Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling.

BACKGROUND: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems-the ubiquitin proteasome system (UPS) and macroautophagy-mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless, how Ate1 operates through this intricate mechanism remains elusive. METHODS: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofluorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform were used as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofluorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy. RESULTS: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specific inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic flux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical significance was assessed using a two-sided unpaired t test with a significance threshold set at P<0.05. CONCLUSIONS: Our findings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.

Previously Unidentified Histone H1-Like Protein Is Involved in Cell Division and Ribosome Biosynthesis in Toxoplasma gondii.

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii ("TgH1-like") that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. IMPORTANCE Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

Overview of protein posttranslational modifications in Arthropoda venoms.

Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study "PTM-functional-venomics", herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.

Functional Study of Leishmania braziliensis Protein Arginine Methyltransferases (PRMTs) Reveals That PRMT1 and PRMT5 Are Required for Macrophage Infection.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other RBP interactions by transferring methyl groups to arginine residues (R-methylation). Herein, we functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that R-methylation profiles vary among Leishmania species and across L. braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes. A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation assay using HA-tagged PRMTs, suggesting a network of putative targets of PRMTs and cooperation between the R-methylation writers. Knockout of each L. braziliensis PRMT led to significant changes in global arginine methylation patterns without affecting cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine levels. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and for axenic amastigote proliferation. The results indicate that R-methylation is modulated across lifecycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

Previously unidentified histone H1-like protein is involved in cell division and ribosome biosynthesis in toxoplasma gondii

Chromatin dynamics can regulate all DNA-dependent processes. Access to DNA within chromatin is orchestrated mainly by histones and their posttranslational modifications (PTMs). Like other eukaryotes, the apicomplexan parasite Toxoplasma gondii encodes four canonical histones and five histone variants. In contrast, the linker histone (H1) has never been identified in apicomplexan parasites. In other eukaryotes, histone H1 compacts the chromatin by linking the nucleosome and increasing the DNA compaction. H1 is a multifunctional protein and can be involved in different steps of DNA metabolism or associated with protein complexes related to distinct biological processes. We have identified a novel protein in T. gondii (“TgH1-like”) that, although lacking the globular domain of mammalian H1, is remarkably like the H1-like proteins of bacteria and trypanosomatids. Our results demonstrate that TgH1-like is a nuclear protein associated with chromatin and other histones. Curiously, TgH1-like is also in the nucleolus and associated with ribosomal proteins, indicating a versatile function in this parasite. Although knockout of the tgh1-like gene does not affect the cell cycle, it causes endopolygeny and asynchronous division. Interestingly, mutation of posttranslationally modified amino acids results in defects in cell division like those in the Δtgh1-like mutant, showing that these sites are important for protein function. Furthermore, in the bradyzoite stage, this protein is expressed only in dividing parasites, reinforcing its importance in cell division. Indeed, the absence of TgH1-like decreases compaction of peripheral chromatin, confirming its role in the chromatin modulation in T. gondii. Histone H1, or linker histone, is an important protein that binds to the nucleosome, aiding chromatin compaction. Here, we characterize for the first time a linker histone in T. gondii, named TgH1-like. It is a small and basic protein that corresponds only to the C-terminal portion of the human H1 but is similar to histone H1 from trypanosomatids and bacteria. TgH1-like is located in the nucleus, interacts with nucleosome histones, and acts in chromatin structure and cell division. Our findings show for the first time the presence of a histone H1 protein in an apicomplexan parasite and will provide new insights into cell division and chromatin dynamics in T. gondii and related parasites.

Overview of protein posttranslational modifications in Arthropoda venoms

Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study "PTM-functional-venomics", herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.(AU)

Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation.

Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.

Recombinant HCV NS3 and NS5B enzymes exhibit multiple posttranslational modifications for potential regulation.

Posttranslational modification (PTM) of proteins is critical to modulate protein function and to improve the functional diversity of polypeptides. In this report, we have analyzed the PTM of both hepatitis C virus NS3 and NS5B enzyme proteins, upon their individual expression in insect cells under the baculovirus expression system. Using mass spectrometry, we present evidence that these recombinant proteins exhibit diverse covalent modifications on certain amino acid side chains, such as phosphorylation, ubiquitination, and acetylation. Although the functional implications of these PTM must be further addressed, these data may prove useful toward the understanding of the complex regulation of these key viral enzymes and to uncover novel potential targets for antiviral design.

Rhodococcus erythropolis as a host for expression, secretion and glycosylation of Mycobacterium tuberculosis proteins.

BACKGROUND: Glycosylation is one of the most abundant posttranslational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. There is growing evidence about the importance of these modifications in host bacteria interactions in tuberculosis. It is known, that the sugars present in some Mycobacterium tuberculosis glycoproteins play an important role in both humoral and cellular immune response against the pathogen. Since this modification is lost in the recombinant proteins expressed in Escherichia coli, it is fundamental to search for host bacteria with the capacity to modify the foreign proteins. Amongst the bacteria that are likely to have this possibility are some members of Rhodococcus genus which are Gram-positive bacteria, with high GC-content and genetically very close related to M. tuberculosis. RESULTS: In this work, apa, pstS1 and lprG genes that coding for M. tuberculosis glycoproteins were cloned and expressed in Rhodococcus erythropolis. All recombinant proteins were mannosylated as demonstrated by their interaction with mannose binding lectin Concanavalin A. In addition, as native proteins recombinants Apa and PstS1 were secreted to the culture medium in contrast with LprG that was retained in the cell wall. CONCLUSIONS: Together these results, point out R. erythropolis, as a new host for expression of M. tuberculosis glycoproteins.

Regulation of RNA binding proteins in trypanosomatid protozoan parasites.

Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

GABA-induced uncoupling of GABA/benzodiazepine site interactions is associated with increased phosphorylation of the GABAA receptor.

The use-dependent regulation of the GABAA receptor occurs under physiological, pathological, and pharmacological conditions. Tolerance induced by prolonged administration of benzodiazepines is associated with changes in GABAA receptor function. Chronic exposure of neurons to GABA for 48 hr induces a downregulation of the GABAA receptor number and an uncoupling of the GABA/benzodiazepine site interactions. A single brief exposure ((t1/2) = 3 min) of rat neocortical neurons to the neurotransmitter initiates a process that results in uncoupling hours later (t(1/2) = 12 hr) without alterations in the number of GABAA receptors and provides a paradigm to study the uncoupling mechanism selectively. Here we report that uncoupling induced by a brief GABAA receptor activation is blocked by the coincubation with inhibitors of protein kinases A and C, indicating that the uncoupling is mediated by the activation of a phosphorylation cascade. GABA-induced uncoupling is accompanied by subunit-selective changes in the GABAA receptor mRNA levels. However, the GABA-induced downregulation of the α3 subunit mRNA level is not altered by the kinase inhibitors, suggesting that the uncoupling is the result of a posttranscriptional regulatory process. GABA exposure also produces an increase in the serine phosphorylation on the GABAA receptor γ2 subunit. Taken together, our results suggest that the GABA-induced uncoupling is mediated by a posttranscriptional mechanism involving an increase in the phosphorylation of GABAA receptors. The uncoupling of the GABAA receptor may represent a compensatory mechanism to control GABAergic neurotransmission under conditions in which receptors are persistently activated.

Recent advances in maize nuclear proteomic studies reveal histone modifications.

The nucleus of eukaryotic organisms is highly dynamic and complex, containing different types of macromolecules including DNA, RNA, and a wide range of proteins. Novel proteomic applications have led to a better overall determination of nucleus protein content. Although nuclear plant proteomics is only at the initial phase, several studies have been reported and are summarized in this review using different plants species, such as Arabidopsis thaliana, rice, cowpea, onion, garden cress, and barrel clover. These include the description of the total nuclear or phospho-proteome (i.e., Arabidopsis, cowpea, onion), or the analysis of the differential nuclear proteome under different growth environments (i.e., Arabidopsis, rice, cowpea, onion, garden cress, and barrel clover). However, only few reports exist on the analysis of the maize nuclear proteome or its changes under various conditions. This review will present recent data on the study of the nuclear maize proteome, including the analysis of changes in posttranslational modifications in histone proteins.