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Early-life respiratory syncytial virus (RSV) infection (eRSV) is one of the leading causes of serious pulmonary disease in children. eRSV is associated with higher risk of developing asthma and compromised lung function later in life. Cadmium (Cd) is a toxic metal, widely present in the environment and in food. We recently showed that eRSV reprograms metabolism and potentiates Cd toxicity in the lung, and our transcriptome-metabolome-wide study showed strong associations between S-palmitoyl transferase expression and Cd-stimulated lung inflammation and fibrosis signaling. Limited information is available on the mechanism by which eRSV reprograms metabolism and potentiates Cd toxicity in the lung. In the current study, we used a mouse model to examine the role of protein S-palmitoylation (Pr-S-Pal) in low dose Cd-elevated lung metabolic disruption and inflammation following eRSV. Mice exposed to eRSV were later treated with Cd (3.3 mg CdCl2/l) in drinking water for 6 weeks (RSV + Cd). The role of Pr-S-Pal was studied using a palmitoyl transferase inhibitor, 2-bromopalmitate (BP, 10 µM). Inflammatory marker analysis showed that cytokines, chemokines, and inflammatory cells were highest in the RSV + Cd group, and BP decreased inflammatory markers. Lung metabolomics analysis showed that pathways including phenylalanine, tyrosine and tryptophan, phosphatidylinositol and sphingolipid were altered across treatments. The BP antagonized metabolic disruption of sphingolipid and glycosaminoglycan metabolism by RSV + Cd, consistent with BP effect on inflammatory markers. This study shows that Cd exposure following eRSV has a significant impact on subsequent inflammatory response and lung metabolism, which is mediated by Pr-S-Pal, and warrants future research for a therapeutic target. [ABSTRACT FROM AUTHOR]

The mitochondrial proteome is subject to abundant post-translational modifications, including lysine acetylation and phosphorylation of serine, threonine, and tyrosine. The biological function of the majority of these protein modifications is unknown. Proteins required for the transcription and translation of mitochondrial DNA (mtDNA) are subject to modification. This suggests that reversible post-translational modifications may serve as a regulatory mechanism for mitochondrial gene transcription, akin to mechanisms controlling nuclear gene expression. We set out to determine whether acetylation or phosphorylation controls the function of mitochondrial RNA polymerase (POLRMT). Mass spectrometry was used to identify post-translational modifications on POLRMT. We analyzed three POLRMT modification sites (lysine 402, threonine 315, threonine 993) found in distinct structural regions. Amino acid point mutants that mimic the modified and unmodified forms of POLRMT were employed to measure the effect of acetylation or phosphorylation on the promoter binding ability of POLRMT in vitro. We found a slight decrease in binding affinity for the phosphomimic at threonine 315. We did not identify large changes in viability, mtDNA content, or mitochondrial transcript level upon overexpression of POLRMT modification mimics in HeLa cells. Our results suggest minimal biological impact of the POLRMT post-translational modifications studied in our system. [ABSTRACT FROM AUTHOR]

Objective·To analyze the full-length protein structure of human-derived lysine acetyltransferase 7 (KAT7) using cryoelectron microscopy (Cryo-EM) and to obtain the profile information of human-derived KAT7. Methods·The recombinant protein expression plasmid pGEX-4T1-GST-KAT7 was constructed by using the pGEX-4T1 vector and the full-length gene of humanderived KAT7, and the KAT7 protein was expressed in the prokaryotic protein expression system BL21 (DE3). The GST-KAT7 recombinant protein was obtained by using GST affinity chromatography. After removing the GST protein tag with TEV protease, KAT7 was further isolated and purified by HiLoad 16/600 Superdex 75 pg volume exclusion chromatography. The obtained protein samples were identified by Western blotting, and the samples were screened. The protein morphology was observed under negativestain electron microscopy, and data were collected by using Cryo-EM. The protein particles were selected and the spatial structure of the full-length KAT7 was analyzed with the Cryo-EM analysis software CryoSparc. The MYST structural domain model (5GK9) in the Protein Data Bank (PDB) and AlphaFold prediction model of KAT7 were matched with the generated structural model by UCSF Chimera software. Results·The full-length protein of human-derived KAT7 was successfully purified by affinity chromatography, and high purity KAT7 was obtained by volume exclusion chromatography. After identifying KAT7 by Western blotting, the spatial structure of KAT7 full-length protein was initially resolved by Cryo-EM and single-particle reconstruction techniques, and a preliminary three-dimensional structure model with a resolution of about 10 Å was obtained by three-dimensional optimization. The spatial structure of KAT7 full-length protein was irregular and semi-loop-shaped, and the existing MYST domain model (PDB: 5GK9) can be matched into the C-terminal part of the KAT7 full-length model. The adjusted AlphaFold prediction model can also match the KAT7 full-length structure model. Conclusion·A preliminary analysis of the spatial structure model of full-length protein of human-derived KAT7 is performed by using Cryo-EM. [ABSTRACT FROM AUTHOR]

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1 , which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132—requiring upregulation of neddylation to restore proteasomal function and proteasomal stress—led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature—delayed closure of the ischiopubic rami—correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects. [Display omitted] The authors describe four individuals with bi-allelic variants in NAE1 , which encodes the NEDD8-activating enzyme E1 regulatory subunit and is essential for neddylation. By focusing on the biology underlying the rarest observed clinical features, they unveil the importance of neddylation for skeletal development and for maintaining cellular integrity during stress. [ABSTRACT FROM AUTHOR]

Tau is a widespread neuroprotein that regulates the cytoskeleton assembly. In some neurological disorders, known as tauopathies, tau is dissociated from the microtubule and forms insoluble neurofibrillary tangles. Tau comprises four pseudorepeats (R1–R4), containing one (R1, R2, R4) or two (R3) histidines, that potentially act as metal binding sites. Moreover, Cys291 and Cys322 in R2 and R3, respectively, might have an important role in protein aggregation, through possible disulfide bond formation, and/or affecting the binding and reactivity of redox-active metal ions, as copper. We, therefore, compare the interaction of copper with octadeca-R3-peptide (R3C) and with the mutant containing an alanine residue (R3A) to assess the role of thiol group. Spectrophotometric titrations allow to calculate the formation constant of the copper(I) complexes, showing a remarkable stronger interaction in the case of R3C (log Kf = 13.4 and 10.5 for copper(I)-R3C and copper(I)-R3A, respectively). We also evaluate the oxidative reactivity associated to these copper complexes in the presence of dopamine and ascorbate. Both R3A and R3C peptides increase the capability of copper to oxidize catechols, but copper-R3C displays a peculiar mechanism due to the presence of cysteine. HPLC-MS analysis shows that cysteine can form disulfide bonds and dopamine-Cys covalent adducts, with potential implication in tau aggregation process. [ABSTRACT FROM AUTHOR]

Lysine crotonylation (Kcr) is increasingly recognized as a key protein post‐translational modification. However, selective detection and enrichment of crotonylated proteins remains a challenging task. Herein we present a covalent binder for the selective recognition of protein crotonylation. Based on proximity‐induced crosslinking, a bacterial sirtuin (CobB) was remodeled with genetically installed thiol‐bearing noncanonical amino acids at the Kcr‐interacting site, which subsequently could react with Kcr sites in a unique NAD+‐dependent manner. The covalent binder has been used to selectively recognize crotonylated proteins in extracted histone samples and in fixed cells. [ABSTRACT FROM AUTHOR]

Lysine crotonylation (Kcr) is increasingly recognized as a key protein post‐translational modification. However, selective detection and enrichment of crotonylated proteins remains a challenging task. Herein we present a covalent binder for the selective recognition of protein crotonylation. Based on proximity‐induced crosslinking, a bacterial sirtuin (CobB) was remodeled with genetically installed thiol‐bearing noncanonical amino acids at the Kcr‐interacting site, which subsequently could react with Kcr sites in a unique NAD+‐dependent manner. The covalent binder has been used to selectively recognize crotonylated proteins in extracted histone samples and in fixed cells. [ABSTRACT FROM AUTHOR]

In nature, the majority of known RNA-protein interactions are transient. Our recent study has depicted a novel mechanism known as RNAylation, which covalently links proteins and RNAs. This novel modification bridges the realms of RNA and protein modifications. This review specifically explores RNAylation catalyzed by bacteriophage T4 ADP-ribosyltransferase ModB, with a focus on its protein targets and RNA substrates in the context of Escherichia coli -bacteriophage T4 interaction. Additionally, we discuss the biological significance of RNAylation and present perspectives on RNAylation as a versatile bioconjugation strategy for RNAs and proteins. [Display omitted] • RNAs and proteins are covalently linked in vivo by an RNAylation reaction. • RNAylation is catalyzed by the bacteriophage T4 ADP-ribosyltransferase ModB. • NAD-capped RNAs are the substrates of ModB for RNAylation. • Ribosomal proteins are specifically RNAylated in vivo. • RNAylation may be used as a bioconjugation strategy to engineer RNA-protein conjugates. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) have emerged as pivotal regulators of the development of cancers, including esophageal squamous cell carcinoma (ESCC). Here, we conducted a comprehensive analysis of PTM-related genetic variants associated with ESCC risk using large-scale genome-wide and exome-wide association datasets. We observed significant enrichment of PTM-related variants in the ESCC risk loci and identified five variants that were significantly associated with ESCC risk. Among them, rs6780013 in PTPN23 exhibited the highest level of significance in ESCC susceptibility in 9,728 ESCC cases and 10,977 controls (odds ratio [OR] = 0.85, 95 % confidence interval [CI] = 0.81– 0.89, P = 9.77 × 10−14). Further functional investigations revealed that PTPN23[Thr] variant binds to EGFR and modulates its phosphorylation at Thr699. PTPN23[Thr] variant substantially inhibited ESCC cell proliferation both in vitro and in vivo. Our findings underscore the critical role of PTPN23[Thr]-EGFR interaction in ESCC development, providing more insights into the pathogenesis of this cancer. Schematic illustration of the biological mechanism of PTPN23[Thr] variant affecting ESCC risk. The transition from rs6780013G > A induces an amino acid substitution from Alanine (Ala) to Threonine (Thr) at position 944 of the PTPN23 protein and enhance the phosphorylation of PTPN23. PTPN23 plays a crucial role in interacting with EGFR and facilitating its dephosphorylation. PTPN23[Thr] variant inhibits ESCC cell proliferation through diminishing EGFRThr669 phosphorylation, in line with the population-based findings indicating that the rs6780013[A] allele contributes to reduced ESCC susceptibility. [Display omitted] • PTM-related variants are enriched in risk loci for ESCC. • The PTPN23 rs6780013 variant shows a significant association with risk of ESCC. • PTPN23[Thr] variant interacts with EGFR, dephosphorylating it at Thr699. • PTPN23[Thr] variant plays a role in inhibiting the proliferation of ESCC cells. [ABSTRACT FROM AUTHOR]

Lysine-specific methyltransferase 7 (KMT7) SET7/9, aka Set7, Set9, or SetD7, or KMT5 was discovered 20 years ago, yet its biological role remains rather enigmatic. In this review, we analyze the particularities of SET7/9 enzymatic activity and substrate specificity with respect to its biological importance, mostly focusing on its two well-characterized biological functions: cellular proliferation and stress response. [ABSTRACT FROM AUTHOR]

The fragile X-related (FXR) family proteins FMRP, FXR1, and FXR2 are RNA binding proteins that play a critical role in RNA metabolism, neuronal plasticity, and muscle development. These proteins share significant homology in their protein domains, which are functionally and structurally similar to each other. FXR family members are known to play an essential role in causing fragile X mental retardation syndrome (FXS), the most common genetic form of autism spectrum disorder. Recent advances in our understanding of this family of proteins have occurred in tandem with discoveries of great importance to neurological disorders and cancer biology via the identification of their novel RNA and protein targets. Herein, we review the FXR family of proteins as they pertain to FXS, other mental illnesses, and cancer. We emphasize recent findings and analyses that suggest contrasting functions of this protein family in FXS and tumorigenesis based on their expression patterns in human tissues. Finally, we discuss current gaps in our knowledge regarding the FXR protein family and their role in FXS and cancer and suggest future studies to facilitate bench to bedside translation of the findings. [ABSTRACT FROM AUTHOR]

In essentially all eukaryotes, proteins can bemodified by the attachment of small ubiquitin-relatedmodifier (SUMO) proteins to lysine side chains to produce branched proteins. This process of 'SUMOylation' plays essential roles in plant and animal development by altering protein function in spatially and temporally controlled ways. In this Primer, we explain the process of SUMOylation and summarize how SUMOylation regulates a number of signal transduction pathways. Next, we discuss multiple roles of SUMOylation in the epigenetic control of transcription. In addition, we evaluate the role of SUMOylation in the etiology of neurodegenerative disorders, focusing on Parkinson's disease and cerebral ischemia. Finally, we discuss the possibility that SUMOylation may stimulate survival and neurogenesis of neuronal stem cells. [ABSTRACT FROM AUTHOR]

• Feeding grass carp with faba bean conferred them with "crisp" muscle texture. • "Crisp" grass carp muscle is characterized by substantial protein N -glycosylation. • N -glycosylated proteins participate in myogenesis, muscle function, & ECM formation. • N -glycosylation regulation is independent with phosphorylation regulation and protein expression. • These findings lay a foundation for optimizing fish fillet texture & quality. Post-translational protein modification affects muscle physiochemistry. To understand the roles of N- glycosylation in this process, the muscle N- glycoproteomes of crisp grass carp (CGC) and ordinary grass carp (GC) were analyzed and compared. We identified 325 N- glycosylated sites with the NxT motif, classified 177 proteins, and identified 10 upregulated and 19 downregulated differentially glycosylated proteins (DGPs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations revealed that these DGPs participate in myogenesis, extracellular matrix content formation, and muscle function. The DGPs partially accounted for the molecular mechanisms associated with the relatively smaller fiber diameter and higher collagen content observed in CGC. Though the DGPs diverged from the identified differentially phosphorylated proteins and differentially expressed proteins detected in previous study, they all shared similar metabolic and signaling pathways. Thus, they might independently alter fish muscle texture. Overall, the present study provides novel insights into the mechanisms underlying fillet quality. [ABSTRACT FROM AUTHOR]

We have shown for the first time that a natural protein (human insulin) can be acylated at the N‐terminus with a β‐amino acid (H‐β3hAla‐), in a process catalyzed by the β‐peptidyl aminopeptidase 3‐2W4‐BapA. This selective modification, which could also be applied for protein labeling and tagging, should be generally useful, also to protect peptides and proteins from attack by common aminopeptidases. [ABSTRACT FROM AUTHOR]

Histoplasma capsulatum , the causative agent of histoplasmosis (also called “Darling's disease”), can affect both immunocompetent and immunocompromised hosts. Post-translational protein modification by lysine succinylation (Ksuc) is a frequent occurrence in eukaryote and prokaryote. Recently, the roles of succinylation and its regulatory enzymes in regulating metabolic pathway in bacteria, mammalian and fungus were highlighted. Here, we report the first global profiling of lysine succinylation, with 463 modification sites in 202 proteins from H. capsulatum NAM1 identified, coupling immune-affinity enrichment using an anti-succinyllysine antibody with mass spectrometry. The bioinformatics results including GO functional and enrichment analysis showed that these succinylated proteins are mainly involved in central metabolism and protein synthesis, consistent with previous reports. 13 lysine succinylation sites on histones including H2A, H2B, H3 and H4 in H. capsulatum were firstly reported. The data is a good resource for further functional characterization of lysine succinylation in H. capsulatum . Biological significance H. capsulatum is the causative agent of lung disease histoplasmosis. The ability of H. capsulatum yeasts to infect and proliferate within macrophages as an intracellular pathogen can be contributed to several virulence factors and metabolic regulation. Lysine succinylation was recently shown to play a critical role in the metabolism regulation of Candida albicans . H. capsulatum succinylated proteins were firstly characterized in this work, and bioinformatics results showed that this modification may also be relevant with central metabolism in H. capsulatum . New succinylation sites on histones were reported. This represents an important resource to address the function of H. capsulatum lysine succinylation. [ABSTRACT FROM AUTHOR]

Retinal neurons use multiple strategies to fine-tune visual signal transduction, including post-translational modifications of proteins, such as addition of an isoprenyl lipid to a carboxyl-terminal cysteine in proteins that terminate with a "CAAX motif." We previously showed that RAS converting enzyme 1 (RCE1)-mediated processing of isoprenylated proteins is required for photoreceptor maintenance and function. However, it is not yet known whether the requirement for the RCE1-mediated protein processing is related to the absence of the endoproteolytic processing step, the absence of the subsequent methylation step by isoprenylcysteine methyltransferase (ICMT), or both. To approach this issue and to understand the significance of protein methylation, we generated mice lacking Icmt expression in the retina. In the absence of Icmt expression, rod and cone light-mediated responses diminished progressively. Lack of ICMT-mediated methylation led to defective association of isoprenylated transducin and cone phosphodiesterase 6 (PDE6α') with photoreceptor membranes and resulted in decreased levels of transducin, PDE6α', and cone G-protein coupled receptor kinase-1 (GRK1). In contrast to our earlier findings with retina-specific Reel knock-out mice, rod PDE6 in Icmt-deficient mice trafficked normally to the photoreceptor outer segment, suggesting that the failure to remove the -AAX is responsible for blocking the movement of PDE6 to the outer segment. Our findings demonstrate that carboxyl methylation of isoprenylated proteins is crucial for maintenance of photoreceptor function. [ABSTRACT FROM AUTHOR]

Minimally processed fruit products such as smoothies are increasingly coming into demand. However, they are often combined with dairy ingredients. In this combination, phenolic compounds, polyphenoloxidases, and amino compounds could interact. In this work, a model approach is presented where apple serves as a source for a high polyphenoloxidase activity for modulating the reactions. The polyphenoloxidase activity ranged from 128 to 333 nakt/mL in different apple varieties. From these, ‘Braeburn’ was found to provide the highest enzymatic activity. The formation and stability of resulting chromogenic conjugates was investigated. The results show that such adducts are not stable and possible degradation mechanisms leading to follow-up products formed are proposed. Finally, apple extracts were used to modify proteins and their functional properties characterized. There were retaining antioxidant properties inherent to phenolic compounds after adduct formation. Consequently, such interactions may also be utilized to improve the textural quality of food products. [ABSTRACT FROM AUTHOR]

The importance of the arginyl- tRNA protein transferase ( ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation ( NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes., We characterize ATE: GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology., ATE protein abundance is spatially and temporally regulated in P. patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss-of-function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants., Our analysis further substantiates the conservation of the N-end rule pathway components in land plants and highlights lineage-specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized in the brain by the formation of amyloid-beta (Aβ)-containing plaques and neurofibrillary tangles containing the microtubule-associated protein tau. Neuroinflammation is another feature of AD and astrocytes are receiving increasing attention as key contributors. Although some progress has been made, the molecular mechanisms underlying the pathophysiology of AD remain unclear. Interestingly, some of the main proteins involved in AD, including amyloid precursor protein (APP) and tau, have recently been shown to be SUMOylated. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to regulate APP and tau and may modulate other proteins implicated in AD. Here we present an overview of recent studies suggesting that protein SUMOylation might be involved in the underlying pathogenic mechanisms of AD and discuss how this could be exploited for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein-protein interactions ( PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine ( pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY- PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes. [ABSTRACT FROM AUTHOR]

The authors focus on a study related to the expression of the mouse Aos1-Uba2-fusion small ubiquitin-related modifier (SUMO)-activating enzyme mAU in a baculovirus-insect cell system. They inform that purification of a hexahistidine-tagged mouse fusion protein has revealed that 1.0 milligram of highly-purified His6-mAU has been obtained from 100 milliliter of Sf9 cell culture.

Summary [ABSTRACT FROM AUTHOR]

Cellular DNA repair is a frontline system that is responsible for maintaining genome integrity and thus preventing premature aging and cancer by repairing DNA lesions and strand breaks caused by endogenous and exogenous mutagens. However, it is also the principal cellular system in cancer cells that counteracts the killing effect of the major cancer treatments, e.g. chemotherapy and ionizing radiation. Although it is clear that an individual's DNA repair capacity varies, the mechanisms involved in the regulation of repair systems that are responsible for such variations are only just emerging. This knowledge gap is impeding the finding of new cancer therapy targets and the development of novel treatment strategies. In recent years the vital role of post-translational modifications of DNA repair proteins, including ubiquitylation and phosphorylation, has been uncovered. This review will cover recent progress in our understanding of the role of ubiquitylation in the regulation of DNA repair. [ABSTRACT FROM AUTHOR]

Small ubiquitin-related modifier (SUMO) is a member of the ubiquitin-related protein family. SUMO modulates protein function through covalent conjugation to lysine residues in a large number of proteins. Once covalently conjugated to a protein, SUMO often regulates that protein's function by recruiting other cellular proteins. Recruitment frequently involves a non-covalent interaction between SUMO and a SUMO-interacting motif (SIM) in the interacting protein. SIMs generally consist of a four-residue-long hydrophobic stretch of amino acids with aliphatic non-polar side chains flanked on one side by negatively charged amino acid residues. The SIM assumes an extended β-strand-like conformation and binds to a conserved hydrophobic groove in SUMO. In addition to hydrophobic interactions between the SIM non-polar core and hydrophobic residues in the groove, the negatively charged residues in the SIM make favorable electrostatic contacts with positively charged residues in and around the groove. The SIM/SUMO interaction can be regulated by the phosphorylation of residues adjacent to the SIM hydrophobic core, which provide additional negative charges for favorable electrostatic interaction with SUMO. The SUMO interactome consists of hundreds or perhaps thousands of SIM-containing proteins, but we do not fully understand how each SUMOylated protein selects the set of SIM-containing proteins appropriate to its function. SIM/SUMO interactions have critical functions in a large number of essential cellular processes including the formation of membraneless organelles by liquid–liquid phase separation, epigenetic regulation of transcription through histone modification, DNA repair, and a variety of host–pathogen interactions. [ABSTRACT FROM AUTHOR]

Transient cerebral ischemia/stroke activates various post-translational protein modifications such as phosphorylation and ubiquitin conjugation that are believed to play a major role in the pathological process triggered by an interruption of blood supply and culminating in cell death. A new system of post-translational protein modification has been identified, termed as small ubiquitin-like modifier (SUMO) conjugation. Like ubiquitin, SUMO is conjugated to the lysine residue of target proteins in a complex process. This review summarizes observations from recent experiments focusing on the effect of cerebral ischemia on SUMO conjugation. Transient global and focal cerebral ischemia both induced a rapid, dramatic and long-lasting rise in levels of SUMO2/3 conjugation. After transient focal cerebral ischemia, SUMO conjugation was particularly prominent in neurons located at the border of the ischemic territory where SUMO-conjugated proteins translocated to the nucleus. Many SUMO conjugation target proteins are transcription factors and sumoylation has been shown to have a major impact on the activity, stability, and cellular localization of target proteins. The rise in levels of SUMO-conjugated proteins is therefore likely to have a major effect on the fate of post-ischemic neurons. The sumoylation process could provide an exciting new target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Modification of cellular proteins by the small ubiquitin-like modifier SUMO is important in regulating various cellular events. Many different nuclear proteins are targeted by SUMO, and the functional consequences of this modification are diverse. For most proteins, however, the functional and structural consequences of modification by specific SUMO isomers are unclear. Conjugation of SUMO to thymine-DNA glycosylase (TDG) induces the dissociation of TDG from its product DNA. Structure determination of the TDG central region conjugated to SUMO-1 previously suggested a mechanism in which the SUMOylation-induced conformational change in the C-terminal region of TDG releases TDG from tight binding to its product DNA. Here, we have determined the crystal structure of the central region of TDG conjugated to SUMO-3. The overall structure of SUMO-3-conjugated TDG is similar to the previously reported structure of TDG conjugated to SUMO-1, despite the relatively low level of amino acid sequence similarity between SUMO-3 and SUMO-1. The two structures revealed that the sequence of TDG that resembles the SUMO-binding motif (SBM) can form an intermolecular β-sheet with either SUMO-1 or SUMO-3. Structural comparison with the canonical SBM shows that this SBM-like sequence of TDG retains all of the characteristic interactions of the SBM, indicating sequence diversity in the SBM. [Copyright &y& Elsevier]

Peptidylarginine deiminases (PADs) convert arginine residues in proteins into citrullines. They are suspected to be involved in multiple sclerosis and rheumatoid arthritis pathophysiology, and they play a role in epidermis homeostasis and possibly in regulation of gene expression through histone modification. In humans, four isoforms encoded by the genes PADI1–4 are known so far. We here report the characterization and comparative analysis of the human (355 kb) and mouse (240 kb) PAD gene clusters on chromosomes 1p35–36 and 4E1, respectively. We characterized an as yet unknown human PADI6 gene, and cloned the corresponding cDNA encoding a 694-amino-acid protein. RT-PCR analysis showed a rather restricted pattern of tissue-specific expression, mainly in ovary, testis and peripheral blood leukocytes. Nucleotide substitution rates suggest that PADI genes are under purifying selection. Comparative analysis of the human and mouse sequences identified 251 conserved non-coding segments predominantly clustered within the promoter regions, the large (>10 kb) first intron of each of the genes PADI1–3, and an 8 kb PADI1–2 intergenic region. The presence of numerous transcription factor binding sites suggests the segments are putative regulatory elements. This study is the first description of the human PADI6 gene and encoded protein, and the first step towards a better understanding of the coordinated regulation of PADI gene expression. [Copyright &y& Elsevier]

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron system involvement, and is epidemiologically subclassified into sporadic, familial and endemic forms. About 20% of ALS families are associated with mutations in the gene for superoxide dismutase-1 (SOD1) encoded on chromosome 21q22.1. Several studies have pointed to a variety of functions of mutant SOD1, which has enhanced catalytic activity of the peroxynitrite-mediated tyrosine nitration, readily releases the reactive Cu ions, induces apoptotic cell death, has enhanced peroxidase activity, damages the mitochondria to release Ca[sup 2+], SOD1-containing aggregates in the cytoplasm. Many of these studies have obtained evidence for increased oxidative damage in ALS. On the other hand, some reports disagree with oxidative damage involvement in SOD1 mutant ALS. In considering the findings of increased oxidative damage in mutant SOD1- expressing transgenic mice, it should be remembered that overexpression of mutant SOD1 may enhance oxidative stress generation from this enzyme. In this review, we present the clinicopathological features of SOD1 mutant familial ALS and its transgenic mouse model, and also discuss SOD1 mutation-related neurotoxicity, including SOD1 protein aggreand forms gation and post-translational protein modification. (ALS 2000; 1:143-161). [ABSTRACT FROM AUTHOR]

Investigating a protein of interest that runs at the same molecular weight as antibody heavy chain is a frequent deterrent to its evaluation by immunoprecipitation. Methods of minimizing the detection of the immunoprecipitating antibody are available. However, these still present a barrier to evaluating if intracellular proteins are modified by the O-GlcNAc post-translation protein modification due to interfering glycosylation on antibodies. IdeZ protease specifically cleaves antibody at the hinge region, allowing collapse of the antibody fragments to 25 kDa after denaturation. Thus, this proteolytic method uniquely allows evaluation of O-GlcNAcylation of proteins of interest formerly obscured by antibody heavy chain. Image 1 • Adding IdeZ protease to immunoprecipitated proteins cleaves antibody heavy chain. • Formerly obscured ~50 kDa proteins and presence of O-GlcNAcylation can be observed. • Conveniently, only the addition of a single, short incubation step is required. [ABSTRACT FROM AUTHOR]

The article discusses the role of SUMOylation in Magnaporthe oryzae pathogenicity. Topics discussed include issues relating to the rice blast; information on the biology of rice blast infection; and the how maintenance of the biotrophic phase of infection is achieved along with the changes in reactive oxygen species.

DNA replication differs from most other processes in biology in that any error will irreversibly change the nature of the cellular progeny. DNA replication initiation, therefore, is exquisitely controlled. Deregulation of this control can result in over-replication characterized by repeated initiation events at the same replication origin. Over-replication induces DNA damage and causes genomic instability. The principal mechanism counteracting over-replication in eukaryotes is a division of replication initiation into two steps—licensing and firing—which are temporally separated and occur at distinct cell cycle phases. Here, we review this temporal replication control with a specific focus on mechanisms ensuring the faultless transition between licensing and firing phases. [ABSTRACT FROM AUTHOR]