Multiple Lysine Residues Research Articles

Epigenetic Regulation of CXC Chemokine Expression by Environmental Electrophiles Through DNA Methyltransferase Inhibition.

Ubiquitously distributed environmental electrophiles covalently modify DNA and proteins, potentially leading to adverse health effects. However, the impacts of specific electrophiles on target proteins and their physiological roles remain largely unknown. In the present study, we focused on DNA methylation, which regulates gene expression and physiological responses. A total of 45 environmental electrophiles were screened for inhibitory effects on the activity of DNA methyltransferase 3B (DNMT3B), a key enzyme in DNA methylation, and four compounds were identified. We focused on 1,2-naphthoquinone (1,2-NQ), an air pollutant whose toxicity has been reported previously. Interestingly, we found that 1,2-NQ modified multiple lysine and histidine residues in DNMT3B, one of which was near the active site in DNMT3B. It was found that 1,2-NQ altered gene expression and evoked inflammatory responses in lung adenocarcinoma cell lines. Furthermore, we found that 1,2-NQ upregulated CXCL8 expression through DNA demethylation of the distal enhancer and promoted cancer cell growth. Our study reveals novel mechanisms of epigenetic regulation by environmental electrophiles through the inhibition of DNMT3B activity and suggests their physiological impact.

Uncovering the Profile of Ubiquitination Motif in Catalytic Proteins using Sequence Contextual Features

Ubiquitin molecules modify their target protein by conjugating the amino group of lysine side chain, thereby regulating the activity and function of most intracellular proteins. However, within multiple lysine residues on a target protein, only particular residues can be modified by ubiquitin molecules. The occurrence of ubiquitination varies on different lysine residues. The ubiquitination of a lysine residue is ubiquitinated depends on the environment of amino acid sequences near the lysine residue; this sequence is called a motif. Although the interaction of motifs with ubiquitin molecules have been extensively studied, there have been no reports on the ubiquitination motifs. In the current study, 133878 ubiquitinated peptide data from 27762 proteins in 30 articles and the mUbiSiDa database in the past decade were collected and analyzed. A total of 208 motifs were found with a score above 6, covering most of the ubiquitination peptides. Of these, six motifs with higher scores and significant regularity were found to belong to zinc finger proteins and serine/threonine kinases, termed zinc finger and serine/threonine kinase motifs, respectively. Analysis on four zinc finger motifs revealed that the ubiquitination sites in zinc finger motifs, xxHxxxxxxE_K_xxxCxxCxxx and xxxKxxHxxx_K_xxxxxExxKx, were located between two zinc finger domains, while the ubiquitination sites in zinc finger motifs, xxxxxxxxCG_K_xFxxxxxxxx and CxxxxxxFxQ_K_xxxxxxxxxx were located within zinc chelation structure near one Cys residue. Different proteins contain different types and quantities of zinc finger motifs. The results of the 3D structure showed that the ubiquitination sites in motif CxxxxxxFxQ_K_xxxxxxxxxx were located in the ring structure of the zinc finger domain. This may affect the interaction between zinc finger domains and DNA. Meanwhile, the ubiquitination sites of the other three zinc finger motifs located outside the interaction structure between zinc finger domains and DNA may not serve to block interaction but degradation. The analysis of two serine/threonine kinase motifs revealed that some residues near the ubiquitination site in two serine/threonine kinase motifs were completely identical in different proteins; however, these conserved residues were completely different in different motifs, suggesting the varied function of the two motifs. Moreover, it was found that no protein simultaneously contains both serine/threonine kinase motifs. Although there were significant differences in the amino acid sequences of these motifs, their spatial structures were highly conserved. These results suggest that the regulatory functions of the two motifs may differ. This is because two motifs may be recognized by different ubiquitinases or deubiquitinases. Overall, these results suggest that the motifs of ubiquitination sites exhibit high regularity and special structural characteristics in different types of proteins. This study sheds light on the profile and regularity of ubiquitination motifs, promoting the prediction of the function, structure, and modified sites of ubiquitinated proteins.

Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy

ABSTRACT Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB. Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: L egionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin.

The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

DUSP22 is a dual-specificity phosphatase that inhibits T cell activation by inactivating the kinase Lck. Here we show that the E3 ubiquitin ligase UBR2 is a positive upstream regulator of Lck during T-cell activation. DUSP22 dephosphorylates UBR2 at specific Serine residues, leading to ubiquitin-mediated UBR2 degradation. UBR2 is also modified by the SCF E3 ubiquitin ligase complex via Lys48-linked ubiquitination at multiple Lysine residues. Single-cell RNA sequencing analysis and UBR2 loss of function experiments showed that UBR2 is a positive regulator of proinflammatory cytokine expression. Mechanistically, UBR2 induces Lys63-linked ubiquitination of Lck at Lys99 and Lys276 residues, followed by Lck Tyr394 phosphorylation and activation as part of TCR signalling. Inflammatory phenotypes induced by TCR-triggered Lck activation or knocking out DUSP22, are attenuated by genomic deletion of UBR2. UBR2-Lck interaction and Lck Lys63-linked ubiquitination are induced in the peripheral blood T cells of human SLE patients, which demonstrate the relevance of the UBR2-mediated regulation of inflammation to human pathology. In summary, we show here an important regulatory mechanism of T cell activation, which finetunes the balance between T cell response and aggravated inflammation.

Non-natural sialic acid derivatives with o-nitrobenzyl alcohol substituents for light-mediated protein conjugation and cell imaging.

We have synthesized two sialic acid derivatives substituted with an ortho-nitrobenzyl alcohol (o-NBA) group that can undergo light-mediated conjugation with primary amines at their 5- or 9-carbon position. The o-NBA derivatives were shown to react with multiple lysine residues of human serum albumin (HSA) when exposed to 365 nm light irradiation within 10 min. The resulting sugar conjugates were characterized by mass spectroscopy and used for fluorescence-based cell imaging.

Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease

In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.

HDAC6 preserves BNIP3 expression and mitochondrial integrity by deacetylating p53 at lysine 320

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.

New Insights into the Complement Receptor of the Ig Superfamily Obtained from Structural and Functional Studies on Two Mutants.

The extracellular region of the complement receptor of the Ig superfamily (CRIg) binds to certain C3 cleavage products (C3b, iC3b, C3c) and inhibits the alternative pathway (AP) of complement. In this study, we provide further insight into the CRIg protein and describe two CRIg mutants that lack multiple lysine residues as a means of facilitating chemical modifications of the protein. Structural analyses confirmed preservation of the native CRIg architecture in both mutants. In contrast to earlier reports suggesting that CRIg binds to C3b with an affinity of ∼1 μM, we found that wild-type CRIg binds to C3b and iC3b with affinities <100 nM, but to C3c with an affinity closer to 1 μM. We observed this same trend for both lysine substitution mutants, albeit with an apparent ∼2- to 3-fold loss of affinity when compared with wild-type CRIg. Using flow cytometry, we confirmed binding to C3 fragment-opsonized Staphylococcus aureus cells by each mutant, again with an ∼2- to 3-fold decrease when compared with wild-type. Whereas wild-type CRIg inhibits AP-driven lysis of rabbit erythrocytes with an IC50 of 1.6 μM, we observed an ∼3-fold reduction in inhibition for both mutants. Interestingly, we found that amine-reactive crosslinking of the CRIg mutant containing only a single lysine results in a significant improvement in inhibitory potency across all concentrations examined when compared with the unmodified mutant, but in a manner sensitive to the length of the crosslinker. Collectively, our findings provide new insights into the CRIg protein and suggest an approach for engineering increasingly potent CRIg-based inhibitors of the AP.

Acetylation-dependent coupling between G6PD activity and apoptotic signaling

Lysine acetylation has been discovered in thousands of non-histone human proteins, including most metabolic enzymes. Deciphering the functions of acetylation is key to understanding how metabolic cues mediate metabolic enzyme regulation and cellular signaling. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is acetylated on multiple lysine residues. Using site-specifically acetylated G6PD, we show that acetylation can activate (AcK89) and inhibit (AcK403) G6PD. Acetylation-dependent inactivation is explained by structural studies showing distortion of the dimeric structure and active site of G6PD. We provide evidence for acetylation-dependent K95/97 ubiquitylation of G6PD and Y503 phosphorylation, as well as interaction with p53 and induction of early apoptotic events. Notably, we found that the acetylation of a single lysine residue coordinates diverse acetylation-dependent processes. Our data provide an example of the complex roles of acetylation as a posttranslational modification that orchestrates the regulation of enzymatic activity, posttranslational modifications, and apoptotic signaling.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

Src family kinases engage differential pathways for encapsulation into extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.

Structure of coagulation factor VIII bound to a patient-derived anti–C1 domain antibody inhibitor

AbstractThe development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in all cases of acquired hemophilia A. KM33 is an anti–C1 domain antibody inhibitor previously isolated from a patient with severe hemophilia A. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, via single-particle cryo-electron microscopy. Structural analysis revealed that the NB33 epitope localizes to the FVIII residues R2090-S2094 and I2158-R2159, which constitute membrane-binding loops in the C1 domain. Further analysis revealed that multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence for engineering FVIII with reduced LRP1–mediated clearance.

Tau Lysine Pseudomethylation Regulates Microtubule Binding and Enhances Prion-like Tau Aggregation.

Alzheimer's disease (AD) and frontotemporal dementia (FTD) can be classified as tauopathies, which are a group of neurodegenerative diseases that develop toxic tau aggregates in specific brain regions. These pathological tau inclusions are altered by various post-translational modifications (PTMs) that include phosphorylation, acetylation, and methylation. Tau methylation has emerged as a target of interest for its potential involvement in tau pathomechanisms. Filamentous tau aggregates isolated from patients with AD are methylated at multiple lysine residues, although the exact methyltransferases have not been identified. One strategy to study the site-specific effects of methylation is to create methylation mimetics using a KFC model, which replaces lysine (K) with a hydrophobic group such as phenylalanine (F) to approximate the effects of lysine methylation (C or methyl group). In this study, tau methylmimetics were used to model several functional aspects of tau methylation such as effects on microtubule binding and tau aggregation in cell models. Overall, several tau methylmimetics displayed impaired microtubule binding, and tau methylmimetics enhanced prion-like seeded aggregation in the context of the FTD tau mutation P301L. Like other PTMs, tau methylation is a contributing factor to tau pathogenesis and could be a potential therapeutic drug target for the treatment of different tauopathies.

The roles of serine hydrolases and serum albumin in alisol B 23-acetate hydrolysis in humans.

Introduction: Alisol B 23-acetate (AB23A), a major bioactive constituent in the Chinese herb Zexie (Rhizoma Alismatis), has been found with multiple pharmacological activities. AB23A can be readily hydrolyzed to alisol B in mammals, but the hydrolytic pathways of AB23A in humans and the key enzymes responsible for AB23A hydrolysis are still unrevealed. This study aims to reveal the metabolic organs and the crucial enzymes responsible for AB23A hydrolysis in human biological systems, as well as to decipher the impact of AB23A hydrolysis on its biological effects. Methods: The hydrolytic pathways of AB23A in human plasma and tissue preparations were carefully investigated by using Q-Exactive quadrupole-Orbitrap mass spectrometer and LC-UV, while the key enzymes responsible for AB23A hydrolysis were studied via performing a set of assays including reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses. Finally, the agonist effects of both AB23A and its hydrolytic metabolite(s) on FXR were tested at the cellular level. Results: AB23A could be readily hydrolyzed to form alisol B in human plasma, intestinal and hepatic preparations, while human butyrylcholinesterase (hBchE) and human carboxylesterases played key roles in AB23A hydrolysis in human plasma and tissue preparations, respectively. It was also found that human serum albumin (hSA) could catalyze AB23A hydrolysis, while multiple lysine residues of hSA were covalently modified by AB23A, suggesting that hSA catalyzed AB23A hydrolysis via its pseudo-esterase activity. Biological tests revealed that both AB23A and alisol B exhibited similar FXR agonist effects, indicating AB23A hydrolysis did not affect its FXR agonist effect. Discussion: This study deciphers the hydrolytic pathways of AB23A in human biological systems, which is very helpful for deep understanding of the metabolic rates of AB23A in humans, and useful for developing novel prodrugs of alisol B with desirable pharmacokinetic behaviors.

E3 ligase Cul2 mediates Drosophila early germ cell differentiation through targeting Bam

Drosophila ovary has been one of the most mature and excellent systems for studying the in vivo regulatory mechanisms of stem cell fate determination. It has been well-known that the bone morphogenetic protein (BMP) signaling released by the niche cells promotes the maintenance of germline stem cells (GSCs) through inhibiting the transcription of the bag-of-marbles (bam) gene, which encodes a key factor for GSC differentiation. However, whether Bam is regulated at the post-translational level remains largely unknown. Here we show that the E3 ligase Cullin-2 (Cul2) is involved in modulating Bam ubiquitination, which occurs probably at multiple lysine residues of Bam's C-terminal region. Genetic evidence further supports the notion that Cul2-mediated Bam ubiquitination and turnover are essential for GSC maintenance and proper germline development. Collectively, our data not only uncovers a novel regulatory mechanism by which Bam is controlled at the post-translational level, but also provides new insights into how Cullin family protein determines the differentiation fate of early germ cells.

The R3-Type MYB Transcription Factor BrMYBL2.1 Negatively Regulates Anthocyanin Biosynthesis in Chinese Cabbage (Brassica rapa L.) by Repressing MYB–bHLH–WD40 Complex Activity

Chinese cabbage (Brassica rapa L.) leaves are purple in color due to anthocyanin accumulation and have nutritional and aesthetic value, as well as antioxidant properties. Here, we identified the R3 MYB transcription factor BrMYBL2.1 as a key negative regulator of anthocyanin biosynthesis. A Chinese cabbage cultivar with green leaves harbored a functional BrMYBL2.1 protein, designated BrMYBL2.1-G, with transcriptional repressor activity of anthocyanin biosynthetic genes. By contrast, BrMYBL2.1 from a Chinese cabbage cultivar with purple leaves carried a poly(A) insertion in the third exon of the gene, resulting in the insertion of multiple lysine residues in the predicted protein, designated BrMYBL2.1-P. Although both BrMYBL2.1 variants localized to the nucleus, only BrMYBL2.1-G interacted with its cognate partner BrTT8. Transient infiltration assays in tobacco leaves revealed that BrMYBL2.1-G, but not BrMYBL2.1-P, actively represses pigment accumulation by inhibiting the transcription of anthocyanin biosynthetic genes. Transient promoter activation assay in Arabidopsis protoplasts verified that BrMYBL2.1-G, but not BrMYBL2.1-P, can repress transcriptional activation of BrCHS and BrDFR, which was activated by co-expression with BrPAP1 and BrTT8. We determined that BrMYBL2.1-P may be more prone to degradation than BrMYBL2.1-G via ubiquitination. Taken together, these results demonstrate that BrMYBL2.1-G blocks the activity of the MBW complex and thus represses anthocyanin biosynthesis, whereas the variant BrMYBL2.1-P from purple Chinese cabbage cannot, thus leading to higher anthocyanin accumulation.

Acetylation, Phosphorylation, Ubiquitination (Oh My!): Following Post-Translational Modifications on the Ubiquitin Road.

Ubiquitination is controlled by a series of E1, E2, and E3 enzymes that can ligate ubiquitin to cellular proteins and dictate the turnover of a substrate and the outcome of signalling events such as DNA damage repair and cell cycle. This process is complex due to the combinatorial power of ~35 E2 and ~1000 E3 enzymes involved and the multiple lysine residues on ubiquitin that can be used to assemble polyubiquitin chains. Recently, mass spectrometric methods have identified that most enzymes in the ubiquitination cascade can be further modified through acetylation or phosphorylation under particular cellular conditions and altered modifications have been noted in different cancers and neurodegenerative diseases. This review provides a cohesive summary of ubiquitination, acetylation, and phosphorylation sites in ubiquitin, the human E1 enzyme UBA1, all E2 enzymes, and some representative E3 enzymes. The potential impacts these post-translational modifications might have on each protein function are highlighted, as well as the observations from human disease.

Effects of Lysine N-ζ-Methylation in Ultrashort Tetrabasic Lipopeptides (UTBLPs) on the Potentiation of Rifampicin, Novobiocin, and Niclosamide in Gram-Negative Bacteria.

Outer membrane (OM) drug impermeability typically associated with a molecular weight above 600 Da and high hydrophobicity prevents accumulation of many antibiotics in Gram-negative bacteria (GNB). Previous studies have shown that ultrashort tetrabasic lipopeptides (UTBLPs) containing multiple lysine residues potentiate Gram-positive bacteria (GPB)-selective antibiotics in GNB by enhancing OM permeability. However, there is no available information on how N-substitution at the ζ-position of lysine in UTBLPs affects antibiotic potentiation in GNB. To study these effects, we prepared a series of branched and linear UTBLPs that differ in the degree of N-ζ-methylation and studied their potentiating effects with GPB-selective antibiotics including rifampicin, novobiocin, niclosamide, and chloramphenicol against wild-type and multidrug-resistant GNB isolates. Our results show that increasing N-ζ-methylation reduces or abolishes the potentiating effects of UTBLPs with rifampicin, novobiocin, and niclosamide against GNB. No trend was observed with chloramphenicol that is largely affected by efflux. We were unable to observe a correlation between the strength of the antibiotic potentiating effect to the increase in fluorescence in the 1-N-phenylnaphthylamine (NPN) OM permeability assay suggesting that other factors besides OM permeability of NPN play a role in antibiotic potentiation. In conclusion, our study has elucidated crucial structure–activity relationships for the optimization of polybasic antibiotic potentiators in GNB.

Lysine Methylation Modulates the Interaction of Archaeal Chromatin Protein Cren7 With DNA.

Cren7 and Sis7d, two chromatin proteins from Sulfolobus islandicus, undergo extensive methylations at multiple lysine residues to various extents. Whether this highly conserved protein serves an epigenetic role in the regulation of the structure and function of the chromosome remains unclear. In the present study, we show that methylation significantly affects Cren7, but not Sis7d, in the ability to bind DNA and to constrain negative DNA supercoils. Strikingly, methylated Cren7 was significantly less efficient in forming oligomers or mediating intermolecular DNA bridging. Single-site substitution mutation with glutamine reveals that methylation of the four lysine residues (K24, K31, K42, and K48) of Cren7 at the protein-DNA interface, which are variably conserved among Cren7 homologues from different branches of the Crenarchaeota, influenced Cren7-DNA interactions in different manners. We suggest that dynamic methylation of Cren7 may represent a potential epigenetic mechanism involved in the chromosomal regulation in crenarchaea.

Acetylated Thioredoxin Reductase 1 Resists Oxidative Inactivation.

Thioredoxin Reductase 1 (TrxR1) is an enzyme that protects human cells against reactive oxygen species generated during oxidative stress or in response to chemotherapies. Acetylation of TrxR1 is associated with oxidative stress, but the function of TrxR1 acetylation in oxidizing conditions is unknown. Using genetic code expansion, we produced recombinant and site-specifically acetylated variants of TrxR1 that also contain the non-canonical amino acid, selenocysteine, which is essential for TrxR1 activity. We previously showed site-specific acetylation at three different lysine residues increases TrxR1 activity by reducing the levels of linked dimers and low activity TrxR1 tetramers. Here we use enzymological studies to show that acetylated TrxR1 is resistant to both oxidative inactivation and peroxide-induced multimer formation. To compare the effect of programmed acetylation at specific lysine residues to non-specific acetylation, we produced acetylated TrxR1 using aspirin as a model non-enzymatic acetyl donor. Mass spectrometry confirmed aspirin-induced acetylation at multiple lysine residues in TrxR1. In contrast to unmodified TrxR1, the non-specifically acetylated enzyme showed no loss of activity under increasing and strongly oxidating conditions. Our data suggest that both site-specific and general acetylation of TrxR1 regulate the enzyme’s ability to resist oxidative damage.