Amino Acid Acceptor Research Articles

Abstract 1834: Discovery of a novel and highly selective PARP7 inhibitor for cancer immunotherapy

Abstract Poly-ADP-ribose polymerase 7 (PARP7) is a member of the wider PARP enzyme family that modulate protein function by using nicotinamide adenine dinucleotide (NAD+) as a substrate to transfer an ADP-ribose monomer onto specific amino acid acceptor residues of target proteins. PARP7 acts as a key repressor of type I interferon (IFN) signaling. Its inhibition combats tumor regression by enhancing antitumor immunity that depends on STING pathway. Therefore, PARP7 is emerging as a promising drug target for new immunotherapy. Using structure-based drug design and optimization, we discovered a novel PARP7 inhibitor, HSN002066, with high inhibitory potency (Enzyme assay IC50 = 0.96 nM) and high selectivity for PARP7 over PARP1/2/12/5A/5B/10 (IC50 =163.4/4.7/345.2/1790/110.2/>10000 nM, respectively). In addition, HSN002066 has excellent ADMET profiles with CYPs inhibition, IC50 >10 μM, human LMS T1/2 > 60 min, hERG inhibition, IC50 > 30 μM,mouse oral bioavailability (F) 98.1%. Notably, HSN002066 and RBN-2397, the only PARP7 inhibitor currently in Ph 1/2 clinical development, displayed differentiated antiproliferative activities in cell line NCI-H1373 (IC50 are 387.8 nM and 15.2 nM, respectively). However, HSN002066 revealed remarkable antitumor effects as monotherapy (TGI 77% at 100 mg/kg) or combination with PD-1 antibody in CT-26 allograft model. The structure of HSN002066 was not presented and will not be disclosed at the time of presentation at AACR meeting. Citation Format: Xiaohui Liu, Ying Wang, Dongliang Xia, Wei Zhang, Shijie Tian, Yan He, Xuerong Feng, Xia Yang, Qi Zhang, Shiyi Jiang, Meifeng Wu, Jianfeng Deng, Yeye Wang, Yuxi Su, Min Yao, Weijiao Yuan, Xuedan You. Discovery of a novel and highly selective PARP7 inhibitor for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1834.

Contribution of γ-Glutamyl-Cysteine Ligases of Limosilactobacillus reuteri to the Formation of Kokumi-Active γ-Glutamyl Dipeptides in Sourdough Bread.

Kokumi-active γ-glutamyl dipeptides accumulate during sourdough fermentation. γ-Glutamylcysteine ligases (Gcls) of Limosilactobacillus reuteri synthesize γ-glutamyl dipeptides during growth in sourdough. This study aimed to evaluate the contribution of Gcls from strains of L. reuteri in the formation of kokumi-active γ-glutamyl dipeptides in sourdough bread. Among 12 acceptor amino acids, the three Gcls of L. reuteri were the most active to Cys. With the acceptor amino acids Ile, Leu, and Phe, Gcl1 was more active than Gcl2 and Gcl3. Accordingly, Gcl1 contributed to the γ-Glu-Ile synthesis in sourdough fermentation. Proofing and baking strongly influenced the concentration of γ-glutamyl dipeptides in bread. The addition of 10% sourdough increased the content of γ-Glu-Leu and γ-Glu-Phe but not of other γ-glutamyl dipeptides in bread. In conclusion, the accumulation of kokumi γ-glutamyl dipeptides in sourdoughs was attributed to the combined activity of cereal enzymes, γ-glutamyl-cysteine ligases, and other microbial enzymes.

Importance of residue 248 in Escherichia coli RNase P RNA mediated cleavage

tRNA genes are transcribed as precursors and RNase P generates the matured 5' end of tRNAs. It has been suggested that residue − 1 (the residue immediately 5ʹ of the scissile bond) in the pre-tRNA interacts with the well-conserved bacterial RNase P RNA (RPR) residue A248 (Escherichia coli numbering). The way A248 interacts with residue − 1 is not clear. To gain insight into the role of A248, we analyzed cleavage as a function of A248 substitutions and N−1 nucleobase identity by using pre-tRNA and three model substrates. Our findings are consistent with a model where the structural topology of the active site varies and depends on the identity of the nucleobases at, and in proximity to, the cleavage site and their potential to interact. This leads to positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. We propose that in addition to be involved in anchoring the substrate the role of A248 is to exclude bulk water from access to the amino acid acceptor stem, thereby preventing non-specific hydrolysis of the pre-tRNA. Finally, base stacking is discussed as a way to protect functionally important base-pairing interactions from non-specific hydrolysis, thereby ensuring high fidelity during RNA processing and the decoding of mRNA.

Abstract B072: Induction of PARP7 creates a vulnerability for growth inhibition by RBN2397 in prostate cancer cells

Abstract The ADP-ribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids. PARP7 has been shown to affect gene expression in prostate cancer cells and certain other cell types by mechanisms that include transcription factor ADP-ribosylation. Here, we use a recently developed catalytic inhibitor to PARP7, RBN2397 (NCT04053673), to study the effects of PARP7 inhibition in androgen receptor-positive (AR+) and androgen receptor-negative (AR-) prostate cancer cells. Ribon Therapeutics developed RBN2397 as a first-in-class mono-ADP-ribosyltransferase inhibitor, and showed that it blocks PARP7 negative regulation of TBK1 [1]. We find that RBN2397 has nanomolar potency for inhibiting androgen-induced ADP-ribosylation of the androgen receptor. RBN2397 inhibits the growth of prostate cancer cells in culture when cells are treated with ligands that activate the androgen receptor (PC3-AR, VCaP, CWR22Rv1), or the aryl hydrocarbon receptor (PC3, DU145, NCI-H660), and induce PARP7 expression. We show that the growth inhibitory effects of RBN2397 are distinct from its enhancement of interferon signaling recently shown to promote tumor immunogenicity in lung cancer models [1]. Chemical inhibitors to PARP1 exert effects on cells by blocking enzyme function, but also via cytotoxic effects attributed to stabilizing PARP1-chromatin interactions in a process termed trapping [2]. Drug-induced trapping of PARP1 can be detected biochemically by immunoblotting the detergent-resistant chromatin fraction. We found that RBN2397 treatment of AR+ and AR- prostate cancer cells induces biochemical trapping of PARP7 within the nucleus, which was also detected by confocal microscopy. Potential therapeutic benefits of RBN2397 are likely to depend on the level of PARP7 expression, given its induction is necessary for growth inhibitory effects of RBN2397 in cell culture. As a first step towards evaluating whether PARP7 levels in human prostate cancer may be actionable with RBN2397, we used computational methods to analyze PARP7 gene expression data from primary prostate tumors and metastatic AR+ and AR- prostate tumors. To assess PARP7 mRNA levels, we used data from the online resource recount3, which uniformly reprocesses publicly available RNA-seq datasets using a Monorail analysis pipeline. Using the level of PARP7 expression in VCaP cells that confers sensitivity to RBN2397 as a threshold, 50% of primary tumors, 41% of metastatic AR- and 11% of AR+ tumors are predicted to have PARP7 expression levels that are sufficient for a response to RBN2397. Because RBN2397 can inhibit the growth of castration-resistant and neuroendocrine prostate cancer cells, PARP7 may be an actionable target in advanced prostate cancer. 1. Gozgit, J.M., et al., PARP7 negatively regulates the Type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell, 2021. 39(9): p. 1214-1226 e10 2. Murai, J., et al., Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Res, 2012. 72(21): p. 5588-99. Citation Format: Chunsong Yang, Krzysztof Wierbilowicz, Natalia M. Dworak, Song Yi Bae, Sachi B. Tengse, Nicki Abianeh, Justin M. Drake, Tarek Abbas, Aakrosh Ratan, David Wotton, Bryce M. Paschal. Induction of PARP7 creates a vulnerability for growth inhibition by RBN2397 in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B072.

A simple geometrical model of the electrostatic environment around the catalytic center of the ribosome and its significance for the elongation cycle kinetics

The central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase center (PTC). Experimental evidence shows that the catalytic activity is affected by the electrostatic environment around the peptidyl transferase center. Here, we set up a minimal geometrical model fitting the available x-ray solved structures of the ribonucleic cavity around the catalytic center of the large subunit of the ribosome. The purpose of this phenomenological model is to estimate quantitatively the electrostatic potential and electric field that are experienced during the peptidyl transfer reaction. At least two reasons motivate the need for developing this quantification. First, we inquire whether the electric field in this particular catalytic environment, made only of nucleic acids, is of the same order of magnitude as the one prevailing in catalytic centers of the proteic enzymes counterparts. Second, the protein synthesis rate is dependent on the nature of the amino acid sequentially incorporated in the nascent chain. The activation energy of the catalytic reaction and its detailed kinetics are shown to be dependent on the mechanical work exerted on the amino acids by the electric field, especially when one of the four charged amino acid residues (R, K, E, D) has previously been incorporated at the carboxy-terminal end of the peptidyl-tRNA. Physical values of the electric field provide quantitative knowledge of mechanical work, activation energy and rate of the peptide bond formation catalyzed by the ribosome. We show that our theoretical calculations are consistent with two independent sets of previously published experimental results. Experimental results for E.coli in the minimal case of the dipeptide bond formation when puromycin is used as the final amino acid acceptor strongly support our theoretically derived reaction time courses. Experimental Ribo-Seq results on E. coli and S. cerevisiae comparing the residence time distribution of ribosomes upon specific codons are also well accounted for by our theoretical calculations. The statistical queueing time theory was used to model the ribosome residence time per codon during nascent protein elongation and applied for the interpretation of the Ribo-Seq data. The hypo-exponential distribution fits the residence time observed distribution of the ribosome on a codon. An educated deconvolution of this distribution is used to estimate the rates of each elongation step in a codon specific manner. Our interpretation of all these results sheds light on the functional role of the electrostatic profile around the PTC and its impact on the ribosome elongation cycle.

Phosphorylation, compartmentalization, and cardiac function.

Protein phosphorylation is a fundamental element of cell signaling. First discovered as a biochemical switch in glycogen metabolism, we now know that this posttranslational modification permeates all aspects of cellular behavior. In humans, over 540 protein kinases attach phosphate to acceptor amino acids, whereas around 160 phosphoprotein phosphatases remove phosphate to terminate signaling. Aberrant phosphorylation underlies disease, and kinase inhibitor drugs are increasingly used clinically as targeted therapies. Specificity in protein phosphorylation is achieved in part because kinases and phosphatases are spatially organized inside cells. A prototypic example is compartmentalization of the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A through association with A-kinase anchoring proteins. This configuration creates autonomous signaling islands where the anchored kinase is constrained in proximity to activators, effectors, and selected substates. This article primarily focuses on A kinase anchoring protein (AKAP) signaling in the heart with an emphasis on anchoring proteins that spatiotemporally coordinate excitation-contraction coupling and hypertrophic responses.

Are PARPs promiscuous?

Post-translational modifications exist in different varieties to regulate diverse characteristics of their substrates, ultimately leading to maintenance of cell health. The enzymes of the intracellular poly(ADP-ribose) polymerase (PARP) family can transfer either a single ADP-ribose to targets, in a reaction called mono(ADP-ribosyl)ation or MARylation, or multiple to form chains of poly(ADP-ribose) or PAR. Traditionally thought to be attached to arginine or glutamate, recent data have added serine, tyrosine, histidine and others to the list of potential ADP-ribose acceptor amino acids. PARylation by PARP1 has been relatively well studied, whereas less is known about the other family members such as PARP7 and PARP10. ADP-ribosylation on arginine and serine is reversed by ARH1 and ARH3 respectively, whereas macrodomain-containing MACROD1, MACROD2 and TARG1 reverse modification of acidic residues. For the other amino acids, no hydrolases have been identified to date. For many PARPs, it is not clear yet what their endogenous targets are. Better understanding of their biochemical reactions is required to be able to determine their biological functions in future studies. In this review, we discuss the current knowledge of PARP specificity in vitro and in cells, as well as provide an outlook for future research.

Processing Condition and Properties of Protein Isolates from Mung Bean (Vigna radiata) and Quinoa (Chenopodium quinoa Willd)

Mung bean and quinoa are good sources of protein from legumes and pseudo cereals respectively. Both sources have relatively high protein content and acceptable amino acids composition. The potential and application of mung bean and quinoa as protein isolates have been discovered in some studies. Several isolation techniques such as wet fractionation (acid-base extraction), micellization, dry fractionation, aqueous separation or combination of some techniques can be used to isolate protein. This review summarized different methods and processing conditions applied during production of mung bean and quinoa protein isolates. Extracting protein from different sources may require different conditions, which results in different compositions and functional properties. Acid-base extraction is the most common method applied in mung bean and quinoa and results in high protein purity and yields. Micellization is an alternative method used in mung bean to produce higher protein content. Dry fractionation is a sustainable option used in quinoa to concentrate protein fractions. Purification methods such as ultrafiltration and aqueous phase separation can be used. Different methods and processing conditions affect functional properties, including solubility, water and oil absorption, emulsification, foaming, thermal properties of isolates, which also affect the suitable application for isolates.
 Keywords: Protein isolates; Quinoa; Mung bean; Wet fractionation; Dry fractionation

The Making and Breaking of Serine-ADP-Ribosylation in the DNA Damage Response.

ADP-ribosylation is a widespread posttranslational modification that is of particular therapeutic relevance due to its involvement in DNA repair. In response to DNA damage, PARP1 and 2 are the main enzymes that catalyze ADP-ribosylation at damage sites. Recently, serine was identified as the primary amino acid acceptor of the ADP-ribosyl moiety following DNA damage and appears to act as seed for chain elongation in this context. Serine-ADP-ribosylation strictly depends on HPF1, an auxiliary factor of PARP1/2, which facilitates this modification by completing the PARP1/2 active site. The signal is terminated by initial poly(ADP-ribose) chain degradation, primarily carried out by PARG, while another enzyme, (ADP-ribosyl)hydrolase 3 (ARH3), specifically cleaves the terminal seryl-ADP-ribosyl bond, thus completing the chain degradation initiated by PARG. This review summarizes recent findings in the field of serine-ADP-ribosylation, its mechanisms, possible functions and potential for therapeutic targeting through HPF1 and ARH3 inhibition.

Degradation of host translational machinery drives tRNA acquisition in viruses.

Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p= 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.

An overall framework for the E. coli γ-glutamyltransferase-catalyzed transpeptidation reactions

γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as flavor enhancers or biologically active compounds. However, their supply, on a large scale and at reasonable costs, remains challenging. Enzymatic synthesis has been recognized as a possible affordable alternative with respect to both isolation procedures from natural sources, burdened by low-yield and by the requirement of massive amount of starting material, and chemical synthesis, inconvenient because of the need of protection/deprotection steps. The E. coli γ-glutamyltransferase (Ec-GGT) has already been proposed as a biocatalyst for the synthesis of various γ-glutamyl derivatives. However, enzymatic syntheses using this enzyme usually provide the desired products in limited yield. Hydrolysis and autotranspeptidation of the donor substrate have been identified as the side reactions affecting the final yield of the catalytic process. In addition, experimental conditions need to be specifically adjusted for each acceptor substrate. Substrate specificity and the fine characterization of the activities exerted by the enzyme over time has so far escaped rationalization. In this work, reactions catalyzed by Ec-GGT between the γ-glutamyl donor glutamine and several representative acceptor amino acids have been finely analyzed with the identification of single reaction products over time. This approach allowed to rationalize the effect of donor/acceptor molar ratio on the outcome of the transpeptidation reaction and on the distribution of the different byproducts, inferring a general scheme for Ec-GGT-catalyzed reactions. The propensity to react of the different acceptor substrates is in agreement with recent findings obtained using model substrates and further supported by x-ray crystallography and will contribute to characterize the still elusive acceptor binding site of the enzyme.

Current Advancements in Sactipeptide Natural Products.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products that benefited from genome sequencing technology in the past two decades. RiPPs are widely distributed in nature and show diverse chemical structures and rich biological activities. Despite the various structural characteristic of RiPPs, they follow a common biosynthetic logic: a precursor peptide containing an N-terminal leader peptide and a C-terminal core peptide; in some cases,a follower peptide is after the core peptide. The precursor peptide undergoes a series of modification, transport, and cleavage steps to form a mature natural product with specific activities. Sactipeptides (Sulfur-to-alpha carbon thioether cross-linked peptides) belong to RiPPs that show various biological activities such as antibacterial, spermicidal and hemolytic properties. Their common hallmark is an intramolecular thioether bond that crosslinks the sulfur atom of a cysteine residue to the α-carbon of an acceptor amino acid, which is catalyzed by a rSAM enzyme. This review summarizes recent achievements concerning the discovery, distribution, structural elucidation, biosynthesis and application prospects of sactipeptides.

Acyl Donor Stringency and Dehydroaminoacyl Intermediates in β-Lactam Formation by a Non-ribosomal Peptide Synthetase

Condensation (C) domains in non-ribosomal peptide synthetases catalyze peptide elongation steps whereby activated amino acid or peptidyl acyl donors are coupled with specific amino acid acceptors. In the biosynthesis of the β-lactam antibiotic nocardicin A, an unusual C domain converts a seryl tetrapeptide into its pentapeptide product containing an integrated β-lactam ring. While indirect evidence for the intermediacy of a dehydroalanyl species has been reported, here we describe observation of the elusive enzyme-bound dehydroamino acyl intermediate generated from the corresponding allo-threonyl tetrapeptide and partitioned into pentapeptide products containing either a dehydrobutyrine residue or an embedded β-lactam. Contrary to trends in the literature where condensation domains have been deemed flexible as to acyl donor structure, this β-lactam synthesizing domain is highly discriminating. The observation of dehydrobutyrine formation links this C domain to related clades associated with natural products containing dehydroamino acid and d-configured residues, suggesting a common mechanistic link.

Whey protein addition and its increased light absorption and tinctorial strength of model solutions colored with anthocyanins

Anthocyanins (ACN) are pigments with vivid colors, but their application as food colorants is restricted by their limited stability and color expression. Anthocyanins exhibit higher stability in dairy systems than in buffers at similar pH, suggesting that pigments may be able to interact with dairy components such as proteins, resulting in improved performance as colorants. Our objective was to determine the type of interaction between whey proteins (WP) and ACN leading to color enhancements and to determine the role of the ACN chemical structure in this interaction. Model solutions colored with semipurified pigments from sources with different ACN profiles (Berberis boliviana, grape skin, purple corn, black carrot, and red cabbage) were mixed with different concentrations of whey protein isolate (WPI) in pH 3 buffer. Absorption spectra of these solutions were acquired using an absorbance microplate reader, and color parameters were calculated from spectral data. Isolated ACN 3-glucosides were used to determine the role of the aglycone structure in the WP-ACN interaction using visible and fluorescence spectroscopy. In silico modeling was used to visualize potential differences in the interaction between β-lactoglobulin and ACN. Addition of WPI resulted in hyperchromic shifts at the wavelength of maximum absorption in the visible range (λvis-max) of up to 19%, and a significant increase in tinctorial strength for all ACN sources (ΔE > 5). Moreover, ACN acylation did not seem to play a significant role in the WP-ACN interaction. When using isolated ACN, WPI addition resulted in hyperchromic shifts at the λvis-max only for methoxylated ACN such as petunidin-3-glucoside (up to 24%), and malvidin-3-glucoside (up to 97%). The bimolecular quenching constant values (Kq > 1010M-1s-1) strongly suggested that the predominant type of quenching interaction was static. Analysis of enthalpy, entropy, and Gibbs free energy showed that this binding was spontaneous; depending on the chemical structure of the ACN, the predominant binding forces could be hydrophobic interactions or hydrogen bonding. Modeling suggested that methoxylations in the B ring of the aglycon structure promoted interactions with electron acceptor amino acids. Overall, WP could be used to enhance the tinctorial strength of select ACN depending on their structural characteristics. Therefore, ACN source selection may play a key role for specific applications in dairy products.

Gas-Phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and Their Implication in Database Searches.

ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and particularly of their acceptor amino acids remains a major challenge. The attachment sites of the modification are difficult to localize by mass spectrometry (MS) because of the labile nature of the linkage and the complex fragmentation pattern of the ADP-ribose in MS/MS experiments. In this study we performed a comprehensive analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine, or lysine residues. In addition to the fragmentation of the peptide backbone, various cleavages of the ADP-ribosylated amino acid side chains were investigated. We focused on gas-phase fragmentations that were specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid, or aspartic acid was the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine was relatively stable. The main cleavage site was the inner bond of the guanidine group, which resulted in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking peptide fragment ions resulting from this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches. Furthermore, the presence of diagnostic ions and of losses of fragments from peptide ions allowed us, in most cases, to distinguish between ADP-ribosylated arginine and serine residues.

Rational Design of Aptamer-Tagged tRNAs.

Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.

Development of allergic airway immunity depends on MARCH1-mediated ubiquitination of MHCII and CD86 in dendritic cells

Abstract Dendritic cells (DCs) play an essential role in the development of type 2 allergic immunity by presenting allergens to cognate naïve T cells inducing production of IL-4 thus priming type 2 T helper (Th2) cells. However, the molecular mechanisms underlying this function are poorly understood. We found that mice deficient in the expression of MARCH1 ubiquitin ligase in DCs were completely resistant to developing Th2 cell inflammation and eosinophilia upon airway exposure to house dust mite allergens. These mice exhibited normal expansion of allergen-specific CD4+ T cells and normal production of IFN-γ from the T cells but demonstrated a significant defect in the production of IL-4, suggesting a specific role of MARCH1 in DC priming of Th2 cells. Remarkably, mice lacking the ubiquitin acceptor amino acids of the MARCH1 substrates, MHCII and CD86, phenocopied MARCH1-deficient mice, indicating that MARCH1 promotes Th2 cell priming by mediating ubiquitination of MHCII and CD86. Ubiquitination of MHCII was important for CD11b+ DCs to transport respiratory allergens to the mediastinal lymph node whereas ubiquitination of CD86 as well as MHCII was involved in transcriptional reprograming of these DCs. In conclusion, dendritic cells depend on ubiquitination of MHCII and CD86 to condition themselves to effectively migrate to the draining lymph node and induce production of IL-4 from allergen-specific naïve CD4+ T cells.

The complete mitochondrial genome of Epeus alboguttatus (Araneae: Salticidae)

The complete mitochondrial genome of Epeus alboguttatus (Araneae: Salticidae) is 14,625 bp in length (GenBank accession number MH922026). It was predicted to consist of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA genes, and a putative control region. The gene composition and orientation of E. alboguttatus were identical to those found in other spider mitogenomes. The nucleotide composition shows heavily biased towards As and Ts, accounting for 77.59%. Twenty-two genes were in majority strand and 15 genes were in minority strand. ATT, ATA, TTA, and TTG were initiation codons, and TAA, TAG, and T were termination codons. Twenty-two reading frame overlaps and four intergenic regions were found in the mitogenome of E. alboguttatus. Ten tRNA genes lost the TΨC arm stems and had unpaired amino acid acceptor arms, whereas three tRNAs lacked the dihydrouracil (DHU) arm. Phylogenetic analysis indicated that E. alboguttatus is nested with Salticidae and recovered as sister to other five salticids.

Archaeal NSUN6 catalyzes m5C72 modification on a wide-range of specific tRNAs.

Human NOL1/NOP2/Sun RNA methyltransferase family member 6 (hNSun6) generates 5-methylcytosine (m5C) at C72 of four specific tRNAs, and its homologs are present only in higher eukaryotes and hyperthermophilic archaea. Archaeal NSun6 homologs possess conserved catalytic residues, but have distinct differences in their RNA recognition motifs from eukaryotic NSun6s. Until now, the biochemical properties and functions of archaeal NSun6 homologs were unknown. In archaeon Pyrococcus horikoshii OT3, the gene encoding the NSun6 homolog is PH1991. We demonstrated that the PH1991 protein could catalyze m5C72 formation on some specific PhtRNAs in vitro and was thus named as PhNSun6. Remarkably, PhNSun6 has a much wider range of tRNA substrates than hNSun6, which was attributed to its tRNA substrate specificity. The mechanism was further elucidated using biochemical and crystallographic experiments. Structurally, the binding pocket for nucleotide 73 in PhNSun6 is specific to accommodate U73 or G73-containing PhtRNAs. Furthermore, PhNSun6 lacks the eukaryotic NSun6-specific Lys-rich loop, resulting in the non-recognition of D-stem region by PhNSun6. Functionally, the m5C72 modification could slightly promote the thermal stability of PhtRNAs, but did not affect the amino acid accepting activity of PhtRNAs.

Characterisation of the complete mitochondrial genome of the black-footed abalone Haliotis iris

ABSTRACTThe complete mitogenome of Haliotis iris, an economically important shellfish endemic to New Zealand, was sequenced for the first time. The mitogenome was 17,131 base pairs (bp) in length and contained 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and a control region. All 13 genes were initiated by the start codon ATG, except for nad5 (ATA). Two typical stop codons, TAA and TAG, were present. All of the tRNAs could be folded into typical cloverleaf secondary structures except tRNASer1 and tRNALys, which lacked a DHU stem and complete amino acid acceptor stem, respectively. The control region was 1132 bp in length and contained six AT tandem repeats. According to the gene order of the mitogenome, the 30 analysed Vetigastropoda species could be classified into three types—type I: over half of the studied species were very similar to the gastropod ancestral gene order, and the rearrangements occurred in five tRNAs; type II: eight species were found to be missing several tRNA genes; type III: Fissurellidae, Lepetodrilidae showed a large inverted fragment.