Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells.

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

Structure and dynamics of a pentameric KCTD5/CUL3/Gßγ E3 ubiquitin ligase complex.

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gßγ and reduces Gßγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gß1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gßγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gßγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gß and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gßγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

Predictive value of the hemispheric magnetic resonance angiography score on the development of indirect pial synangiosis after combined revascularization surgery for adult moyamoya disease.

PURPOSE: It is difficult to precisely predict indirect bypass development in the context of combined bypass procedures in moyamoya disease (MMD). We aimed to investigate the predictive value of magnetic resonance angiography (MRA) signal intensity in the peripheral portion of the major cerebral arteries for indirect bypass development in adult patients with MMD. METHODS: We studied 93 hemispheres from 62 adult patients who underwent combined direct and indirect revascularization between 2005 and 2019 and genetic analysis for RNF213 p.R4810K. The signal intensity of the peripheral portion of the major intracranial arteries during preoperative MRA was graded as a hemispheric MRA score (0-3 in the middle cerebral artery and 0-2 in the anterior cerebral and posterior cerebral arteries, with a high score representing low visibility) according to each vessel's visibility. Postoperative bypass development was qualitatively evaluated using MRA, and we evaluated the correlation between preoperative factors, including the hemispheric MRA score and bypass development, using univariate and multivariate analyses. RESULTS: A good indirect bypass was observed in 70% of the hemispheres. Hemispheric MRA scores were significantly higher in hemispheres with good indirect bypass development than in those with poor indirect bypass development (median: 3 vs. 1; p < 0.0001). Multiple logistic regression analysis revealed hemispheric MRA score as an independent predictor of good indirect bypass development (odds ratio, 2.1; 95% confidence interval, 1.3-3.6; p < 0.01). The low hemispheric MRA score (< 2) and wild-type RNF213 predicted poor indirect bypass development with a specificity of 0.92. CONCLUSION: Hemispheric MRA score was a predictive factor for indirect bypass development in adult patients who underwent a combined bypass procedure for MMD. Predicting poor indirect bypass development may lead to future tailored bypass surgeries for MMD.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

Pellino1 orchestrates gut-kidney axis to perpetuate septic acute kidney injury through activation of STING pathway and NLRP3 inflammasome.

AIMS: Intestinal barrier dysfunction is the initial and propagable factor of sepsis in which acute kidney injury (AKI) has been considered as a common life-threatening complication. Our recent study identifies the regulatory role of Pellino1 in tubular death under inflammatory conditions in vitro. The objective of our current study is to explore the impact of Pellino1 on gut-kidney axis during septic AKI and uncover the molecular mechanism (s) underlying this process. MATERIALS AND METHODS: Immunohistochemistry (IHC) was conducted to evaluate Pellino1 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) levels in renal biopsies from critically ill patients with a clinical diagnosis of sepsis. Functional and mechanistic studies were characterized in septic models of the Peli-knockout (Peli1-/-) mice by histopathological staining, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence, biochemical detection, CRISPR/Cas9-mediated gene editing and intestinal organoid. KEY FINDINGS: Pellino1, together with NLRP3, are highly expressed in renal biopsies from critically ill patients diagnosed with sepsis and kidney tissues of septic mice. The Peli1-/- mice with sepsis become less prone to develop AKI and have markedly compromised NLRP3 activation in kidney. Loss of Peli1 endows septic mice refractory to intestinal inflammation, barrier permeability and enterocyte apoptosis that requires stimulator of interferons genes (STING) pathway. Administration of STING agonist DMXAA deteriorates AKI and mortality of septic Peli1-/- mice in the presence of kidney-specific NLRP3 reconstitution. SIGNIFICANCE: Our studies suggest that Pellino1 has a principal role in orchestrating gut homeostasis towards renal pathophysiology, thus providing a potential therapeutic target for septic AKI.

Complex genetic interaction between glucose sensor HXK1 and E3 SUMO ligase SIZ1 in regulating plant morphogenesis.

Sugar signaling forms the basis of metabolic activities crucial for an organism to perform essential life activities. In plants, sugars like glucose, mediate a wide range of physiological responses ranging from seed germination to cell senescence. This has led to the elucidation of cell signaling pathways involving glucose and its counterparts and the mechanism of how these sugars take control over major hormonal pathways such as auxin, ethylene, abscisic acid and cytokinin in Arabidopsis. Plants use HXK1(Hexokinase) as a glucose sensor to modulate changes in photosynthetic gene expression in response to high glucose levels. Other proteins such as SIZ1, a major SUMO E3 ligase have recently been implicated in controlling sugar responses via transcriptional and translational regulation of a wide array of sugar metabolic genes. Here, we show that these two genes work antagonistically and are epistatic in controlling responsiveness toward high glucose conditions.

RNF20 Reduces Cell Proliferation and Warburg Effect by Promoting NLRP3 Ubiquitination in Liver Cancer.

The present study explored that the effects and its possible mechanisms of ring finger protein 20 (RNF20) in Postoperative survival rate of liver cancer in clinical. All the serum samples were collected from our hospital. Quantitative polymerase chain reaction (PCR) and microarray analysis, and RNA pull down assay were used in this study. We found that the serum RNF20 mRNA expression level in patients with liver cancer were down-regulated. Postoperative survival rate of RNF20 high expression was higher than that of RNF20 low expression. Then, over-expression of RNF20 diminished liver cancer cell proliferation and metastasis. RNF20 reduced Warburg effect of liver cancer. RNF20 expression regulated NOD-like receptor protein 3 (NLRP3) expression and increased NLRP3 Ubiquitination. NLRP3 participated in the effects of RNF20 on cell proliferation, and not affected on Warburg effect of liver cancer. Our study demonstrated that the serum RNF20 expression level was down-regulated in liver cancer, and promoted postoperative survival rate. RNF20 can reduce cancer progression of liver cancer by NLRP3 signal pathway, suggesting that it may prove to be a potential therapeutic target for postoperative survival rate of liver cancer.

Sulfatase-Induced In Situ Formulation of Antineoplastic Supra-PROTACs.

Proteolysis targeting chimera (PROTAC) technology is an innovative strategy for cancer therapy, which, however, suffers from poor targeting delivery and limited capability for protein of interest (POI) degradation. Here, we report a strategy for the in situ formulation of antineoplastic Supra-PROTACs via intracellular sulfatase-responsive assembly of peptides. Coassembling a sulfated peptide with two ligands binding to ubiquitin VHL and Bcl-xL leads to the formation of a pro-Supra-PROTAC, in which the ratio of the two ligands is rationally optimized based on their protein binding affinity. The resulting pro-Supra-PROTAC precisely undergoes enzyme-responsive assembly into nanofibrous Supra-PROTACs in cancer cells overexpressing sulfatase. Mechanistic studies reveal that the pro-Supra-PROTACs selectively cause apparent cytotoxicity to cancer cells through the degradation of Bcl-xL and the activation of caspase-dependent apoptosis, during which the rationally optimized ligand ratio improves the bioactivity for POI degradation and cell death. In vivo studies show that in situ formulation enhanced the tumor accumulation and retention of the pro-Supra-PROTACs, as well as the capability for inhibiting tumor growth with excellent biosafety when coadministrating with chemodrugs. Our findings provide a new approach for enzyme-regulated assembly of peptides in living cells and the development of PROTACs with high targeting delivering and POI degradation efficiency.

Monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation.

Posttranslational modification of histone proteins is critical for memory formation. Recently, we showed that monoubiquitination of histone H2B at lysine 120 (H2Bub) is critical for memory formation in the hippocampus. However, the transcriptome controlled by H2Bub remains unknown. Here, we found that fear conditioning in male rats increased or decreased the expression of 86 genes in the hippocampus but, surprisingly, siRNA-mediated knockdown of the H2Bub ligase, Rnf20, abolished changes in all but one of these genes. These findings suggest that monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation.

Identification and structural characterization of small molecule inhibitors of PINK1.

Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

Suppression of AGRN enhances CD8+ T cell recruitment and inhibits breast cancer progression.

Breast cancer (BC) stands as a prominent contributor to global cancer-related mortality, with an increasing incidence annually. This study aims to investigate AGRN gene expression in BC, as well as explore its influence on the tumor immune microenvironment. AGRN displayed a pronounced upregulation in BC tissues relative to paracancerous tissues. Single-cell RNA analysis highlighted AGRN-specific elevation within cancer cell clusters and also showed expression expressed in stromal as well as immune cell clusters. AGRN upregulation was positively correlated with clinicopathological stage and negatively correlated with BC prognosis. As revealed by the in vitro experiment, AGRN knockdown effectively hinders BC cells in terms of proliferation, invasion as well as migration. AGRN protein, which may interact with EXT1, LRP4, RAPSN, etc., was primarily distributed in the cell cytoplasm. Notably, immune factors might interact with AGRN in BC, evidenced by its discernible associations with immunofactors like IL10, CD274, and PVRL2. Mass spectrometry and immunohistochemistry revealed that the reduction of AGRN led to an increase in CD8+ T cells with triple-negative breast cancer (TNBC). Mechanistically, the connection between TRIM7 and PD-L1 is improved by AGRN, acting as a scaffold, thereby facilitating the accelerated degradation of PD-L1 by TRIM7. Downregulation of AGRN inhibits BC progression and increases CD8+ T cell recruitment. Targeting AGRN may contribute to BC treatment. The biomarker AGRN, serving as a therapeutic target for BC, emerges as a prospective avenue for enhancing both diagnosis and prognosis in BC cases.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Pepper U-Box E3 ubiquitin ligase 24, CaPUB24, negatively regulates drought stress response.

Under stress conditions, plants modulate their internal states and initiate various defence mechanisms to survive. The ubiquitin-proteasome system is one of the critical modules in these mechanisms, and Plant U-Box proteins play an important role in this process as E3 ubiquitin ligases. Here, we isolated the Plant U-box 24 gene CaPUB24 (Capsicum annuum Plant U-Box 24) from pepper and characterized its functions in response to drought stress. We found that, compared to the other CaPUBs in the same group, the expression of CaPUB24 was significantly induced by drought stress. We also found that CaPUB24 was localized to the nucleus and cytoplasm and had E3 ubiquitin ligase activity. To investigate the biological role of CaPUB24 in response to drought stress further, we generated CaPUB24-silenced pepper plants and CaPUB24-overexpressing Arabidopsis transgenic plants. CaPUB24-silenced pepper plants exhibited enhanced drought tolerance compared to the control plants due to reduced transpirational water loss and increased abscisic acid (ABA) sensitivity. In contrast, CaPUB24-overexpressing Arabidopsis transgenic plants exhibited reduced drought tolerance and ABA-insensitive phenotypes. Our findings suggest that CaPUB24 negatively modulates drought stress response in an ABA-dependent manner.

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Central precocious puberty (CPP) is a developmental disorder caused by early activation of the hypothalamic-pituitary-gonadal axis. The incidence of CPP is rapidly increasing, but the underlying mechanisms are not fully understood. Previous studies have shown that gain-of-function mutations in the KISS1R and KISS1 genes and loss-of-function mutations in the MKRN3, LIN28, and DLK1 genes may lead to early initiation of pubertal development. Recent research has also revealed the significant role of epigenetic factors such as DNA methylation and microRNAs in the regulation of gonadotropin-releasing hormone neurons, as well as the modulating effect of gene networks involving multiple variant genes on pubertal initiation. This review summarizes the genetic etiology and pathogenic mechanisms underlying CPP.

STUB1 is acetylated by KAT5 and alleviates myocardial ischemia-reperfusion injury through LATS2-YAP-ß-catenin axis.

Myocardial ischemia-reperfusion injury (MIRI) is involved in the pathogenesis of multiple cardiovascular diseases. This study elucidated the biological function of lysine acetyltransferase 5 (KAT5) in cardiomyocyte pyroptosis during MIRI. Oxygen-glucose deprivation/reoxygenation and left anterior descending coronary artery ligation were used to establish MIRI models. Here we show, KAT5 and STIP1 homology and U-box-containing protein 1 (STUB1) were downregulated, while large tumor suppressor kinase 2 (LATS2) was upregulated in MIRI models. KAT5/STUB1 overexpression or LATS2 silencing repressed cardiomyocyte pyroptosis. Mechanistically, KAT5 promoted STUB1 transcription via acetylation modulation, and subsequently caused ubiquitination and degradation of LATS2, which activated YAP/ß-catenin pathway. Notably, the inhibitory effect of STUB1 overexpression on cardiomyocyte pyroptosis was abolished by LATS2 overexpression or KAT5 depletion. Our findings suggest that KAT5 overexpression inhibits NLRP3-mediated cardiomyocyte pyroptosis to relieve MIRI through modulation of STUB1/LATS2/YAP/ß-catenin axis, providing a potential therapeutic target for MIRI.

The E3 ubiquitin ligase ITCH negatively regulates intercellular communication via gap junctions by targeting connexin43 for lysosomal degradation.

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development.

A case of polyglucosan body myopathy caused by an RBCK1 gene variant and literature review.

OBJECTIVE: To analyze the clinical and genetic characteristics of a patient with Polyglucosan body myopathy 1 (PGBM1) caused by a novel compound heterozygous variant in the RBCK1 gene. METHODS: The clinical data of the patient were collected, next-generation sequencing technology was used to determine the exome sequence of the patient, and the suspected pathogenic locus was verified by Sanger sequencing. RESULTS: Through whole-exome sequencing, we found that there were c.919G>T; p. (Glu307*) and c.723_730dup; p. (Glu244fs) variants of the RBCK1 gene in the patient, inherited from his parents, constituting a compound heterozygous variation. According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), the two variants were rated as pathogenic, but there were no comparable cases. Previous literature reported 24 patients with RBCK1 gene variants, involving a total of 20 myocardial and 18 skeletal muscle cases. CONCLUSIONS: The patient was twice diagnosed with cardiac insufficiency, neglecting the usual manifestations of muscle weakness, resulting in misdiagnosis. Later, novel variants in the RBCK1 gene were discovered through whole-exome sequencing, and symptomatic treatment was given after diagnosis. The importance of whole-exome sequencing technology in disease diagnosis and genetic counseling was emphasized.

The clinical value of optical genome mapping in the rapid characterization of RB1 duplication and 15q23q24.2 triplication, for more appropriate prenatal genetic counselling.

BACKGROUND: Despite recent advances in prenatal genetic diagnosis, medical geneticists still face considerable difficulty in interpreting the clinical outcome of copy-number-variant duplications and defining the mechanisms underlying the formation of certain chromosomal rearrangements. Optical genome mapping (OGM) is an emerging cytogenomic tool with proved ability to identify the full spectrum of cytogenetic aberrations. METHODS: Here, we report on the use of OGM in a prenatal diagnosis setting. Detailed breakpoint mapping was used to determine the relative orientations of triplicated and duplicated segments in two unrelated foetuses harbouring chromosomal aberrations: a de novo 15q23q24.2 triplication and a paternally inherited 13q14.2 duplication that overlapped partially with the RB1 gene. RESULTS: OGM enabled us to suggest a plausible mechanism for the triplication and confirmed that the RB1 duplication was direct oriented and in tandem. This enabled us to predict the pathogenic consequences, refine the prognosis and adapt the follow-up and familial screening appropriately. CONCLUSION: Along with an increase in diagnostic rates, OGM can rapidly highlight genotype-phenotype correlations, improve genetic counselling and significantly influence prenatal management.

Rhes, a striatal enriched protein, regulates post-translational small-ubiquitin-like-modifier (SUMO) modification of nuclear proteins and alters gene expression.

Rhes (Ras homolog enriched in the striatum), a multifunctional protein that regulates striatal functions associated with motor behaviors and neurological diseases, can shuttle from cell to cell via the formation of tunneling-like nanotubes (TNTs). However, the mechanisms by which Rhes mediates diverse functions remain unclear. Rhes is a small GTPase family member which contains a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes SUMO post-translational modification of proteins (SUMOylation) by promoting "cross-SUMOylation" of the SUMO enzyme SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). Nevertheless, the identity of the SUMO substrates of Rhes remains largely unknown. Here, by combining high throughput interactome and SUMO proteomics, we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene expression. Rhes increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, while decreasing SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 6 different lysine residues (K32, K110, K114, K120, K124, and K245). Furthermore, Rhes regulated the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide evidence for a previously undescribed role for Rhes in regulating the SUMOylation of nuclear targets and in orchestrating striatal gene expression via SUMOylation.

Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.