Lysine In Position Research Articles

Post-translational modifications on protein VII are important during the early stages of adenovirus infection.

Due to the importance of post-translational modification (PTM) in cellular function, viruses have evolved to both take advantage of and be susceptible to such modification. Adenovirus encodes a multifunctional protein called protein VII, which is packaged with the viral genome in the core of virions and disrupts host chromatin during infection. Protein VII has several PTMs whose addition contributes to the subnuclear localization of protein VII. Here, we used mutant viruses that abrogate or mimic these PTMs on protein VII to interrogate their impact on protein VII function during adenovirus infection. We discovered that acetylation of the lysine in positions 2 or 3 (K2 or K3) is deleterious during early infection as mutation to alanine led to greater intake of protein VII to the nucleus and enhanced early gene expression. Furthermore, we determined that protein VII is acetylated at alternative residues late during infection which may compensate for the mutated sites. Lastly, due to the role of the early viral protein E1A in viral gene activation, we investigated the interaction between protein VII and E1A and demonstrated that protein VII interacts with E1A through a chromatin-mediated interaction. Together, these results emphasize that the complexity of virus-host interactions is intimately tied to post-translational modification.

A single viral amino acid shapes the root system architecture of a plant host upon virus infection

BackgroundGrapevine fanleaf virus (GFLV) is one of the most detrimental viral pathogens of grapevines worldwide but no information is available on its effect on the root system architecture (RSA) of plant hosts. We used two wildtype GFLV strains and their single amino acid mutants to assess RSA traits in infected Nicotiana benthamiana and evaluate transcriptomic changes in host root gene expression in replicated time course 3’RNA-Seq experiments. Mutations targeted the multi-functional GFLV-encoded protein 1EPol*/Sd, a putative RNA-dependent RNA polymerase and determinant of foliar symptoms in N. benthamiana plants.ResultsPlant infection with wildtype GFLV strain GHu and mutant GFLV strain F13 1EPolG802K, both carrying a lysine in position 802 of protein 1EPol*/Sd, resulted in a significantly lower number of root tips (-30%), and a significantly increased average root diameter (+ 20%) at 17 days post inoculation (dpi) in comparison with roots of mock inoculated plants. In contrast, the RSA of plants infected with wildtype GFLV strain F13 and mutant GFLV strain GHu 1EPolK802G, both carrying a glycine in position 802 of protein 1EPol*/Sd, resembled that of mock inoculated plants. Modifications of RSA traits were not associated with GFLV titer. Root tissue transcriptome analysis at 17 dpi indicated dysregulation of pattern recognition receptors, plant hormones, RNA silencing, and genes related to the production of reactive oxygen species (ROS). For wildtype GFLV strain GHu, RSA modifications were correlated with an abundant accumulation of ROS in the pericycle of primary roots at 7 dpi and the duration of vein clearing symptom expression in apical leaves. Dysegulation of a hypersensitive response was an overarching gene ontology found through enrichment analyses of 3’RNA-Seq data.ConclusionsOur findings revealed the causative role of lysine in position 802 of protein 1EPol*/Sd in a novel RSA phenotype during viral infection and documented GFLV-N. benthamiana interactions at the root level based on (i) antiviral response, (ii) receptor mediated production of ROS, and (iii) hormone regulation. A correlation between above and below ground symptoms was reported for the first time in plants infected with wildtype GFLV strain GHu. Further work is warranted to test whether the modified RSA of a plant host might impact GFLV acquisition and transmission by the ectoparasitic dagger nematode Xiphinema index.

Biosynthesis of Strained Amino Acids by a PLP‐Dependent Enzyme through Cryptic Halogenation

AbstractAmino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non‐ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non‐canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal‐5′‐phosphate‐dependent enzyme, PazB, via an SN2‐like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane‐containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Biosynthesis of Strained Amino Acids by a PLP-Dependent Enzyme through Cryptic Halogenation.

Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Structure of the complete Saccharomyces cerevisiae Rpd3S-nucleosome complex

Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S complex plays a critical role in the suppression of spurious transcription by removing histone acetylation from actively transcribed genes. The S. cerevisiae Rpd3S complex has five subunits (Rpd3, Sin3, Rco1, Eaf3, and Ume1) but its subunit stoichiometry and how the complex engages nucleosomes to achieve substrate specificity remains elusive. Here we report the cryo-EM structure of the complete Rpd3S complex bound to a nucleosome. Sin3 and two copies of subunits Rco1 and Eaf3 encircle the deacetylase subunit Rpd3 and coordinate the positioning of Ume1. The Rpd3S complex binds both trimethylated H3 tails at position lysine 36 and makes multiple additional contacts with the nucleosomal DNA and the H2A–H2B acidic patch. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity.

SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway

Interferon-gamma (IFN-γ) signaling is necessary for the proinflammatory activation of macrophages but IFN-γ-independent pathways, for which the initiating stimuli and downstream mechanisms are lesser known, also contribute. Here we identify, by high-content screening, SEPTIN2 (SEPT2) as a negative regulation of IFN-γ-independent macrophage autoactivation. Mechanistically, endoplasmic reticulum (ER) stress induces the expression of SEPT2, which balances the competition between acetylation and ubiquitination of heat shock protein 5 at position Lysine 327, thereby alleviating ER stress and constraining M1-like polarization and proinflammatory cytokine release. Disruption of this negative feedback regulation leads to the accumulation of unfolded proteins, resulting in accelerated M1-like polarization, excessive inflammation and tissue damage. Our study thus uncovers an IFN-γ-independent macrophage proinflammatory autoactivation pathway and suggests that SEPT2 may play a role in the prevention or resolution of inflammation during infection.

Issue Information

New PhytologistVolume 238, Issue 3 p. 917-920 Cover and Issue InformationFree Access Issue Information First published: 30 March 2023 https://doi.org/10.1111/nph.18237AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Cover Legend Toluidine blue-stained cross-section through a stem of an Arabidopsis plant accumulating a variant of Histone 3 devoid of Lysine in position 27. Image courtesy of Kateryna Fal, LPCV-IRIG, UGA (Fal et al., pp. 1085–1100). Volume238, Issue3May 2023Pages 917-920 RelatedInformation

Studying lysine acetylation of citric acid cycle enzymes by genetic code expansion.

Lysine acetylation is one of the most abundant post-translational modifications in nature, affecting many key biological pathways in both prokaryotes and eukaryotes. It has not been long since technological advances led to understanding of the roles of acetylation in biological processes. Most of those studies were based on proteomic analyses, which have identified thousands of acetylation sites in a wide range of proteins. However, the specific role of individual acetylation event remains largely unclear, mostly due to the existence of multiple acetylation and dynamic changes of acetylation levels. To solve these problems, the genetic code expansion technique has been applied in protein acetylation studies, facilitating the incorporation of acetyllysine into a specific lysine position to generate a site-specifically acetylated protein. By this method, the effects of acetylation at a specific lysine residue can be characterized with minimal interferences. Here, we summarized the development of the genetic code expansion technique for lysine acetylation and recent studies on lysine acetylation of citrate acid cycle enzymes in bacteria by this approach, providing a practical application of the genetic code expansion technique in protein acetylation studies.

Molecular fingerprint in hyaluronan-peptide interactions: Cooperation of electrostatics and side-chain specificity in enhanced arginine effect.

Hyaluronan is a sugar polymer found on the extracellular side, close to the plasma membrane. It is the scaffold of the extracellular matrix due to its high solubility, despite its low negative charge density and unusual length. Hyaluronan is also a known ligand of several glycan-binding proteins. We can improve our understanding of these interactions by characterizing hyaluronan affinity to model amino acid sequences of reduced complexity. Extracted features and fingerprints allow for identifying new potential binding partners and expand the current understanding of hyaluronan's biological role. Here, we use a combination of NMR and molecular dynamics simulations (MD) to study hyaluronan binding to short oligopeptides. We focus on arginine as a known critical amino acid for this interaction and compare it to lysine and glycine to highlight its distinctive features. We can test the binding using 1H NMR chemical shift perturbation (CSP) by titrating hyaluronan with a peptide. CSP not only estimates binding strength but also pinpoints the binding motif involved. Our findings are further explained by MD, which provide an atomistic fingerprint of the hyaluronan-peptide complex. Interestingly, we could not find any NOE signal confirming the CSP observed interactions. The combination of positive CSP with no visible NOEs indicates the presence of a dynamic binding mode in agreement with our MD simulations. This agreement with simulations was only achieved using the prosECCo force field for accounting for electronic polarizability. Overall, we find that arginine, a positively charged amino acid, binds the strongest to the negative hyaluronan, followed by the positive lysine. The hyaluronan's carboxyl and amide groups are the driving force for interacting with the peptides. We prove the importance of the electrostatics interaction but also a specific sidechain affinity that favors arginine.

Peptide-Membrane Binding: Effects of the Amino Acid Sequence.

An understanding of how the amino acid sequence affects the interaction of peptides with lipid membranes remains mostly unknown. This type of knowledge is required to rationalize membrane-induced toxicity of amyloid peptides and to design peptides that can interact with lipid bilayers. Here, we perform a systematic study of how variations in the sequence of the amphipathic Ac-(FKFE)2-NH2 peptide affect its interaction with zwitterionic lipid bilayers using extensive all-atom molecular dynamics simulations in explicit solvent. Our results show that peptides with a net positive charge bind more frequently to the lipid bilayer than neutral or negatively charged sequences. Moreover, neutral amphipathic peptides made with the same numbers of phenylalanine (F), lysine (K), and glutamic (E) amino acids at different positions in the sequence differ significantly in their frequency of binding to the membrane. We find that peptides bind with a higher frequency to the membrane if their positive lysine side chains are more exposed to the solvent, which occurs if they are located at the extremity (as opposed to the middle) of the sequence. Non-polar residues play an important role in accounting for the adsorption of peptides onto the membrane. In particular, peptides made with less hydrophobic non-polar residues (e.g., valine and alanine) are significantly less adsorbed to the membrane compared to peptides made with phenylalanine. We also find that sequences where phenylalanine residues are located at the extremities of the peptide have a higher tendency to be adsorbed.

Potential Antifungal Targets for Aspergillus sp. from the Calcineurin and Heat Shock Protein Pathways.

Aspergillus species, especially A. fumigatus, and to a lesser extent others (A. flavus, A. niger, A. terreus), although rarely pathogenic to healthy humans, can be very aggressive to immunocompromised patients (they are opportunistic pathogens). Although survival rates for such infections have improved in recent decades following the introduction of azole derivatives, they remain a clinical challenge. The fact that current antifungals act as fungistatic rather than fungicide, that they have limited safety, and that resistance is becoming increasingly common make the need for new, more effective, and safer therapies to become more acute. Over the last decades, knowledge about the molecular biology of A. fumigatus and other Aspergillus species, and particularly of calcineurin, Hsp90, and their signaling pathway proteins, has progressed remarkably. Although calcineurin has attracted much interest, its adverse effects, particularly its immunosuppressive effects, make it less attractive than it might at first appear. The situation is not very different for Hsp90. Other proteins from their signaling pathways, such as protein kinases phosphorylating the four SPRR serine residues, CrzA, rcnA, pmcA-pmcC (particularly pmcC), rfeF, BAR adapter protein(s), the phkB histidine kinase, sskB MAP kinase kinase, zfpA, htfA, ctfA, SwoH (nucleoside diphosphate kinase), CchA, MidA, FKBP12, the K27 lysine position from Hsp90, PkcA, MpkA, RlmA, brlA, abaA, wetA, other heat shock proteins (Hsp70, Hsp40, Hsp12) currently appear promising and deserve further investigation as potential targets for antifungal drug development.

An in silico study of how histone tail conformation affects the binding affinity of ING family proteins.

BackgroundDue to its intrinsically disordered nature, the histone tail is conformationally heterogenic. Therefore, it provides specific binding sites for different binding proteins or factors through reversible post-translational modifications (PTMs). For instance, experimental studies stated that the ING family binds with the histone tail that has methylation on the lysine in position 4. However, numerous complexes featuring a methylated fourth lysine residue of the histone tail can be found in the UniProt database. So the question arose if other factors like the conformation of the histone tail affect the binding affinity.MethodsThe crystal structure of the PHD finger domain from the proteins ING1, ING2, ING4, and ING5 are docked to four histone H3 tails with two different conformations using Haddock 2.4 and ClusPro. The best four models for each combination are selected and a two-sample t-test is performed to compare the binding affinities of helical conformations vs. linear conformations using Prodigy. The protein-protein interactions are examined using LigPlot.ResultsThe linear histone conformations in predicted INGs-histone H3 complexes exhibit statistically significant higher binding affinity than their helical counterparts (confidence level of 99%). The outputs of predicted models generated by the molecular docking programs Haddock 2.4 and ClusPro are comparable, and the obtained protein-protein interaction patterns are consistent with experimentally confirmed binding patterns.ConclusionThe results show that the conformation of the histone tail is significantly affecting the binding affinity of the docking protein. Herewith, this in silico study demonstrated in detail the binding preference of the ING protein family to histone H3 tail. Further research on the effect of certain PTMs on the final tail conformation and the interaction between those factors seem to be promising for a better understanding of epigenetics.

Hydroxamic Acid-Modified Peptide Library Provides Insights into the Molecular Basis for the Substrate Selectivity of HDAC Corepressor Complexes.

Targeting the lysine deacetylase activity of class I histone deacetylases (HDACs) is potentially beneficial for the treatment of several diseases including human immunodeficiency virus (HIV) infection, Alzheimer’s disease, and various cancers. It is therefore important to understand the function and mechanism of action of these enzymes. Class I HDACs act as catalytic components of seven large, multiprotein corepressor complexes. Different HDAC corepressor complexes have specific, nonredundant roles in the cell. It is likely that their specific functions are at least partly influenced by the substrate specificity of the complexes. To address this, we developed chemical tools to probe the specificity of HDAC complexes. We assessed a library of acetyl-lysine-containing substrate peptides and hydroxamic acid-containing inhibitor peptides against the full range of class I HDAC corepressor complexes. The results suggest that site-specific HDAC corepressor complex activity is driven in part by the recognition of the primary amino acid sequence surrounding a particular lysine position in the histone tail.

Identification of the initial reactive sites of micellar and non-micellar casein exposed to microbial transglutaminase

To investigate the influence of the internal micellar structure on the course of enzymatic cross-linking especially in the initial phase of the reaction, casein micelles isolated from raw milk via ultracentrifugation were incubated with microbial transglutaminase (mTG) in comparison with non-micellar sodium caseinate. Reactive lysine and glutamine residues were identified using a label-free approach, based on the identification of isopeptides within tryptic hydrolysates by targeted HRMS as well as manual monitoring of fragmentation spectra. Identified reactive sites were furthermore weighted by tracking the formation of isopeptides over an incubation time of 15, 30, 45 and 60 min, respectively. Fifteen isopeptides formed in the early stage of mTG cross-linking of caseins were identified and further specified concerning the position of lysine and glutamine residues involved in the reaction. The results revealed lysine K176 and glutamine Q175 of β-casein as the most reactive residues, which might be located in a highly flexible region of the molecule based on different possible reaction partners identified in this study. Except for the isopeptide αs1 K34–αs2 Q101 in sodium caseinate (SC), all reactive sites were detected in micellar and in non-micellar casein, indicating that the initial phase of enzymatic cross-linking is not affected by micellar aggregation of caseins.Graphical abstract

H3K27m3 overexpression as a new, BCL2 independent diagnostic tool in follicular and cutaneous follicle center lymphomas

Approximately 15% of follicular lymphomas (FL) lack overexpression of BCL2 and the underlying translocation t(14;18). These cases can be diagnostically challenging, especially regarding follicular hyperplasia (FH). In a subset of FL, mutations in genes encoding for epigenetic modifiers, such as the histone-lysine N-methyltransferase EZH2 (enhancer of zeste homolog 2), were found, which might be used diagnostically. These molecular alterations can lead to an increased tri-methylation of histone H3 at position lysine 27 (H3K27m3) that, in turn, can be visualized immunohistochemically. The aim of this study was to analyze the expression of H3K27m3 in FL, primary cutaneous follicle center lymphomas (PCFCL), and pediatric-type FL (PTFL) in order to investigate its value in the differential diagnosis to FH and other B cell lymphomas and to correlate it to BCL2 expression and the presence of t(14;18). Additionally, the mutational profile of selected cases was considered to address H3K27m3’s potential use as a surrogate parameter for mutations in genes encoding for epigenetic modifiers. Eighty-nine percent of FL and 100% of PCFCL cases overexpressed H3K27m3, independently of BCL2, EZH2, and the presence of mutations. In contrast, 95% of FH and 100% of PTFL cases lacked H3K27m3 overexpression. Other B cell lymphomas considered for differential diagnosis also showed overexpression of H3K27m3 in the majority of cases. In summary, overexpression of H3K27m3 can serve as a new, BCL2 independent marker in the differential diagnosis of FL and PCFCL, but not PTFL, to FH, while being not of help in the differential diagnosis of FL to other B cell lymphomas.

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in peach reproductive tissues

BackgroundPerennial fruit trees display a growth behaviour characterized by annual cycling between growth and dormancy, with complex physiological features. Rosaceae fruit trees represent excellent models for studying not only the fruit growth/patterning but also the progression of the reproductive cycle depending upon the impact of climate conditions. Additionally, current developments in high‐throughput technologies have impacted Rosaceae tree research while investigating genome structure and function as well as (epi)genetic mechanisms involved in important developmental and environmental response processes during fruit tree growth. Among epigenetic mechanisms, chromatin remodelling mediated by histone modifications and other chromatin-related processes play a crucial role in gene modulation, controlling gene expression. Chromatin immunoprecipitation is an effective technique to investigate chromatin dynamics in plants. This technique is generally applied for studies on chromatin states and enrichment of post-transcriptional modifications (PTMs) in histone proteins.ResultsPeach is considered a model organism among climacteric fruits in the Rosaceae family for studies on bud formation, dormancy, and organ differentiation. In our work, we have primarily established specific protocols for chromatin extraction and immunoprecipitation in reproductive tissues of peach (Prunus persica). Subsequently, we focused our investigations on the role of two chromatin marks, namely the trimethylation of histone H3 at lysine in position 4 (H3K4me3) and trimethylation of histone H3 at lysine 27 (H3K27me3) in modulating specific gene expression. Bud dormancy and fruit growth were investigated in a nectarine genotype called Fantasia as our model system.ConclusionsWe present general strategies to optimize ChIP protocols for buds and mesocarp tissues of peach and analyze the correlation between gene expression and chromatin mark enrichment/depletion. The procedures proposed may be useful to evaluate any involvement of histone modifications in the regulation of gene expression during bud dormancy progression and core ripening in fruits.

Small-molecule albumin ligand modification to enhance the anti-diabetic ability of GLP-1 derivatives

Glucagon-like peptide-1 (GLP-1) receptor agonists modified with albumin ligands which can specificity bind to the human serum albumin (HSA) was an efficient strategy to prolong the half-time of GLP-1. Herein, we investigated the effect of small-molecule albumin ligand modification on the hypoglycemic activities of GLP-1 derivatives. Two GLP-1 derivatives MPA-C12-GLP-1 and Rhein-C12-GLP-1 were achieved by modification of the side chain amino of lysine in position 26 of the Arg34-GLP-1(7−37)-OH with Rhein and 3-Maleimidopropionic acid respectively using 12-aminolauric acid as a linker, and its specific albumin-conjugating characteristics, pharmaceutical characterization, and the antidiabetic effects were investigated. In vitro level, two GLP-1 derivatives demonstrated a higher binding capacity to GLP-1 receptor than that of Arg34-GLP-1(7−37)-OH. Interestingly, although the binding ability of MPA-C12-GLP-1 was equal to liraglutide, the binding ability of Rhein-C12-GLP-1 was 10-fold higher than liraglutide. In vivo level, the two GLP-1 derivatives can significantly increase their glucose tolerance and prolong their half-life in ICR mice, and they were also superior to GLP-1 in controlling glucose homeostasis and suppression of food intake and water consumption in db/db mice. Importantly, the two GLP-1 derivatives showed comparable efficacy to liraglutide for the therapy of type 2 diabetes mellitus. The in vitro INS-1 cells toxicity and the in vivo hepatotoxicity indicated that the Rhein-C12-GLP-1 was a safe candidate for the therapy of type 2 diabetes, and the serum biomarkers determination results showed that the Rhein-modified GLP-1 could significantly improve the HbA1c and blood lipids, and the H&E stain exhibited that the Rhein-C12-GLP-1 can effectively promote β-cell proliferation and differentiation. In conclusion, the 3-Maleimidopropionic acid or Rhein-modified GLP-1derivatives have great potential for development as a Type 2 diabetes mellitus therapeutic drug.

Derivative of Scorpion Neurotoxin BeM9 Is Selective for Insect Voltage-Gated Sodium Channels

Scorpion α-toxins are small proteins inhibiting the inactivation of voltage-gated sodium channels. They can selectively act on either mammalian (mammal toxins) or insect channels (insect toxins), or affect both types of channels (α-like toxins). Currently no model has been proposed that fully explains the dependence of selectivity upon amino acid sequence, but some patterns have already been established. Thus, most mammal toxins have an aspartic acid residue in position 8, which is involved in the formation of the nest motif, but it is still not clear whether this residue interacts directly with channels. The objective of our study was to obtain a derivative of the α-like toxin BeM9 with the replacement of lysine in position 8 by glutamate (K8E), changing the charge, but excluding the formation of the nest motif. In addition, we replaced the tyrosine in position 17 with glycine (Y17G), which is characteristic of mammal toxins. Surprisingly, the double-mutant derivative BeM9EG lost its activity on mammalian channels, becoming an insect toxin. To explain these changes, we constructed models of BeM9 and BeM9EG complexes with channels, and also performed molecular dynamics of isolated toxins. Analysis of intermolecular contacts in the complexes did not explain the reason for the selectivity change. Nevertheless, the structure of intramolecular contacts and data on molecular mobility indicate an important role of residues K8 and Y17 in stabilizing a certain conformation of BeM9 loops. We assume that the replacement of these residues allosterically affects the efficiency of toxin binding to channels.

CLLU1 Expression Is Not Confined to Chronic Lymphocytic Leukemia Cells; Putative Role of Gene-Body Hypomethylation in Regulating Splice Variant-Specific Transcriptional Dynamics

Background: Alternative splicing of the CLLU1 gene generates 6 transcript variants (accession numbers from the National Center for Biotechnology Information: AJ845162-AJ845167), two of which comprise a putative coding DNA sequence (AJ845165 and AJ845166, CDS transcripts). Previous studies showed that CLLU1 overexpression is highly confined to chronic lymphocytic leukemia (CLL) cells and that only the AJ845162 is expressed in both high- and low-risk CLL patients, while expression of the CDS-transcripts is confined to the high-risk subgroup.Pair-wise linear regression analysis revealed a powerful linear relation between the expression levels of all the splice variants, thereby it was deduced that CLLU1 transcription is orchestrated by a common promoter, which is located upstream of CLLU1 exon 1 (Buhl et al., Leukemia 2009). We recently documented (Papamichos et al., Haematologica 2016; s1) that this promoter was evolutionarily derived by an endogenous retrovirus sequence.Herein we argue that CLLU1 expression is not restricted to CLL cells while also we highlight that transcription of CDS transcripts is regulated by an alternative intronic promoter.Methods:CLLU1 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 51 human tissues and 2 human cell lines, generated by the Genotype-Tissue Expression (GTEx) project of the US National Institutes of Health Common Fund (GTEx Analysis Release V6p), were analyzed via GTEx Portal. CLLU1 GeneChip expression data (AJ845164 transcript-specific probe set 1558185_at from Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array), generated by Genevestigator project, were downloaded from Genevisible portal.CLLU1 chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) as well as transcription factor (TF) ChIP-Seq data were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, which is available in UCSC Genome Browser Database. Data on CLLU1 gene-body methylation status in 63 cell lines from ENCODE were downloaded from the HAIB Methyl450 ENCODE track.Results:CLLU1 GeneChip expression data show that the AJ845164 transcript (corresponding to the RP11-693J15.4 long non-coding RNA) is promiscuously expressed in various normal B-cell subpopulations andin mature B-cell neoplasms other than CLL, especially in mantle cell lymphoma.RNA-Seq data analysis reveals that AJ845162 transcript is expressed in normal somatic tissues such as skeletal muscle, salivary gland, and esophageal mucosa. Vice versa, expression of CLLU1 CDS transcripts is confined to transformed lymphocytes.Structure interrogation of CDS transcripts indicates that these variants initiate from an alternative transcriptional start site which is located 2429 nucleotides downstream from the canonical one. H3K4me3 ChIP-Seq data signify the presence of an alternative intronic promoter in this genomic segment. TF Chip-Seq data reveal that the alternative promoter region comprises high affinity binding sites for signal transducer and activator of transcription 3 (STAT3) and Activator protein 1 (AP-1). Analysis of CLLU1 -body methylation status in five lymphoblastoid cell lines reveals that the alternative promoter remains heavily methylated in these cell types. This finding is in accordance with previous studies reporting that CLLU1 gene body is hypermethylated in B-cells from healthy individuals and in IgVH-mutated (IgVH-M) CLL cases while appears hypomethylated in IgVH-unmutated (IgVH-UM) CLL cells.Conclusion: Not all CLLU1 transcripts are equal in discriminating CLL from other mature B-cell malignancies.Whilst of retroviral origin, CLLU1 upstream promoter is not subjected to stringent epigenetic repression but remains active in somatic cells, a tantalizing finding that warrants further investigation.The level of intragenic DNA methylation represents a decisive regulator of alternative promoter usage (Neri et al., Nature 2017). It would be tantalizing to speculate that CLLU1 -body hypomethylation, occuring exclusively in IgVH-unmutated CLL, is mechanistically linked to the epigenetic derepression of the alternative intronic CLLU1 promoter and the subsequent high expression of CLLU1 CDS transcripts in high-risk CLL. DisclosuresNo relevant conflicts of interest to declare.

Endogenous Retrovirus Derepression Drives Ectopic UGT2B17 Overexpression in Multiple Myeloma Cells: Molecular Sequelae and Pathophysiological Implications

Introduction: UGT2B17 is a phase II detoxification enzyme which catalyzes the glucuronidation of C19 steroid hormones and lipid-soluble drugs. UGT2B17 gene is ubiquitously expressed in hepatic tissues as well as in steroid target tissues including breast, uterus, and prostate. UGT2B17 mRNA levels directly correlate with functional glucuronidation activity toward vorinostat (Gruber et al., Blood 2013), which is used against a variety of cancers including multiple myeloma (MM).Progression from monoclonal gammopathy of undetermined significance to MM is associated with increased genomic hypomethylation. Importantly it is transposable elements (TEs), remaining stochastically methylated in normal cells, that lend themselves predominantly susceptible to hypomethylation during myelomatogenesis. Hypomethylated TEs could subsequently acquire the potential to exert a significant impact on host gene expression. Herein we present evidence that an aberrantly activated long terminal repeat (LTR) of the ERV1 family of endogenous retroviruses (ERVs) drives the lineage-inordinate expression of UGT2B17 in MM cells.Methods:UGT2B17 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 27 normal tissues from 95 human individuals, generated from the PRJEB4337 BioProject, were analyzed via the National Center for Biotechnology Information (NCBI) portal. Publicly available UGT2B17 RNA-Seq data coupled with ribosome profiling (Ribo-Seq) data across 17 cell types were downloaded from GWIPS-viz ribo-seq genome browser. UGT2B17 Transcription factor (TF) chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) data as well as ChIP-Seq data on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, publicly available in UCSC Genome Browser Database. RepeatMasker software was employed to reveal the presence of integrated TEs in the genomic segment comprising UGT2B17 on chromosome 4.Results: Data analysis from the PRJEB4337 BioProject reveals low-level ectopic UGT2B17 transcription initiation in 5 of 27 normal human tissues (stomach, colon, appendix, spleen, and white blood cells) from a novel exon (E0) which is located ~7500 nucleotides (nts) upstream of the canonical UGT2B17 first exon (E1). Notably, H3K4me3 ChIP-Seq data indicate the presence of a highly potent promoter sequence immediately upstream of E0. RepeatMasker scan reveals that the genomic segment that comprises UGT2B17 E0 was evolutionary derived from a human-specific Harlequin-int LTR of the ERV1 family.Data from the GWIPS-viz genome browser show that the LTR-driven UGT2B17 transcription is minimal in 15 cell types, including normal somatic cells, cancer cells, and embryonic stem cells. In stark contrast, specifically in EBV-immortalized lymphocytes and the MM.1 myeloma cell line, UGT2B17 E0 presents higher transcription rate and also higher ribosome coverage compared to the UGT2B17 E1.TF ChIP-seq data from ENCODE reveal that UGT2B17 E0 comprises a binding site for RNA polymerase II subunit A (POLR2A) which, amongst several cell types examined, is functional specifically in B-lineage cells. Furthermore the intragenic segment that is located between E0 and E1 comprises a high-affinity binding site for RELA proto-oncogene. Of note, adjacent to the POLR2A and RELA binding sites, located are two binding sites for the CCCTC-binding factor (CTCF), which is known to act as a transcriptional insulator restricting activation of ectopic promoters.Conclusion: A retroviral-derived promoter that exhibits only basal activity in most normal or malignant somatic cells has the potential to initiate transcription of the adjacent UGT2B17 locus specifically in EBV-immortalized lymphocytes and MM cells. It is highly likely that ectopic UGT2B17 expression, restricted in B-lineage transformed and cancer cells, is induced by a lineage-specific aberration of CTCF function. Furthermore, the RELA binding site may serve as a potent enhancer of UGT2B17 inordinate transcription.The UGT2B17 LTR-driven misexpression in B-lineage transformed and cancer cell types may impact the effectiveness of vorinostat-based interventions in MM patients, a tantalizing scenario warranting further interrogation in primary MM samples. DisclosuresNo relevant conflicts of interest to declare.