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ABSTRACT SUMOylation involves covalent attachment of small ubiquitin‐like modifier (SUMO) proteins to specific lysine residues on target proteins and regulates various aspects of their function. Sentrin‐specific proteases (SENPs) are key players in both the conjugation reaction of SUMO proteins to their targets and the subsequent deconjugation of SUMO‐conjugated substrates. Here, we provide the first comprehensive prenatal description of a lethal syndrome linked to a novel homozygous stop‐gain variant in SENP7 c.745C>T; p.(Arg249*) in a consanguineous Egyptian family with a history of three fetal deaths, all presenting with multiple congenital anomalies. Similar anomalies were observed through ultrasound assessment of the current fetus, including arthrogryposis multiplex congenita, CNS malformations, congenital heart disease, and renal anomalies. Singleton exome sequencing (ES) of fetal DNA revealed a SENP7 variant allele, which results in a premature termination codon, and the mutant mRNA is predicted to be degraded by nonsense‐mediated decay (NMD). This leads to impaired gene function, particularly disrupting SENP7's isopeptidase activity in deconjugating polySUMO chains. Our findings broaden the molecular spectrum of SENP7 variants and emphasize their essential role in the development of the nervous, musculoskeletal, cardiovascular, and renal systems. This study offers new insights into the genotype–phenotype correlations observed in lethal congenital contracture syndromes and severe embryological abnormalities. [ABSTRACT FROM AUTHOR]

The article from PLoS Pathogens discusses the role of SUMOylation in plants, a posttranslational modification process that can influence protein function and interactions. The study highlights how SUMOylation is involved in plant defense responses against pathogens, such as bacteria, and how viruses can manipulate the SUMO machinery to promote their own replication and evade host defenses. The complex interplay between SUMOylation and viral infections in plants underscores the importance of understanding these interactions for advancements in plant virology and agricultural biotechnology. [Extracted from the article]

The article discusses the enhancement of salt tolerance in wheat through the overexpression of the TaDSU gene, which encodes a SUMO protease. The study shows that TaDSU overexpression leads to increased growth and yield in wheat under saline soil conditions by reducing Na+ content, increasing K+ levels, and enhancing metabolic markers of salt tolerance. The research also highlights the role of the TaDSU gene in deSUMOylating the transcription factor TaMYC2, which contributes to salt tolerance in wheat and Arabidopsis. The study suggests a conserved mechanism of salt tolerance mediated by TaDSU through MYC2 deSUMOylation, providing insights for genetic strategies to improve wheat under saline conditions. [Extracted from the article]

LL-37 is an antimicrobial protein expressed by the CAMP gene in humans. This protein has various antibacterial, antiviral and anticancer effects. Expressing LL-37 as a heterologous protein in E. coli cells has its challenges. LL-37 is a peptide that is so small that it must be engineered with a fusion protein to increase its size solubility and prevent proteolysis of the target protein in cells. Factor Xa is the protease chosen to cleave LL-37 from its fusion protein in this research due to leucine binding factor Xa quite strongly. The aim of this study is, therefore, to express LL-37 as a fusion protein with Histaq_SUMO at the N-terminus linked to LL-37 at the C-terminus through Factor Xa cleavage site. Then, the fusion protein was cleaved by Factor Xa to obtain pure LL-37. In this study, LL-37 was produced by recombinant DNA technology, starting with the construction, expression, purification and cleavage of the fusion protein. The constructed genes consisted of 6xHis, SUMO, the factor X cleavage site, and LL-37, a total of 450 bp inserted in the pD451-SR vector plasmid. The results of this study yielded a SUMO_LL-37 protein with a molecular weight of approximately 17.34 kDa, which could be purified by Ni-NTA under native purification conditions. Based on ImageJ SUMO_LL-37 quantification, it was 1.65 µg/µL. LL-37 can be cleaved by factor Xa from SUMO with an enzyme-to-substrate ratio of 1:12.5 at 37°C with a 24-hour incubation time and results as much as 0.144 µg/µL. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is a common and serious micro-vascular complication of diabetes and a leading cause of end-stage renal disease globally. This disease primarily affects middle-aged and elderly individuals, especially those with a diabetes history of over 10 years and poor long-term blood glucose control. Small ubiquitin-related modifiers (SUMOs) are a group of reversible post-translational modifications of proteins that are widely expressed in eukaryotes. SUMO proteins intervene in the progression of DN by modulating various signaling cascades, such as Nrf2-mediated oxidative stress, NF-κB, TGF-β, and MAPK pathways. Recent advancements indicate that natural products regulating SUMOylation hold promise as targets for intervening in DN. In a previous article published in 2022, we reviewed the mechanisms by which SUMOylation intervenes in renal fibrosis and presented a summary of some natural products with therapeutic potential. Therefore, this paper will focus on DN. The aim of this review is to elucidate the mechanism of action of SUMOylation in DN and related natural products with therapeutic potential, thereby summarising the targets and candidate natural products for the treatment of DN through the modulation of SUMOylation, such as ginkgolic acid, ginkgolide B, resveratrol, astragaloside IV, etc., and highlighting that natural product-mediated modulation of SUMOylation is a potential therapeutic strategy for the treatment of DN as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is a common and serious micro-vascular complication of diabetes and a leading cause of end-stage renal disease globally. This disease primarily affects middle-aged and elderly individuals, especially those with a diabetes history of over 10 years and poor long-term blood glucose control. Small ubiquitin-related modifiers (SUMOs) are a group of reversible post-translational modifications of proteins that are widely expressed in eukaryotes. SUMO proteins intervene in the progression of DN by modulating various signaling cascades, such as Nrf2-mediated oxidative stress, NF-κB, TGF-β, and MAPK pathways. Recent advancements indicate that natural products regulating SUMOylation hold promise as targets for intervening in DN. In a previous article published in 2022, we reviewed the mechanisms by which SUMOylation intervenes in renal fibrosis and presented a summary of some natural products with therapeutic potential. Therefore, this paper will focus on DN. The aim of this review is to elucidate the mechanism of action of SUMOylation in DN and related natural products with therapeutic potential, thereby summarising the targets and candidate natural products for the treatment of DN through the modulation of SUMOylation, such as ginkgolic acid, ginkgolide B, resveratrol, astragaloside IV, etc., and highlighting that natural product-mediated modulation of SUMOylation is a potential therapeutic strategy for the treatment of DN as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Zinc (Zn) transporters contribute to the maintenance of intracellular Zn homeostasis in vertebrate, whose activity and function are modulated by post-translational modification. However, the function of small ubiquitin-like modifier (SUMOylation) in Zn metabolism remains elusive. Here, compared with low Zn group, a high-Zn diet significantly increases hepatic Zn content and upregulates the expression of metal-response element-binding transcription factor-1 (MTF-1), Zn transporter 6 (ZnT6) and deSUMOylation enzymes (SENP1, SENP2, and SENP6), but inhibits the expression of SUMO proteins and the E1, E2, and E3 enzymes. Mechanistically, Zn triggers the activation of the MTF-1/SENP1 pathway, resulting in the reduction of ZnT6 SUMOylation at Lys 409 by small ubiquitin-like modifier 1 (SUMO1), and promoting the deSUMOylation process mediated by SENP1. SUMOylation modification of ZnT6 has no influence on its localization but reduces its protein stability. Importantly, deSUMOylation of ZnT6 is crucial for controlling Zn export from the cytosols into the Golgi apparatus. In conclusion, for the first time, we elucidate a novel mechanism by which SUMO1-catalyzed SUMOylation and SENP1-mediated deSUMOylation of ZnT6 orchestrate the regulation of Zn metabolism within the Golgi apparatus. [ABSTRACT FROM AUTHOR]

Oxidative stress is employed to depict a series of responses detrimental to normal cellular functions resulting from an imbalance between intracellular oxidants, mainly reactive oxygen species and antioxidant defenses. Oxidative stress often contributes to the development of various diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases. In this process, the relationship between small ubiquitin‐like modifier (SUMO) and oxidative stress has garnered significant attention, with its posttranslational modification (PTM) frequently serving as a marker of oxidative stress status. Sentrin/SUMO‐specific proteases (SENPs), affected by alternative splicing, PTMs such as phosphorylation and ubiquitination, and various protein interactions, are crucial molecules in the SUMO process. The human SENP family has six members (SENP1–3, SENP5–7), which are classified into two categories based on sequence similarity, substrate specificity, and subcellular location. They have two core functions in the human body: first, by cleaving the precursor SUMO and exposing the C‐terminal glycine, they initiate the SUMO process; second, they can specifically recognize and dissociate SUMO proteins bound to substrates, a process known as deSUMOylation. However, the connection between deSUMOylation and oxidative stress remains a relatively unexplored area despite their strong association with oxidative diseases such as cancer and cardiovascular disease. This article aims to illustrate the significant contribution of SENPs to the oxidative stress pathway through deSUMOylation by reviewing their structure and classification, their roles in oxidative stress, and the changes in their expression and activity in several typical oxidative stress‐related diseases. [ABSTRACT FROM AUTHOR]

Cytokinins regulate plant growth, development, and responses to environmental stresses such as cold via phosphorelay from cytokinin receptors to the ARABIDOPSIS RESPONSE REGULATORs (ARRs). However, the molecular mechanisms underlying the activation of type-B ARR transcriptional activity in Arabidopsis (Arabidopsis thaliana) remain unclear. Here, we show that the E3 SUMO ligase HIGH PLOIDY2 SUMOylates ARR1, a type-B ARR, at K236, triggering its activation. Cold- or cytokinin-induced phosphorylation of ARR1 at D89 is crucial for its interaction with HPY2. Lysine 236 is critical for ARR1's transactivation without compromising its DNA-binding ability, while D89 is crucial for ARR1's binding to target gene promoters. Cytokinin enhances ARR1's chromatin binding, but cold does not. ARR1 K236 plays a critical role in promoting histone H3 acetylation in response to both cytokinin and cold without affecting chromatin binding. The K236R mutation in ARR1 reduces target gene expression and alters cytokinin and cold response phenotypes. This study unveils a mechanism of ARR1 activation wherein phosphorylated ARR1 interacts with HPY2 and binds to chromatin in response to cytokinin. Cold triggers a phosphorelay targeting chromatin-bound ARR1. HPY2 then catalyzes ARR1 SUMOylation at K236, enhancing histone H3 acetylation and leading to transcriptional activation of ARR1 in response to both cold and cytokinin. The conjugation of SUMO proteins to the transcription factor ARR1 by HIGH PLOIDY2 activates ARR1, triggering two-component signaling in Arabidopsis. [ABSTRACT FROM AUTHOR]

CRM197 (Cross Reacting Material 197) is an inactive form of the C. diphtheriae exotoxin used as a carrier protein for the development and production of conjugated polysaccharide vaccines and immunotherapeutic drugs. However, the development of these research areas is not possible without an efficient and cost-effective technology to produce CRM197 of the proper quality. In this study, we developed a highly efficient method to produce recombinant CRM197 as a fusion with SUMO protein, yielding more than three grams per liter in the form of inclusion bodies. We examined the significant effect of the type of expression vector, the heterologous gene expression conditions, and cultivation on its solubility. Using a combination of reduced cultivation temperature and the promoter of the gene encoding the heat shock protein CspA, we achieved an increase in the solubility level of SUMO-CRM197 of more than 30%, with an overall biosynthesis level of more than two grams per liter. Coexpression of the target gene with the DsbC disulfide isomerase gene allowed us to obtain the target protein completely in the soluble state with a yield of more than 1.4 grams per liter. The results obtained may become the basis for the development of a promising domestic technology for the production of CRM197. [ABSTRACT FROM AUTHOR]

Introduction: SUMOylation is an important post-translational modification that regulates the expression, localization, and activity of substrate proteins, thereby participating in various important cellular processes such as the cell cycle, cell metabolism, gene transcription, and antiviral activity. However, the function of SUMOylation in phytopathogenic fungi has not yet been adequately explored. Methods: A comprehensive analysis composed of proteomics, affinity pulldown, molecular and cellular approaches was performed to explore the roles of SUMOylation in Cryphonectria parasitica, the fungal pathogen responsible for chestnut blight. Results and discussion: CpSmt3, the gene encoding the SUMO protein CpSmt3 in C. parasitica was identified and characterized. Deletion of the CpSmt3 gene resulted in defects in mycelial growth and hyphal morphology, suppression of sporulation, attenuation of virulence, weakening of stress tolerance, and elevated accumulation of hypovirus dsRNA. The 1CpSmt3 deletion mutant exhibited an increase in mitochondrial ROS, swollen mitochondria, excess autophagy, and thickened cell walls. About 500 putative SUMO substrate proteins were identified by affinity pull-down, among which many were implicated in the cell cycle, ribosome, translation, and virulence. Proteomics and SUMO substrate analyses further revealed that deletion of CpSmt3 reduced the accumulation of CpRho1, an important protein that is involved in TOR signal transduction. Silencing of CpRho1 resulted in a phenotype similar to that of 1CpSmt3, while overexpression of CpRho1 could partly rescue some of the prominent defects in 1CpSmt3. Together, these findings demonstrate that SUMOylation by CpSmt3 is vitally important and provide new insights into the SUMOylation-related regulatory mechanisms in C. parasitica. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Expressed protein ligation (EPL) provides a powerful tool to access large‐size proteins with precise structures. Existing methods for constructing the critical protein thioester for EPL have predominantly relied on the recombinant intein fusion expressed in Escherichia coli (E. coli). Despite its powerful applications, the expression of thioester derived from eukaryotic protein in E. coli inherently suffers from its limited solubility, the inactivity of intein, premature hydrolysis and low yields. To overcome these obstacles, we present herein the facile one‐flask synthesis of inaccessible protein α‐thioester via a SUMO‐protein‐intein (SPI) sandwich model. The utility of SUMO enhances the protein fusion yield and solubility, prevents premature hydrolysis and simplifies the purification process. The inaccessible protein thioester with internal Cys residues can be readily produced and is compatible with the EPL‐desulfurization protocol used to prepare complex proteins, which is otherwise difficult to obtain using traditional methods. Its utility has been highlighted through the synthesis of human granulocyte colony‐stimulating factor (G‐CSF). [ABSTRACT FROM AUTHOR]

Nuclear Piwi/Piwi‐interacting RNA complexes mediate co‐transcriptional silencing of transposable elements by inducing local heterochromatin formation. In Drosophila, sumoylation plays an essential role in the assembly of the silencing complex; however, the molecular mechanism by which the sumoylation machinery is recruited to the transposon loci is poorly understood. Here, we show that the Drosophila E3 SUMO‐ligase Su(var)2‐10 directly binds to the Piwi protein. This interaction is mediated by the SUMO‐interacting motif‐like (SIM‐like) structure in the C‐terminal domain of Su(var)2‐10. We demonstrated that the SIM‐like structure binds to a special region found in the MID domain of the Piwi protein, the structure of which is highly similar to the SIM‐binding pocket of SUMO proteins. Abrogation of the Su(var)2‐10‐binding surface of the Piwi protein resulted in transposon derepression in the ovary of adult flies. Based on our results, we propose a model in which the Piwi protein initiates local sumoylation in the silencing complex by recruiting Su(var)2‐10 to the transposon loci. [ABSTRACT FROM AUTHOR]

Protein inhibitors of activated STATs (PIAS) are proteins for cytokine signaling that activate activator-mediated gene transcription. These proteins, as versatile cellular regulators, have been described as regulators of approximately 60 proteins. Dysregulation of PIAS is associated with inappropriate gene expression that promotes oncogenic signaling in multiple cancers. Multiple lines of evidence have revealed that PIAS family members show modulated expressions in cancer cells. Most frequently reported PIAS family members in cancer development are PIAS1 and PIAS3. SUMOylation as post-translational modifier regulates several cellular machineries. PIAS proteins as SUMO E3 ligase factor promotes SUMOylation of transcription factors tangled cancer cells for survival, proliferation, and differentiation. Attenuated PIAS-mediated SUMOylation mechanism is involved in tumorigenesis. This review article provides the PIAS/SUMO role in the modulation of transcriptional factor control, provides brief update on their antagonistic function in different cancer types with particular focus on PIAS proteins as a bonafide therapeutic target to inhibit STAT pathway in cancers, and summarizes natural activators that may have the ability to cure cancer. [ABSTRACT FROM AUTHOR]

The document titled "The materials of the XVIII Ukrainian Conference of Young Scientists of IMBG of NAS of Ukraine" discusses a study on the association between genotypes and allelic variants of the I/D polymorphism of the ACE1 gene and the severity of COVID-19 in children from Ukraine. The study found no significant difference in genotypes and alleles between children with COVID-19 and the control group, but allele I was more common in patients with lung damage. Additionally, a statistically significant increase in the frequency of genotype I/I was found in children with a history of juvenile idiopathic arthritis and mild COVID-19. Another article in the document discusses the development of a continuous monitoring system integrated with an enzyme inhibition-based biosensor to detect heavy metal ions in water. The system showed high sensitivity, stability, and reproducibility, and has potential for analyzing heavy metals. [Extracted from the article]

Plants being fixed in one place are exposed to various episodes of different abiotic stresses such as drought, salinity, cold, and heat. SUMOylation is one of the ignored arsenals that help plants to develop tolerance to these external abiotic stresses. SUMOylation of target protein generally leads to changes in its transportation, transcriptional regulation, apoptosis, stability, and response to different stresses. de-SUMOylation of substrate proteins by SUMO proteases also play a crucial role in maintaining the cellular pool of SUMO. This review highlights different components of SUMOylation and their role in different abiotic stresses and their ability to contribute to plant abiotic stress tolerance. Furthermore, the current perspective of SUMOylation in phytochrome signaling, nutrient and ROS homeostasis is discussed. The full potential of SUMOylation in combination with other molecular approaches to combat abiotic stresses in plants is not yet realized. As research in this area continues to advance, it is crucial to explore the interplay between SUMOylation and other signaling networks, as well as the crosstalk with different stress-responsive pathways. Additionally, understanding the specificity and dynamics of SUMOylation in response to specific stressors can provide valuable insights for designing targeted interventions to enhance plant stress tolerance. In conclusion, the review highlights the emerging significance of SUMOylation in plant stress responses and its potential in contributing to plant resilience against abiotic stresses. [ABSTRACT FROM AUTHOR]

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin-nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

Acting as a cysteine protease, small ubiquitin-like modifier (SUMO)/sentrinspecific protease1 (SENP1) involved in multiple physiological and pathological processes through processing the precursor SUMO protein into mature form and deSUMOylating target protein. It has been reported that SENP1 is highly expressed and plays a carcinogenic role in various cancers. In this paper, we mainly explore the function and mechanism of SENP1 in tumor cell proliferation, apoptosis, invasion, metastasis, stemness, angiogenesis, metabolism and drug resistance. Furthermore, the research progress of SENP1 inhibitors for cancer treatment is introduced. This study aims to provide theoretical references for cancer therapy by targeting SENP1. [ABSTRACT FROM AUTHOR]

SUMO (small ubiquitin-like modifier) conjugation or SUMOylation, a post-translational modification, is a crucial regulator of protein function and cellular processes. In the context of neural stem cells (NSCs), SUMOylation has emerged as a key player, affecting their proliferation, differentiation, and survival. By modifying transcription factors, such as SOX1, SOX2, SOX3, SOX6, Bmi1, and Nanog, SUMOylation can either enhance or impair their transcriptional activity, thus impacting on NSCs self-renewal. Moreover, SUMOylation regulates neurogenesis and neuronal differentiation by modulating key proteins, such as Foxp1, Mecp2, MEF2A, and SOX10. SUMOylation is also crucial for the survival and proliferation of NSCs in both developing and adult brains. By regulating the activity of transcription factors, coactivators, and corepressors, SUMOylation acts as a molecular switch, inducing cofactor recruitment and function during development. Importantly, dysregulation of NSCs SUMOylation has been implicated in various disorders, including embryonic defects, ischemic cerebrovascular disease, glioma, and the harmful effects of benzophenone-3 exposure. Here we review the main findings on SUMOylation-mediated regulation of NSCs self-renewal, differentiation and survival. Better understanding NSCs SUMOylation mechanisms and its functional consequences might provide new strategies to promote neuronal differentiation that could contribute for the development of novel therapies targeting neurodegenerative diseases. • SUMOylation regulates several protein function and cellular processes. • SUMOylation affects NSCs proliferation, differentiation, and survival. • SUMOylation enhances or impairs transcriptional factors controlling NSCs self-renewal. • SUMOylation regulates neurogenesis and neuronal differentiation processes. • Dysregulation of NSCs SUMOylation is linked to disorders. [ABSTRACT FROM AUTHOR]

The correct inheritance of chromatin structure is key for maintaining genome function and cell identity and preventing cellular transformation. DEK, a conserved non-histone chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis and stemness, but a characteristic oncogenic mechanism has remained elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK that regularly occur at specific, yet unidentified, sites of heterochromatin replication exclusively in late S-phase. In these bodies, DEK localizes in direct proximity to active replisomes in agreement with a function in the early maturation of heterochromatin. A high-throughput siRNA screen, supported by mutational and biochemical analyses, identifies SUMO as one regulator of DEK body formation, linking DEK to the complex SUMO protein network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress, and delineates an avenue of further study for unraveling the contribution of DEK to cancer development. [ABSTRACT FROM AUTHOR]

Regulation by the small modifier SUMO is heavily dependent on spatial control of enzymes that mediate the attachment and removal of SUMO on substrate proteins. Here, we show that in the fission yeast Schizosaccharomyces pombe, delocalisation of the SUMO protease Ulp1 from the nuclear envelope results in centromeric defects that can be attributed to hyper-SUMOylation at the nuclear periphery. Unexpectedly, we find that although this localised hyper-SUMOylation impairs centromeric silencing, it can also enhance centromere clustering. Moreover, both effects are at least partially dependent on SUMOylation of the inner nuclear membrane protein Lem2. Lem2 has previously been implicated in diverse biological processes, including the promotion of both centromere clustering and silencing, but how these distinct activities are coordinated was unclear; our observations suggest a model whereby SUMOylation serves as a regulatory switch, modulating Lem2 interactions with competing partner proteins to balance its roles in alternative pathways. Our findings also reveal a previously unappreciated role for SUMOylation in promoting centromere clustering. [ABSTRACT FROM AUTHOR]

Crossing over is essential for chromosome segregation during meiosis. Protein modification by SUMO is implicated in crossover control, but pertinent targets have remained elusive. Here we identify Msh4 as a target of SUMO-mediated crossover regulation. Msh4 and Msh5 constitute the MutSγ complex, which stabilizes joint-molecule (JM) recombination intermediates and facilitates their resolution into crossovers. Msh4 SUMOylation enhances these processes to ensure that each chromosome pair acquires at least one crossover. Msh4 is directly targeted by E2 conjugase Ubc9, initially becoming mono-SUMOylated in response to DNA double-strand breaks, then multi/poly-SUMOylated forms arise as homologs fully engage. Mechanistically, SUMOylation fosters interaction between Msh4 and Msh5. We infer that initial SUMOylation of Msh4 enhances assembly of MutSγ in anticipation of JM formation, while secondary SUMOylation may promote downstream functions. Regulation of Msh4 by SUMO is distinct and independent of its previously described stabilization by phosphorylation, defining MutSγ as a hub for crossover control.

This document is an editorial titled "Molecular and genetic mechanisms of plant architecture regulation" published in the journal Frontiers in Plant Science. The editorial discusses the impact of environmental stressors on sugarcane crops and the molecular and genetic mechanisms that regulate plant architecture. It highlights the effects of temperature, water availability, and light intensity on sugarcane growth and development. The editorial also explores the accumulation of reactive oxygen species (ROS) and the role of stress-related genes and proteins in plant adaptation to environmental stress. Strategies to mitigate environmental stress, such as breeding stress-resistant cultivars and implementing irrigation scheduling, are also discussed. The document emphasizes the importance of molecular breeding methods in improving sugarcane selection and breeding efficiency. [Extracted from the article]

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Thyroid hormone (TH) and thyroid hormone receptor (THR) regulate stem cell proliferation and differentiation during development, as well as during tissue renewal and repair in the adult. THR undergoes posttranslational modification by small ubiquitin-like modifier (SUMO). We generated the THRA (K283Q/K288R)-/- mouse model for in vivo studies and used human primary preadipocytes expressing the THRA sumoylation mutant (K283R/K288R) and isolated preadipocytes from mutant mice for in vitro studies. THRA mutant mice had reduced white adipose stores and reduced adipocyte cell diameter on a chow diet, compared to wild-type, and these differences were further enhanced after a high fat diet. Reduced preadipocyte proliferation in mutant mice, compared to wt, was shown after in vivo labeling of preadipocytes with EdU and in preadipocytes isolated from mice fat stores and studied in vitro. Mice with the desumoylated THRA had disruptions in cell cycle G1/S transition and this was associated with a reduction in the availability of cyclin D2 and cyclin-dependent kinase 2. The genes coding for cyclin D1, cyclin D2, cyclin-dependent kinase 2 and Culin3 are stimulated by cAMP Response Element Binding Protein (CREB) and contain CREB Response Elements (CREs) in their regulatory regions. We demonstrate, by Chromatin Immunoprecipitation (ChIP) assay, that in mice with the THRA K283Q/K288R mutant there was reduced CREB binding to the CRE. Mice with a THRA sumoylation mutant had reduced fat stores on chow and high fat diets and reduced adipocyte diameter.

Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation. NuSAP1 regulates these processes by interacting with several protein partners. Its abundance, activity and interactions are therefore tightly regulated during mitosis. Protein conjugation with SUMO (Small Ubiquitin-like MOdifier peptide) is a reversible post-translational modification that modulates rapid changes in the structure, interaction(s) and localization of proteins. NuSAP1 was previously found to interact with RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilizing activity, but how this interaction affects NuSAP1 activity has remained elusive. Here, we show that NuSAP1 interacts with RANBP2 and forms proximity ligation products with SUMO2/3 peptides in a RANBP2-dependent manner at key mitotic sites. A bioinformatic search identified two putative SUMO consensus sites in NuSAP1, within the DNA-binding and the microtubule-binding domains, respectively. Site-specific mutagenesis, and mitotic phenotyping in cell lines expressing each NuSAP1 mutant version, revealed selective roles of each individual site in control of NuSAP1 localization and in generation of specific mitotic defects and distinct fates in daughter cells. These results identify therefore two new regulatory sites for NuSAP1 functions and implicate RANBP2 in control of NuSAP1 activity. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD), diagnosed by reduced lung function, is a leading cause of morbidity and mortality. We performed whole genome sequence (WGS) analysis of lung function and COPD in a multi-ethnic sample of 11,497 participants from population- and family-based studies, and 8499 individuals from COPD-enriched studies in the NHLBI Trans-Omics for Precision Medicine (TOPMed) Program. We identify at genome-wide significance 10 known GWAS loci and 22 distinct, previously unreported loci, including two common variant signals from stratified analysis of African Americans. Four novel common variants within the regions of PIAS1, RGN (two variants) and FTO show evidence of replication in the UK Biobank (European ancestry n ~ 320,000), while colocalization analyses leveraging multi-omic data from GTEx and TOPMed identify potential molecular mechanisms underlying four of the 22 novel loci. Our study demonstrates the value of performing WGS analyses and multi-omic follow-up in cohorts of diverse ancestry.

BACKGROUND: Cardiac fibroblasts have crucial roles in the heart. In particular, fibroblasts differentiate into myofibroblasts in the damaged myocardium, contributing to scar formation and interstitial fibrosis. Fibrosis is associated with heart dysfunction and failure. Myofibroblasts therefore represent attractive therapeutic targets. However, the lack of myofibroblast-specific markers has precluded the development of targeted therapies. In this context, most of the noncoding genome is transcribed into long noncoding RNAs (lncRNAs). A number of lncRNAs have pivotal functions in the cardiovascular system. lncRNAs are globally more cell-specific than protein-coding genes, supporting their importance as key determinants of cell identity. METHODS: In this study, we evaluated the value of the lncRNA transcriptome in very deep single-cell RNA sequencing. We profiled the lncRNA transcriptome in cardiac nonmyocyte cells after infarction and probed heterogeneity in the fibroblast and myofibroblast populations. In addition, we searched for subpopulation-specific markers that can constitute novel targets in therapy for heart disease. RESULTS: We demonstrated that cardiac cell identity can be defined by the sole expression of lncRNAs in single-cell experiments. In this analysis, we identified lncRNAs enriched in relevant myofibroblast subpopulations. Selecting 1 candidate we named FIXER (fibrogenic LOX-locus enhancer RNA), we showed that its silencing limits fibrosis and improves heart function after infarction. Mechanitically, FIXER interacts with CBX4, an E3 SUMO protein ligase and transcription factor, guiding CBX4 to the promoter of the transcription factor RUNX1 to control its expression and, consequently, the expression of a fibrogenic gene program. FIXER is conserved in humans, supporting its translational value. CONCLUSIONS: Our results demonstrated that lncRNA expression is sufficient to identify the various cell types composing the mammalian heart. Focusing on cardiac fibroblasts and their derivatives, we identified lncRNAs uniquely expressed in myofibroblasts. In particular, the lncRNA FIXER represents a novel therapeutic target for cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Small ubiquitin-like modifier (SUMO) E3 ligases that facilitate the conjugation of SUMO proteins to target substrates contain an SP-RING domain which is like the RING domain found in ubiquitin E3 ligases. In this study, we isolated and characterized the Oryza sativa protein inhibitor of activated STAT like1 (OsPIAL1) containing SP-RING domains, as the rice homolog of Arabidopsis PIALs. OsPIAL1 interacts with OsSUMO proteins but does not interact with rice SUMO-conjugating enzymes (OsSCEs). An analysis of transgenic rice plant shows that OsPIAL1 is involved in SUMO conjugation to SCEs but not in SUMO conjugation to substrates. In addition, this OsPIAL1 activity requires drought stress conditions. Expression profiles show that the OsPIAL1 gene is induced by only drought stress in the leaves, whereas it is repressed by ABA and abiotic stresses in the roots. Salt stress leads to the fastest decrease in OsPIAL1 transcripts in the roots. Furthermore, the stress experiments indicate that the transgenic rice plants overexpressing OsPIAL1 exhibit a drought stress-tolerant phenotype but a salt stress hypersensitive phenotype. Our results and those from Arabidopsis pial mutants suggest that PIALs act as a positive regulator in the drought stress response but as a negative regulator in the salt stress response. [ABSTRACT FROM AUTHOR]

The developmental biology underlying the morphogenesis of mushrooms remains poorly understood despite the essential role of fungi in the terrestrial environment and global carbon cycle. The mushroom Coprinopsis cinerea is a leading model system for the molecular and cellular basis of fungal morphogenesis. The dikaryotic vegetative hyphae of this fungus grow by tip growth with clamp cell formation, conjugate nuclear division, septation, subapical peg formation, and fusion of the clamp cell to the peg. Studying these processes provides many opportunities to gain insights into fungal cell morphogenesis. Here, we report the dynamics of five septins, as well as the regulators CcCla4, CcSpa2, and F-actin, visualized by tagging with fluorescent proteins, EGFP, PA-GFP or mCherry, in the growing dikaryotic vegetative hyphae. We also observed the nuclei using tagged Sumo proteins and histone H1. The five septins colocalized at the hyphal tip in the shape of a dome with a hole (DwH). CcSpa2-EGFP signals were observed in the hole, while CcCla4 signals were observed as the fluctuating dome at the hyphal tip. Before septation, CcCla4-EGFP was also occasionally recruited transiently around the future septum site. Fluorescent protein-tagged septins and F-actin together formed a contractile ring at the septum site. These distinct specialized growth machineries at different sites of dikaryotic vegetative hyphae provide a foundation to explore the differentiation program of various types of cells required for fruiting body formation. [ABSTRACT FROM AUTHOR]

Сhromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. We found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression in Drosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.

Structural studies with tryptophan synthase (TS) bienzyme complex (α2β2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated β-subunit allowed the purification of high amounts of pure subunits and α2β2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme β-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α2β2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α2β2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α2β2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α2β2 StTS complexes.

Background: Some peptides are targets for degradation when heterologously expressed as fusion proteins in E. coli, which can limit yields after isolation and purification. We recently reported that peptide degradation may be prevented by production of a "sandwiched" SUMO-peptide-intein (SPI) fusion protein, which protects the target peptide sequence from truncation and improves yield. This initial system required cloning with two commercially available vectors. It used an N-terminal polyhistidine tagged small ubiquitin-like modifier (SUMO) protein and a C-terminal engineered Mycobacterium xenopii DNA Gyrase A intein with an inserted chitin binding domain (CBD) to create "sandwiched" fusion proteins of the form: His6-SUMO-peptide-intein-CBD. However, the major drawback of this previously reported fusion protein "sandwich" approach is the increased time and number of steps required to complete the cloning and isolation procedures, relative to the simple procedures to produce recombinant peptides in E. coli from a single (non-"sandwiched") fusion protein system. Results: In this work we generate the plasmid pSPIH6, which improves upon the previous system by encoding both the SUMO and intein proteins and allows facile construction of a SPI protein in a single cloning step. Additionally, the Mxe GyrA intein encoded in pSPIH6 contains a C-terminal polyhistidine tag, resulting in SPI fusion proteins of the form: His6-SUMO-peptide-intein-CBD-His6. The dual polyhistidine tags greatly simplify isolation procedures compared to the original SPI system, which we have here demonstrated with two linear bacteriocin peptides: leucocin A and lactococcin A. The yields obtained for both peptides after purification were also improved compared to the previous SPI system as a result of this streamlined protocol. Conclusions: This modified SPI system and its simplified cloning and purification procedures described here may be generally useful as a heterologous E. coli expression system to obtain pure peptides in high yield, especially when degradation of the target peptide is an issue. [ABSTRACT FROM AUTHOR]

Small ubiquitin-like modifier (SUMO) conjugation to target proteins is an important post-translational modification, which regulates plant tolerance to biotic and abiotic stresses. SIZ1, a well-characterized SUMO E3 ligase, facilitates the conjugation of SUMO to target proteins. Here, a SIZ/PAIS-type protein SlSIZ2 was identified in tomato (Solanum lycopersicum) that is a homolog of AtSIZ1 and SlSIZ1. SlSIZ2 was expressed in tomato vegetative and reproductive tissues, and induced by ABA and NaCl. Nucleus-localized SlSIZ2 partially rescued atsiz1-2 dwarfism and also alleviated the sensitivity of atsiz1-2 to ABA and NaCl, suggesting the functional replacement of SlSIZ2 to AtSIZ1. Moreover, SlSIZ2-overexpressing Arabidopsis has higher cotyledon expansion rate, lateral root density and survival rate under salinity stress. These results suggested the contribution of SlSIZ2 to the tolerance of salinity stress. [ABSTRACT FROM AUTHOR]

SUMOylation is a protein modification process that involves the covalent attachment of a small ubiquitin-like modifier (SUMO) to a specific lysine residue on the target protein. This modification can influence the function, localization, stability, and interactions of proteins, thereby regulating various cellular processes. Altering the SUMOylation of certain proteins is expected to be a potential approach for treating specific cancers and diseases. Among these, SENP3 can affect target proteins by regulating the deSUMOylation process, which in turn influences the transcriptional activity of downstream genes, playing a role in either promoting or inhibiting cancer. SENP3 regulates the SUMO status of proteins in numerous signaling pathways, modulating the activity of specific signaling molecules to impact cellular responses and tumor progression. Additionally, SENP3 promotes cell growth and division by deSUMOylating key cyclins. In the context of DNA repair, SENP3 regulates the activity of proteins associated with DNA repair by deSUMOylating repair factors, thereby enhancing DNA repair and maintaining genome stability. Furthermore, SENP3 has specific functions in various other diseases. The complex roles of SENP3 indicate its potential as both a therapeutic target and a biomarker. [ABSTRACT FROM AUTHOR]

SUMOylation is a post-translational modification essential for various biological processes. SUMO proteins bind to target substrates in a three-step enzymatic pathway, which is rapidly reversible by the action of specific proteases, known as SENPs. Studies have shown that SUMOylation is dysregulated in several human disorders, including neurodegenerative diseases that are characterized by the progressive loss of neurons, mitochondrial dysfunction, deficits in autophagy, and oxidative stress. Considering the potential neuroprotective roles of SUMOylation, the aim of this study was to investigate the effects of SENP3 knockdown in H4 neuroglioma cells exposed to rotenone, an in vitro model of cytotoxicity that mimics dopaminergic loss in Parkinson's disease (PD). The current data show that SENP3 knockdown increases SUMO-2/3 conjugates, which is accompanied by reduced levels of the mitochondrial fission protein Drp1 and increased levels of the mitochondrial fusion protein OPA1. Of high interest, SENP3 knockdown prevented rotenone-induced superoxide production and cellular death. Taken together, these findings highlight the importance of SUMOylation in maintaining mitochondrial homeostasis and the neuroprotective potential of this modification in PD. [Display omitted] • SENP3 knockdown increases SUMO-2/3 conjugates in H4 neuroglioma cells exposed to rotenone; • SENP3 knockdown reduces mitochondrial fission protein Drp1 and increases mitochondrial fusion protein OPA1; • SENP3 knockdown preventes rotenone-induced superoxide production and cellular death. [ABSTRACT FROM AUTHOR]

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.

Mechanisms underlying anteroposterior body axis differences during adult tissue maintenance and regeneration are poorly understood. Here, we identify that post-translational modifications through the SUMO (Small Ubiquitin-like Modifier) machinery are evolutionarily conserved in the Lophotrocozoan Schmidtea mediterranea. Disruption of SUMOylation in adult animals by RNA-interference of the only SUMO E2 conjugating enzyme Ubc9 leads to a systemic increase in DNA damage and a remarkable regional defect characterized by increased cell death and loss of the posterior half of the body. We identified that Ubc9 is mainly expressed in planarian stem cells (neoblasts) but it is also transcribed in differentiated cells including neurons. Regeneration in Ubc9(RNAi) animals is impaired and associated with low neoblast proliferation. We present evidence indicating that Ubc9-induced regional cell death is preceded by alterations in transcription and spatial expression of repressors and activators of the Hedgehog signaling pathway. Our results demonstrate that SUMOylation acts as a regional-specific cue to regulate cell fate during tissue renewal and regeneration.

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. [ABSTRACT FROM AUTHOR]

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 SUMOdependent 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 PQCassociated diseases. [ABSTRACT FROM AUTHOR]

The Ufd1/Npl4/Cdc48 complex is a universal protein segregase that plays key roles in eukaryotic cellular processes. Its functions orchestrating the clearance or removal of polyubiquitylated targets are established; however, prior studies suggest that the complex also targets substrates modified by the ubiquitin-like protein SUMO. Here, we show that interactions between Ufd1 and SUMO enhance unfolding of substrates modified by SUMO-polyubiquitin hybrid chains by the budding yeast Ufd1/Npl4/Cdc48 complex compared to substrates modified by polyubiquitin chains, a difference that is accentuated when the complex has a choice between these substrates. Incubating Ufd1/Npl4/Cdc48 with a substrate modified by a SUMO-polyubiquitin hybrid chain produced a series of single-particle cryo-EM structures that reveal features of interactions between Ufd1/Npl4/Cdc48 and ubiquitin prior to and during unfolding of ubiquitin. These results are consistent with cellular functions for SUMO and ubiquitin modifications and support a physical model wherein Ufd1/Npl4/Cdc48, SUMO, and ubiquitin conjugation pathways converge to promote clearance of proteins modified with SUMO and polyubiquitin. [ABSTRACT FROM AUTHOR]

Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein (pal) gene. Ulp1403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δpal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1403-621 approximately four fold. Moreover, extracellular Ulp1403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus. The specific activity of extracellular Ulp1403-621 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 106 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. Key points: • First report of extracellular production of Ulp1403-621 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1403-621 approximately four fold. • The specific activity of extracellular Ulp1403-621 produced in this study reached 2.0 × 106 U/L. [ABSTRACT FROM AUTHOR]

In the last decades, the post-translational modification system by covalent attachment of the SUMO polypeptide to proteins has emerged as an essential mechanism controlling virtually all the physiological processes in the eukaryotic cell. This includes vertebrate development. In the nervous system, SUMO plays crucial roles in synapse establishment and it has also been linked to a variety of neurodegenerative diseases. However, to date, the involvement of the modification of specific targets in key aspects of nervous system development, like patterning and differentiation, has remained largely elusive. A number of recent works confirm the participation of target-specific SUMO modification in critical aspects of nervous system development. Here, we review pioneering and new findings demonstrating the essential role SUMO plays in neurogenesis and other facets of neurodevelopment, which will help to precisely understand the variety of mechanisms SUMO utilizes to control most fundamental processes in the cell. [ABSTRACT FROM AUTHOR]

Post-translational modification by Small Ubiquitin-like Modifier (SUMO) proteins regulates numerous cellular processes. This modification involves the covalent and reversible attachment of SUMO to target proteins through an isopeptide bond, using a cascade of E1, E2 and E3 SUMOylation enzymes. Most functions of SUMO depend on the establishment of non-covalent protein-protein interactions between SUMOylated substrates and their binding partners. The vast majority of these interactions involve a conserved surface in the SUMO protein and a SUMO interacting motif (SIM), a short stretch of hydrophobic amino acids and an acidic region, in the interactor protein. Despite single SUMO-SIM interactions are relatively weak, they can have a huge impact at different levels, altering the activity, localization and stability of proteins, triggering the formation of macromolecular assemblies or inducing phase separation. Moreover, SUMO-SIM interactions are ubiquitous in most enzymes of the SUMO pathway, and play essential roles in SUMO conjugation and deconjugation. Here, we analyze the role of SUMO-SIM contacts in SUMO enzymes and targets and discuss how this humble interaction participates in SUMOylation reactions and mediates the outcome of this essential post-translational modification. [ABSTRACT FROM AUTHOR]

Glycolipids are complex molecules involved in important cellular processes. Among them, the glycosphingolipid α-galactosylceramide has proven to be of interest in biomedicine for its immunostimulatory capabilities. Given its structural requirements, the use of ceramide glycosyltransferase enzymes capable of synthesizing this molecule under in vivo or in vitro conditions is a potential production strategy. Several GT4 enzymes from Bacteroides fragilis were considered as potential candidates in addition to the known BF9343_3149, but only this one showed glycolipid synthase activity. The enzyme was expressed as a SUMO fusion protein to produce soluble protein. It is a non-processive glycosyltransferase that prefers UDP-Gal over UDP-Glc as a donor substrate, and maximum activity was found at pH 7.3 and around 30–35 °C. It does not require metal cations for activity as other GT4 enzymes, but Zn2+ inactivates the enzyme. The reaction occurs when the ceramide lipid acceptor is solubilized with BSA (100% conversion) but not when it is presented in mixed micelles, and anionic lipids do not increase activity, as in other membrane-associated glycolipid synthases. Further protein engineering to increase stability and activity can make feasible the enzymatic synthesis of α-GalCer for biomedical applications. [ABSTRACT FROM AUTHOR]