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Cellular prion protein (PrP C ) is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. The mature cell-surface PrP C is internalized and subsequently degraded by lysosomes. Although, proteasomes are proposed to be involved, the precise mechanism of PrP C degradation remains uncertain. Given that proteins are ubiquitinated primarily on lysine residues, we sought to determine whether lysine residues within PrP C are involved in the ubiquitination and subsequent degradation of PrP C . We generated a plasmid vector expressing a mutant PrP C (called lysine-null PrP C ) in which all lysine residues were replaced with arginine residues. Subsequently, we established stably transformed cell lines (designated HpL2-1 PrP-WT and HpL2-1 PrP-K/R, respectively) using the mouse PrP C -deficient neuronal cell line (HpL2-1) and plasmids expressing wild-type (WT) or lysine-null PrP C (PrP-K/R). We found that HpL2-1 PrP-WT and HpL2-1 PrP-K/R cells correctly expressed their respective PrP C which translocated efficiently to the plasma membrane. Subsequently, using immunoblotting and confocal microscopy, we found that treatment with cycloheximide (CHX; a protein synthesis inhibitor) significantly reduced PrP C expression in both these transformed cell lines, indicating that WT and lysine-null PrP C are degraded similarly. Taken together, these results indicate that the lysine residues of PrP C do not regulate its degradation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cationic polymers that bind with the plasmids to form polyplexes protect the DNA from enzymatic degradation and improve cellular and tissue uptake. Complete or near complete gel retardation of the polyplex is an important assay to determine the optimal polymer: plasmid ratio for in vitro and in vivo studies. Nevertheless, despite minimal to moderate gel retardation of histidine-lysine (HK) polyplexes formed with low peptide: plasmid DNA ratios (1:2 and 1:4; w:w), the polyplexes effectively targeted the tumor in vivo. To understand the lack of predictability of the initial gel mobility shift assays, we revisited the retardation and stability of polyplexes with these electrophoresis assays. Because the histidine component with a pKa of about 6.0 will have a greater positive charge and may bind plasmids with a higher affinity at lower pHs, we compared the retardation of the two HK polyplexes when the pH of the running buffer of the gel mobility shift assay was altered. Both HK polyplexes were retarded significantly more when the running buffer had a pH of 7.3 instead of the standard pH of 8.3. Indeed, the HK polyplexes formed at the 1:2 ratio showed complete retardation at pH 7.3. Consequently, while both HK polyplexes formed at these low ratios targeted the tumor, the polyplex formed with the 1:2 ratio had reduced tumor gene expression variability and lower lung and liver values. Thus, the selection of the optimal ratios for the linear HK and plasmid for transfection studies in vivo was improved with a running buffer pH of 7.3., Competing Interests: Declaration of competing interest The authors of this manuscript declare that there are no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

HDAC6 has been reported as a deacetylase of p53 at multiple lysine residues, associated with the canonical functions of p53, such as apoptosis and tumor suppression. We have previously reported that p53 acetylation at the lysine 320 site accumulates due to the genetic ablation of HDAC6 in mice liver. However, the biological processes affected by K320 acetylation of p53 are yet to be elucidated. In this study, we demonstrate that K320 acetylation of p53 is regulated by HDAC6 deacetylase activity. HDAC6 knockout mouse brains exhibit a significant accumulation of K320 acetylated p53 compared to other tissues. The level of K320 acetylation of p53 inversely correlates with the level of BNIP3, a direct target of p53 and essential for mitophagy. Notably, overexpressing the deacetylation mimic K320R mutant p53 restored BNIP3 expression in HDAC6 knockout MEFs. Furthermore, we observed that neurons are particularly susceptible to the genetic ablation of HDAC6, impacting BNIP3 expression, which inversely correlates with the accumulation of abnormal mitochondria characterized by swollen cristae. Our findings suggest that HDAC6 plays a crucial role in maintaining BNIP3 expression by deacetylating p53 at the K320 site, which is linked to the structural integrity of mitochondria., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Joo-Yong Lee reports financial support was provided by National Research Foundation of Korea, Ministry of Health and Welfare, Republic of Korea, and Chungnam National University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

ASH2L (Absent-Small-Homeotic-2-Like protein) is a core subunit of the COMPASS (COMplex of Proteins ASsociated with Set1) complex, the most notable writer of the methylation of histone H3 lysine 4 (H3K4). The COMPASS complex regulates active promoters or enhancers for gene expression, and its dysfunction is associated with aberrant development and disease. Here, we demonstrated that ASH2L mediated the cell invasion and migration activity of triple-negative breast cancer cells through the interaction with the COMPASS components and the target genomic regions. Transcriptome analysis indicated a potential correlation between ASH2L and the genes involved in inflammatory/immune responses. Among them, we found that the intrinsic expression of IL1B (interleukin 1 beta), an essential proinflammatory gene, was directly regulated by ASH2L. These results revealed a novel role of ASH2L on the maintenance of breast cancer malignancy possibly through H3K4 methylation of the target inflammatory/immune responsive genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Oxpecker, the homolog of Rhino/HP1D, exclusively expressed in Drosophila ovaries, belongs to the Heterochromatin Protein 1 family, as does Rhino. Rhi recognizes piRNA clusters enriched with the heterochromatin marker H3K9me3 via its N-terminal chromodomain and recruits Deadlock via its C-terminal chromoshadow domain, further recruits Moonshiner, a paralog of the TATA box-binding protein-related factor 2 large subunits, to promote transcription of piRNA precursors, thereby protecting the genome. Despite Oxp possessing only the chromodomain, its loss leads to the upregulation of transposons in the female germline. In this study, we solved the crystal structure of the Oxp chromodomain in complex with the histone H3K9me3 peptide. As the Oxp chromodomain dimerizes, two H3K9me3 peptides bind to the Oxp chromodomain in an antiparallel manner. ITC experiments and site-directed mutagenesis experiments showed that E44 determines Oxp's five-fold stronger binding ability to H3K9me3 than that of Rhi. In addition, we found that Oxp and Rhi can form a heterodimer, which may shed light on the molecular mechanism by which Oxp regulates transposon silencing in the absence of CSD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Given our previous finding that certain tumor-suppressing functions of p53 are exerted by the p53/p21 complex, rather than p53 alone, cells may have a system to regulate the p53/p21 interaction. As p53 binds to p21 via its C-terminal domain, which contains acetylable lysine residues, we investigated whether the C-terminal acetylation of p53 influences the p53/p21 interaction. Indeed, the p53/p21 interaction was reduced when various types of cells (HCT116 colon cancer, A549 lung cancer, and MCF7 breast cancer cells) were treated with MS-275, an inhibitor of SIRT1 (a p53 deacetylase), or with SIRT1-targeting small interfering RNAs. These treatments also increased the acetylation levels of the five lysine residues (K370, K372, K373, K381, K382) in the C-terminal domain of p53. The p53/p21 interaction was also reduced when these lysine residues were substituted with glutamine (an acetylation memetic), but not arginine (an unacetylable lysine analog). While the inhibitory effect of the lysine-to-glutamine substitution was evident upon the substitution of all the five lysine residues, the substitution of only two (K381, K382) or three residues (K370, K372, K373) was less effective. Consistently, the five substitutions reduced the ability of p53 to regulate cell invasion and death by liberating Bax from Bcl-w. Overall, our data suggest that the acetylation, especially the hyperacetylation, of the p53 C-terminal domain suppresses the p53/p21-complex-dependent functions of p53 by inhibiting the p53/p21 interaction. We propose that cellular components involved in the acetylation or deacetylation of the p53 C-terminus are critical regulators of the formation of p53/p21 complex., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histone deacetylase 6 (HDAC6) is known to deacetylate amino acid lysine in alpha-tubulin. However, the functional role of HDAC6 in the progression of cardiac disease remains uncertain. The functional role of HDAC6 in the hearts was examined using transgenic (TG) mice expressing either human wild-type HDAC6, deacetylase inactive HDAC6 (HDAC6 H216A, H611A ), and human HDAC6 replaced all serine or threonine residues with aspartic acid at N-terminal 1- 43 amino acids (HDAC6 NT-allD ) specifically in the hearts. Overexpression of wild-type HDAC6 significantly reduced acetylated tubulin levels, and overexpression of HDAC6 H216A, H611A significantly increased it in the mouse hearts. Detectable acetylated tubulin disappeared in HDAC6 NT-allD TG mouse hearts. Neither histological alteration nor alteration of cardiac function was observed in the HDAC6 TG mouse hearts. To analyze the role of HDAC6 and acetylated tubulin in disease conditions, we examined HDAC6 in isoprenaline-induced hypertrophy or pressure-overload hypertrophy (TAC). No obvious alteration in the heart weight/body weight ratio or gene expressions of hypertrophic markers between NTG and HDAC6 NT-allD mice was observed following treatment with isoprenaline. In contrast, a marked reduction in the shortening fraction and dilated chamber dilatation was detected in the HDAC6 NT-allD TG mouse hearts 2 weeks after TAC. A sustained low level of acetylated tubulin and acetylated cortactin was observed in the TAC HDAC6 NT-allD TG mouse hearts. Cardiac HDAC6 activity that can regulate acetylated levels of tubulin and cortactin may be critical factors involved in cardiac disease such as pressure-overload hypertrophy., Competing Interests: Declaration of conflicting interest None., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone lysine crotonylation (Kcr) is a novel hydrophobic histone acylation modification, and we recently report its crucial roles in neural differentiation. However, it is still unclear how histone Kcr involve in early neural commitment. Here, we systematically investigate the H3K9cr landscapes during neuroectodermal differentiation of pluripotent P19 embryonal carcinoma cells (ECCs). We reveal that the genome-wide changes in H3K9cr favor neural fate specification, and identify potential co-factors binding H3K9cr. We also uncover that H3K9cr collaborates with H3K9ac to regulate gene expression changes. Our results provide novel insights into the epigenetic mechanisms underlying neural commitment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Although post-translational modifications (-PTMs) of some histone H3 lysine residues are well studied, the PTMs of histone H3 lysine 37 in mammalian cells remain largely unknown. In this study, we provide evidence to show that SMYD family member 5 (SMYD5) is a histone H3-specfic methyltransferase that catalyzes mono-methylation of H3 lysine 36 and 37 (H3K36/K37me1) in vitro. The site-mutagenesis analysis shows that a species-conserved histidine in its catalytic SET domain is required for its histone methyltransferase activity. Genetic deletion of Smyd5 in murine embryonic stem cells (mESCs) partially reduces the global histone H3K37me1 level in cells, suggesting SMYD5 is one of histone methyltransferases catalyzing histone H3K37me1 in vivo. Hence, our study reveals that SMYD5 is a histone H3-specific methyltransferase that mediates histone H3K36/K37me1, which provides a biochemical basis for further studying its functions in mammalian cells., Competing Interests: Declaration of competing interest Authors declares that they have no conflict of interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteocytes are master regulators of skeletal homeostasis. However, little is known about the molecular mechanism of their differentiation. Epigenetic regulations, especially H3K27me3 modification, play critical roles in cell differentiation. Here, we found that H3K27me3 in the loci of osteocyte-expressing genes decreased during osteocyte differentiation and that H3K27me3 demethylase, Utx, was bound to the loci of those genes. To investigate the physiological functions of Utx in vivo, we generated late osteoblast-to-osteocyte specific Utx knockout mice using Dmp1-cre mice (Utx ΔOcy/ΔOcy ). Micro CT analyses showed that Utx ΔOcy/ΔOcy displayed osteopenic phenotypes with lower bone volume and trabecular number, and greater trabecular separation. Bone histomorphometric analysis showed that bone mineralization and formation were significantly lower in Utx ΔOcy/ΔOcy . Furthermore, Dmp1 expression and the number of osteocytes were significantly decreased in Utx ΔOcy/ΔOcy . These results suggest that Utx in Dmp1-expressing osteoblast/osteocyte positively regulates osteoblast-to-osteocyte differentiation through H3K27me3 modifications in osteocyte genes. Our results provide new insight into the molecular mechanism of osteocyte differentiation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lysine crotonylation (Kcr) is a newly identified protein translational modification and is involved in major biological processes including glycolysis, but its role in colorectal cancer (CRC) is unknown. Here, we found that the Kcr of α enolase (ENO1) was significantly elevated in human CRC tissues compared with the paratumoral tissues. CREB-binding protein (CBP) functioned as a crotonyltranferase of ENO1, and SIRT2 was involved in the decrotonylation of ENO1. Using quantitative mass spectrometry for crotonylomics analysis, we further found that K420 was the main Kcr site of ENO1 and ENO1 K420 Kcr promoted the growth, migration, and invasion of CRC cells in vitro by enhancing the activity of ENO1 and regulating the expression of tumor-associated genes. Our study reveals an important mechanism by which ENO1 regulates CRC through crotonylation., Competing Interests: Declaration of competing interest The authors declare there is no conflict of interests., (Copyright © 2021. Published by Elsevier Inc.)

The NSD proteins, namely NSD1, NSD2 and NSD3, are lysine methyltransferases, which catalyze mono- and di-methylation of histone H3K36. They are multi-domain proteins, including two PWWP domains (PWWP1 and PWWP2) separated by some other domains. These proteins act as potent oncoproteins and are implicated in various cancers. However the biological functions of these PWWP domains are still largely unknown. To better understand the functions of these proteins' PWWP domains, we cloned, expressed and purified all the PWWP domains of these NSD proteins to characterize their interactions with methylated histone peptides and dsDNA by quantitative binding assays and crystallographic analysis. Our studies indicate that all these PWWP domains except NSD1_PWWP1 bind to trimethylated H3K36, H3K79 peptides and dsDNA weakly. Our crystal structures uncover that the NDS3_PWWP2 and NSD2_PWWP1 domains, which hold an extremely long α-helix and α-helix bundle, respectively, need a conformation adjustment to interact with nucleosome., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Reversible acylation of lysine ε-amino groups, e.g., acetylation, succinylation, maronylation, and myristoylation, is involved in basic physiological processes such as metabolism, cell signaling and aging. In this study, we developed a novel enrichment method for acylated peptides without the use of antibodies, in which endogenously acylated peptides are deacylated by recombinant lysine deacylases based on the enzyme-substrate relationship and enriched by N-hydroxysuccinimidyl chemistry for identification of the acylated sites by nanoscale liquid chromatography-tandem mass spectrometric analysis. To demonstrate the validity of this acylomics platform, we used it to identify acylated sites on chemically acylated model protein samples. We also applied it to the nuclei of HeLa cells to identify endogenous acylated sites., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes not only canonical (noncyclic) but also cyclic K48-linked ubiquitin chains. Although the cyclic conformation is expected to repress molecular recognition by ubiquitin binding proteins due to restricting the flexibility of the ubiquitin subunits in a chain, multiple proteins are reported to associate with cyclic ubiquitin chains similar to noncyclic chains. However, the molecular mechanism of how cyclic ubiquitin chains are recognized remains unclear. Here we investigated the effect of cyclization on ubiquitin-chain cleavage and molecular recognition by a K48-linkage specific deubiquitinating enzyme OTUB1 for cyclic diubiquitin by NMR spectroscopic analyses. Compared to noncyclic diubiquitin, we observed slow but unambiguously detectable cleavage of cyclic diubiquitin to monoubiquitin by OTUB1. Intriguingly, upon ubiquitin chain cleavage, cyclic diubiquitin appeared to alter its "autoinhibited" conformation to an incompletely but partially accessible conformation, induced by interaction with OTUB1 via the ubiquitin-subunit specific recognition patches and adjacent surfaces. These data imply that cyclic ubiquitin chains may exist stably in cells in spite of the presence of deubiquitinating enzymes and that these chains can be recognized by intracellular proteins in a manner distinct from that of noncyclic ubiquitin chains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Protein lysine propionylation (Kpr) modification is a novel post-translational modification (PTM) of prokaryotic cells that was recently discovered; however, it is not clear how this modification regulates bacterial life. In this study, the protein Kpr modification profile in Aeromonas hydrophila was identified by high specificity antibody-based affinity enrichment combined with high resolution LC MS/MS. A total of 98 lysine-propionylated peptides with 59 Kpr proteins were identified, most of which were associated with energy metabolism, transcription and translation processes. To further understand the role of Kpr modified proteins, the K168 site on malate dehydrogenase (MDH) and K608 site on acetyl-coenzyme A synthetase (AcsA) were subjected to site-directed mutation to arginine (R) and glutamine (Q) to simulate deacylation and propionylation, respectively. Subsequent measurement of the enzymatic activity showed that the K168 site of Kpr modification on MDH may negatively regulate the MDH enzymatic activity while also affecting the survival of mdh derivatives when using glucose as the carbon source, whereas Kpr modification of K608 of AcsA does not. Overall, the results of this study indicate that protein Kpr modification plays an important role in bacterial biological functions, especially those involved in the activity of metabolic enzymes., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aspirin can efficiently inhibit the glycolysis and proliferation of cancer cells, however, the underlying mechanism is poorly understood. Here, we report that aspirin attenuates the glycolysis and proliferation of hepatoma cells through modulating the levels of lysine 2-hydroxyisobutyrylation (Khib) of enolase 1 (ENO1). We found that aspirin decreased the levels of glucose consumption and lactate production in hepatoma cells. Moreover, 4 mM aspirin reduced the activities of ENO1, a key enzyme of glycolysis, and decreased the levels of ENO1 Khib in the cells. Interestingly, we identified that 4 mM aspirin could decrease the levels of Khib on many proteins by using pan Khib antibody in the cells. Interestingly, the activities of ENO1 could be rescued by the transient overexpression of ENO1, but not by ENO1 mutant (K281R). Moreover, we identified that the C646, an inhibitor of p300 which is a writer of Khib, could reduce the levels of ENO1 Khib, resulting in the decrease of ENO1 activities. The treatment with PDTC, an inhibitor of NF-κB which is a target of aspirin, could work well as C646 in the cells. Both of aspirin and C646 (or PDTC) displayed a stronger effect than the single treatment in the system. Functionally, ENO1, but not ENO1 mutant (K281R), could rescue the aspirin-induced inhibition of proliferation of liver cancer cells in vitro, suggesting that ENO1K281 is involved in the aspirin-mediated inhibition of liver cancer. Our finding provides new insights into the mechanism by which aspirin attenuates the glycolysis and proliferation of hepatoma cells., Competing Interests: Declaration of competing interest Ying Yuan, Hong-feng Yuan and Yu Geng performed experiments, and analyzed the data. Li-na Zhao, Hao-lin Yun, Yu-fei Wang contributed to the interpretation of the results. Xiao-dong Zhang designed, conceived, wrote the manuscript, and supervised the study. All authors read and approved the manuscript., (Copyright © 2021. Published by Elsevier Inc.)

Glucosyl-galactosyl-hydroxylation (GGH) is one type of post-translational modification, which is mainly observed in collagen-like domain-containing proteins. Using LC-MS/MS analysis, we found a GGH-like modification at Lys65 of fibrinogen-like protein 1 (FGL1), although it does not contain a collagen-like domain. To identify the glycosyltransferases responsible for this modification, we established LH3/GLT25D1-knockout FGL1-overexpressing HT1080 cell lines. The result showed that knockout of LH3 or GLT25D1 significantly inhibited the glycosylation. Furthermore, deficiency of GGH by point mutation of the FGL1 protein or knockout of the GGH-related glycosyltransferase reduced FGL1 protein levels. Taken together, these data indicate that Lys65 of FGL1 is glucosyl-galactosyl-hydroxylated by LH3 and GLT25D1. Our results provide novel insights to regulate various FGL1 functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background and Aims: Hypercholesterolemia is characterized by the elevation of plasma total cholesterol level, especially low-density lipoprotein (LDL) cholesterol. This disease is usually caused by a mutation in genes such as LDL receptor, apolipoprotein B, or proprotein convertase subtilisin/kexin type 9. However, a considerable number of patients with hypercholesterolemia do not have any mutation in these candidate genes. In this study, we examined the difference in the metabolic level between patients with hypercholesterolemia and healthy subjects, and screened the potential biomarkers for this disease., Methods: Analysis of plasma metabolomics in hypercholesterolemia patients and healthy controls was performed by gas chromatography-mass spectrometry and metabolic correlation networks were constructed using Gephi-0.9.2., Results: First, metabolic profile analysis confirmed the distinct metabolic footprints between the patients and the healthy ones. The potential biomarkers screened by orthogonal partial least-squares discrimination analysis included l-lactic acid, cholesterol, phosphoric acid, d-glucose, urea, and d-allose (Variable importance in the projection > 1). Second, arginine and methionine metabolism were significantly perturbed in hypercholesterolemia patients. Finally, we identified that l-lactic acid, l-lysine, l-glutamine, and l-cysteine had high scores of centrality parameters in the metabolic correlation network., Conclusion: Plasma l-lactic acid could be used as a sensitive biomarker for hypercholesterolemia. In addition, arginine biosynthesis and cysteine and methionine metabolism were profoundly altered in patients with hypercholesterolemia., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

High mobility group A2 (HMGA2) is a chromatin-associated protein involved in the regulation of stem cell function, embryogenesis and cancer development. Although the protein does not contain a consensus SUMOylation site, it is shown to be SUMOylated. In this study, we demonstrate that the first lysine residue in the reported K 66 KAE SUMOylation motif in HMGA2 can be methylated in vitro and in vivo by the Set7/9 methyltransferase. By editing the lysine, the increased hydrophobicity of the resulting 6-N-methyl-lysine transforms the sequence into a consensus SUMO motif. This post-translational editing dramatically increases the subsequent SUMOylation of this site. Furthermore, similar putative methylation-dependent SUMO motifs are found in a number of other chromatin factors, and we confirm methylation-dependent SUMOylation of a site in one such protein, the Polyhomeotic complex 1 homolog (PHC1). Together, these results suggest that crosstalk between methylation and SUMOylation is a general mode for regulation of chromatin function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ribosomal protein synthesis is a central process of the modern biological world. Because the ribosome contains proteins itself, it is very important to understand its precursor and evolution. Small ribozymes have demonstrated the principle of "RNA world" hypothesis, but protein free peptide ligase remains elusive. In this report, we have identified two fragments in the peptidyl transfer center that can synthesize a 9-mer poly-lysine in a solution contains Mg 2+ . This result is deduced from isotope-shifting in high resolution MS. To our best knowledge, this is the longest peptide oligo that can be synthesized by a pure ribozyme. Via single molecule FRET experiments, we have demonstrated the ligase mechanism was probably by substrate proximity via dimerization. We prospect that these RNA fragments can be useful to synthesize template free natural and non-natural peptides, to be model system for peptidyl transfer reaction mechanism and can shed light to the evolution of ribosome., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Reacted with methylglyoxal (MGO), murine Aβ(1-40) (mAβ) produced significantly less superoxide anion (O 2 •- ) compared to human Aβ (1-40) (hAβ). The reactions of MGO with mAβ(R13H), hAβ(H13F), N α -acetyl-l-lysine, and N α -acetyl-l-arginine implied that the lack of His13 in mAβ prohibits its Lys16 residue from reacting to produce cross-linked reaction products and O 2 •- . Our results suggest that murine brains are under less oxidative stress than human brains, which may be one of the reasons why rodents do not develop AD-like symptoms, and which provides further insight into a chemical mechanism for the development of AD in humans., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanistic target of rapamycincomplex 1 (mTORC1) integrates various environmental signals to regulate cell growth and metabolism. mTORC1 activity is sensitive to changes in amino acid levels. Here, we investigated the effect of lysine on mTORC1 activity in non-small cell lung cancer (NSCLC) cells. Lysine deprivation suppressed mTORC1 activity and lysine replenishment restored the decreased mTORC1 activity in lysine-deprived cells. Supplementing growth factors, such as insulin growth factor-1 or insulin restored the decreased mTORC1 activity in serum-deprived cells. However, in serum/lysine-deprived cells, supplementing growth factors was not sufficient to restore mTORC1 activity, suggesting thatgrowth factors could not activate mTORC1 efficiently in the absence of lysine. General control nonderepressible 2 and AMP-activated protein kinase were involved in lysine deprivation-mediated inhibition of mTORC1. Taken together, these results suggest that lysine might play role in the regulation of mTORC1 activation in NSCLC cells., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mitochondria play a central role in biological oxidation that inevitably generates reactive oxygen species (ROS) as by-products. Maintenance of mitochondrial redox balance status requires NADPH, which is primarily generated by the mitochondrial matrix protein isocitrate dehydrogenase 2 (IDH2). The activity of IDH2 is regulated by post-translational modifications (PTMs). In this study, we found IDH2 is modified by small ubiquitin-like modifier 1 (SUMO1) at lysine 45. SUMO specific protease 1 (SENP1) is responsible for deSUMOylation of IDH2. SUMOylation of IDH2 is induced by oxidants and enhances the antioxidant activity of IDH2 to protect cells against oxidative stress. Mutation of the SUMOylation site impairs the enzymatic activity of IDH2 and hence decreases levels of α-ketoglutarate (α-KG), NADPH and GSH. Cells with SUMOylation deficient IDH2 suffer more apoptosis than that with wild type IDH2 under oxidative stress. These results indicate that SUMOylation is an important way to regulate IDH2 activity to maintain mitochondrial redox balance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Despite significant process in ubiquitin modification by using traditional genetic methods, chemical small molecules that directly target and modify ubiquitin are little reported. Here, we find that a fluorescigenic pyrazoline derivative (FPD5) could do so effectively. Molecule docking revealed that lysine 11 of ubiquitin was the key contact residue. FPD5, with stronger fluorescence, elevated the ubiquitination of beclin 1 (BECN1) and promoted autophagy. This study highlights that targeting ubiquitin by chemical small molecules enables us to modulate ubiquitination and the downstream signaling in the ubiquitin system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2020 Elsevier Inc. All rights reserved.)

The outbreak of corona virus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is spreading globally and quickly, leading to emerging health issues. SARS-CoV-2 enters into and infects host cells through its spike glycoprotein recognizing the cell receptor Angiotensin-converting enzyme II (ACE2). Here, we noticed that ACE2 was further enhanced by SARS-CoV-2 infection. Human germ cells and early embryos express high level of ACE2. Notably, RNA-seq result showed that reduction of H3K27me3, but not H3K4/9/36me3, led to upregulation of Ace2 expression in mouse germ cell line GC-2. In agreement with this result, we found in human embryonic stem cells that ACE2 expression was significantly increased in absence of EZH2, the major enzyme catalyzing H3K27me3. ChIP-seq analysis further confirmed decrease of H3K27me3 signal and increase of H3K27ac signal at ACE2 promoter upon EZH2 knockout. Therefore, we propose that EZH2-mediated H3K27me3 at ACE2 promoter region inhibits ACE2 expression in mammalian cells. This regulatory pattern may also exist in other human cells and tissues. Our discovery provides clues for pathogenesis and targeted drug therapy towards ACE2 expression for prevention and adjuvant therapy of COVID-19., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Lysine-specific methyltransferase Set7/9 (KMT7) belongs to the SET domain family of proteins. Besides the SET domain, Set7/9 also contains a so-called MORN (Membrane Occupation and Recognition Nexus) domain whose function in high eukaryotes is largely unknown. Set7/9 has been shown to specifically methylate both histones H1 and H3 as well as a number of non-histone substrates, including p53, E2F1, RelA, AR, and other important transcription factors. However, despite the ever growing list of potential substrates of Set7/9, the question of its substrate specificity is still debatable. To gain a better understanding of the Set7/9 substrate specificity and to clarify the importance of structural domains of Set7/9 for protein-protein interactions (PPIs) we determined interactomes for both MORN and SET domains of Set7/9 by pull-down assay coupled with mass-spectrometry. Importantly, we demonstrated that most of PPIs of Set7/9 are mediated via its MORN domain. The latter has preference towards positively charged amino acids that are often found in RNA-binding proteins. One of the Set7/9-interacting proteins was identified as Sam68, an RNA splicing protein with a KH (heterogeneous nuclear ribonucleoprotein K (hnRNP K) homology) domain. Importantly, the RG-rich domain of Sam68 that is also present in many splicing factors was found to interact with Set7/9. We revealed that Set7/9 not only co-immunoprecipitated with Sam68, but also methylated the latter on K208. Functionally, knockout of Set7/9 decreased the protein level of Sam68 in cells resulting in altered regulation of cell cycle and apoptosis. Finally, the bioinformatics analysis established a correlation between the high levels of Sam68/Set7/9 co-expression and better survival rates of patients with colon cancer., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive occlusion of the internal carotid arteries. Genetic studies originally identified RNF213 as an MMD susceptibility gene that encodes a large 591 kDa protein with a functional RING domain and dual AAA+ ATPase domains. As the functions of RNF213 and its relationship to MMD onset are unknown, we set out to characterize the ubiquitin ligase activity of RNF213, and the effects of MMD patient mutations on these activities and on other cellular processes. In vitro ubiquitination assays, using the RNF213 RING domain, identified Ubc13/Uev1A as a key ubiquitin conjugating enzyme that together generate K63-linked polyubiquitin chains. However, nearly all MMD patient mutations in the RING domain greatly reduced this activity. When full-length proteins were overexpressed in HEK293T cells, patient mutations that abolished the ubiquitin ligase activities conversely enhanced nuclear factor κB (NFκB) activation and induced apoptosis accompanied with Caspase-3 activation. These induced activities were dependent on the RNF213 AAA+ domain. Our results suggest that the NFκB- and apoptosis-inducing functions of RNF213 may be negatively regulated by its ubiquitin ligase activity and that disruption of this regulation could contribute towards MMD onset., Competing Interests: Declaration of competing interest Dr. Akio Koizumi has a patent for RNF213 (P130009545), which does not alter adherence to all BBRC policies on sharing data and materials. Other authors declare no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cereblon (CRBN) was identified as a gene that causes intellectual disabilities. The encoded CRBN protein, containing 442 amino acids, is located in several organs. Cytosolic CRBN was reported to mainly act as a component of the E3 ubiquitin ligase complex. CRBN is one of the substrate receptors of the E3 ubiquitin ligase complex and promotes the degradation of targeted proteins. Studies have reported that CRBN recognizes the C-terminal region of the amyloid precursor protein (APP), a protein known for its involvement in the development of Alzheimer's disease. Although CRBN may interact with the C-terminal region of APP in mice, the CRBN-mediated degradation mechanism of human APP remains unclear. Here, we analyzed the CRBN-mediated degradation mechanism of human APP via the ubiquitin-proteasome system. Immunoprecipitation experiments showed that CRBN interacts with human full-length APP via its C-terminal region. Next, we examined CRBN-mediated degradation of APP in the ubiquitin-proteasome system. CRBN recognizes Lys 751 in human APP and ubiquitinates it in SH-SY5Y cells. Overexpression of CRBN decreased wild-type APP expression levels. In contrast, the expression level of K751R APP remained unchanged by CRBN overexpression, while knockdown of endogenous CRBN increased APP levels. As such, our results suggest that CRBN ubiquitinates Lys 751 of human APP thereby degrading it via the ubiquitin-proteasome system., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hepatic ischemia-reperfusion (IR) injury can cause serious liver damage, leading to liver dysfunction after liver surgery, which is associated with NF-κB-mediated inflammation. The K63-linked auto-polyubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB. Here, we found that OTU domain-containing protein 4 (OTUD4), a deubiquitinating enzyme (DUB), interacts with TRAF6 and decreases the K63 auto-polyubiquitination of TRAF6. In addition, the data showed that NF-κB activation was impaired and inflammatory factor levels were reduced after overexpressing OTUD4 in a hypoxia/reoxygenation (HR) model and a hepatic IR model. Additionally, the liver inflammatory response and tissue damage were ameliorated in mice overexpressing OTUD4.Taken together, these results show that OTUD4 can negatively regulate NF-κB activation by suppressing the K63-linked ubiquitination of TRAF6, thus alleviating hepatic ischemia-reperfusion injury., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues., Aim: We sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin., Methods: We analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of β-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM β-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer., Results: Eleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with β-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with β-d-glucose or ASA. Incubation with concomitant ASA and β-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM β-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM β-d-glucose was associated the absence of acetylation at that site., Conclusion: Human α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Enhancers can regulate gene transcription from afar. Many enhancers are located in genes. Although the regulatory roles of several individual intragenic enhancers have been elaborated, a genome-wide insight into intragenic enhancers remains to be elucidated. We found that active intragenic enhancers have a preference for being located in expressed genes. Unlike intergenic enhancers, active intragenic enhancers are enriched of H3K79me2 epigenetic signal, and depleted of variant histone H2A.Z. Moreover, eRNAs of active intragenic enhancers show lower degradation rates than those of the other enhancers. Our findings will have implications in understanding functions of intragenic enhancers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Single-nucleotide polymorphisms (SNPs) identified by Genome-Wide Association Studies (GWASs) have been determined to closely connect with multiple diseases. Previous studies revealed one underlying mechanism that SNPs located within the regulatory elements could affect the encoding gene expression through long-range regulation. SNP rs6854845 was suggested to be a risk of colon cancer in human population. Nevertheless, the underlying molecular mechanism for colon carcinogenesis remains largely unknown. In present study, rs6854845 with G > T mutation in situ in FHC, HCT-116 and SW-480 cells were generated by Crispr/Cas9. The nearby chromatin organization was investigated by chromatin conformation capture (3C). And the expression of coding gene regulated by super-enhancer (SE) was detected by real-time PCR. We observed a significantly different pattern of the genome organization upon rs6854845 generation in colon epithelial cells but not in colon cancer cells. Moreover, we observed the shifted enrichment of H3K4me1 and H3K27ac at the SE (chr4:75.7M-76.0 M) where rs6854845 located. Furthermore, we observed that the transcription of the gene clusters regulated by SE were affected by rs6854845 in colon cells. Overall, our results demonstrated that SNP rs6854845 located in SE could destroy the long-range chromosomal interaction between SE and target gene clusters thereby affecting the transcription of these genes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The bestrophin family comprises well-known Ca 2+ -activated chloride channels (CaCC) that are expressed in a variety tissues including the brain, eye, gastrointestinal tract, and muscle tissues. Among the family members, bestrophin-1 (BEST1) is known to exist mainly in retinal pigment epithelium cells, but we recently reported that BEST1 mediates Ca 2+ -activated Cl - currents in hippocampal astrocytes. Despite its critical roles in physiological processes, including tonic γ-aminobutyric acid release and glutamate transport, the mechanisms that regulate BEST1 are poorly understood. In this study, we identified NEDD4L (NEDD4-2), an E3 ubiquitin ligase, as a binding partner of BEST1. A series of deletion constructs revealed that the WW3-4 domains of NEDD4L were important for interaction with BEST1. We observed that BEST1 underwent ubiquitin-dependent proteolysis and found that the conserved lysine370 residue in the C-terminus of BEST1 was important for its ubiquitination. Finally, we demonstrated that NEDD4L inhibited whole cell currents mediated by BEST1 but not by the BEST1(K370R) mutant. Collectively, our data demonstrated that NEDD4L played a critical role in regulating the surface expression of BEST1 by promoting its internalization and degradation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein-protein interaction, including protein homo-oligomerization, is commonly believed to occur through a specific interface made of a limited number of amino acid residues. Here our systematic in vivo photo-crosslinking analysis via genetically incorporated unnatural amino acids unexpectedly shows that the dimerization of HdeA, an acid stress chaperone, is mediated by the residues along its whole polypeptide. These include those "forbidden" residues that are far away from the dimerization interface as judged according to the reported 3-D structure. We demonstrate that such dimerization, though intriguing, is neither a result of protein over-expression nor of any structural disturbance caused by the residue replacement. Similar unexpected dimerization also occurs for two other oligomeric proteins, IbpB (a molecular chaperone existing as polydispersed oligomers in vitro) and DegP (a protease existing as hexamers in vitro). In contrast to these three proteins, dimerization of a few other oligomeric proteins (e.g., OmpF, LamB, SurA, FtsZ and FkpA) that we similarly examined in living cells seems to be mediated only by specific residues. Together, our unexpected observations suggest that, for some oligomeric proteins such as HdeA, IbpB and DegP, their subunit interactions in living cells can also be mediated by residues other than those located at the interfaces as revealed by in vitro structure determination. Our observations might be partially explained by the formation of "encounter complex" or by protein conformational dynamics. Our findings provide new insights on understanding protein-protein interactions and encounter complex formation in living cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Virus-induced signaling adaptor (VISA), which mediates the production of type I interferon, is crucial for the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes double-stranded viral RNA and interacts with VISA to mediate antiviral innate immunity. However, the mechanisms underlying RIG/VISA-mediated antiviral regulation remain unclear. In this study, we confirmed that receptor for activated C kinase 1 (RACK1) interacts with VISA and attenuates the RIG/VISA-mediated antiviral innate immune signaling pathway. Overexpression of RACK1 inhibited the interferon-β (IFN-β) promoter; interferon-stimulated response element (ISRE); nuclear factor kappa B (NF-κB) activation; and dimerization of interferon regulatory factor 3 (IRF3) mediated by RIG-I, VISA, and TANK-binding kinase 1 (TBK1). A reduction in RACK1 expression level upon small interfering RNA knockdown increased RIG/VISA-mediated antiviral transduction. Additionally, RACK1 disrupted formation of the VISA-tumor necrosis factor receptor-associated factor 2 (TRAF2), VISA-TRAF3, and VISA-TRAF6 complexes during RIG-I/VISA-mediated signal transduction. Additionally, RACK1 enhanced K48-linked ubiquitination of VISA, attenuated its K63-linked ubiquitination, and decreased VISA-mediated antiviral signal transduction. Together, these results indicate that RACK1 interacts with VISA to repress downstream signaling and downregulates virus-induced IFN-β production in the RIG-I/VISA signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

STAM-binding protein, STAMBP, is a JAMM-family deubiquitinating enzyme containing the microtubule-interacting/transport domain and STAM-binding domain. Although the biological importance of STAMBP in development has been recognized because the microcephaly-capillary malformation syndrome in human is caused by its somatic mutations, the role of STAMBP in cancer has not yet been determined. In this study, we demonstrate that STAMBP is a key molecule for regulating melanoma migration and invasion, but not survival, by knocking down STAMBP in vitro. STAMBP regulates SLUG expression through a post-transcriptional mechanism to control protein stability and further contributes to the in vivo metastatic potential of melanoma. Collectively, these results indicate the importance of STAMBP in melanoma metastasis by regulating SLUG. It is therefore a potential therapeutic target., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ubiquitin-conjugating enzymes (E2) form thioester bonds with ubiquitin (Ub), which are subsequently transferred to target proteins for cellular progress. Ube2K/E2-25K (a class II E2 enzyme) contains a C-terminal ubiquitin-associated (UBA) domain that has been suggested to control ubiquitin recognition, dimerization, or poly-ubiquitin chain formation. Ube2K is a special E2 because it synthesizes K48-linked poly-ubiquitin chains without E3 ubiquitin ligase. We found that a novel interaction between the acceptor di-Ub (Ub 2 ) and the auxiliary Ube2K promotes the discharging reaction and production of tri-Ub (Ub 3 ), probably by guiding and positioning the K48 (in the distal Ub) of the acceptor Ub 2 in the active site. We also determined the crystal structure of Ube2K-Ub 2 at 2.47 Å resolution. Based on our structural and biochemical data, we proposed a structural model of Ub 3 synthesis by Ube2K without E3., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Prion diseases are fatal neurodegenerative diseases associated with structural conversion of α-helical prion protein (PrP) into its β-sheet rich isoform (PrP Sc ). Previous genetic analyses have indicated that several amino acid residues involved in the hydrophobic core of PrP (such as V180, F198, and V210) play a critical role in the development of prion diseases. To understand how these hydrophobic residues would contribute to the α-to-β conversion process of PrP, we substituted the V210 residue with bulkier (V210F, V210I, and V210L), smaller (V210A), and charged amino acids (V210K) and characterized its effects. Interestingly, although most of the mutations had little or no effect on the biochemical properties of PrP, the V210K mutation induced structural conversion of PrP into a β-structure. The β-inducing effect was prominent and observed even under a physiological condition (i.e., in the absence of denaturant, acidic pH, reducing agent, and high temperature) in contrast to the disease-associated mutations in the PrP gene. We also examined structural features of V210K PrP using guanidine-hydrochloride unfolding, dynamic light scattering, 8-anilino-1-naphthalene sulfonate fluorescence, and electron microscopy, and revealed that V210K PrP assembles into a non-fibrillar β-rich oligomer. Thus, the α-to-β conversion can be induced by introduction of a charged residue into the hydrophobic core, which provide novel insight into the structural dynamics of PrP., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dihydropyrimidinase (DHPase) is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase (DHOase), hydantoinase, and imidase. Almost all of these zinc metalloenzymes possess a binuclear metal center in which two metal ions are bridged by a post-translational carbamylated Lys. Crystal structure of Tetraodon nigroviridis DHPase reveals that one zinc ion is sufficient to stabilize Lys carbamylation. In this study, we found that one metal coordination was not sufficient to fix CO 2 to the Lys in bacterial DHPase. We prepared and characterized mono-Zn DHPase from Pseudomonas aeruginosa (PaDHPase), and the catalytic activity of mono-Zn PaDHPase was not detected. The crystal structure of mono-Zn PaDHPase determined at 2.23 Å resolution (PDB entry 6AJD) revealed that Lys150 was no longer carbamylated. This finding indicated the decarbamylation of the Lys during the metal chelating process. To confirm the state of Lys carbamylation in mono-Zn PaDHPase in solution, mass spectrometric (MS) analysis was carried out. The MS result was in agreement with the theoretical value for uncarbamylated PaDHPase. Crystal structure of the human DHOase domain (huDHOase) K1556A mutant was also determined (PDB entry 5YNZ), and the structure revealed that the active site of huDHOase K1556A mutant contained one metal ion. Like mono-Zn PaDHPase, oxygen ligands of the carbamylated Lys were not required for Znα binding. Considering the collective data from X-ray crystal structure and MS analysis, mono-Zn PaDHPase in both crystalline state and solution was not carbamylated. In addition, structural evidences indicated that post-translational carbamylated Lys was not required for Znα binding in PaDHPase and in huDHOase., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Acetylation of histones and other proteins plays crucial roles in transcriptional regulation, chromatin organization, and other biological processes. It has been recently reported that the nucleoprotein (NP) of influenza virus is acetylated in infected cells, and this modification contributes to the RNA polymerization activity of the virus. As the influenza virus, the Ebolavirus contains single-stranded negative-sense RNA as its viral genome, which interacts with NP and other viral proteins. In this study, we performed a series of biochemical experiments and revealed that the recombinant Ebolavirus NP and the viral matrix protein VP40, which binds with NP, were acetylated by eukaryotic histone acetyltransferases, such as P300/CREB-binding protein (P300/CBP) and P300/CBP-associated factor (PCAF), in vitro. Mass spectrometry was used to identify the lysine residues that were potential acetylation targets in NP and VP40. The identified lysine residues in NP were located in the RNA-binding cleft and the VP35-binding domain. Potentially acetylated lysine targets in VP40 were identified in the basic patch, which is necessary for constructing oligomers. These results suggest that the acetylation of these lysine residues is involved in the interactions between viral proteins., (Copyright © 2018 Elsevier Inc. All rights reserved.)

FAM122A is a highly conserved protein in mammals. Here, we identify that FAM122A can be sumoylated at lysine 89, which can be de-conjugated by SENP1. Furthermore, the sumoylation of FAM122A reduces the PP2A-Cα protein level together with the reduced phosphatase activity of PP2A, which suppresses cell proliferation. Collectively, our results suggest that the sumoylation of FAM122A may have a significant role in cellular function., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Krüppel-like factor 5 (KLF5) is a basic transcription factor that regulates diverse cellular processes during tumor development. Acetylation of KLF5 at lysine 369 (K369) reverses its function from promoting to suppressing cell proliferation and tumor growth. In this study, we examined the regulation of KLF5 by histone deacetylases in the prostate cancer cell line DU 145. While confirming the functions of HDAC1/2 in KLF5 deacetylation and the promotion of cell proliferation, we found that the knockdown of HDAC1/2 upregulated KLF5 protein but not KLF5 mRNA, and the increase in KLF5 protein level by silencing HDAC1/2 was at least in part due to decreased proteasomal degradation. Deacetylase activity was required for HDAC1/2-mediated KLF5 degradation, and mutation of KLF5 to an acetylation-mimicking form prevented its degradation, even though the mutation did not affect the binding of KLF5 with HDAC1/2. Mutation of K369 to arginine, which prevents acetylation, did not affect the binding of KLF5 to HDAC1 or the response of KLF5 to HDAC1/2-promoted degradation. These findings provide a novel mechanistic association between the acetylation status of KLF5 and its protein stability. They also suggest that maintaining KLF5 in a deacetylated form may be an important mechanism by which KLF5 and HDACs promote cell proliferation and tumor growth., (Published by Elsevier Inc.)

Plant abiotic resistance in cultivated species features limited variability. Using genes of wild species serves as a valid approach for improving abiotic resistance of cultivated plants. In this study, we uncovered a previously uncharacterized proline-, lysine-, and glutamic-rich protein gene (SpPKE1), which was isolated from drought-resistant wild tomato species Solanum pennellii (LA0716). When M82, which is a drought-sensitive tomato cultivar, was engineered to overexpress SpPKE1, its tolerance under drought stress was significantly improved by the accumulation of more chlorophyll, proline, and limited malondialdehyde compared with that in RNA interference (RNAi)-suppression lines, which were more sensitive than the wild-type plants. Several ion transporter genes, abiotic-related transcriptional factors, and reactive oxygen species-scavenging genes were upregulated in PKE1 overexpression (OE) lines but downregulated in RNAi plants. OE of SpPKE1 enhanced drought tolerance in tobacco. Screening results of yeast two-hybrid protein-protein interaction revealed that SpPKE1 can bind to an F-box protein that plays an important role in plant drought resistance. We posited that PKE1 enhanced drought tolerance by modulating the expressions of stress-responsive genes and interacting with the F-box protein., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Describing the regulation of chromatin segments by protein recognition events constitute a major goal in biology and biotechnology. Despite astonishing experimental developments, achieving nearly atomistic spatial/temporal resolution on such macromolecular systems remains a big challenge owing to the intrinsic flexibility of large biological assemblies. Although computer simulations have become a reliable complement to experimental techniques, computational cost limits their routine applications to relatively small systems. However, the development of accurate and cost-effective coarse-grained (CG) models helps to bridge the gap between molecular dynamics simulations and biologically relevant scales. Performing an exhaustive search on a set of well-resolved crystallographic protein-DNA complexes, we introduced improvements on the CG SIRAH force field to describe protein-DNA interfaces. Modifications were validated against a set of non redundant structures and applied to the simulation of the longest DNA segment in complex with proteins that has been crystallized to date, i.e. a tetranucleosome. Multimicrosecond simulation of this small chromatin segment evidences a large mobility of the external DNA filaments, which is consistent with results from FRET experiments in solution. Moreover, we found that the sub-microsecond dynamics of DNA is strongly modulated by the quaternary structure, partially overcoming the intrinsic dynamics dictated by the primary structure., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Zscan4 is an early embryonic gene cluster expressed in mouse embryonic stem and induced pluripotent stem cells where it plays critical roles in genomic stability, telomere maintenance, and pluripotency. Zscan4 expression is transient, and characterized by infrequent high expression peaks that are quickly down-regulated, suggesting its expression is tightly controlled. However, little is known about the protein degradation pathway responsible for regulating the human ZSCAN4 protein levels. In this study we determine for the first time the ZSCAN4 protein half-life and degradation pathway, including key factors involved in the process, responsible for the regulation of ZSCAN4 stability. We demonstrate lysine 48 specific polyubiquitination and subsequent proteasome dependent degradation of ZSCAN4, which may explain how this key factor is efficiently cleared from the cells. Importantly, our data indicate an interaction between ZSCAN4 and the E3 ubiquitin ligase RNF20. Moreover, our results show that RNF20 depletion by gene knockdown does not affect ZSCAN4 transcription levels, but instead results in increased ZSCAN4 protein levels. Further, RNF20 depletion stabilizes the ZSCAN4 protein half-life, suggesting that RNF20 negatively regulates ZSCAN4 stability. Due to the significant cellular functions of ZSCAN4, our results have important implications in telomere regulation, stem cell biology, and cancer., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Our previous study showed that the level of glutamate carboxypeptidase II (GCPII) protein is regulated by valproic acid, a histone deacetylase (HDAC) inhibitor, through acetylation of lysine residue in the GCPII protein in human astrocytes, U-87MG. The present study further investigated which HDAC subtype is involved in the acetylation of GCPII. The results revealed that GCPII interacted with HDAC1 but not with HDAC2, HDAC3, HDAC4, HDAC5, and HDAC6. Overexpression of catalytic domain (1-56 aa)-deleted HDAC1, which poorly binds to GCPII, enhanced lysine acetylation in GCPII and increased the level of GCPII protein when compared with that of the wild-type HDAC1. Further experiments showed that HDAC1 regulated the stability of GCPII protein. These data suggest that acetylation of GCPII is facilitated by HDAC1, and the acetylated GCPII is more stable than the non-acetylated GCPII. Additional experiments using siRNA HDAC1 and by HDAC1 overexpression confirmed the role of HDAC1 in regulating the stability of GCPII protein. Further, database search of acetylation and ubiquitination sites showed four candidate lysine sites in human GCPII protein that can be both acetylated and ubiquitinylated (K207, K479, K491, and K699). Mutation (lysine residues to arginine (R)) analysis showed that in the presence of cycloheximide K479R- and K491R-hGCPII mutants were less ubiquitinylated and degraded, and decrease in the level of GCPII protein by HDAC1 was significantly blocked by K479R mutants. These data suggest that K479 is a possible site of acetylation or ubiquitination. Furthermore, the results also demonstrate that the stability of GCPII protein is regulated by HDAC1 through acetylation at the lysine 479 residue., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ubiquitination of target proteins is accomplished by isopeptide bond formation between the carboxy group of the C-terminal glycine (Gly) residue of ubiquitin (Ub) and the ɛ-amino group of lysine (Lys) on the target proteins. The formation of an isopeptide bond between Ubs that gives rise to a poly-Ub chain on the target proteins and the types of poly-Ub chains formed depend on which of the seven Lys residues or N-terminal methionine (Met) residue on Ub is used for chain elongation. To understand the linkage specificity mechanism of Ub chains on E3, the previous study established an assay to monitor the formation of a free diubiquitin chain (Ub 2 chain synthesis assay) by HECT type E3 ligase. In this study, we investigated Ub 2 chain specificity using E6AP HECT domain. We here demonstrate the importance of the N-terminal domain of full length E6AP for Ub 2 chain specificity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The cluster of differentiation 36 (CD36) is implicated in the intake of long-chain fatty acids and fat storage in various cell types including the pancreatic beta cell, thus contributing to the pathogenesis of metabolic stress and diabetes. Recent evidence indicates that CD36 undergoes post-translational modifications such as acetylation-deacetylation. However, putative roles of such modifications in its functional activation and onset of beta cell dysregulation under the duress of glucolipotoxicity (GLT) remain largely unknown. Using pharmacological approaches, we validated, herein, the hypothesis that acetylation-deacetylation signaling steps are involved in CD36-mediated lipid accumulation and downstream apoptotic signaling in pancreatic beta (INS-1832/13) cells under GLT. Exposure of these cells to GLT resulted in significant lipid accumulation without affecting the CD36 expression. Sulfo-n-succinimidyl oleate (SSO), an irreversible inhibitor of CD36, significantly attenuated lipid accumulation under GLT conditions, thus implicating CD36 in this metabolic step. Furthermore, trichostatin A (TSA) or valproic acid (VPA), known inhibitors of lysine deacetylases, markedly suppressed GLT-associated lipid accumulation with no discernible effects on CD36 expression. Lastly, SSO or TSA prevented caspase 3 activation in INS-1832/13 cells exposed to GLT conditions. Based on these findings, we conclude that an acetylation-deacetylation signaling step might regulate CD36 functional activity and subsequent lipid accumulation and caspase 3 activation in pancreatic beta cells exposed to GLT conditions. Identification of specific lysine deacetylases that control CD36 function should provide novel clues for the prevention of beta-cell dysfunction under GLT., (Published by Elsevier Inc.)