Functional interplay between arginyl-tRNA synthetases and arginyltransferase

Protein arginylation, mediated by arginyltransferase ATE1, is a posttranslational modification of emerging biological importance that consists of transfer of the amino acid Arg to protein and peptide substrates. ATE1 utilizes charged tRNAArg as the donor of the arginyl group, which depends on the activity of Arg-tRNA synthetases (RARS) and is also utilized in translation. The mechanisms that regulate the functional balance between ATE1, RARS and translation are unknown. Here we addressed the functional interplay between these mechanisms using intracellular arginylation sensor in cell lines with overexpression or deletion of ATE1 and RARS isoforms. We find that arginylation levels depend on the physiological state of the cells but are not directly affected by translation activity or availability of RARS isoforms. However, displacement of RARS from the multi-synthetase complex leads to an increase in intracellular arginylation independently of RARS enzymatic activity. This effect is accompanied by ATE1 redistribution into the cytosol. Our results provide the first comprehensive analysis of the interdependence between translation, arginyl-tRNA synthesis, and arginylation.

Molecular Evolution of Tubulins in Diatoms

Microtubules are formed by α- and β-tubulin heterodimers nucleated with γ-tubulin. Tubulins are conserved eukaryotic proteins. Previously, it was shown that microtubules are involved in diatom silica frustule morphogenesis. Diatom frustules are varied, and their morphology is species-specific. Despite the attractiveness of the problem of elucidating the molecular mechanisms of genetically programmed morphogenesis, the structure and evolution of diatom tubulins have not been studied previously. Based on available genomic and transcriptome data, we analyzed the phylogeny of the predicted amino acid sequences of diatom α-, β- and γ-tubulins and identified five groups for α-tubulins, six for β-tubulins and four for γ-tubulins. We identified characteristic amino acids of each of these groups and also analyzed possible posttranslational modification sites of diatom tubulins. According to our results, we assumed what changes occurred in the diatom tubulin structures during their evolution. We also identified which tubulin groups are inherent in large diatom taxa. The similarity between the evolution of diatom tubulins and the evolution of diatoms suggests that molecular changes in α-, β- and γ-tubulins could be one of the factors in the formation of a high morphological diversity of diatoms.

Clostridioides difficile Phosphoproteomics Shows an Expansion of Phosphorylated Proteins in Stationary Growth Phase

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile . To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile .

Photocaged Dicarbonyl Probe Provides Spatiotemporal Control Over Protein Glycation

Protein glycation is a disease associated, non-enzymatic, posttranslational modification generated by endogenous dicarbonyl metabolites. Currently, there is a lack of chemical tools capable of studying protein adducts caused by this...

Availability of Arg, but Not tRNA, Is a Rate-Limiting Factor for Intracellular Arginylation

Protein arginylation, mediated by arginyltransferase ATE1, is a posttranslational modification of emerging biological importance that consists of transfer of the amino acid Arg from tRNA to protein and peptide targets. ATE1 can bind tRNA and exhibits specificity toward particular tRNA types, but its dependence on the availability of the major components of the arginylation reaction has never been explored. Here we investigated key intracellular factors that can potentially regulate arginylation in vivo, including several tRNA types that show strong binding to ATE1, as well as availability of free Arg, in an attempt to identify intracellular rate limiting steps for this enzyme. Our results demonstrate that, while modulation of tRNA levels in cells does not lead to any changes in intracellular arginylation efficiency, availability of free Arg is a potentially rate-limiting factor that facilitates arginylation if added to the cultured cells. Our results broadly outline global pathways that may be involved in the regulation of arginylation in vivo.

Poly(ADP-ribosyl)ating pathway regulates development from stem cell niche to longevity control

The regulation of poly(ADP-ribose) polymerase, the enzyme responsible for the synthesis of homopolymer ADP-ribose chains on nuclear proteins, has been extensively studied over the last decades for its involvement in tumorigenesis processes. However, the regulation of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for removing this posttranslational modification, has attracted little attention. Here we identified that PARG activity is partly regulated by two phosphorylation sites, ph1 and ph2, in Drosophila. We showed that the disruption of these sites affects the germline stem-cells maintenance/differentiation balance as well as embryonic and larval development, but also the synchronization of egg production with the availability of a calorically sufficient food source. Moreover, these PARG phosphorylation sites play an essential role in the control of fly survivability from larvae to adults. We also showed that PARG is phosphorylated by casein kinase 2 and that this phosphorylation seems to protect PARG protein against degradation in vivo. Taken together, these results suggest that the regulation of PARG protein activity plays a crucial role in the control of several developmental processes.

FOXO1 Is a Critical Switch Molecule for Autophagy and Apoptosis of Sow Endometrial Epithelial Cells Caused by Oxidative Stress

Oxidative stress (OS) is involved in various reproductive diseases and can induce autophagy and apoptosis, which determine the different fates of cells. However, the sequence and the switch mechanism between autophagy and apoptosis are unclear. Here, we reported that chronic restraint stress (CRS) induced OS (decreased T-AOC, T-SOD, CAT and GSH-Px and increased MDA) and then disturbed the endocrine environment of sows during early pregnancy, including the hypothalamic-pituitary-ovarian (HPO) and the hypothalamic-pituitary-adrenal (HPA) axes. Meanwhile, after CRS, the KEAP1/NRF2 pathway was inhibited and attenuated the antioxidative ability to cause OS of the endometrium. The norepinephrine (NE) triggered β2-AR to activate the FOXO1/NF-κB pathway, which induced endometrial inflammation. CRS induced the caspase-dependent apoptosis pathway and caused MAP1LC3-II accumulation, SQSTM1/p62 degradation, and autophagosome formation to initiate autophagy. Furthermore, in vitro, a cellular OS model was established by adding hydrogen peroxide into cells. Low OS maintained the viability of endometrial epithelial cells by triggering autophagy, while high OS induced cell death by initiating caspase-dependent apoptosis. Autophagy preceded the occurrence of apoptosis, which depended on the subcellular localization of FOXO1. In the low OS group, FOXO1 was exported from the nucleus to be modified into Ac-FOXO1 and bound to ATG7 in the cytoplasm, which promoted autophagy to protect cells. In the high OS group, FOXO1 located in the nucleus to promote transcription of proapoptotic proteins and then induce apoptosis. Here, FOXO1, as a redox sensor switch, regulated the transformation of cell autophagy and apoptosis. In summary, the posttranslational modification of FOXO1 may become the target of OS treatment.

Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease. Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, will inform our understanding of this disease and its treatment.

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

Pathogen-Host Interaction Repertoire at Proteome and Posttranslational Modification Levels During Fungal Infections

Prevalence of fungal diseases has increased globally in recent years, which often associated with increased immunocompromised patients, aging populations, and the novel Coronavirus pandemic. Furthermore, due to the limitation of available antifungal agents mortality and morbidity rates of invasion fungal disease remain stubbornly high, and the emergence of multidrug-resistant fungi exacerbates the problem. Fungal pathogenicity and interactions between fungi and host have been the focus of many studies, as a result, lots of pathogenic mechanisms and fungal virulence factors have been identified. Mass spectrometry (MS)-based proteomics is a novel approach to better understand fungal pathogenicities and host–pathogen interactions at protein and protein posttranslational modification (PTM) levels. The approach has successfully elucidated interactions between pathogens and hosts by examining, for example, samples of fungal cells under different conditions, body fluids from infected patients, and exosomes. Many studies conclude that protein and PTM levels in both pathogens and hosts play important roles in progression of fungal diseases. This review summarizes mass spectrometry studies of protein and PTM levels from perspectives of both pathogens and hosts and provides an integrative conceptual outlook on fungal pathogenesis, antifungal agents development, and host–pathogen interactions.