SUMOylated Golgin45 associates with PML-NB to transcriptionally regulate lipid metabolism genes during heat shock stress.

Golgin tethers are known to mediate vesicular transport in the secretory pathway, whereas it is relatively unknown whether they may mediate cellular stress response within the cell. Here, we describe a cellular stress response during heat shock stress via SUMOylation of a Golgin tether, Golgin45. We found that Golgin45 is a SUMOylated Golgin via SUMO1 under steady state condition. Upon heat shock stress, the Golgin enters the nucleus by interacting with Importin-ß2 and gets further modified by SUMO3. Importantly, SUMOylated Golgin45 appears to interact with PML and SUMO-deficient Golgin45 mutant functions as a dominant negative for PML-NB formation during heat shock stress, suppressing transcription of lipid metabolism genes. These results indicate that Golgin45 may play a role in heat stress response by transcriptional regulation of lipid metabolism genes in SUMOylation-dependent fashion.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

SUMO modifies GßL and mediates mTOR signaling.

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

SENP1 inhibits ferroptosis and promotes head and neck squamous cell carcinoma by regulating ACSL4 protein stability via SUMO1.

Head and neck squamous cell carcinoma (HNSCC) is currently one of the most common malignancies with a poor prognosis worldwide. Meanwhile, small ubiquitin­like modifier (SUMO) specific peptidase 1 (SENP1) was associated with ferroptosis. However, the specific functions and underlying mechanisms of action of SENP1 in ferroptosis and tumor progression of HNSCC remain to be established. The findings of the present study implicated a novel ferroptosis pathway in the initiation and progression of HNSCC, providing new functional targets to guide future therapy. In the present study, The Cancer Genome Atlas database was employed to establish a gene model related to ferroptosis and verified SENP1 as a key gene via transcriptome sequencing. Expression of SENP1 in HNSCC tissue and CAL­27 cells was detected based on reverse transcription­quantitative PCR and western blot analysis. Proliferation and migration abilities of cells were determined using Cell Counting Kit­8, wound healing and Transwell experiments. Expression levels of iron, glutathione (GSH) and lipid peroxidation end­product malondialdehyde (MDA) under conditions of silencing of SENP1 with shRNA lentivirus were assayed. Additionally, the relationship between SENP1 and long­chain acyl­coenzyme A synthase 4 (ACSL4) was validated with the aid of immunoblotting and co­immunoprecipitation (co­IP). Finally, the influence of shSENP1 on the expression of key ferroptosis proteins, glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11, was evaluated via western blotting. It was revealed that SENP1 was significantly overexpressed in HNSCC and associated with low patient survival. Silencing of SENP1 led to significant suppression of cell proliferation, migration and invasion, increase in the contents of iron ions and MDA and decline in GSH levels in HNSCC cells, thereby enhancing ferroptosis and inhibiting disease progression. Conversely, overexpression of SENP1 suppressed ferroptosis and promoted progression of HNSCC. Co­IP and western blot analyses revealed a SUMOylation link between SENP1 and ACSL4. SENP1 reduced the stability of ACSL4 protein through deSUMOylation, leading to inhibition of ferroptosis. SENP1 silencing further inhibited the expression of the key iron death protein, GPX4, to regulate ferroptosis. Taken together, SENP1 deficiency promoted ferroptosis and inhibited tumor progression through reduction of SUMOylation of ACSL4 in HNSCC. The collective results of the present study supported the utility of SENP1 as an effective predictive biomarker for targeted treatment of HNSCC.

SUMO-3 promotes the ubiquitin-dependent turnover of TRIM55.

Human muscle-specific RING fingers (MURFs) are members of the tripartite motif (TRIM) family of proteins characterized by their C-terminal subgroup one signature domain. MURFs play a role in sarcomere formation and microtubule dynamics. It was previously established that some TRIMs undergo post-translational modification by small ubiquitin-like modifier (SUMO). In this study, we explored the putative SUMOylation of MURF proteins as well as their interactions with SUMO. MURF proteins (TRIM54, TRIM55, and TRIM63) were not found to be SUMOylated. However, TRIM55 turnover by proteasomal and lysosomal degradation was higher upon overexpression of SUMO-3 but not of SUMO-1. Furthermore, it is predicted that TRIM55 contains two potential SUMO-interacting motifs (SIMs). We found that SIM1- and SIM2-mutated TRIM55 were more stable than the wild-type (WT) protein partly due to decreased degradation. Consistently, SIM-mutated TRIM55 was less polyubiquitinated than the WT protein, despite similar monoubiquitination levels. Using IF microscopy, we observed that SIM motifs influenced TRIM55 subcellular localization. In conclusion, our results suggest that SUMO-3 or SUMO-3-modified proteins modulate the localization, stability, and RING ubiquitin ligase activity of TRIM55.

Monoclonal antibody-based systematic identification of SUMO1-modification sites reveals TFII-I SUMOylation is involved in tumor growth.

SUMOylation plays an essential role in diverse physiological and pathological processes. Identification of wild-type SUMO1-modification sites by mass spectrometry is still challenging. In this study, we produced a monoclonal SUMO1C-K antibody recognizing SUMOylated peptides and proposed an efficient streamline for identification of SUMOylation sites. We identified 471 SUMOylation sites in 325 proteins from five raw data. These identified sites exhibit a high positive rate when evaluated by mutation-verified SUMOylation sites. We identified many SUMOylated proteins involved in mitochondrial metabolism and non-membrane-bounded organelles formation. We proposed a SUMOylation motif, ΨKXD/EP, where proline is required for efficient SUMOylation. We further revealed SUMOylation of TFII-I was stimulated by growth signals and was required for nucleus-localization of p-ERK1/2. Mutation of SUMOylation sites of TFII-I suppressed tumor cell growth in vitro and in vivo. Taken together, we provided a strategy for personalized identification of wild-type SUMO1-modification sites and revealed the physiological significance of TFII-I SUMOylation in this study.

SUMO1-regulated DBC1 promotes p53-dependent stress-induced apoptosis of lens epithelial cells.

Deleted in breast cancer 1 (DBC1) was initially identified from a homozygously deleted region in human chromosome 8p21. It has been well established that DBC1 plays a dual role during cancer development. Depending on the physiological context, it can promote or inhibit tumorigenesis. Whether it plays a role in lens pathogenesis remains elusive. In the present study, we demonstrated that DBC1 is highly expressed in lens epithelial cells from different vertebrates and in retina pigment epithelial cells as well. Moreover, DBC1 is SUMOylated through SUMO1 conjugation at K591 residue in human and mouse lens epithelial cells. The SUMOylated DBC1 is localized in the nucleus and plays an essential role in promoting stress-induced apoptosis. Silence of DBC1 attenuates oxidative stress-induced apoptosis. In contrast, overexpression of DBC1 enhances oxidative stress-induced apoptosis, and this process depends on p53. Mechanistically, DBC1 interacts with p53 to regulate its phosphorylation status at multiple sites and the SUMOylation of DBC1 enhances its interaction with p53. Together, our results identify that DBC1 is an important regulator mediating stress-induced apoptosis in lens, and thus participates in control of lens cataractogenesis.

Splenic SUMO1 controls systemic inflammation in experimental sepsis.

Introduction: The recent discovery of TAK981(Subasumstat), the first-in-class selective inhibitor of SUMOylation, enables new immune treatments. TAK981 is already in clinical trials to potentiate immunotherapy in metastatic tumors and hematologic malignancies. Cancer patients have more than ten times higher risk of infections, but the effects of TAK981 in sepsis are unknown and previous studies on SUMO in infections are conflicting. Methods: We used TAK981 in two sepsis models; polymicrobial peritonitis (CLP) and LPS endotoxemia. Splenectomy was done in both models to study the role of spleen. Western blotting of SUMO-conjugated proteins in spleen lysates was done. Global SUMO1 and SUMO3 knockout mice were used to study the specific SUMO regulation of inflammation in LPS endotoxemia. Splenocytes adoptive transfer was done from SUMO knockouts to wild type mice to study the role of spleen SUMOylation in experimental sepsis. Results and discussion: Here, we report that inhibition of SUMOylation with TAK981 improved survival in mild polymicrobial peritonitis by enhancing innate immune responses and peritoneal bacterial clearance. Thus, we focused on the effects of TAK981 on the immune responses to bacterial endotoxin, showing that TAK981 enhanced early TNFα production but did not affect the resolution of inflammation. Splenectomy decreased serum TNFα levels by nearly 60% and TAK981-induced TNFα responses. In the spleen, endotoxemia induced a distinct temporal and substrate specificity for SUMO1 and SUMO2/3, and both were inhibited by TAK981. Global genetic depletion of SUMO1, but not SUMO3, enhanced TNFα production and metabolic acidosis. The transfer of SUMO1-null, but not wild-type, splenocytes into splenectomized wild-type mice exacerbated TNFα production and metabolic acidosis in endotoxemia. Conclusion: These results suggest that specific regulation of splenic SUMO1 can modulate immune and metabolic responses to bacterial infection.

SUMO1 degrader induces ER stress and ROS accumulation through deSUMOylation of TCF4 and inhibition of its transcription of StarD7 in colon cancer.

Small molecule degraders of small ubiquitin-related modifier 1 (SUMO1) induce SUMO1 degradation in colon cancer cells and inhibits the cancer cell growth; however, it is unclear how SUMO1 degradation leads to the anticancer activity of the degraders. Genome-wide CRISPR-Cas9 knockout screen has identified StAR-related lipid transfer domain containing 7 (StarD7) as a critical gene for the degrader's anticancer activity. Here, we show that both StarD7 mRNA and protein are overexpressed in human colon cancer and its knockout significantly reduces colon cancer cell growth and xenograft progression. The treatment with the SUMO1 degrader lead compound HB007 reduces StarD7 mRNA and protein levels and increases endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production in colon cancer cells and three-dimensional (3D) organoids. The study further provides a novel mechanism of the compound anticancer activity that SUMO1 degrader-induced decrease of StarD7 occur through degradation of SUMO1, deSUMOylation and degradation of T cell-specific transcription 4 (TCF4) and thereby inhibition of its transcription of StarD7 in colon cancer cells, 3D organoids and patient-derived xenografts (PDX).

The SMC5/6 complex subunit MMS21 regulates stem cell proliferation in rice.

KEY MESSAGE: SMC5/6 complex subunit OsMMS21 is involved in cell cycle and hormone signaling and required for stem cell proliferation during shoot and root development in rice. The structural maintenance of chromosome (SMC)5/6 complex is required for nucleolar integrity and DNA metabolism. Moreover, METHYL METHANESULFONATE SENSITIVITY GENE 21 (MMS21), a SUMO E3 ligase that is part of the SMC5/6 complex, is essential for the root stem cell niche and cell cycle transition in Arabidopsis. However, its specific role in rice remains unclear. Here, OsSMC5 and OsSMC6 single heterozygous mutants were generated using CRISPR/Cas9 technology to elucidate the function of SMC5/6 subunits, including OsSMC5, OsSMC6, and OsMMS21, in cell proliferation in rice. ossmc5/ + and ossmc6/ + heterozygous single mutants did not yield homozygous mutants in their progeny, indicating that OsSMC5 and OsSMC6 both play necessary roles during embryo formation. Loss of OsMMS21 caused severe defects in both the shoot and roots in rice. Transcriptome analysis showed a significant decrease in the expression of genes involved in auxin signaling in the roots of osmms21 mutants. Moreover, the expression levels of the cycB2-1 and MCM genes, which are involved the cell cycle, were significantly lower in the shoots of the mutants, indicating that OsMMS21 was involved in both hormone signaling pathways and the cell cycle. Overall, these findings indicate that the SUMO E3 ligase OsMMS21 is required for both shoot and root stem cell niches, improving the understanding of the function of the SMC5/6 complex in rice.

Alternative splicing of the SUMO1/2/3 transcripts affects cellular SUMOylation and produces functionally distinct SUMO protein isoforms.

Substantial increases in the conjugation of the main human SUMO paralogs, SUMO1, SUMO2, and SUMO3, are observed upon exposure to different cellular stressors, and such increases are considered important to facilitate cell survival to stress. Despite their critical cellular role, little is known about how the levels of the SUMO modifiers are regulated in the cell, particularly as it relates to the changes observed upon stress. Here we characterize the contribution of alternative splicing towards regulating the expression of the main human SUMO paralogs under normalcy and three different stress conditions, heat-shock, cold-shock, and Influenza A Virus infection. Our data reveal that the normally spliced transcript variants are the predominant mature mRNAs produced from the SUMO genes and that the transcript coding for SUMO2 is by far the most abundant of all. We also provide evidence that alternatively spliced transcripts coding for protein isoforms of the prototypical SUMO proteins, which we refer to as the SUMO alphas, are also produced, and that their abundance and nuclear export are affected by stress in a stress- and cell-specific manner. Additionally, we provide evidence that the SUMO alphas are actively synthesized in the cell as their coding mRNAs are found associated with translating ribosomes. Finally, we provide evidence that the SUMO alphas are functionally different from their prototypical counterparts, with SUMO1α and SUMO2α being non-conjugatable to protein targets, SUMO3α being conjugatable but targeting a seemingly different subset of protein from those targeted by SUMO3, and all three SUMO alphas displaying different cellular distributions from those of the prototypical SUMOs. Thus, alternative splicing appears to be an important contributor to the regulation of the expression of the SUMO proteins and the cellular functions of the SUMOylation system.

Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control.

Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs. Smt3-K38A/K40A mutant strains also exhibited a defect in the turnover of a soluble PQC model substrate containing the CL1 degron (NES-GFP-Ura3-CL1) localized in the cytoplasm, but not the nucleus. Using human U2OS SUMO1- and SUMO2-KO cell lines, we observed a similar SUMO-dependent pathway for degradation of the mammalian degron-containing PQC reporter protein, GFP-CL1, also only in the cytoplasm but not the nucleus. Moreover, we found that turnover of GFP-CL1 in the cytoplasm was uniquely dependent on SUMO1 but not the SUMO2 paralogue. Additionally, we showed that turnover of GFP-CL1 in the cytoplasm is dependent on the AAA-ATPase, Cdc48/p97. Cellular fractionation studies and analysis of a SUMO1-GFP-CL1 fusion protein revealed that SUMO1 promotes cytoplasmic misfolded protein degradation by maintaining substrate solubility. Collectively, our findings reveal a conserved and previously unrecognized role for SUMO1 in regulating cytoplasmic PQC and provide valuable insights into the roles of sumoylation in PQC-associated diseases.

SUMO1-modified DNA methyltransferase 1 induces DNA hypermethylation of VWC2 in the development of colorectal cancer.

Aberrant DNA methylation of genes is closely linked to many aspects of tumor development. This study focuses on the effect of DNA hypermethylation of von Willebrand factor C domain containing 2 (VWC2) on colorectal cancer (CRC) progression and the underpinning mechanism. According to data in the bioinformatic systems, VWC2 had the highest degree of DNA methylation in colonic adenocarcinoma, and it showed DNA hypermethylation in rectal adenocarcinoma as well. CRC and the para-tumorous tissues were collected from 86 patients. VWC2 was expressed at low levels in CRC samples and inversely correlated with tumor stage and tumor biomarker expression. DNA hypermethylation and reduced expression of VWC2 were also detected in CRC cell lines HCT-116 and HT29. VWC2 overexpression suppressed the malignant growth of cells in vitro and in vivo. Co-immunoprecipitation and western blot assays showed that small ubiquitin-like modifier 1 (SUMO1) mediated SUMOylation of DNA methyltransferase 1 (DNMT1) and strengthened its protein stability, which promoted DNA methylation and suppression of the VWC2 gene. In summary, this study demonstrates that SUMO1-mediated activation of DNMT1 induces DNA methylation and downregulation of VWC2 in CRC to augment cancer development.

Salmonella pathogenicity island 1 knockdown confers protection against myocardial fibrosis and inflammation in uremic cardiomyopathy via down-regulation of S100 Calcium Binding Protein A8/A9 transcription.

BACKGROUND/AIM: Uremic cardiomyopathy (UCM) is a characteristic cardiac pathology that is commonly found in patients with chronic kidney disease. This study dissected the mechanism of SPI1 in myocardial fibrosis and inflammation induced by UCM through S100A8/A9. METHODS: An UCM rat model was established, followed by qRT-PCR and western blot analyses of SPI1 and S100A8/A9 expression in myocardial tissues. After alterations of SPI1 and S100A8/A9 expression in UCM rats, the blood specimens were harvested from the cardiac apex of rats. The levels of creatine phosphokinase-MB (CK-MB), blood creatinine, blood urea nitrogen (BUN), and inflammatory cytokines (interleukin [IL]-6, IL-1ß, and tumor necrosis factor-α [TNF-α]) were examined in the collected blood. Collagen fibrosis was assessed by Masson staining. The expression of fibrosis markers [transforming growth factor (TGF)-ß1, α-smooth muscle actin (SMA), Collagen 4a1, and Fibronectin], IL-6, IL-1ß, and TNF-α was measured in myocardial tissues. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were conducted to test the binding relationship between SPI1 and S100A8/A9. RESULTS: S100A8/A9 and SPI1 were highly expressed in the myocardial tissues of UCM rats. Mechanistically, SPI1 bound to the promoter of S100A8/A9 to facilitate S100A8/A9 transcription. S100A8/A9 or SPI1 knockdown reduced myocardial fibrosis and inflammation and the levels of CK-MB, blood creatinine, and BUN, as well as the expression of TGF-ß1, α-SMA, Collagen 4a1, Fibronectin, IL-6, TNF-α, and IL-1ß in UCM rats. CONCLUSION: SPI1 knockdown diminished S100A8/A9 transcription, thus suppressing myocardial fibrosis and inflammation caused by UCM.

Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1.

SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO-SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.

Insights into the Allosteric Effect of SENP1 Q597A Mutation on the Hydrolytic Reaction of SUMO1 via an Integrated Computational Study.

Small ubiquitin-related modifier (SUMO)-specific protease 1 (SENP1) is a cysteine protease that catalyzes the cleavage of the C-terminus of SUMO1 for the processing of SUMO precursors and deSUMOylation of target proteins. SENP1 is considered to be a promising target for the treatment of hepatocellular carcinoma (HCC) and prostate cancer. SENP1 Gln597 is located at the unstructured loop connecting the helices α4 to α5. The Q597A mutation of SENP1 allosterically disrupts the hydrolytic reaction of SUMO1 through an unknown mechanism. Here, extensive multiple replicates of microsecond molecular dynamics (MD) simulations, coupled with principal component analysis, dynamic cross-correlation analysis, community network analysis, and binding free energy calculations, were performed to elucidate the detailed mechanism. Our MD simulations showed that the Q597A mutation induced marked dynamic conformational changes in SENP1, especially in the unstructured loop connecting the helices α4 to α5 which the mutation site occupies. Moreover, the Q597A mutation caused conformational changes to catalytic Cys603 and His533 at the active site, which might impair the catalytic activity of SENP1 in processing SUMO1. Moreover, binding free energy calculations revealed that the Q597A mutation had a minor effect on the binding affinity of SUMO1 to SENP1. Together, these results may broaden our understanding of the allosteric modulation of the SENP1-SUMO1 complex.

Associations of genetic variation in E3 SUMO-protein ligase CBX4 with noise-induced hearing loss.

Noise-induced hearing loss (NIHL) is a multifactorial disease caused by environmental, genetic and epigenetic variables. SUMOylation is a post-translational modification that regulates biological processes. The objective of this study was to determine the link between genetic variation in the chromobox 4 (CBX4) and the risk of NIHL. This study applied a case-control design with 588 cases and 582 controls, and the sample was predominantly male (93.76%). The T allele of CBX4 rs1285250 was found to be significantly linked with NIHL (P = 0.002) and showed strong associations in both the codominant and recessive models (TT versus CC, P = 0.005; TT/TC versus CC, P = 0.009). By constructing a mouse model of hearing loss because of noise exposure, changes in hearing thresholds were observed in noise-exposed mice, along with a decrease in the number of cochlear hair cells. Furthermore, noise promotes cochlear hair cell apoptosis by inducing SP1/CBX4 pathway activation. Further functional studies demonstrated that SP1 has an influence on the promoter activity of the CBX4 rs1285250 intron, with the promoter activity of the T allele being higher than that of the C allele. Knockdown of transcription factor SP1 reduced the expression of CBX4 expression and simultaneously reduced apoptosis in HEI-OC1 cells. Together, our findings have shown that CBX4 genetic polymorphism rs1285250 T-allele was associated with increased risk of NIHL and might be used as biomarkers for male workers exposed to noise. Furthermore, we speculate that the CBX4 of rs1285250 T-allele leads to a stronger potential enhancer activity from a predicted gain of stronger SP1 binding.

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease.

The CAG expansion of huntingtin (mHTT) associated with Huntington disease (HD) is a ubiquitously expressed gene, yet it prominently damages the striatum and cortex, followed by widespread peripheral defects as the disease progresses. However, the underlying mechanisms of neuronal vulnerability are unclear. Previous studies have shown that SUMO1 (small ubiquitin-like modifier-1) modification of mHtt promotes cellular toxicity, but the in vivo role and functions of SUMO1 in HD pathogenesis are unclear. Here, we report that SUMO1 deletion in Q175DN HD-het knockin mice (HD mice) prevented age-dependent HD-like motor and neurological impairments and suppressed the striatal atrophy and inflammatory response. SUMO1 deletion caused a drastic reduction in soluble mHtt levels and nuclear and extracellular mHtt inclusions while increasing cytoplasmic mHtt inclusions in the striatum of HD mice. SUMO1 deletion promoted autophagic activity, characterized by augmented interactions between mHtt inclusions and a lysosomal marker (LAMP1), increased LC3B- and LAMP1 interaction, and decreased interaction of sequestosome-1 (p62) and LAMP1 in DARPP-32-positive medium spiny neurons in HD mice. Depletion of SUMO1 in an HD cell model also diminished the mHtt levels and enhanced autophagy flux. In addition, the SUMOylation inhibitor ginkgolic acid strongly enhanced autophagy and diminished mHTT levels in human HD fibroblasts. These results indicate that SUMO is a critical therapeutic target in HD and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.

The cross-talk between PARylation and SUMOylation in C/EBPß at K134 site participates in pathological cardiac hypertrophy.

Poly(ADP-ribosyl)ation (PARylation) and SUMO modification (SUMOylation) are novel post-translational modifications (PTMs) mainly induced by PARP1 and SUMO1. Growing evidence has revealed that C/EBPß plays multiple roles in biological processes and participates in cardiovascular diseases. However, the cross-talk between C/EBPß PARylation and SUMOylation during cardiovascular diseases is unknown. This study aims to investigate the effects of C/EBPß PTMs on cardiac hypertrophy and its underlying mechanism. Abdominal aortic constriction (AAC) and phenylephrine (PE) were conducted to induce cardiac hypertrophy. Intramyocardial delivery of recombinant adenovirus (Ad-PARP1) was taken to induce PARP1 overexpression. In this study, we found C/EBPß participates in PARP1-induced cardiac hypertrophy. C/EBPß K134 residue could be both PARylated and SUMOylated individually by PARP1 and SUMO1. Moreover, the accumulation of PARylation on C/EBPß at K134 site exhibits downregulation of C/EBPß SUMOylation at the same site. Importantly, C/EBPß K134 site SUMOylation could decrease C/EBPß protein stability and participates in PARP1-induced cardiac hypertrophy. Taken together, these findings highlight the importance of the cross-talk between C/EBPß PTMs at K134 site in determining its protein level and function, suggesting that multi-target pharmacological strategies inhibiting PARP1 and activating C/EBPß SUMOylation would be potential for treating pathological cardiac hypertrophy.

Soluble expression of bioactive recombinant porcine-human chimeric uricase mutant employing MBP-SUMO fusion system.

Urate oxidase is a promising biological medicine for hyperuricemia treatment, but immunogenicity obstructs the development of its clinical application. The recombinant porcine-human chimeric uricase mutant named dHU-wPU is a humanized chimeric uricase based on wild porcine uricase (wPU), which can effectively reduce the limitation of potential immunogenicity with a high homology (92.76%) to deduced human uricase (dHU). Unfortunately, the insoluble expression form of dHU-wPU in E. coli increases the difficulty of production. In this study, we described a more convenient method to efficiently obtain recombinant dHU-wPU protein from E. coli. Combination small ubiquitin-related modifier protein (SUMO) and maltose-binding protein (MBP) was employed to achieve the soluble expression of dHU-wPU. MBP-SUMO-dHU-wPU fusion protein was not only overexpressed in a soluble form, but also showed high purification and cleavage efficiency. Subsequently, we optimized the culture conditions of shake flasks and expanded the production of MBP-SUMO-dHU-wPU fusion protein in a 5 L bioreactor. Finally, about 15 mg of recombinant dHU-wPU was obtained from 1 L M9 fermentation culture by using two-step affinity chromatography, with a SDS-PAGE purity over 90%. In vitro activity analysis showed that dHU-wPU had better ability to catalyze uric acid than wPU.