SUMOylation Research Articles

DNA Damage Induction Alters the Expression of Ubiquitin and SUMO Regulators in Preimplantation Stage Pig Embryos.

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo.

Small ubiquitin-related modifier-fused bacteriophage tail fiber protein with favorable aqueous solubility for lateral flow assay of Pseudomonas aeruginosa

Rapid and sensitive assay of pathogenic bacteria is critical for minimizing the risk of infectious diseases. Inspired by the interaction between bacteriophages and host bacteria, we obtained a gene sequence of tail fiber protein (TFP) from Pseudomonas aeruginosa (P. aeruginosa) bacteriophage. Then the gene sequence was used to express a recombinant TFP, which can act as a potential capture molecule for P. aeruginosa. Small ubiquitin-related modifier (SUMO) tag was fused onto the TFP fragment to overcome its unfavorable aqueous solubility. The obtained SUMO tag-fused TFP (STFP) can be uniformly distributed onto a nitrocellulose membrane to form a test line due to the improved aqueous solubility, which facilities fabrication of a lateral flow assay strip. Thus we developed a lateral flow assay method by using STFP as a capture molecule and AuCo nanoparticles-labeled aptamer as a signal tracer for point-of-care testing of P. aeruginosa. By using the test strip, P. aeruginosa can be semi quantified with color band and quantified with chemiluminescent (CL) signal catalyzed by AuCo nanoparticles. The concentration range for quantification is 3.3 × 102 CFU/mL to 3.3 × 107 CFU/mL. The test strip was applied to assay P. aeruginosa in different sample matrixes including cerebrospinal fluid, physiological salt solution, drinking water and pear juice. The results demonstrate the application potential of the STFP-based lateral flow assay for medical diagnosis, food and drug safety monitoring.

Isoform-specific mutation in Dystonin-b gene causes late-onset protein aggregate myopathy and cardiomyopathy.

Dystonin (DST), which encodes cytoskeletal linker proteins, expresses three tissue-selective isoforms: neural DST-a, muscular DST-b, and epithelial DST-e. DST mutations cause different disorders, including hereditary sensory and autonomic neuropathy 6 (HSAN-VI) and epidermolysis bullosa simplex; however, etiology of the muscle phenotype in DST-related diseases has been unclear. Because DST-b contains all of the DST-a-encoding exons, known HSAN-VI mutations could affect both DST-a and DST-b isoforms. To investigate the specific function of DST-b in striated muscles, we generated a Dst-b-specific mutant mouse model harboring a nonsense mutation. Dst-b mutant mice exhibited late-onset protein aggregate myopathy and cardiomyopathy without neuropathy. We observed desmin aggregation, focal myofibrillar dissolution, and mitochondrial accumulation in striated muscles, which are common characteristics of myofibrillar myopathy. We also found nuclear inclusions containing p62, ubiquitin, and SUMO proteins with nuclear envelope invaginations as a unique pathological hallmark in Dst-b mutation-induced cardiomyopathy. RNA-sequencing analysis revealed changes in expression of genes responsible for cardiovascular functions. In silico analysis identified DST-b alleles with nonsense mutations in populations worldwide, suggesting that some unidentified hereditary myopathy and cardiomyopathy are caused by DST-b mutations. Here, we demonstrate that the Dst-b isoform is essential for long-term maintenance of striated muscles.

Protective Effect of Curcuma Extract in an Ex Vivo Model of Retinal Degeneration via Antioxidant Activity and Targeting the SUMOylation.

Retinal degeneration is the major and principal cause behind many incurable blindness diseases. Several studies indicated the neuroprotective effect of Curcuma longa in eye pathologies, specifically retinopathy. However, the molecular mechanism behind its effect has not been completely elucidated. Using an ex vivo model of retinal degeneration obtained from an ex vivo optic nerve cut (ONC), we demonstrated that Curcuma extract (Cur) exerted a neuroprotective effect. Importantly, Cur was able to modulate apoptosis and MAPK signaling pathway activation and prevent retinal ganglion cell (RGC) loss. Other well-known neuroprotective pharmacological tools, including memantine (Mem), citicoline (Cit), and ginkgolic acid (GA), were used to compare the potential mechanisms of Cur. The antioxidant activity of retinas treated with Cur following optic nerve cut was significantly higher than control, but Cur failed to change the retina glutamate content. Considering the antioxidant effect of Cur and taking advantage of our recent findings on the crosstalk between oxidative stress and post-translational protein modifiers, in particular, small ubiquitin-related modifier (SUMO), we were interested in exploring the effect of Cur on SUMOylation. We found that Cur significantly prevented the increase of protein SUMOylation, confirming our previous in vitro data indicating the cytoprotective effect of curcumin through modulating the oxidative stress and SUMO-JNK axis. Altogether, these results suggest that Curcuma protects the retina from degeneration via antioxidant activity and targets SUMOylation. Therefore, it might be considered for the combination therapy with other neuroprotective agents with different mechanisms in preclinical studies on retinal degeneration.

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.

Targeting DNA-Protein Crosslinks via Post-Translational Modifications

Covalent binding of proteins to DNA forms DNA-protein crosslinks (DPCs), which represent cytotoxic DNA lesions that interfere with essential processes such as DNA replication and transcription. Cells possess different enzymatic activities to counteract DPCs. These include enzymes that degrade the adducted proteins, resolve the crosslinks, or incise the DNA to remove the crosslinked proteins. An important question is how DPCs are sensed and targeted for removal via the most suited pathway. Recent advances have shown the inherent role of DNA replication in triggering DPC removal by proteolysis. However, DPCs are also efficiently sensed and removed in the absence of DNA replication. In either scenario, post-translational modifications (PTMs) on DPCs play essential and versatile roles in orchestrating the repair routes. In this review, we summarize the current knowledge of the mechanisms that trigger DPC removal via PTMs, focusing on ubiquitylation, small ubiquitin-related modifier (SUMO) conjugation (SUMOylation), and poly (ADP-ribosyl)ation (PARylation). We also briefly discuss the current knowledge gaps and emerging hypotheses in the field.

GDF11 inhibits abnormal adipogenesis of condylar chondrocytes in temporomandibular joint osteoarthritis.

AimsAbnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear.MethodsSix-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis.ResultsAbnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice.ConclusionAbnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ.Cite this article: Bone Joint Res 2022;11(7):453–464.

Utilization of SUMO Tag and Freeze-thawing Method for a High-level Expression and Solubilization of Recombinant Human Angiotensinconverting Enzyme 2 (rhACE2) Protein in E. coli

SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor for entering the host cells. Production of the ACE2 molecule is important because of its potency to use as a blocker and therapeutic agent against SARS-CoV-2 for the prophylaxis and treatment of COVID-19. The recombinant human ACE2 (rhACE2) is prone to form an inclusion body when expressed in the bacterial cells. We used the SUMO tag fused to the rhACE2 molecule to increase the expression level and solubility of the fusion protein. Afterward, the freeze-thawing method plus 2 M urea solubilized aggregated proteins. Subsequently, the affinity of solubilized rhACE2 to the receptor binding domain (RBD) of the SARS-CoV-2 spike was assayed by ELISA and SPR methods. SUMO protein succeeded in increasing the expression level but not solubilization of the fusion protein. The freeze-thawing method could solubilize and recover the aggregated fusion proteins significantly. Also, ELISA and SPR assays confirmed the interaction between solubilized rhACE2 and RBD with high affinity. The SUMO tag and freeze- Conclusion: The SUMO tag and freeze-thawing method would be utilized for high-level expression and solubilization of recombinant rhACE2 protein.

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.

Promyelocytic leukemia nuclear body-like structures can assemble in mouse oocytes.

ABSTRACTPromyelocytic leukemia (PML) nuclear bodies (PML-NBs), a class of membrane-less cellular organelles, participate in various biological activities. PML-NBs are known as the core-shell-type nuclear body, harboring ‘client’ proteins in their core. Although multiple membrane-less organelles work in the oocyte nucleus, PML-NBs have been predicted to be absent from oocytes. Here, we show that some well-known PML clients (but not endogenous PML) co-localized with small ubiquitin-related modifier (SUMO) protein in the nucleolus and peri-centromeric heterochromatin of maturing oocytes. In oocytes devoid of PML-NBs, endogenous PML protein localized in the vicinity of chromatin. During and after meiotic resumption, PML co-localized with SUMO gathering around chromosomes. To examine the benefit of the PML-NB-free intranuclear milieu in oocytes, we deliberately assembled PML-NBs by microinjecting human PML-encoding plasmids into oocytes. Under conditions of limited SUMO availability, assembled PML-NBs tended to cluster. Upon proteotoxic stress, SUMO delocalized from peri-centromeric heterochromatin and co-localized with SC35 (a marker of nuclear speckles)-positive large compartments, which was disturbed by pre-assembled PML-NBs. These observations suggest that the PML-NB-free intranuclear environment helps reserve SUMO for emergent responses by redirecting the flux of SUMO otherwise needed to maintain PML-NB dynamics.

Tissue-specific inhibition of protein sumoylation uncovers diverse SUMO functions during C. elegans vulval development.

The sumoylation (SUMO) pathway is involved in a variety of processes during C. elegans development, such as gonadal and vulval fate specification, cell cycle progression and maintenance of chromosome structure. The ubiquitous expression and pleiotropic effects have made it difficult to dissect the tissue-specific functions of the SUMO pathway and identify its target proteins. To overcome these challenges, we have established tools to block protein sumoylation and degrade sumoylated target proteins in a tissue-specific and temporally controlled manner. We employed the auxin-inducible protein degradation system (AID) to down-regulate the SUMO E3 ligase GEI-17 or the SUMO ortholog SMO-1, either in the vulval precursor cells (VPCs) or in the gonadal anchor cell (AC). Our results indicate that the SUMO pathway acts in multiple tissues to control different aspects of vulval development, such as AC positioning, basement membrane (BM) breaching, VPC fate specification and morphogenesis. Inhibition of protein sumoylation in the VPCs resulted in abnormal toroid formation and ectopic cell fusions during vulval morphogenesis. In particular, sumoylation of the ETS transcription factor LIN-1 at K169 is necessary for the proper contraction of the ventral vulA toroids. Thus, the SUMO pathway plays several distinct roles throughout vulval development.

Electroporation of SUMO-His-Cre protein triggers a specific recombinase-mediated cassette exchange in HEK 293T cells

Cre recombinase is a widely-used genetic manipulation of genomic DNA. However, the conventional transfection of the DNA vectors expressing the Cre recombinase or viral transduction method yields low transfection efficiencies or insertion mutagenesis. The present paper evaluated whether the direct protein delivery of Cre recombinase through electroporation can induce the Cre-mediated recombination in the HEK 293T cells. Here, the small ubiquitin-related modifier (SUMO) -tagged His-Cre fusion protein was expressed in a soluble pattern in the Eschrichria coli (E.coli) cells, purified using affinity chromatography, and finally electroporated into the HEK 293T cells. These cells were previously transfected with three different Cre reporter vectors. The electroporation of the HEK 293T cells revealed either the activation of EGFP expression, or a decrease in RFP expression, and a concomitant increase in EGFP expression, indicating a desired recombinase-mediated cassette exchange (RMCE) event (conversion of RFP to EGFP), and a biological activity of the purified SUMO-His-Cre protein. The fusion protein is expected to serve in the Easi-CRISPR-LoxP-mediated genome editing to generate transgenic animal models.

Activation of NLR-Mediated Autoimmunity in Arabidopsis Early in Short Days 4 Mutant

From a reverse genetic screen using CRISPR/Cas9 gene editing tool, we unintentionally identified an autoimmune mutant. Map-based cloning and whole-genome sequencing revealed that it contains a deletion in SMALL UBIQUITIN-RELATED MODIFIER (SUMO) protease encoding gene EARLY IN SHORT DAYS 4 (ESD4). Previous studies reported that esd4 mutants accumulate elevated levels of plant defense hormone salicylic acid (SA). However, upregulated PATHOGENESIS-RELATED GENE 1 (PR1) expression in esd4 only partly relies on SA level. In this study, we show that plant metabolite N-hydroxypipecolic acid (NHP) biosynthetic genes are upregulated in esd4, and NHP biosynthesis mutant flavin-dependent-monooxygenase 1 (fmo1) partially suppresses the autoimmune phenotypes of esd4, suggestive of a requirement of NHP signaling for the autoimmunity in esd4. As activation of nucleotide-binding leucine-rich repeat immune receptors (NLRs) are associates with the biosynthesis of SA and NHP and lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) is a key component downstream of many NLRs, we examined the relationship between EDS1 and ESD4 by analyzing the eds1 esd4 double mutant. We found that eds1 largely suppresses esd4 autoimmunity and blocks the elevated expressions of SA and NHP biosynthesis-related genes in esd4. Overall, our study provides evidence supporting the hypothesis that SUMO protease ESD4 likely targets a yet to be identified guardee of NLR by removing its SUMO modification to avoid recognition by the cognate NLR. Loss of ESD4 results in activation of NLR-mediated autoimmunity.

Activated SUMOylation restricts MHC class I antigen presentation to confer immune evasion in cancer.

Activated SUMOylation is a hallmark of cancer. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved function of activated SUMOylation, which attenuated the immunogenicity of tumor cells. Activated SUMOylation allowed cancer cells to evade CD8+ T cell–mediated immunosurveillance by suppressing the MHC class I (MHC-I) antigen-processing and presentation machinery (APM). Loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, and the pharmacological inhibition of SUMOylation (SUMOi) resulted in reduced activity of the transcriptional repressor scaffold attachment factor B (SAFB) and induction of the MHC-I APM. Consequently, SUMOi enhanced the presentation of antigens and the susceptibility of tumor cells to CD8+ T cell–mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T cells and thereby drove a feed-forward loop amplifying the specific antitumor immune response. In summary, we showed that activated SUMOylation allowed tumor cells to evade antitumor immunosurveillance, and we have expanded the understanding of SUMOi as a rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.

A SUMO‐dependent Pathway for Cytosolic Protein Quality Control

Protein quality control (PQC) maintains proteostasis in cells, whereas PQC dysregulation is found in diseases such as cancers, cardiovascular diseases, and neurodegenerative disorders. Over the past few decades, remarkable progress has been made in understanding compartmentalized PQC pathways that operate inside the nucleus and the endoplasmic reticulum (ER). The small ubiquitin‐related modifiers (SUMOs), for example, were recently found to participate in a nuclear PQC pathway that involves selective sumoylation of misfolded proteins by the SUMO E3 ligase PML, and subsequent recognition and ubiquitylation by the SUMO‐targeted ubiquitin E3 ligase RNF4 (Guo et al., Mol Cell, 2014). Roles of SUMOs in cytoplasmic PQC, however, have not previously been reported. Using yeast mutants and human knockout cell lines, our lab has recently identified a conserved SUMO‐dependent pathway for degradation of a cytosolic, misfolded protein model substrate. The substrate consists of GFP fused to an N‐terminal nuclear export signal (NES) and a C‐terminal hydrophobic degron known as CL1 (NES‐GFP‐CL1). Using protein stability assays, we observed a decrease in the turnover rate of NES‐GFP‐CL1 in a yeast SUMO mutant strain and a human U2OS SUMO1 knockout cell line. Further exploration using nuclear and ER‐localized substrates revealed that this pathway is specific for soluble, cytosolic proteins. In addition, analysis of a U2OS SUMO2 knockout cell line revealed that the observed PQC pathway is specific for the SUMO1 paralogue. This suggests a distinct mechanism from the previously characterized nuclear PML‐SUMO‐RNF4 pathway, which targets misfolded proteins modified by SUMO2/3 polymeric chains. Using cellular fractionation studies, we observed an increase in levels of insoluble NES‐GFP‐CL1 specifically in SUMO1 knockout cells compared to wild type cells. We therefore propose a model where SUMO1 modification of misfolded cytosolic proteins promotes their turnover by enhancing solubility and preventing aggregation. Results from our studies reveal a novel cytosolic PQC regulatory network and provide insights into roles for sumoylation in PQC‐associated diseases.

Optimization of expression and purification of SUMO‐tagged LL‐37 peptides

Antimicrobial peptides (AMPs) are a class of naturally occurring biomolecules found in all multicellular organisms, and are used as a first line of defense for plants and animals. AMPs have been found to cause bacterial membrane rupture and in some cases they also inhibit intracellular function, in both cases resulting in bacterial lysis and death. A broad collection of AMPs have been discovered that can selectively target bacteria, yeasts, fungi, viruses, and even cancer cells. For example, AMPs that target bacteria are typically positively charged, and therefore interact via electrostatic interactions with negatively charged bacterial membranes. Cathelicidins, are small, cationic, amphiphilic AMPs found in many mammalian species. Cathelicidins exibit a broad range of antimicrobial activity against fungi, bacteria, and enveloped viruses, allowing for innate immunity in the skin. LL‐37 is the sole member of the human cathelicidins. Numerous studies have investigated the structure and function of LL‐37, in an effort to develop antimicrobial therapeutics based on its core structure. Many of these potential therapeutics rely on automated peptide synthesis. We seek to contribute to the understanding of the LL‐37 peptide structure and function by further investigating key residues which are required for antimicrobial activity, and optimize the expression of recombinant LL‐37‐based peptides in E.coli. A structure‐based design strategy informed the construction of a series of mutant LL‐37 peptides, that were generated using site‐directed mutagenesis. The following mutants were investigated: a series of single amino acid substitutions, G14P, K12R, K18R, K25R, addition of a hydrophobic tail (LLAA) and hydrophilic tail (LRQA). Cloning was achieved using the ThermoFisher Champion pET SUMO Protein Expression System, and sequences were confirmed using Sanger sequencing. The 11‐kD SUMO (small ubiquitin‐related modifier) on the C‐terminus of the recombinant protein enhances the expression and solubility of the LL‐37 peptide in E. coli. The His‐tagged, LL‐37‐SUMO peptides are expressed in BL21(DE3) E. coli, and following removal of the SUMO and His tags, ongoing studies examining the activity of the recombinant peptides against gram‐positive and gram‐negative pathogens continues.

SUMO Regulates Histone mRNA Processing and Polyadenylation

Replication‐dependent histone genes encode the only non‐polyadenylated mRNAs in mammals. Histone pre‐mRNAs, which do not contain introns, therefore require only one processing step that involves 3’ end recognition and cleavage. Both histone gene expression and pre‐mRNA processing take place in specialized nuclear organelles known as histone locus bodies. Recent studies have revealed that polyadenylation of histone mRNAs does occur, however, under specific physiological conditions. These include during normal terminal cellular differentiation, tumorigenesis, and in disease patients with Aicardi–Goutières syndrome. Despite these links between polyadenylation of histone mRNAs, human physiology, and disease, the triggers, the mechanisms, and the consequences of histone mRNA polyadenylation remain poorly defined. In recent studies of small ubiquitin‐related modifier (SUMO) knockout cell lines, we have obtained evidence that SUMO1 and SUMO2 play roles in regulating histone pre‐mRNA 3’ end processing and polyadenylation. This evidence includes alterations in histone locus body size and structure in SUMO1 and SUMO2 knockout cells, as well as mislocalization of key pre‐mRNA processing factors required for normal processing. Collectively, our findings provide new insights into control of histone pre‐mRNA processing that involves posttranslational protein modifications by SUMO1 and SUMO2.

Heterologous expression of novel SUMO proteases from Schizosaccharomyces pombe in E. coli: Catalytic domain identification and optimization of product yields

Small ubiquitin-related modifier (SUMO) proteins are efficiently used to target the soluble expression of various difficult-to-express proteins in E. coli. However, its utilization in large scale protein production is restricted by the higher cost of Ulp, which is required to cleave SUMO fusion tag from protein-of-interest to generate an authentic N-terminus. This study identified and characterized two novel SUMO proteases i.e., Ulp1 and Ulp2 from Schizosaccharomyces pombe. Codon-optimized gene sequences were cloned and expressed in E. coli. The sequence and structure of SpUlp1 and SpUlp2 catalytic domains were deduced using bioinformatics tools. Protein-protein interaction studies predicted the higher affinity of SpUlp1 towards SUMO compared to its counterpart from Saccharomyces cerevisiae (ScUlp1). The catalytic domain of SpUlp1 was purified using Ni-NTA chromatography with 83.33% recovery yield. Moreover, In vitro activity data further confirmed the fast-acting nature of SpUlp1 catalytic domain, where a 90% cleavage of fusion proteins was obtained within 1 h of incubation, indicating novelty and commercial relevance of S. pombe Ulp1. Biophysical characterization showed 8.8% α-helices, 36.7% β-sheets in SpUlp1SD. From thermal CD and fluorescence data, SpUlp1SD Tm was found to be 45 °C. Further, bioprocess optimization using fed-batch cultivation resulted in 3.5 g/L of SpUlp1SD production with YP/X of 77.26 mg/g DCW and volumetric productivity of 205.88 mg/L/h.

Clinical value of lncRNA SOX2-OT in pulmonary arterial hypertension and its role in pulmonary artery smooth muscle cell proliferation, migration, apoptosis, and inflammatory

BackgroundNon-coding RNA is confirmed to be involved in pulmonary arterial hypertension (PAH). ObjectivesThis study investigated the clinical value and potential mechanisms of the long noncoding RNA (lncRNA) SRY-box transcription factor 2 overlapping transcript (SOX2-OT) in PAH. MethodsSOX2-OT levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in serum of 82 patients with PAH and 76 healthy controls. Receiver operating characteristic (ROC) analysis was performed to assess the diagnostic value of SOX2-OT. Human pulmonary arterial smooth muscle cells (hPASMCs) were treated by hypoxia to construct PAH cell models. Proliferation, migration, apoptosis, and inflammatory cytokines levels of hPASMCs were examined by CCK-8, Transwell, flow cytometry, and ELISA assay. Dual-luciferase reporter gene assays were performed to verify the target relationships between miR-455-3p and SOX2-OT, as well as small ubiquitin-related modifier 1 (SUMO1). ResultsSerum SOX2-OT was highly expressed in patients with PAH (P < 0.05). And elevated SOX2-OT levels significantly differentiated PAH patients from healthy controls, confirming high diagnostic feasibility. What's more, SOX2-OT was increased in hypoxia-induced hPASMCs in a time-dependent manner. Silencing SOX2-OT could reverse hypoxia-induced proliferation, migration, anti-apoptosis, and inflammation of hPASMCs (P < 0.05). However, rescue experiments showed that this reversal effect of silencing SOX2-OT was attenuated by suppressed miR-455-3p, which was presumably achieved by SUMO1 (P < 0.05). ConclusionsElevated SOX2-OT is a feasible diagnostic marker for PAH, and its silencing may attenuated hypoxia-induced hPASMCs proliferation, migration, anti-apoptosis, and inflammation by modulating the miR-455-3p/SUMO1 axis, preventing vascular remodeling and PAH progression. Our research provided new insights for PAH treatment.

Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia coli at High Productivity Using SUMO Fusion and an Adjusted Expression Strategy.

CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 μM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 μM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 μg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. These findings established the utility of the method developed in this study to produce CRM197 for medical applications.