target for PCa treatment.

Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

#1225 Lactate aggravates mitochondrial dysfunction via ALDH2 lactylation in acute kidney injury

Abstract Background and Aims Elevated lactate levels have been recognized as an independent risk factor in acute kidney injury (AKI) contributed by mitochondrial dysfunction. We previously proved the crucial role of acetaldehyde dehydrogenase 2 (ALDH2) in AKI but not focused on the lactate and lactylation of ALDH2. Method We first established a maleic acid (MA) induced AKI model (MA-AKI) in C57BL/6J mice, administrating 2-DG i.p. (a glycolysis inhibitor) or oxamate (a lactate dehydrogenase A inhibitor) to suppress the lactate production. We measured the renal function, mitochondrial function, and lactate in serum and urine, then evaluated the ALDH2 lactylation by co-immunoprecipitation assays, and identified the specific lactation site by mass spectrum. The role of ALDH2 lactylation was explored through point mutation in HEK293 cells. Furthermore, an ALDH2 lactylation eraser was identified, and its function was elucidated in mice and HK-2 cells. Results The elevated lactate in the kidney, serum, and urine showed a significant positive correlation to the serum creatinine (Scr, R2 = 0.83, P < 0.0001) in MA-induced mice. Both 2-DG and oxamate partly abolished the Scr increasing and the proximal tubular histopathological injuries. Increased pan-lactylation in AKI mice was effectively inhibited by oxamate, parallel with increased ALDH2 lactylation in MA-stimulated HK-2 cells. Mass spectrometry identified a single lactylate lysine residue in ALDH2 (K52). The introduction of a point mutation (K52 to R) in ALDH2, mimicking the delactylated state, led to partial recovery of ALDH2 activity (1.11 ± 0.07 vs. 0.68 ± 0.15, P = 0.0029) and mitochondrial function in MA-stimulated HK-2 cells. Additionally, sirt3 was identified as an ALDH2 lactylation eraser, and Honokiol (a sirt3 activator) demonstrated enhanced renal function and mitigation of mitochondrial dysfunction in both MA-induced mice and HK-2 cells. Conclusion Lactate played a crucial role in mitochondrial dysfunction in AKI partly via induction of ALDH2 lactylation, which shed light on the potential target for the therapy.

Mitotic deacetylase complex (MiDAC) recognizes the HIV-1 core promoter to control activated viral gene expression.

The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.

Tripartite motif-containing protein 21 is involved in IFN-γ-induced suppression of hepatitis B virus by regulating hepatocyte nuclear factors.

The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.

#2062 Protein interactome and trafficking of uromodulin in HEK and MDCK cell model

Abstract Background and Aims Uromodulin (UMOD) is a kidney-specific glycoprotein and the most abundant urinary protein in healthy individuals. Genome-wide association studies have shown an association between UMOD variants, kidney diseases, and hypertension. UMOD exhibits bidirectional secretion as it releases into the renal interstitium and circulation. However, the molecular trafficking of UMOD and its interaction remains poorly defined. We aimed to establish a stable Human Embryonic Kidney (HEK293) cell line expressing human UMOD to identify the potential regulators of UMOD expression or secretion. This study will provide significant implications for conditions such as hypertension, chronic kidney diseases, acute kidney injury, or UMOD-autosomal dominant tubulointerstitial kidney disease (UMOD-ADTKD). Method A HEK-UMOD-expressing cell line was generated by introducing human UMOD transcript 1 expression and using a tetracycline-inducible expression vector (pcDNA™FRT⁄TO vector) with yellow fluorescent protein tag at the C-terminal and previously established MDCK-UMOD cell line was also used. An untargeted proteomic approach was used to identify proteins, using GFP trap co-immunoprecipitation assay followed by mass spectrometry analysis. Results We identified 1367 and 1140 UMOD interacting proteins in HEK and MDCK cell models respectively. There were 620 proteins common to both these models. Through gene ontology, we unravel the molecular mechanisms involved in the biosynthesis, folding, and transport of UMOD. We observed several proteins associated with clathrin-mediated transporting. To study the role of clathrin in UMOD transport, we treated the polarised MDCK-UMOD cells with pitstop 2. There was significantly more secretion of UMOD with pitstop 2 treatment in the apical compartment compared to untreated cells however there was no significant difference in UMOD secretion in the basolateral compartment. Conclusion These results suggested that clathrin-coated vesicles are involved in transporting and sorting UMOD. Further studies need to investigate the functional importance of these proteins in UMOD trafficking and their implications in different physiological processes.

CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

Bone formation and homeostasis are greatly dependent on the osteogenic differentiation of human bone marrow stem cells (BMSCs). Therefore, revealing the mechanisms underlying osteogenic differentiation of BMSCs will provide new candidate therapeutic targets for osteoporosis. The osteogenic differentiation of BMSCs was measured by analyzing ALP activity and expression levels of osteogenic markers. Cellular Fe and ROS levels and cell viability were applied to evaluate the ferroptosis of BMSCs. qRT-PCR, Western blotting, and co-immunoprecipitation assays were harnessed to study the molecular mechanism. The mRNA level of CRYAB was decreased in the plasma of osteoporosis patients. Overexpression of CRYAB increased the expression of osteogenic markers including OCN, OPN, RUNX2, and COLI, and also augmented the ALP activity in BMSCs, on the contrary, knockdown of CRYAB had opposite effects. IP-MS technology identified CRYAB-interacted proteins and further found that CRYAB interacted with ferritin heavy chain 1 (FTH1) and maintained the stability of FTH1 via the proteasome mechanism. Mechanically, we unraveled that CRYAB regulated FTH1 protein stability in a lactylation-dependent manner. Knockdown of FTH1 suppressed the osteogenic differentiation of BMSCs, and increased the cellular Fe and ROS levels, and eventually promoted ferroptosis. Rescue experiments revealed that CRYAB suppressed ferroptosis and promoted osteogenic differentiation of BMSCs via regulating FTH1. The mRNA level of FTH1 was decreased in the plasma of osteoporosis patients. Downregulation of CRYAB boosted FTH1 degradation and increased cellular Fe and ROS levels, and finally improved the ferroptosis and lessened the osteogenic differentiation of BMSCs.

The flavohemoglobin Yhb1 is a new interacting partner of the heme transporter Str3.

Nitric oxide (˙NO) is a free radical that induces nitrosative stress, which can jeopardize cell viability. Yeasts have evolved diverse detoxification mechanisms to effectively counteract ˙NO-mediated cytotoxicity. One mechanism relies on the flavohemoglobin Yhb1, whereas a second one requires the S-nitrosoglutathione reductase Fmd2. To investigate heme-dependent activation of Yhb1 in response to ˙NO, we use hem1Δ-derivative Schizosaccharomyces pombe strains lacking the initial enzyme in heme biosynthesis, forcing cells to assimilate heme from external sources. Under these conditions, yhb1+ mRNA levels are repressed in the presence of iron through a mechanism involving the GATA-type transcriptional repressor Fep1. In contrast, when iron levels are low, the transcription of yhb1+ is derepressed and further induced in the presence of the ˙NO donor DETANONOate. Cells lacking Yhb1 or expressing inactive forms of Yhb1 fail to grow in a hemin-dependent manner when exposed to DETANONOate. Similarly, the loss of function of the heme transporter Str3 phenocopies the effects of Yhb1 disruption by causing hypersensitivity to DETANONOate under hemin-dependent culture conditions. Coimmunoprecipitation and bimolecular fluorescence complementation assays demonstrate the interaction between Yhb1 and the heme transporter Str3. Collectively, our findings unveil a novel pathway for activating Yhb1, fortifying yeast cells against nitrosative stress.

BRD4 promotes gouty arthritis through MDM2-mediated PPARγ degradation and pyroptosis

BackgroundGouty arthritis (GA) is characterized by monosodium urate (MSU) crystal accumulation that instigates NLRP3-mediated pyroptosis; however, the underlying regulatory mechanisms have yet to be fully elucidated. The present research endeavors to elucidate the regulatory mechanisms underpinning this MSU-induced pyroptotic cascade in GA.MethodsJ774 cells were exposed to lipopolysaccharide and MSU crystals to establish in vitro GA models, whereas C57BL/6 J male mice received MSU crystal injections to mimic in vivo GA conditions. Gene and protein expression levels were evaluated using real-time quantitative PCR, Western blotting, and immunohistochemical assays. Inflammatory markers were quantified via enzyme-linked immunosorbent assays. Pyroptosis was evaluated using immunofluorescence staining for caspase-1 and flow cytometry with caspase-1/propidium iodide staining. The interaction between MDM2 and PPARγ was analyzed through co-immunoprecipitation assays, whereas the interaction between BRD4 and the MDM2 promoter was examined using chromatin immunoprecipitation and dual-luciferase reporter assays. Mouse joint tissues were histopathologically evaluated using hematoxylin and eosin staining.ResultsIn GA, PPARγ was downregulated, whereas its overexpression mitigated NLRP3 inflammasome activation and pyroptosis. MDM2, which was upregulated in GA, destabilized PPARγ through the ubiquitin–proteasome degradation pathway, whereas its silencing attenuated NLRP3 activation by elevating PPARγ levels. Concurrently, BRD4 was elevated in GA and exacerbated NLRP3 activation and pyroptosis by transcriptionally upregulating MDM2, thereby promoting PPARγ degradation. In vivo experiments showed that BRD4 silencing ameliorated GA through this MDM2–PPARγ–pyroptosis axis.ConclusionBRD4 promotes inflammation and pyroptosis in GA through MDM2-mediated PPARγ degradation, underscoring the therapeutic potential of targeting this pathway in GA management.

TDP43 Interacts with MLH1 and MSH6 Proteins in A DNA Damage-Inducible Manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complimentary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 hr treatment of 10μM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS affected neurons.