RING Finger Protein Research Articles

RNF38 promotes gilteritinib resistance in acute myeloid leukemia via inducing autophagy by regulating ubiquitination of LMX1A

BackgroundGilteritinib is a commonly used targeted drug for acute myeloid leukemia (AML), but the emergence of gilteritinib resistance greatly reduces the therapeutic effect. RING finger protein 38 (RNF38), a protein with RING Finger domain and E3 ubiquitin ligase activity, has been implicated in tumorigenesis and drug resistance. However, the role and mechanism of RNF38 in the gilteritinib resistance of AML remains unclear.MethodsNormal AML cells were treated with gilteritinib to construct gilteritinib-resistant cells (MV4-11/Gilteritinib and MOLM-13/Gilteritinib). CCK8 assay, TUNEL staining and EdU assay were used to assess gilteritinib resistance, cell apoptosis and proliferation. The protein levels of autophagy-related markers, RNF38 and LIM homeobox transcription factor 1 alpha (LMX1A) were determined by western blot. Also, RNF38 and LMX1A mRNA levels were tested using qRT-PCR. Autophagic flux was assessed using mRFP-GFP-LC3 labeling, and autophagosome numbers was counted under transmission electron microscopy. Co-IP assay was employed to analyze interaction between RNF38 and LMX1A. The effects of LMX1A and RNF38 on AML tumorigenesis were analyzed by in vivo experiments.ResultsIn gilteritinib-resistant AML cells, autophagy-related markers, mRFP-GFP-LC3 signals and autophagosome numbers were significantly enhanced. Autophagy inhibitor 3-MA could suppress gilteritinib resistance in AML cells. RNF38 knockdown inhibited gilteritinib resistance and autophagy in AML cells. Mechanistically, RNF38 reduced LMX1A expression by inducing its ubiquitination. RNF38 overexpression reversed the inhibitory effect of LMX1A on gilteritinib resistance and autophagy in AML cells, as well as AML tumor growth in vivo, while these effects could be abolished by proteasome inhibitor MG132.ConclusionsRNF38 induced autophagy to promote gilteritinib resistance in AML by increasing the ubiquitination of LMX1A.Graphical 1. Autophagy is activated in gilteritinib-resistant AML cells.2. Reduction of autophagy suppresses gilteritinib resistance in AML cells.3. RNF38 knockdown inhibits gilteritinib resistance and autophagy in AML cells.4. RNF38 reduces LMX1A expression via inducing its ubiquitination.

RNF144A inhibits autophagy by targeting BECN1 for degradation during L. monocytogenes infection

ABSTRACT Listeria monocytogenes (L. monocytogenes, Lm) is widely used in the laboratory as an infection model for the research on pathogenesis and host defense against gram-positive intracellular bacteria. Macroautophagy (called simply “autophagy” hereafter), is important in the host defense against pathogens, such as bacteria, viruses, and parasites. BECN1 plays a pivotal role in the initiation of autophagy and accumulating evidence indicates that post-translational modifications of BECN1 provide multiple strategies for autophagy regulation. In this study, we demonstrated that the RING1-IBR-RING2 (RBR) family member RNF144A (ring finger protein 144A), which was induced by Lm infection, promoted Lm infection in an autophagy-dependent but STING1-independent pattern. rnf144a deficiency in mice protected mice from Lm infection with inhibited innate immune responses. Interestingly, RNF144A decreased Lm-induced autophagosome accumulation. Mechanistic investigation indicated that RNF144A interacted with BECN1 and promoted its K48-linked ubiquitination, leading to the subsequent proteasome-dependent degradation of BECN1 and reduced autophagosome accumulation. Further study demonstrated that RNF144A promoted the ubiquitination of BECN1 at K117 and K427, and these two ubiquitination sites were essential to the role of BECN1 in autophagy and Lm infection. Thus, our findings suggested a new regulator in intracellular bacterial infection and autophagy, which may contribute to our understanding of host defense against intracellular bacterial infection via autophagy. Abbreviations: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG10: autophagy related 10; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; Baf A1: bafilomycin A1; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cell; BMDM: bone marrow-derived macrophage; CFUs: colony-forming units; CHX: cycloheximide; CQ: chloroquine; CXCL10/IP-10: C-X-C motif chemokine ligand 10; EBSS: Earle’s balanced salt solution; ELISA: enzyme-linked immunosorbent assay; IFIT1/ISG56: interferon induced protein with tetratricopeptide repeats 1; IFNB/IFN-β: interferon beta; IL6: interleukin 6; IRF3, interferon regulatory factor 3; Lm: L. monocytogenes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; PLA: proximity ligation assay; PMA: phorbol myristate acetate; PMA-THP1, PMA-differentiated THP1; PMs: peritoneal macrophages; PTMs: posttranslational modifications; RBR: RING1-IBR-RING2; RNF144A: ring finger protein 144A; STING1, stimulator of interferon response cGAMP interactor 1; TBK1, TANK binding kinase 1; TNF/TNF-α: tumor necrosis factor;

ZFP36, an RNA-binding protein promotes hBMSCs osteogenic differentiation via binding with JUN

Osteoporosis (OP) is a metabolic bone disease characterized by progressive decline of bone mass and bone quality, leading to bone fragility and an increased risk of fracture. The osteogenic differentiation of bone mesenchymal stem cells (BMSCs) is crucial to maintain the balance of osteoblast and osteoclast. Bioinformatics prediction indicates that ZFP36 ring finger protein (ZFP36), an RNA-binding protein, is a potential target of OP. Herein, we sought to probe the regulatory role and mechanisms of ZFP36 in the progression of OP. Overexpression of ZFP36 enhanced osteoblast viability, differentiation and mineralization of human BMSCs (hBMSCs). RNA immunoprecipitation qPCR (RIP-qPCR) assays demonstrated that ZFP36 could inhibit the translation of JUN, which was also verified with dual luciferase reporter gene assay. Furthermore, administration with T-5224, a transcription factor c-Fos/activator protein (AP)-1 inhibitor, which specifically inhibits the DNA binding activity of c-Fos/JUN, abolished the effect of ZFP36 knockdown on the behaviors of hBMSCs, suggesting that ZFP36 might promotes osteogenic differentiation through regulating JUN. These findings provide insights into the progression and a potential therapeutic target of OP.

RNF13 protects neurons against ischemia-reperfusion injury via stabilizing p62-mediated Nrf2/HO-1 signaling pathway

BackgroundCerebral ischemia/reperfusion injury (CIRI), a common, universal clinical problem that costs a large proportion of the economic and disease burden. Identifying the key regulators of cerebral I/R injury could provide potential strategies for clinically improving the prognosis of stroke. Ring finger protein 13 (RNF13) has been proven to be involved in the inflammatory response. Here, we aimed to identify the role of RNF13 in cerebral I/R injury and further reveal its immanent mechanisms.MethodsThe CRISPR/Cas9 based knockout mice, RNA sequencing, mass spectrometry, co-immunoprecipitation, GST-pull down, immunofluorescent staining, western blot, RT-PCR were used to investigate biodistribution, function and mechanism of RNF13 during cerebral I/R injury.ResultsIn the present study, we found that RNF13 was significantly up-regulated in patients, mice and primary neurons after I/R injury. Deficiency of RNF13 aggravated I/R-induced neurological impairment, inflammatory response and apoptosis while overexpression of RNF13 inhibited I/R injury. Mechanistically, this inhibitory effect of RNF13 during I/R injury was confirmed to be dependent on the blocking of TRIM21-mediated autophagy-dependent degradation of p62 and the stabilization of the p62-mediated Nrf2/HO-1 signaling pathway.ConclusionRNF13 is a crucial regulator of cerebral I/R injury that plays its role in inhibiting cell apoptosis and inflammatory response by preventing the autophagy-medicated degradation of the p62/Nrf2/HO-1 signaling pathway via blocking the interaction of TRIM21-p62 complex. Therefore, RNF13 represents a potential pharmacological target in acute ischemia stroke therapy.

Transcription factor PagWRKY33 regulates gibberellin signaling and immune receptor pathways in Populus.

Enhanced autoimmunity often leads to impaired plant growth and development, and the coordination of immunity and growth in Populus remains elusive. In this study, we have identified the transcription factors PagWRKY33a/b as key regulators of immune response and growth maintenance in Populus. The disruption of PagWRKY33a/b causes growth issues and autoimmunity while conferring resistance to anthracnose caused by Colletotrichum gloeosporioides. PagWRKY33a/b binds to the promoters of N requirement gene 1.1 (NRG1.1) and Gibberellic Acid-Stimulated in Arabidopsis (GASA14)during infection, activating their transcription. This process maintains disease resistance and engages in GA signaling to reduce growth costs from immune activation. The oxPagWRKY33a/nrg1.1 mutant results in reduced resistance to C. gloeosporioides. Further, PagWRKY33a/b is phosphorylated and activated by Mitogen-Activated Protein Kinase Kinase 1 (MKK1), which inhibits Respiratory Burst Oxidase Homolog D (RBOHD) and Respiratory Burst Oxidase Homolog I (RBOHI) transcription, causing ROS bursts in wrky33a/b double mutants. This leads to an upregulation of PagNRG1.1 in the absence of pathogens. However, the wrky33a/b/nrg1.1 and wrky33a/b/rbohd triple mutants show compromised defense responses, underscoring the complexity of WRKY33 regulation. Additionally, the stability of PagWRKY33 is modulated by Ring Finger Protein 5 (PagRNF5)-mediated ubiquitination, balancing plant immunity and growth. Together, our results provide key insights into the complex function of WRKY33 in Populus autoimmunity and its impact on growth and development.

RNF19A inhibits bladder cancer progression by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway

BackgroundThe role of the RING finger protein superfamily in carcinogenesis has been widely studied, but one member of this family, RNF19A, has not yet been thoroughly explored in bladder cancer (BCa).MethodsThe expression levels of RNF19A in BCa samples and cell lines were analysed through data mining of public resources and further experiments. BCa cells in which RNF19A was stably overexpressed or knocked down were generated through lentivirus infection. The effects of RNF19A on cell proliferation, migration, and invasion were explored by performing a series of in vitro experiments, including CCK-8, colony formation, wound healing, and Transwell invasion assays. Using bioinformatics methods and multiple experiments, including western blot, qRT‒PCR, immunoprecipitation, cycloheximide, ubiquitination, and rescue assays, the mechanism underlying the effect of RNF19A on the progression of BCa was investigated.ResultsHere, we found that RNF19A expression was reduced in BCa samples and cell lines and that lower RNF19A expression predicted shorter overall survival of BCa patients. Functionally, forced expression of RNF19A suppressed BCa cell proliferation, migration, and invasion by inactivating the AKT/mTOR signalling pathway, whereas silencing RNF19A had the opposite effects. Mechanistically, RNF19A could directly interact with ILK and promote its ubiquitination and degradation. Rescue experiments revealed that forced ILK expression partially rescued the decreased phosphorylation of AKT, mTOR, and S6K1 caused by RNF19A overexpression and that the increased levels of the p-AKT, p-mTOR, and p-S6K1 proteins induced by RNF19A knockdown were eliminated after silencing ILK. Similarly, the effects of RNF19A overexpression or knockdown on the phenotypes of cell proliferation, migration, and invasion could also be restored by forced or decreased ILK expression.ConclusionsRNF19A suppressed the proliferation, migration, and invasion abilities of BCa cells by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway. RNF19A might be a potential prognostic biomarker and promising therapeutic target for BCa.

CXCL12/CXCR4 pathway as a novel therapeutic target for RNF213-associated pulmonary arterial hypertension

Genetic backgrounds of patients with pulmonary arterial hypertension (PAH) were not fully investigated. A variant of c.14429G > A (p.Arg4810Lys) in the ring finger protein 213 gene (RNF213) was recently identified as a risk allele for poor treatment response and poor clinical prognosis in patients with PAH. However, the molecular mechanisms of the RNF213 p.Arg4810Lys variant in development of PAH are unknown. We investigated the underlying molecular mechanisms of RNF213-associated vasculopathy using an in vivo mouse model. RNF213+/p.Arg4828Lys mice, harboring the heterozygous RNF213 p.Arg4828Lys variant corresponding to the p.Arg4810Lys variant in humans, were created using the CRISPR-Cas9 system to recapitulate the genetic status of PAH patients. RNF213+/p.Arg4828Lys mice had a significant elevation of the right ventricular systolic pressure, hypertrophy of the right ventricle, and increased thickness of the pulmonary arterial medial wall compared with wild-type mice after 3 months of exposure to a hypoxic environment. C-X-C motif chemokine ligand 12 (CXCL12), a C-X-C chemokine receptor type 4 (CXCR4) ligand, was significantly elevated in the lungs of RNF213+/p.Arg4828Lys mice, and PAH was ameliorated by the administration of a CXCR4 antagonist. CXCL12-CXCR4 is an angiogenic chemokine axis, and immunohistochemistry demonstrated an increase in CXCR4 in vimentin-positive spindle-shaped cells in adventitia and interstitial lesions in RNF213+/p.Arg4828Lys mice and lung specimens from severe PAH patients with the RNF213 p.Arg4810Lys variant. We confirmed a cause-and-effect relationship between the RNF213 p.Arg4810Lys variant and PAH via the CXCL12-CXCR4 pathway. The findings in this study suggest that targeting this pathway might be a novel therapeutic strategy for RNF213-associated vasculopathy.

Impaired inducibility of immune regulatory capacity of peripheral B cells of patients with recurrent pregnancy loss.

The pathogenesis of recurrent pregnancy loss (RPL) is unclear. RPL may have an association with disruption of immune tolerance. The aim of this study is to characterize the inducibility of immune regulatory ability in peripheral naïve B cells of patients with RPL. In this study, blood samples were taken from patients with RPL. B220+ B cells were isolated by flow cytometry cell sorting. The gene profile of B cells was analyzed using RNA sequencing (RNAseq). The results showed that peripheral B220+ B cells of RPL patients had lower expression of IL10 and exacerbated ER stress. The induction of IL10 expression in peripheral B220+ B cells of RPL patients were impaired. High ubiquitination of c-Maf inducing protein (CMIP) was detected in RPL B cells. Exposure to thapsigargin (an ER stress agonist) decreased the amount of CMIP in B cells. The effects of ER stress on reducing CMIP quantity in B cells were mediated by the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20). Inhibition of RNF20 or ER stress restored the inducibility of immune regulatory functions of B220+ B cells of RPL patients. In summary, peripheral B cells in patients with RPL show impaired immune regulation capacity, in which exacerbated ER stress plays a crucial role. Regulation of ER stress or inhibition of RNF20 can restore the immune regulatory capacity in the B cells.

E3 Ubiquitin Ligase Ring Finger Protein 2 Alleviates Cerebral Ischemia-Reperfusion Injury by Stabilizing Mesencephalic Astrocyte-Derived Neurotrophic Factor Through Monoubiquitination.

Cerebral ischemic stroke (IS) is one of the leading causes of morbidity and mortality globally. However, the mechanisms underlying IS injury remain poorly understood. Ring finger protein 2 (RNF2), the member of the polycomb family (PcG), has been implicated in diverse biological and pathological conditions. However, whether RNF2 plays a role in IS progression is not clarified. This study aims to investigate the potential effects of RNF2 on IS. The effects of RNF2 were studied in human postmortem IS brains, a rat model of IS, tunicamycin (TM)-induced mouse neuroblastoma neuro2a (N2a) cells, and oxygen-glucose deprivation/reperfusion (OGD/R)-induced SH-SY5Y cells. Here, we demonstrated that RNF2 was markedly upregulated both in human postmortem IS brains and ischemic rat brains and RNF2 overexpression alleviated brain injury induced by middle cerebral artery occlusion by reducing neuron apoptosis. Mechanistically, we found that RNF2 is an E3 ubiquitin ligase for the mesencephalic astrocyte-derived neurotrophic factor (MANF), which confers protection against brain ischemia. RNF2 interacted with MANF and promoted the monoubiquitination of MANF, consequently facilitating its stability and nuclear localization. Collectively, RNF2 is identified as a critical inhibitor of IS injury by stabilizing MANF through monoubiquitination, suggesting that RNF2 is a potential therapeutic target for IS.

Muscle RING-finger proteins (MuRF) regulate protein kinase A activity via retrograde vesicular transport of PKA RI-alpha

Abstract Background Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins. Methods and Results Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as a cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation. Conclusion MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.

Lipopolysaccharide-regulated RNF31/NRF2 axis in colonic epithelial cells mediates homeostasis of the intestinal barrier in ulcerative colitis

BackgroundAlthough previous studies have shown that the Ring Finger Protein 31 (RNF31) gene confers susceptibility to inflammatory disease and colorectal cancer, the exact function of this protein in ulcerative colitis (UC) has not been determined. MethodsA mouse dextran sulfate sodium (DSS)-induced experimental colitis model was used to study RNF31 and NRF2 in colitis. RNF31 silencing or overexpression in vitro was applied to address the role of RNF31 in colonic mucosal barrier damage. Immunohistochemistry and silico analysis was performed to investigate the expression of RNF31 via taking advantage of UC tissue samples and Gene Expression Omnibus (GEO) data, respectively. The cycloheximide (CHX)-chase experiment and Co-Immunoprecipitation (Co-IP) assays were conducted to explore the association of RNF31 protein with NRF2 and P62. ResultsRNF31 is highly expressed in UC patients, in inflamed murine colon induced DSS and Lipopolysaccharide (LPS)-treated epithelial cells, while the express of NRF2 was Tabdecreased. RNF31-knockdown mice in the DSS-induced colitis model had a less severe phenotype, which was associated with a more integrated barrier of colon epithelial cells. While depletion of NRF2 in colitis model exacerbated intestinal inflammation. Mechanistically, RNF31 promoted the degradation of NRF2 by regulating its ubiquitination. Upon stimulation by RNF31, NRF2 is K63 ubiquitinated, which is associated with the C871 residue of RNF31. Moreover, downregulated NRF2 mediates inflammation by promoting the secretion of IL1β and IL18, leading to damage of the intestinal barrier. Upon LPS stimulation, the interaction of the PUB domain of RNF31 with the UBA domain of P62 increased, resulting in decreased degradation of the RNF31 protein via autophagy. ConclusionOverall, depletion of RNF31 effectively relieves DSS-induced colitis in mice by inhibiting NRF2 degradation, suggesting that RNF31 may be a potential therapy for human ulcerative colitis.

TMEM209 promotes hepatocellular carcinoma progression by activating the Wnt/β-catenin signaling pathway through KPNB1 stabilization

Hepatocellular carcinoma (HCC) is the most common malignancy in the liver, with a poor prognosis. Transmembrane protein 209 (TMEM209) involves multiple biological processes, such as substance transportation and signal transduction, and is abundantly expressed in tumor tissues. However, the relationship between TMEM209 and HCC has not been comprehensively elucidated. In this study, we aimed to illustrate this issue by in vitro and in vivo experiments. Bioinformatic analysis and clinical sample validation revealed that TMEM209 was upregulated in HCC and correlated with reduced survival duration. Functionally, TMEM209 promoted the proliferation, migration, invasion, and EMT of HCC cells in vitro and facilitated tumor growth and metastasis in xenograft models. Mechanistically, TMEM209 promoted the proliferation and metastasis of HCC in a KPNB1-dependent manner. Specifically, TMEM209 could bind to KPNB1, thereby competitively blocking the interaction between KPNB1 and the E3 ubiquitin ligase RING finger and CHY zinc finger domain-containing protein 1 (RCHY1) and preventing K48-associated ubiquitination degradation of KPNB1. Ultimately, the Wnt/β-catenin signaling pathway was activated, contributing to the progression of the malignant phenotype of HCC. In conclusion, the molecular mechanism underlying the TMEM209/KPNB1/Wnt/β-catenin axis in HCC progression was elucidated. TMEM209 is a potential biomarker and therapeutic target for HCC.

Golgi-localized Ring Finger Protein 121 is necessary for MYCN-driven neuroblastoma tumorigenesis

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development.

7381 Overexpression of Makorin Ring Finger Protein 3 in the Arcuate Nucleus of Postpubertal Female Mice Impacts Neurokinin B and Kisspeptin Co-Expressing Neurons

Abstract Disclosure: S.A. Roberts: None. A. Fontes: None. S. Blau: None. M. Magnuson: None. K.M. McKnight: None. M. Sena-Esteves: None. R.S. Carroll: None. U.B. Kaiser: None. Background: Makorin ring finger protein 3 (MKRN3), an upstream inhibitor of GnRH release, is an important regulator of the age of menarche. Loss-of-function mutations in MKRN3 are the most common genetic etiology of central precocious puberty. However, its potential role in delayed puberty or hypogonadism is not well defined. In mice, expression of Mkrn3 in the hypothalamic arcuate nucleus (ARC), where kisspeptin, neurokinin B and dynorphin (KNDy) co-expressing neurons are located, is high early in life and declines before pubertal onset. Neonatal hypothalamic overexpression of Mkrn3 leads to delayed puberty in female mice and decreased neurokinin B (NKB) and kisspeptin protein levels in the ARC peripubertally. Conversely, Mkrn3-deficient peripubertal mice exhibit increased NKB protein levels in the ARC. We hypothesized that Mkrn3 overexpression in the ARC of postpubertal female mice would result in suppression of the HPG axis, manifested as hypogonadotropic hypogonadism. Methods and Results: Twelve week old adult wild type female mice were injected stereotaxically into the ARC with a novel adeno-associated virus overexpressing Mkrn3 (AAV-Mkrn3-IRES-EGFP; denoted AAV-Mkrn3) or a control virus (AAV-EGFP). Mean body weight did not differ between groups post-injection. AAV-Mkrn3 injected mice spent significantly more time (62.7%) in diestrus three to six weeks post-injection compared to controls (41.3%; P<0.01). Eight weeks post-injection, gonad-intact females were injected with an NKB receptor (NK3R) agonist, senktide (5 nmol or 10 nmol ip). AAV-Mkrn3 mice had a decreased peak LH response twenty minutes post-injection, compared to controls (P<0.001) at both doses. Peak LH levels were also reduced in response to senktide (5 nmol ip) in ovariectomized and estradiol capsule replaced (OVX+E2) AAV-Mkrn3 injected females, compared to controls (P< 0.001). In contrast, in both gonad-intact and OVX+E2 female mice, kisspeptin-10 (1 nmol ip) resulted in similar peak serum LH levels in AAV-Mkrn3 injected mice compared to controls. Immunohistochemistry in the ARC of intact and OVX+E2 AAV-Mkrn3 injected females showed significantly decreased NKB protein expression in AAV-Mkrn3 mice compared to controls. In response to OVX, serum LH levels increased similarly between AAV-Mkrn3 and control-injected mice. LH pulsatility in OVX females, assessed over 3 hours, showed that mean basal LH levels, number of pulses, pulse amplitude and area under the curve did not differ significantly between groups. Conclusions: These findings indicate that MKRN3 can exert inhibitory effects on the reproductive axis in female mice beyond the juvenile period, which has implications as a potential therapeutic target. The blunted response to senktide, and decreased NKB protein levels in the setting of Mkrn3 overexpression, further supports the impact of Mkrn3 on the neuropeptides and/or receptors of KNDy neurons. Presentation: 6/2/2024

RNF144A as a potential risk gene for endometrial carcinoma: Insights from Mendelian randomization, bulk RNA sequencing, single-cell RNA, and experimental analysis.

Endometrial carcinoma (EC) is a prevalent gynecological malignancy that poses a significant threat to women's health worldwide. However, its pathogenesis and underlying mechanisms remains unclear. In this study, expression quantitative trait loci data, Mendelian randomization analysis, and differential expression analysis were performed to identify potential targets. A prognostic risk signature was subsequently constructed for EC patients based on the expression of these genes. Four bioinformatics algorithms, including generalized linear model, extreme gradient boosting, support vector machine, and random forest, were used to identify hub genes in EC. The expression of ring finger protein 144A (RNF144A) was validated using quantitative real-time polymerase chain reaction. Cellular proliferation and migration ability were evaluated using CCK-8 and Transwell assays, respectively. The genes RNF144A, ketohexokinase, and Rab interacting lysosomal protein like 2 were identified as potential targets for EC. Their differential expression was observed in EC patients, and Mendelian randomization analysis revealed a negative correlation between these genes and the development of EC. Mechanistic analyses suggested a strong association between these genes and the tumor immune microenvironment. The constructed risk signature was significantly associated with the prognosis, age, cancer stage, and grade of EC patients. Furthermore, based on interacted model algorithms, RNF144A was identified as a hub gene in EC. It was found to be significantly downregulated in EC samples, and its expression was positively correlated with the stage and grade of EC patients. In vitro experiments showed that overexpression of RNF144A significantly promoted cell growth and migration in EC cells. In conclusion, this study provides insights into the molecular mechanisms underlying EC progression and identifies preliminary candidate biomarkers for the development of EC treatment strategies.

Identification of a disease-associated germline mutation (A64T) of the ring finger protein 186 gene (RNF186) in Korean patients with ulcerative colitis

Identification of a disease-associated germline mutation (A64T) of the ring finger protein 186 gene (RNF186) in Korean patients with ulcerative colitis

S431 Association Between Ring Finger Protein 213 (RNF213) Mutation and Increased Risk of Early Onset Colorectal Cancer

S431 Association Between Ring Finger Protein 213 (RNF213) Mutation and Increased Risk of Early Onset Colorectal Cancer

Blended Phenotype of NOTCH3 and RNF213 Variants With Accelerated Large and Small Artery Crosstalk: A Case Report and Literature Review.

Recent advancements in genome research have revealed not only the importance of variants associated with cerebrovascular diseases but also a notably high frequency of carriers harboring multiple variants, presenting with an elusive blended phenotype. In this study, we report the case of a 66-year-old man who experienced 3 stroke episodes over a 4-year period, starting at the age of 62 years. The patient presented with isolated infarcts in the left temporal pole with progressive stenosis in the ipsilateral middle cerebral artery based on large and small artery crosstalk. Exons 2-24 of the NOTCH3 gene were analyzed by direct genomic DNA sequencing. The presence of the p.Arg4810Lys variant of the ring finger protein 213 (RNF213) gene was evaluated using real-time PCR. Diagnoses of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy and RNF213-related vasculopathy were made based on the early-onset recurrent stroke episode, progressive intracranial artery stenosis, and presence of the heterozygous NOTCH3 p.Cys1250Arg and RNF213 p.Arg4810Lys variants. Temporal pole infarcts could represent a blended phenotype of both variants. This case highlights the importance of large and small artery crosstalk and the pivotal role of genetic analysis in determining the pathogenesis of stroke and dementia.

Deciphering MARCH5’s impact on multiple myeloma: insights into autophagy regulation and AKT-FOXO3 signaling

AbstractMultiple myeloma (MM) stands as a formidable blood malignancy, necessitating innovative therapeutic approaches. Excessive immunoglobulin production within myeloma cells leads to a buildup of toxic proteins, and autophagy plays a crucial role in their survival by degrading toxic aggregates and generating energy. Membrane-associated RING finger protein 5 (MARCH5) is an E3-ligase positioned at the outer mitochondrial membrane and has been shown to regulate autophagy by competing for MIR30A. Given the fundamental significance of autophagy in promoting the survival of myeloma cells, coupled with the regulatory role of MARCH5 in autophagic activity, we hypothesized that MARCH5 plays an essential function in MM and holds a pivotal position in the pathogenesis and progression of MM. We identified MARCH5’s unique dependencies in MM cells by analyzing the Cancer Dependency Map, thereby establishing its significance in MM biology. Examining various data sets, including CoMMpass and HOVON, demonstrated a correlation between MARCH5 expression and patient outcomes. Knockdown of MARCH5 revealed a substantial reduction in MM cell viability, which was associated with a decrease in autophagic activity. Mechanistically, we unraveled a novel MARCH5/AKT/FOXO3 axis, wherein MARCH5 regulates autophagy through the AKT-mediated degradation of FOXO3. Compromised MM cell viability observed with MARCH5 knockdown was recapitulated in FOXO3 knockdown experiments, validating the pivotal role of FOXO3 in mediating MARCH5’s effects. In conclusion, this research highlights the crucial role of MARCH5 in MM, and the identified MARCH5/AKT/FOXO3 axis enhances our understanding of MM biology and provides a foundation for developing targeted therapies.

The modulatory effects of Lonicera caerulea L. extract and omega-3 on sarcopenia via regulating PI3K and FOXO signaling pathways in rats

Introduction: Sarcopenia is an inflammatory disease caused by a disruption of muscle homeostasis. Lonicera caerulea L. (Haskap berry) (HB) extract and omega-3 (ω−3) possess antioxidant and anti-inflammatory activities. This study assesses the potential ameliorating effects of HB extract and ω−3 supplementation on dexamethasone (DEXA)-induced sarcopenia. Methods: Rats were divided into five groups; the negative control group was injected with saline (i.p.); groups 2, 3, 4, and 5 were injected with DEXA (2 mg/kg/d, i.p.); groups 3 and 4 also received 400 mg/kg/d and 100 mg/kg/d of HB extract and ω−3, respectively, while group 5 received both treatments daily for 21 days. The ameliorative effects of treatments were investigated by measuring lysosomal proteolytic enzyme cathepsin activity, phosphatidylinositol 3 kinase (PI3K) activity, nuclear factor kappa beta (NF-κB) level, and heme oxygenase-1 (HO-1) activity. The gene expression levels of muscle ring-finger protein (MuRF) and forkhead box O (FOXO) transcription factor were also evaluated. Biochemical and histological examinations were done on muscle tissues. Results: DEXA caused a significant elevation (P<0.05) in NF-κβ level and cathepsin activity in muscle tissue. Also, it significantly upregulated the muscle atrophy markers. Meanwhile, PI3K and HO-1 activities were significantly reduced. HB extract and ω−3 administration significantly (P≤0.05) reversed these effects and downregulated the mRNA expression levels of MuRF and FOXO. Also, the histopathological examination confirmed these results. Conclusion: The current study proved the ameliorative effects of HB extract and ω−3 on sarcopenia parameters. Therefore, they might be potential therapeutic agents for myopathy/sarcopenia.