New insight into arginine and tryptophan metabolism in macrophage activation during tuberculosis.

Arginine and tryptophan are pivotal in orchestrating cytokine-driven macrophage polarization and immune activation. Specifically, interferon-gamma (IFN-γ) stimulates inducible nitric oxide synthase (iNOS) expression), leading to the conversion of arginine into citrulline and nitric oxide (NO), while Interleukin-4 (IL4) promotes arginase activation, shifting arginine metabolism toward ornithine. Concomitantly, IFN-γ triggers indoleamine 2,3-dioxygenase 1 (IDO1) and Interleukin-4 induced 1 (IL4i1), resulting in the conversion of tryptophan into kynurenine and indole-3-pyruvic acid. These metabolic pathways are tightly regulated by NAD+-dependent sirtuin proteins, with Sirt2 and Sirt5 playing integral roles. In this review, we present novel insights that augment our understanding of the metabolic pathways of arginine and tryptophan following Mycobacterium tuberculosis infection, particularly their relevance in macrophage responses. Additionally, we discuss arginine methylation and demethylation and the role of Sirt2 and Sirt5 in regulating tryptophan metabolism and arginine metabolism, potentially driving macrophage polarization.

Structural insights into the regulation of protein-arginine kinase McsB by McsA.

In gram-positive bacteria, phosphorylated arginine functions as a protein degradation signal in a similar manner as ubiquitin in eukaryotes. The protein-arginine phosphorylation is mediated by the McsAB complex, where McsB possesses kinase activity and McsA modulates McsB activity. Although mcsA and mcsB are regulated within the same operon, the role of McsA in kinase activity has not yet been clarified. In this study, we determined the molecular mechanism by which McsA regulates kinase activity. The crystal structure of the McsAB complex shows that McsA binds to the McsB kinase domain through a second zinc-coordination domain and the subsequent loop region. This binding activates McsB kinase activity by rearranging the catalytic site, preventing McsB self-assembly, and enhancing stoichiometric substrate binding. The first zinc-coordination and coiled-coil domains of McsA further activate McsB by reassembling the McsAB oligomer. These results demonstrate that McsA is the regulatory subunit for the reconstitution of the protein-arginine kinase holoenzyme. This study provides structural insight into how protein-arginine kinase directs the cellular protein degradation system.

Loss-of-function mutation in PRMT9 causes abnormal synapse development by dysregulation of RNA alternative splicing.

Protein arginine methyltransferase 9 (PRMT9) is a recently identified member of the PRMT family, yet its biological function remains largely unknown. Here, by characterizing an intellectual disability associated PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncover an important function of PRMT9 in neuronal development. The G189R mutation abolishes PRMT9 methyltransferase activity and reduces its protein stability. Knockout of Prmt9 in hippocampal neurons causes alternative splicing of ~1900 genes, which likely accounts for the aberrant synapse development and impaired learning and memory in the Prmt9 cKO mice. Mechanistically, we discover a methylation-sensitive protein-RNA interaction between the arginine 508 (R508) of the splicing factor 3B subunit 2 (SF3B2), the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element that is critical for RNA splicing. Additionally, using human and mouse cell lines, as well as an SF3B2 arginine methylation-deficient mouse model, we provide strong evidence that SF3B2 is the primary methylation substrate of PRMT9, thus highlighting the conserved function of the PRMT9/SF3B2 axis in regulating pre-mRNA splicing.

The twin-arginine translocation (Tat) system.

In this Quick guide, Palmer and Berks introduce the twin-arginine translocation (Tat) systems. Tats are found in a variety of microbes and microbe-derived organelles, and are known to translocate folded substrate proteins across biological membranes.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase.

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

Standardized ileal digestibility of amino acids in Chinese fermented soybean meal from different sources fed to mid and late-gestating sows.

We determined apparent ileal digestibility (AID) and standardized ileal digestibility (SID) values of crude protein (CP) and amino acids (AA) in fermented soybean meal from five different sources (FSBM 1 to 5) in China when fed to mid and late-gestating sows. Twenty-four parity four sows (12 at 30 d in gestation and 12 at 80 d in gestation) were fitted with a T-cannula in the distal ileum and used in this experiment. Sows were randomly assigned to a replicated 6 × 3 Youden square design including six diets and three periods. Six diets were provided for sows in mid and late gestation, including a nitrogen-free diet and five test diets containing 26% FSBM from different sources. Results showed that there were differences in AID and SID of CP among the different FSBM samples, but no differences between sow physiological stages were observed. Specifically, when mid-gestating sows were fed FSBM 2, the AID of CP was the lowest, whereas FSBM 3 exhibited a greater AID of CP when compared to the other FSBM samples (P < 0.01). Furthermore, during late gestation, FSBM 3 consistently had greater SID of CP when compared to other FSBM samples (P < 0.01). The ileal digestibility of most AA varied with different FSBM samples. In both mid and late gestation, differences (P < 0.05) were observed for AID of lysine, tryptophan, histidine, and arginine across different FSBM samples. Similarly, the AID of dispensable AA (cysteine, glutamine, and serine) also exhibited differences (P < 0.05) across different FSBM samples in both mid and late-gestating sows. For mid-gestating sows, SID differences relating to lysine, phenylalanine, tryptophan, threonine, and arginine were observed among different diets (P < 0.05). In late-gestating sows, SID values for lysine, tryptophan, leucine, and arginine differed across diets (P < 0.05). Furthermore, the ileal digestibility of some dispensable AA was influenced by physiological stage, as evidenced by greater AID and SID values for glycine, glutamine, cysteine, and serine in late-gestating sows when compared to mid-gestating sows (P < 0.01). In summary, our study determined AA ileal digestibility of different FSBM fed to mid and late-gestating sows. We observed that the AA ileal digestibility differed among five FSBM samples, but the physiological stage of sows did not affect the ileal digestibility of CP and most AA. Additionally, when formulating diets for sows, it is crucial to consider the nutritional value differences of FSBM.

Fermented soybean meal (FSBM) is obtained from the microbial fermentation of soybean meal, which reduces anti-nutritional factor levels and enhances other nutrient content. Substituting soybean meal with FSBM in piglet and growing pig diets improves nutrient digestibility. However, its nutritional value for sows remains unclear. Therefore, five sources of FSBM were fed to sows in mid and late gestation to evaluate apparent ileal digestibility (AID) and standardized ileal digestibility (SID) values of amino acids (AA). We found that different FSBM samples impacted the SID value of AA when fed to gestating sows. Additionally, sow physiological stage influenced the SID of some dispensable AA. These findings provide valuable insights into the incorporation of FSBM into sow diets.

Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

L-arginine alleviates heat stress-induced mammary gland injury through modulating CASTOR1-mTORC1 axis mediated mitochondrial homeostasis.

As global warming intensifies, extreme heat is becoming increasingly frequent. These extreme heatwaves have decreased the milk production of dairy animals such as cows and goats and have caused significant damage to the entire dairy industry. It is known that heat stress (HS) can induce the apoptosis and autophagy of mammary epithelial cells (MECs), leading to a decrease in lactating MECs. L-arginine can effectively attenuate HS-induced decreases in milk yield, but the exact mechanisms are not fully understood. In this study, we found that HS upregulated the arginine sensor CASTOR1 in mouse MECs. Arginine activated mTORC1 activity through CASTOR1 and promoted mitochondrial biogenesis through the mTORC1/PGC-1α/NRF1 pathway. Moreover, arginine inhibited mitophagy through the CASTOR1/PINK1/Parkin pathway. Mitochondrial homeostasis ensures ATP synthesis and a stable cellular redox state for MECs under HS, further alleviating HS-induced damage and improving the lactation performance of MECs. In conclusion, these findings reveal the molecular mechanisms by which L-arginine relieves HS-induced mammary gland injury, and suggest that the intake of arginine-based feeds or feed additives is a promising method to increase the milk yield of dairy animals in extreme heat conditions.

Gene expression reprogramming of Pseudomonas alloputida in response to arginine through the transcriptional regulator ArgR.

Different bacteria change their life styles in response to specific amino acids. In Pseudomonas putida (now alloputida) KT2440, arginine acts both as an environmental and a metabolic indicator that modulates the turnover of the intracellular second messenger c-di-GMP, and expression of biofilm-related genes. The transcriptional regulator ArgR, belonging to the AraC/XylS family, is key for the physiological reprogramming in response to arginine, as it controls transport and metabolism of the amino acid. To further expand our knowledge on the roles of ArgR, a global transcriptomic analysis of KT2440 and a null argR mutant growing in the presence of arginine was carried out. Results indicate that this transcriptional regulator influences a variety of cellular functions beyond arginine metabolism and transport, thus widening its regulatory role. ArgR acts as positive or negative modulator of the expression of several metabolic routes and transport systems, respiratory chain and stress response elements, as well as biofilm-related functions. The partial overlap between the ArgR regulon and those corresponding to the global regulators RoxR and ANR is also discussed.

Reduced production of Ethyl Carbamate in wine by regulating the accumulation of arginine in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a multisite carcinogenic compound, is naturally produced from urea and ethanol in alcoholic beverages. In order to reduce the content of EC in wine, the accumulation of arginine in Saccharomyces cerevisiae was regulated by genetic modifying genes involved in arginine transport and synthesis pathways to reduce the production of urea. Knockout of genes encoding arginine permease (Can1p) and amino acid permease (Gap1p) on the cell membrane as well as argininosuccinate synthase (Arg1) respectively resulted in a maximum reduction of 66.88% (9.40 µg/L) in EC, while overexpressing the gene encoding amino acid transporter (Vba2) reduced EC by 52.94% (24.13 µg/L). Simultaneously overexpressing Vba2 and deleting Arg1 showed the lowest EC production with a decrease of 68% (7.72 µg/L). The yield of total higher alcohols of the mutants all decreased compared with that of the original strain. Comprehensive consideration of flavor compound contents and sensory evaluation results indicated that mutant YG21 obtained by deleting two allele coding Gap1p performed best in must fermentation of Cabernet Sauvignon with the EC content low to 9.40 µg/L and the contents of total higher alcohols and esters of 245.61 mg/L and 41.71 mg/L respectively. This study has provided an effective strategy for reducing the EC in wine.

Arginine inhibits the arginine biosynthesis rate-limiting enzyme and leads to the accumulation of intracellular aspartate in Synechocystis sp. PCC 6803.

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.

KEY MESSAGE: This study revealed the catalytic efficiency and inhibition of cyanobacterial argininosuccinate synthetase by arginine and demonstrated that a strain overexpressing this enzyme grew faster than the wild-type strain.

Inhibition of PRMT5/MEP50 Arginine Methyltransferase Activity Causes Cancer Vulnerability in NDRG2low Adult T-Cell Leukemia/Lymphoma.

N-myc downstream-regulated gene 2 (NDRG2), which is a tumour suppressor, is frequently lost in many types of tumours, including adult T-cell leukaemia/lymphoma (ATL). The downregulation of NDRG2 expression is involved in tumour progression through the aberrant phosphorylation of several important signalling molecules. We observed that the downregulation of NDRG2 induced the translocation of protein arginine methyltransferase 5 (PRMT5) from the nucleus to the cytoplasm via the increased phosphorylation of PRMT5 at Serine 335. In NDRG2low ATL, cytoplasmic PRMT5 enhanced HSP90A chaperone activity via arginine methylation, leading to tumour progression and the maintenance of oncogenic client proteins. Therefore, we examined whether the inhibition of PRMT5 activity is a drug target in NDRG2low tumours. The knockdown of PRMT5 and binding partner methylsome protein 50 (MEP50) expression significantly demonstrated the suppression of cell proliferation via the degradation of AKT and NEMO in NDRG2low ATL cells, whereas NDRG2-expressing cells did not impair the stability of client proteins. We suggest that the relationship between PRMT5/MEP50 and the downregulation of NDRG2 may exhibit a novel vulnerability and a therapeutic target. Treatment with the PRMT5-specific inhibitors CMP5 and HLCL61 was more sensitive in NDRG2low cancer cells than in NDRG2-expressing cells via the inhibition of HSP90 arginine methylation, along with the degradation of client proteins. Thus, interference with PRMT5 activity has become a feasible and effective strategy for promoting cancer vulnerability in NDRG2low ATL.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells.

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

Arginine (315) is required for the PLIN2-CGI-58 interface and plays a functional role in regulating nascent LDs formation in bovine adipocytes.

Perilipin-2 (PLIN2) can anchor to lipid droplets (LDs) and play a crucial role in regulating nascent LDs formation. Bimolecular fluorescence complementation (BiFC) and flow cytometry were examined to verify the PLIN2-CGI-58 interaction efficiency in bovine adipocytes. GST-Pulldown assay was used to detect the key site arginine315 function in PLIN2-CGI-58 interaction. Experiments were also examined to research these mutations function of PLIN2 in LDs formation during adipocytes differentiation, LDs were measured after staining by BODIPY, lipogenesis-related genes were also detected. Results showed that Leucine (L371A, L311A) and glycine (G369A, G376A) mutations reduced interaction efficiencies. Serine (S367A) mutations enhanced the interaction efficiency. Arginine (R315A) mutations resulted in loss of fluorescence in the cytoplasm and disrupted the interaction with CGI-58, as verified by pulldown assay. R315W mutations resulted in a significant increase in the number of LDs compared with wild-type (WT) PLIN2 or the R315A mutations. Lipogenesis-related genes were either up- or downregulated when mutated PLIN2 interacted with CGI-58. Arginine315 in PLIN2 is required for the PLIN2-CGI-58 interface and could regulate nascent LD formation and lipogenesis. This study is the first to study amino acids on the PLIN2 interface during interaction with CGI-58 in bovine and highlight the role played by PLIN2 in the regulation of bovine adipocyte lipogenesis.

RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone.

RNA-guided enzymes must quickly search a vast sequence space for their targets. This search is aided by chaperones such as Hfq, a protein that mediates regulation by bacterial small RNAs (sRNAs). How RNA binding proteins enhance this search is little known. Using single-molecule Förster resonance energy transfer, we show that E. coli Hfq performs a one-dimensional scan in which compaction of the target RNA delivers sRNAs to sites distant from the location of Hfq recruitment. We also show that Hfq can transfer an sRNA between different target sites in a single mRNA, favoring the most stable duplex. We propose that compaction and segmental transfer, combined with repeated cycles of base pairing, enable the kinetic selection of optimal sRNA targets. Finally, we show that RNA compaction and sRNA transfer require conserved arginine patches. We suggest that arginine patches are a widespread strategy for enabling the movement of RNA across protein surfaces.

Treating liver cancer through arginine depletion.

Liver cancer, the sixth most common cancer globally and the second-leading cause of cancer-related deaths, presents a critical public health threat. Diagnosis often occurs in advanced stages of the disease, aligning incidence with fatality rates. Given that established treatments, such as stereotactic body radiation therapy and transarterial radioembolization, face accessibility and affordability challenges, the emerging focus on cancer cell metabolism, particularly arginine (Arg) depletion, offers a promising research avenue. Arg-depleting enzymes show efficacy against Arg-auxotrophic cancers, including hepatocellular carcinoma (HCC). Thus, in this review, we explore the limitations of current therapies and highlight the potential of Arg depletion, emphasizing various Arg-hydrolyzing enzymes in clinical development.

Orphan lysosomal solute carrier MFSD1 facilitates highly selective dipeptide transport.

Orphan solute carrier (SLC) represents a group of membrane transporters whose exact functions and substrate specificities are not known. Elucidating the function and regulation of orphan SLC transporters is not only crucial for advancing our knowledge of cellular and molecular biology but can potentially lead to the development of new therapeutic strategies. Here, we provide evidence for the biological function of a ubiquitous orphan lysosomal SLC, the Major Facilitator Superfamily Domain-containing Protein 1 (MFSD1), which has remained phylogenetically unassigned. Targeted metabolomics revealed that dipeptides containing either lysine or arginine residues accumulate in lysosomes of cells lacking MFSD1. Whole-cell patch-clamp electrophysiological recordings of HEK293-cells expressing MFSD1 on the cell surface displayed transport affinities for positively charged dipeptides in the lower mM range, while dipeptides that carry a negative net charge were not transported. This was also true for single amino acids and tripeptides, which MFSD1 failed to transport. Our results identify MFSD1 as a highly selective lysosomal lysine/arginine/histidine-containing dipeptide exporter, which functions as a uniporter.

The unique dual targeting of AGO1 by two types of PRMT enzymes promotes phasiRNA loading in Arabidopsis thaliana.

Arginine/R methylation (R-met) of proteins is a widespread post-translational modification (PTM), deposited by a family of protein arginine/R methyl transferase enzymes (PRMT). Regulations by R-met are involved in key biological processes deeply studied in metazoan. Among those, post-transcriptional gene silencing (PTGS) can be regulated by R-met in animals and in plants. It mainly contributes to safeguard processes as protection of genome integrity in germlines through the regulation of piRNA pathway in metazoan, or response to bacterial infection through the control of AGO2 in plants. So far, only PRMT5 has been identified as the AGO/PIWI R-met writer in higher eukaryotes. We uncovered that AGO1, the main PTGS effector regulating plant development, contains unique R-met features among the AGO/PIWI superfamily, and outstanding in eukaryotes. Indeed, AGO1 contains both symmetric (sDMA) and asymmetric (aDMA) R-dimethylations and is dually targeted by PRMT5 and by another type I PRMT in Arabidopsis thaliana. We showed also that loss of sDMA didn't compromise AtAGO1 subcellular trafficking in planta. Interestingly, we underscored that AtPRMT5 specifically promotes the loading of phasiRNA in AtAGO1. All our observations bring to consider this dual regulation of AtAGO1 in plant development and response to environment, and pinpoint the complexity of AGO1 post-translational regulation.

Identification of novel tail-anchored membrane proteins integrated by the bacterial twin-arginine translocase.

The twin-arginine protein transport (Tat) system exports folded proteins across the cytoplasmic membranes of prokaryotes and the energy transducing-membranes of plant thylakoids and mitochondria. Proteins are targeted to the Tat machinery by N-terminal signal peptides with a conserved twin-arginine motif, and some substrates are exported as heterodimers where the signal peptide is present on one of the partner proteins. A subset of Tat substrates is found in the membrane. Tat-dependent membrane proteins usually have large globular domains and a single transmembrane helix present at the N- or C-terminus. Five Tat substrates that have C-terminal transmembrane helices have previously been characterized in the model bacterium Escherichia coli. Each of these is an iron-sulfur cluster-containing protein involved in electron transfer from hydrogen or formate. Here we have undertaken a bioinformatic search to identify further tail-anchored Tat substrates encoded in bacterial genomes. Our analysis has revealed additional tail-anchored iron-sulfur proteins associated in modules with either a b-type cytochrome or a quinol oxidase. We also identified further candidate tail-anchored Tat substrates, particularly among members of the actinobacterial phylum, that are not predicted to contain cofactors. Using reporter assays, we show experimentally that six of these have both N-terminal Tat signal peptides and C-terminal transmembrane helices. The newly identified proteins include a carboxypeptidase and a predicted protease, and four sortase substrates for which membrane integration is a prerequisite for covalent attachment to the cell wall.

Development of Complementary Photo-arginine/lysine to Promote Discovery of Arg/Lys hPTMs Interactomes.

Arginine and lysine, frequently appearing as a pair on histones, have been proven to carry diverse modifications and execute various epigenetic regulatory functions. However, the most context-specific and transient effectors of these marks, while significant, have evaded study as detection methods have thus far not reached a standard to capture these ephemeral events. Herein, a pair of complementary photo-arginine/δ-photo-lysine (R-dz/K-dz) probes is developed and involve these into histone peptide, nucleosome, and chromatin substrates to capture and explore the interactomes of Arg and Lys hPTMs. By means of these developed tools, this study identifies that H3R2me2a can recruit MutS protein homolog 6 (MSH6), otherwise repelDouble PHD fingers 2 (DPF2), Retinoblastoma binding protein 4/7 (RBBP4/7). And it is disclosed that H3R2me2a inhibits the chromatin remodeling activity of the cBAF complex by blocking the interaction between DPF2 (one component of cBAF) and the nucleosome. In addition, the novel pairs of H4K5 PTMs and respective readers are highlighted, namely H4K5me-Lethal(3)malignant brain tumor-like protein 2 (L3MBTL2), H4K5me2-L3MBTL2, and H4K5acK8ac-YEATS domain-containing protein 4 (YEATS4). These powerful tools pave the way for future investigation of related epigenetic mechanisms including but not limited to hPTMs.