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Necroptosis is a form of programmed necrosis that is mediated by various cytokines and pattern recognition receptors (PRRs). Cells dying by necroptosis show necrotic phenotypes, including swelling and membrane rupture, and release damage-associated molecular patterns (DAMPs), inflammatory cytokines, and chemokines, thereby mediating extreme inflammatory responses. Studies on gene knockout or necroptosis-specific inhibitor treatment in animal models have provided extensive evidence regarding the important roles of necroptosis in inflammatory diseases. The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the necroptosis process, these proteins are fine-tuned by posttranslational regulation via phosphorylation, ubiquitination, glycosylation, and protein–protein interactions. Herein, we review recent findings on the molecular regulatory mechanisms of necroptosis., Inflammation: Understanding cell death opens therapeutic possibilities A better understanding of the signaling pathways that drive an inflammatory mode of cell death known as necroptosis could lead to new therapies for autoimmune and neuroinflammatory conditions. Necroptosis is a regulated form of cell death often triggered by infections, tissue injuries, and chronic inflammatory conditions. It leads cells to rupture and disperse their contents, which in turn spurs an inflammatory immune response that can sometimes fuel disease. A team from South Korea led by Jaewhan Song of Yonsei University in Seoul discusses the molecular mechanisms of this process. They focus on several key mediators of the necroptosis signaling pathway and how those proteins in turn are modified to regulate their function. Several drug inhibitors of these proteins are already in clinical development for the treatment of psoriasis and other inflammatory disorders.

Cyanobacteria are the only prokaryotes that perform plant-like oxygenic photosynthesis. They evolved an inorganic carbon-concentrating mechanism to adapt to low CO2 conditions. Quantitative phosphoproteomics was applied to analyze regulatory features during the acclimation to low CO2 conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Overall, more than 2500 proteins were quantified, equivalent to c. 70% of the Synechocystis theoretical proteome. Proteins with changing abundances correlated largely with mRNA expression levels. Functional annotation of the noncorrelating proteins revealed an enrichment of key metabolic processes fundamental for maintaining cellular homeostasis. Furthermore, 105 phosphoproteins harboring over 200 site-specific phosphorylation events were identified. Subunits of the bicarbonate transporter BCT1 and the redox switch protein CP12 were among phosphoproteins with reduced phosphorylation levels at lower CO2 , whereas the serine/threonine protein kinase SpkC revealed increased phosphorylation levels. The corresponding ΔspkC mutant was characterized and showed decreased ability to acclimate to low CO2 conditions. Possible phosphorylation targets of SpkC including a BCT1 subunit were identified by phosphoproteomics. Collectively, our study highlights the importance of posttranscriptional regulation of protein abundances as well as posttranslational regulation by protein phosphorylation for the successful acclimation towards low CO2 conditions in Synechocystis and possibly among cyanobacteria.

The transcription factor nuclear factor erythroid-derived 2-like 2 (NRF2) participates in the activation of the antioxidant cytoprotective pathway and other important physiological processes to maintain cellular homeostasis. The dysregulation of NRF2 activity plays a role in various diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. Thus, NRF2 activity is tightly regulated through multiple mechanisms, among which phosphorylation by kinases is critical in the posttranslational regulation of NRF2. For instance, PKC, casein kinase 2, and AMP-activated kinase positively, while GSK-3 negatively regulates NRF2 activity through phosphorylation of different sites. Here, we provide an overview of the phosphorylation regulation pattern of NRF2 and discuss the therapeutic potential of interventions targeting NRF2 phosphorylation.

Context Lappaol F (LAF), a natural lignan from Arctium lappa Linné (Asteraceae), inhibits tumour cell growth by inducing cell cycle arrest. However, its underlying anticancer mechanism remains unclear. Objective The effects of LAF on the Hippo-Yes-associated protein (YAP) signalling pathway, which plays an important role in cancer progression, were explored in this study. Materials and methods Cervical (HeLa), colorectal (SW480), breast (MDA-MB-231) and prostate (PC3) cancer cell lines were treated with LAF at different concentrations and different durations. BALB/c nude mice bearing colon xenografts were intravenously injected with vehicle, LAF (10 or 20 mg/kg) or paclitaxel (10 mg/kg) for 15 days. The expression and nuclear localisation of YAP were analysed using transcriptome sequencing, quantitative PCR, western blotting and immunofluorescence. Results LAF suppressed the proliferation of HeLa, MDA-MB-231, SW480 and PC3 cells (IC50 values of 41.5, 26.0, 45.3 and 42.9 μmol/L, respectively, at 72 h), and this was accompanied by significant downregulation in the expression of YAP and its downstream target genes at both the mRNA and protein levels. The expression of 14-3-3σ, a protein that causes YAP cytoplasmic retention and degradation, was remarkably increased, resulting in a decrease in YAP nuclear localisation. Knockdown of 14-3-3σ with small interfering RNA partially blocked LAF-induced YAP inhibition and anti-proliferation effects. In colon xenografts, treatment with LAF led to reduced YAP expression, increased tumour cell apoptosis and tumour growth inhibition. Conclusion LAF was shown to be an inhibitor of YAP. It exerts anticancer activity by inhibiting YAP at the transcriptional and post-translational levels.

BubR1 is an essential component of the spindle assembly checkpoint (SAC) during mitosis where it functions to prevent anaphase onset to ensure proper chromosome alignment and kinetochore-microtubule attachment. Loss or mutation of BubR1 results in aneuploidy that precedes various potential pathologies, including cancer and mosaic variegated aneuploidy (MVA). BubR1 is also progressively downregulated with age and has been shown to be directly involved in the aging process through suppression of cellular senescence. Post-translational modifications, including but not limited to phosphorylation, acetylation, and ubiquitination, play a critical role in the temporal and spatial regulation of BubR1 function. In this review, we discuss the currently characterized post-translational modifications to BubR1, the enzymes involved, and the biological consequences to BubR1 functionality and implications in diseases associated with BubR1. Understanding the molecular mechanisms promoting these modifications and their roles in regulating BubR1 is important for our current understanding and future studies of BubR1 in maintaining genomic integrity as well as in aging and cancer.

Developmental regulation of the vertebrate visual system has been a focus of investigation for generations as understanding this critical time period has direct implications on our understanding of congenital blinding disease. The majority of studies to date have focused on transcriptional regulation mediated by morphogen gradients and signaling pathways. However, recent studies of post translational regulation during ocular development have shed light on the role of the ubiquitin proteasome system (UPS). This rather ubiquitous yet highly diverse system is well known for regulating protein function and localization as well as stability via targeting for degradation by the 26S proteasome. Work from many model organisms has recently identified UPS activity during various milestones of ocular development including retinal morphogenesis, retinal ganglion cell function as well as photoreceptor homeostasis. In particular work from flies and zebrafish has highlighted the role of the E3 ligase enzyme family, Seven in Absentia Homologue (Siah) during these events. In this review, we summarize the current understanding of UPS activity during Drosophila and vertebrate ocular development, with a major focus on recent findings correlating Siah E3 ligase activity with two major developmental stages of vertebrate ocular development, retinal morphogenesis and photoreceptor specification and survival.

Autophagy is a conserved catabolic process involving the degradation and recycling of damaged biomacromolecules or organelles through lysosomal-dependent pathways and plays a crucial role in maintaining cell homeostasis. Consequently, abnormal autophagy is associated with multiple diseases, such as infectious diseases, neurodegenerative diseases and cancer. Currently, autophagy is considered to be a dual regulator in cancer, functioning as a suppressor in the early stage while supporting the growth and metastasis of cancer cells in the later stage and may also produce therapeutic resistance. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level by silencing targeted mRNA. MiRNAs have great regulatory potential for several fundamental biological processes, including autophagy. In recent years, an increasing number of studies have linked miRNA dysfunction to the growth, metabolism, migration, metastasis, and responses of cancer cells to therapy. Therefore, the study of autophagy-related miRNAs in cancer will provide insights into cancer biology and lead to the development of novel anti-cancer strategies. In the present review, we summarise the current knowledge of miRNA dysregulation during autophagy in cancer, focusing on the relationship between autophagy and miRNAs, and discuss their involvement in cancer biology and cancer treatment.

Influenza viruses continue evolving and have the ability to cause a global pandemic, so it is very important to elucidate its pathogenesis and find new treatment methods. In recent years, proteomics has made important contributions to describing the dynamic interaction between influenza viruses and their hosts, especially in posttranslational regulation of a variety of key biological processes. Protein posttranslational modifications (PTMs) increase the diversity of functionality of the organismal proteome and affect almost all aspects of pathogen biology, primarily by regulating the structure, function, and localization of the modified proteins. Considerable technical achievements in mass spectrometry-based proteomics have been made in a large number of proteome-wide surveys of PTMs in many different organisms. Herein we specifically focus on the proteomic studies regarding a variety of PTMs that occur in both the influenza viruses, mainly influenza A viruses (IAVs), and their hosts, including phosphorylation, ubiquitination and ubiquitin-like modification, glycosylation, methylation, acetylation, and some types of acylation. Integration of these data sets provides a unique scenery of the global regulation and interplay of different PTMs during the interaction between IAVs and their hosts. Various techniques used to globally profiling these PTMs, mostly MS-based approaches, are discussed regarding their increasing roles in mechanical regulation of interaction between influenza viruses and their hosts.

Double-strand breaks that are induced postreplication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break site and genome-wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome-wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin-dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28. Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo. We also demonstrated that Eco1 acetylated polymerase η in vitro. Certain nonacetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation, and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as post-translational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.

Fibroblast growth factor 23 (FGF23) is a key phosphate- and vitamin D-regulating hormone. FGF23 circulates as an intact 251 amino acid protein or N- and C-terminal degradation products. Hormone activity resides in the intact molecule, but it has been suggested that high levels of the C-terminal protein can interfere with intact FGF23 (iFGF23) activity. New evidence points to involvement of the hypoxia-inducible factor (HIF)/erythropoietin (EPO)/iron pathway as important in FGF23 physiology. Exactly how this pathway regulates FGF23 is not clear. Various in vitro, in vivo, and clinical studies involving perturbations in this pathway at various points have yielded conflicting results. Many of these studies are complicated by the confounding, independent effect of renal insufficiency on FGF23. To gain insight into FGF23 physiology, we studied 8 patients with a rare paraganglioma/somatostatinoma syndrome who had elevated blood EPO levels as a result of somatic gain-of-function mutations in HIF2A (EPAS1) that stimulate tumoral EPO production. All patients had normal renal function. EPO levels varied; most were very elevated and highly correlated with C-terminal FGF23 (cFGF23) levels that were also markedly elevated. Blood phosphate and intact FGF23 levels were normal. These data from patients with normal renal function in whom HIF activation was the inciting event suggest a direct role of the HIF/EPO pathway in FGF23 transcription and translation. They also demonstrate that posttranslational regulation was finely tuned to maintain normal blood phosphate levels. Additionally, normal phosphate and intact FGF23 levels in the setting of markedly increased C-terminal FGF23 levels suggest intact FGF23 action is not attenuated by C-terminal FGF23. Published 2020. This article is a U.S. Government work and is in the public domain in the USA. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Early-life stress is correlated with the development of anxiety-related behavior in adolescence, but underlying mechanisms remain poorly known. The α1A-adrenergic receptor (AR) is linked to mood regulation and its function is assumed to be regulated by β-arrestins (βArrs) via desensitization and downregulation. Here, we investigated correlation between changes in α1A-AR and βArr2 levels in the prefrontal cortex (PFC) and hippocampus of adolescent and adult male rats subjected to maternal separation (MS) and their relationship with anxiety-like behavior in adolescence. MS was performed 3 h per day from postnatal days 2–11 and anxiety-like behavior was evaluated in the elevated plus-maze and open field tests. The protein levels were examined using western blot assay. MS decreased α1A-AR expression and increased βArr2 expression in both brain regions of adolescent rats, while induced reverse changes in adulthood. MS adolescent rats demonstrated higher anxiety-type behavior and lower activity in behavioral tests than controls. Decreased α1A-AR levels in MS adolescence strongly correlated with reduced time spent in the open field central area, consistent with increased anxiety-like behavior. An anxiety-like phenotype was mimicked by acute and chronic treatment of developing rats with prazosin, an α1A-AR antagonist, suggesting α1A-AR downregulation may facilitate anxiety behavior in MS adolescent rats. Together, our results indicate a negative correlation between α1A-AR neurotransmission and βArr2 levels in both adults and anxious-adolescent rats and suggest that increased βArr2 levels may contribute to posttranslational regulation of α1A-AR and modulation of anxiety-like behavior in adolescent rats. This may provide a path to develop more effective anxiolytic treatments.

Plant roots are sustained through meristem activity at the root tip. Two transcriptional pathways, one mediated by PLETHORAs (PLTs) and the other by SHORTROOT and SCARECROW, play major roles in root meristem development. The role of PLTs during root meristem development requires a concentration gradient, which is not only contributed by posttranslational regulation such as growth dilution and intercellular movement but also likely by a largely unknown fine-tuned transcriptional regulatory mechanism. We report here that Arabidopsis (Arabidopsis thaliana) JANUS positively regulates PLT1 expression in the root meristem by recruiting RNA polymerase II (Pol II) to PLT1 and by interacting with PLT1. JANUS-dependent recruitment of Pol II is inhibited through the competitive binding of JANUS by GRF-INTERACTING FACTOR1 (GIF1)/ANGUSTIFOLIA3, a transcriptional cofactor that negatively regulates PLT1 expression. Finally, GIF1 and JANUS, the antagonistic regulators of PLT1, both depend on Arabidopsis IMPORTIN β4 for their nuclear accumulation. The combination of an importin and its two antagonistic cargos in PLT1 transcription may have logistic benefits in fine-tuning the transcription of PLT1 in root meristem.

Ethylene plays a critical regulatory role in climacteric fruit ripening, and its biosynthesis is fine-tuned at the transcriptional and posttranslational levels. Nevertheless, the mechanistic link between transcriptional and posttranslational regulation of ethylene biosynthesis during fruit ripening is largely unknown. This study uncovers a coordinated transcriptional and posttranslational mechanism of controlling ethylene biosynthesis during banana (Musa acuminata) fruit ripening. NAC (NAM, ATAF, and CUC) proteins MaNAC1 and MaNAC2 repress the expression of MaERF11, a protein previously known to negatively regulate ethylene biosynthesis genes MaACS1 and MaACO1. A RING E3 ligase MaXB3 interacts with MaNAC2 to promote its ubiquitination and degradation, leading to the inhibition of MaNAC2-mediated transcriptional repression. In addition, MaXB3 also targets MaACS1 and MaACO1 for proteasome degradation. Further evidence supporting the role of MaXB3 is provided by its transient and ectopic overexpression in banana fruit and tomato (Solanum lycopersicum), respectively, which delays fruit ripening via repressing ethylene biosynthesis and thus ethylene response. Strikingly, MaNAC1 and MaNAC2 directly repress MaXB3 expression, suggesting a feedback regulatory mechanism that maintains a balance of MaNAC2, MaACS1, and MaACO1 levels. Collectively, our findings establish a multilayered regulatory cascade involving MaXB3, MaNACs, MaERF11, and MaACS1/MaACO1 that controls ethylene biosynthesis during climacteric ripening.

BACKGROUND: The emergence of reactive stroma is a hallmark of PCa progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen type proteins in PCa prostatectomies. METHODS: Prostatectomies from 9 patients were divided into zones containing 0, 5, 20, 70–80% glandular tissue and 0, 5, 25, 70% by mass of prostate cancer tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation. High resolution accurate mass (HRAM) collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen type regulation corresponding to pathologist defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of inflammation compared to zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared to zones with inflammation and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared to zones of inflammation (2.59 ± 0.29 p-value 0.015; 3.75 ± 0.96 p-value 0.036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist defined tumor regions showed significant increases in COL1A1 HYP modifications compared to COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist defined tumor and inflammatory regions. CONCLUSIONS: Site-specific post-translational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and post-translational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.

C4photosynthesis evolved multiple times independently from ancestral C3photosynthesis in a broad range of flowering land plant families and in both monocots and dicots. The evolution of C4photosynthesis entails the recruitment of enzyme activities that are not involved in photosynthetic carbon fixation in C3plants to photosynthesis. This requires a different regulation of gene expression as well as a different regulation of enzyme activities in comparison to the C3context. Further, C4photosynthesis relies on a distinct leaf anatomy that differs from that of C3, requiring a differential regulation of leaf development in C4. We summarize recent progress in the understanding of C4-specific features in evolution and metabolic regulation in the context of C4photosynthesis.

High-mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that plays essential roles in embryonic development and cancer progression. However, the mechanism of HMGA2 regulation remains largely uncharacterized. Here, we demonstrate that HMGA2 can be modulated by hepatitis B X-interacting protein (HBXIP), an oncogenic transcriptional coactivator, in esophageal squamous cell carcinoma (ESCC). HMGA2 expression was positively associated with HBXIP expression in clinical ESCC tissues, and their high levels were associated with advanced tumor stage and reduced overall and disease-free survival. We found that oncogenic HBXIP could posttranslationally upregulate HMGA2 protein level in ESCC cells. HBXIP induced HMGA2 acetylation at the lysine 26 (K26), resulting in HMGA2 protein accumulation. In this process, HBXIP increased the acetyltransferase p300/CBP-associated factor (PCAF) phosphorylation and activation via the Akt pathway, then PCAF directly interacted with HMGA2, leading to HMGA2 acetylation in the cells. HMGA2 K26 acetylation enhanced its DNA binding capacity and blocked its ubiquitination and then inhibited proteasome-dependent degradation. Functionally, HBXIP-stabilized HMGA2 could promote ESCC cell growth in vitro and in vivo. Strikingly, aspirin suppressed ESCC growth by inhibiting HBXIP and HMGA2. Collectively, our findings disclose a new mechanism for the posttranslational regulation of HMGA2 mediated by HBXIP in ESCC.

Francisella tularensis is a Gram-negative bacterium that causes the zoonotic disease tularemia. The historical development of tularemia as a biological weapon has led to it being characterized by the CDC as a category A biothreat agent. Neither posttranslational modification (PTM) of proteins, in particular lysine acetylation, in Francisella nor its subsequent regulation of the protein activity has been well studied. In this work, we analyze N-e-lysine acetylation of the F. tularensis ssp. novicida proteome by mass spectrometry for the first time. To create a comprehensive acetylation profile, we enriched protein acetylation using two approaches: (1) the addition of glucose or acetate into the culture medium and (2) direct chemical acetylation of N-e-lysines with acetyl phosphate. We discovered 280 acetylated proteins with 1178 acetylation sites in the F. tularensis ssp. novicida strain U112. Lysine acetylation is an important PTM that regulates multiple cellular processes in bacteria, including metabolism, transcription, translation, stress response, and protein folding. We discovered that Francisella chitinases A and B are acetylated naturally and when chemically induced by acetyl phosphate. Moreover, chemical overacetylation of chitinases results in silencing of the enzymatic activity. Our findings suggest a novel mechanism of posttranslational regulation of the chitinase activity and that acetylation may play a role in Francisella's regulation of the protein activity.

Background Primary Ovarian Insufficiency (POI), a public health problem, affects 1-3.7% of women under 40 yield-ing infertility and a shorter lifespan. Most causes are unknown. Recently, genetic causes were identified, mostly in single families. We studied an unprecedented large cohort of POI to unravel its molecular pathophysiology.Methods 375 patients with 70 families were studied using targeted (88 genes) or whole exome sequencing with pathogenic/likely-pathogenic variant selection. Mitomycin-induced chromosome breakages were studied in patients' lymphocytes if necessary. Findings A high-yield of 29.3% supports a clinical genetic diagnosis of POI. In addition, we found strong evidence of pathogenicity for nine genes not previously related to a Mendelian phenotype or POI: ELAVL2, NLRP11, CENPE, SPATA33, CCDC150, CCDC185, including DNA repair genes: C17orf53(HROB), HELQ, SWI5 yielding high chromo-somal fragility. We confirmed the causal role of BRCA2, FANCM, BNC1, ERCC6, MSH4, BMPR1A, BMPR1B, BMPR2, ESR2, CAV1, SPIDR, RCBTB1 and ATG7 previously reported in isolated patients/families. In 8.5% of cases, POI is the only symptom of a multi-organ genetic disease. New pathways were identified: NF-kB, post-translational regulation, and mitophagy (mitochondrial autophagy), providing future therapeutic targets. Three new genes have been shown to affect the age of natural menopause supporting a genetic link.Interpretation We have developed high-performance genetic diagnostic of POI, dissecting the molecular pathogene-sis of POI and enabling personalized medicine to i) prevent/cure comorbidities for tumour/cancer susceptibility genes that could affect life-expectancy (37.4% of cases), or for genetically-revealed syndromic POI (8.5% of cases), ii) predict residual ovarian reserve (60.5% of cases). Genetic diagnosis could help to identify patients who may benefit from the promising in vitro activation-IVA technique in the near future, greatly improving its success in treating infertility.Funding Universite? Paris Saclay, Agence Nationale de Biome?decine.Copyright (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Histone methylation is a key epigenetic modification that regulates eukaryotic transcription. It is involved in many cellular processes, including growth, development, and differentiation, and its dysregulation is linked to the aetiology and progression of disease. In the budding yeast and model eukaryote, Saccharomyces cerevisiae, there are six histone lysine methylation sites (H3K4, H3K36, H3K79, H4K5, H4K8, and H4K12) that are controlled by an evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. While the histone targets of these enzymes are known, their regulation is poorly understood. To this end, we performed a systematic investigation into the post-translational regulation of the histone lysine methylation network in S. cerevisiae, with a particular focus on the role of phosphorylation and its connection with upstream signalling pathways. First, we used targeted mass spectrometry to identify a total of 75 phosphorylation sites occurring on yeast histone methyltransferase and demethylase enzymes in vivo. To investigate the functional effects of these sites, we constructed and screened a phosphosite mutant library, consisting of 82 genomically-edited yeast strains, using mass spectrometry, immunoblotting, and spot plate growth assays. This revealed the importance of phosphosites on methyltransferase Set2p, at residues S6, S8, S10, and T127, and demethylase Jhd1p, at residue S44, in the regulation of H3K36 methylation and in the coordination of specific stress response pathways. Finally, we employed in vitro and in vivo kinase discovery approaches to show that the mitogen-activated protein kinase, Hog1p, phosphorylates methyltransferase Dot1p at multiple proline-adjacent sites. Taken together, our findings establish phosphorylation as a key regulator of histone methylation enzymes in budding yeast, and contextualise the histone-based gene regulatory system within the signalling system of the cell.

Prokineticins are a new class of chemokine-like peptides that bind their G protein-coupled receptors, PKR1 and PKR2, and promote chemotaxis and the production of pro-inflammatory cytokines following tissue injury or infection. This review summarizes the major cellular and biochemical mechanisms of prokineticins pathway regulation that, like other chemokines, include: genetic polymorphisms; mRNA splice modulation; expression regulation at transcriptional and post-transcriptional levels; prokineticins interactions with cell-surface glycosaminoglycans; PKRs degradation, localization, post-translational modifications and oligomerization; alternative signaling responses; binding to pharmacological inhibitors. Understanding these mechanisms, which together exert substantial biochemical control and greatly enhance the complexity of the prokineticin-receptor network, leads to novel opportunities for therapeutic intervention. In this way, besides targeting prokineticins or their receptors directly, it could be possible to indirectly influence their activity by modulating their expression and localization or blocking the downstream signaling pathways.

PURPOSE OF THE REVIEW Lipoprotein lipase (LPL) is the rate-limiting enzyme for intravascular processing of circulating triglyceride-rich lipoproteins (TRLs). One emerging strategy for therapeutic lowering of plasma triglyceride levels aims at increasing the longevity of LPL activity by attenuating its inhibition from angiopoietin-like proteins (ANGPTL) 3, 4 and 8. This mini-review focuses on recent insights into the molecular mechanisms underpinning the regulation of LPL activity in the intravascular unit by ANGPTLs with special emphasis on ANGPTL4. RECENT FINDINGS Our knowledge on the molecular interplays between LPL, its endothelial transporter GPIHBP1, and its inhibitor(s) ANGPTL4, ANGPTL3 and ANGPTL8 have advanced considerably in the last 2 years and provides an outlined on how these proteins regulate the activity and compartmentalization of LPL. A decisive determinant instigating this control is the inherent protein instability of LPL at normal body temperature, a property that is reciprocally impacted by the binding of GPIHBP1 and ANGPTLs. Additional layers in this complex LPL regulation is provided by the different modulation of ANGPTL4 and ANGPTL3 activities by ANGPTL8 and the inhibition of ANGPTL3/8 complexes by apolipoprotein A5 (APOA5). SUMMARY Posttranslational regulation of LPL activity in the intravascular space is essential for the differential partitioning of TRLs across tissues and their lipolytic processing in response to nutritional cues.

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography-tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.

Intervening proteins, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and N-chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis in vitro. Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. IMPORTANCE Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.

Microbial production of natural compounds has attracted extensive attention due to their high value in pharmaceutical, cosmetic, and food industries. Constructing efficient microbial cell factories for biosynthesis of natural products requires the fine-tuning of gene expressions to minimize the accumulation of toxic metabolites, reduce the competition between cell growth and product generation, as well as achieve the balance of redox or co-factors. In this review, we focus on recent advances in fine-tuning gene expression at the DNA, RNA, and protein levels to improve the microbial biosynthesis of natural products. Commonly used regulatory toolsets in each level are discussed, and perspectives for future direction in this area are provided.

Η μετα-μεταφραστική ρύθμιση αποτελεί ένα σημαντικό, γρήγορο και ενεργειακά αποδοτικό επίπεδο της κυτταρικής ρύθμισης, για το οποίο έχουν αναπτυχθεί πολλές ομικές τεχνολογίες μεγάλης κλίμακας. Η μετα-μεταφραστική τροποποίηση των αμινοξέων και ειδικότερα η πρωτεϊνική φωσφορυλίωση και μεθυλίωση έχουν κεντρικό ρόλο. Αυτή η διδακτορική διατριβή εστίασε σε δημοσιευμένα δεδομένα φωσφο-πρωτεωμικής και μεθυλ-πρωτεωμικής με σκοπό να αναπτύξει υπολογιστικά εργαλεία και βιοπληροφορικές μεθόδους/αναλύσεις που θα μπορούν να τα αναλύσουν και να εξάγουν γνώση για τις ιδιότητες αυτού του επιπέδου ρύθμισης. Κατά την διάρκεια αυτής της διατριβής, συλλέχθησαν, φιλτραρίστηκαν, αποθηκεύτηκαν και οργανώθηκαν δημοσιευμένα δεδομένα, με τη βοήθεια ενός υπολογιστικού εργαλείου διαχείρισης της Βιβλιογραφίας και μιας βάσης δεδομένων που ανέπτυξα. Επιπλέον, και άλλα ομικά και εξελικτικά δεδομένα ενσωματώθηκαν με σκοπό να πραγματοποιηθούν βιοπληροφορικές αναλύσεις σε βάθος. Στατιστικές αναλύσεις επέτρεψαν να εκτιμηθεί το σύνολο των πρωτεϊνών και των αμινοξέων ενός ευκαρυωτικού οργανισμού που υφίστανται φωσφορυλίωση. Mια εις βάθος βιοπληροφορική ανάλυση επέτρεψε να αποκαλυφθεί η σημασία της πρωτεϊνικής φωσφορυλίωσης στην ρύθμιση του κεντρικού μεταβολισμού του ζυμομύκητα S. cerevisiae όπως επίσης και οι θέσεις φωσφορυλίωσης με πιθανές βιοτεχνολογικές εφαρμογές, σε περίπτωση στοχευμένης μετάλλαξής τους. Επιπλέον, αναπτύχθηκαν νευρωνικά δίκτυα για την πρόβλεψη θέσεων φωσφορυλίωσης, μεθυλίωσης, καθώς επίσης και συνδυαστικών μοριακών διακοπτών. Τα εργαλεία και οι μέθοδοι/αναλύσεις που αναπτύχθηκαν/εφαρμόστηκαν κατά την πραγματοποίηση αυτής της διατριβής δύνανται να εξελιχθούν ώστε να επιτρέψουν την ενσωμάτωση επιπλέον μετα-μεταφραστικών τροποποιήσεων στο μέλλον.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind and are the causative agents of the neuroparalytic disease botulism. Despite the overall importance of BoNTs in public health and safety, as a bioterrorism concern, and in pharmaceutical development, little is known about the molecular mechanisms mediating BoNT stability and degradation in various environments. Previous studies using Clostridium botulinum strain ATCC 3502 revealed that high levels of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold. In the present study, the mechanisms of toxin reduction in arginine-enriched cultures of C. botulinum strain Hall A-hyper, which we have previously genetically manipulated using ClosTron technology, were explored. Cultures were grown in toxin production medium (TPM) and TPM enriched with arginine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), changes in pH, and BoNT formation and stability. Our data indicate that arginine enrichment of C. botulinum strain Hall A-hyper cultures results in a pH shift that induces pH-dependent posttranslational control mechanisms. We further show that independent of arginine, maintenance of an acidic culture pH during growth of C. botulinum strain Hall A-hyper plays a central role in toxin stability and that an extracellular metalloprotease produced by the culture results in BoNT degradation at pH levels between ⁓6.5 and 8.0. IMPORTANCE Botulinum neurotoxin (BoNT) is a public health and bioterrorism concern as well as an important and widely used pharmaceutical, yet the regulation of its synthesis by BoNT-producing clostridia is not well understood. This paper highlights the role of environmentally controlled posttranslational regulatory mechanisms influencing processing and stability of biologically active BoNTs produced by C. botulinum. The results of this work will help enhance public health and safety measures and our ability to evaluate safety risks of novel BoNTs and improve production and quality of BoNTs for pharmaceutical use.

SHANK‐associated RH domain interacting protein (SHARPIN) plays an important role in carcinogenesis, as well as inflammation and immunity. Our study explored the effects and underlying mechanisms of SHARPIN in clear cell renal cell carcinoma (ccRCC). By analyzing The Cancer Genome Atlas database, we found that upregulated SHARPIN in patients with ccRCC led to a poor prognosis. Semiquantitative immunohistochemical analysis of clinical samples was carried out and the results suggested the positive association between SHARPIN and hypoxia‐induced factor‐2α (HIF‐2α). Von Hippel‐Lindau protein (pVHL) is a tumor suppressor that contributes to degrading HIF‐2α. Mechanically, SHARPIN promoted the ubiquitination and proteasomal degradation of pVHL, resulting in the sustained activation of HIF‐2α. The α and β domains of pVHL and ubiquitin‐like domain of SHARPIN are required for the interaction. The knockdown of SHARPIN effectively inhibited acquired sorafenib resistance in ccRCC cell lines and tumor growth in xenograft models. In conclusion, our work reveals a novel posttranslational regulation of SHARPIN on pVHL, indicating that SHARPIN could be a potential target for ccRCC treatment., SHARPIN promotes the ubiquitination and proteasomal degradation of von Hippel‐Lindau protein (pVHL) through the interaction between the ubiquitin‐like domain of SHARPIN and the α and β domains of pVHL, resulting in the sustained activation of hypoxia‐induced factor‐2α. The knockdown of SHARPIN effectively inhibited acquired sorafenib resistance in clear cell renal cell carcinoma cell lines and tumor growth in xenograft models.

The dysregulation of NLRP3 inflammasome plays a critical role in pathogenesis of various human inflammatory diseases, thus NLRP3 inflammasome activation must be tightly controlled at multiple levels. However, the underlying mechanism regulating NLRP3 inflammasome activation remains unclear. Herein, the effects of Tripartite motif-containing protein 65 (TRIM65) on NLRP3 inflammasome activation and the underlying molecular mechanism were investigated in vitro and in vivo. Inhibition or deletion of Trim65 could significantly strengthen agonist induced NLRP3 inflammasome activation in THP-1 cells and BMDMs, indicated by increased caspase-1 activation and interleukin-1β secretion. However, TRIM65 had no effect on poly (dA: dT)-induced AIM2 inflammasome activation or flagellin-induced IPAF inflammasome activation. Mechanistically, immunoprecipitation assays demonstrated that TRIM65 binds to NACHT domain of NLRP3, promotes lys48- and lys63- linked ubiquitination of NLRP3 and restrains the NEK7-NLRP3 interaction, thereby inhibiting NLRP3 inflammasome assembly, caspase-1 activation, and IL-1β secretion. In vivo, three models of inflammatory diseases were used to confirm the suppression role of TRIM65 in NLRP3 inflammasome activation. TRIM65-deficient mice had a higher production of IL-1β induced by lipopolysaccharide in sera, and more IL-1β secretion and neutrophil migration in the ascites, and more severity of joint swelling and associated IL-1β production induced by monosodium urate, suggesting that TRIM65 deficiency was susceptible to inflammation. Therefore, the data elucidate a TRIM65-dependent negative regulation mechanism of NLRP3 inflammasome activation and provide potential therapeutic strategies for the treatment of NLRP3 inflammasome-related diseases.

Summary Circadian protein oscillations are maintained by the lifelong repetition of protein production and degradation in daily balance. It comes at the cost of ever-replayed, futile protein synthesis each day. This biosynthetic cost with a given oscillatory protein profile is relievable by a rhythmic, not constant, degradation rate that selectively peaks at the right time of day but remains low elsewhere, saving much of the gross protein loss and of the replenishing protein synthesis. Here, our mathematical modeling reveals that the rhythmic degradation rate of proteins with circadian production spontaneously emerges under steady and limited activity of proteolytic mediators and does not necessarily require rhythmic post-translational regulation of previous focus. Additional (yet steady) post-translational modifications in a proteolytic pathway can further facilitate the degradation's rhythmicity in favor of the biosynthetic cost saving. Our work is supported by animal and plant circadian data, offering a generic mechanism for potentially widespread, time-dependent protein turnover., Graphical abstract, Highlights • Rhythmic degradation of circadian proteins lowers the cost of protein synthesis • This rhythmic degradation emerges without rhythmic post-translational regulation • Extra, yet steady post-translational modifications enhance degradation rhythmicity • This mechanism hints at how organisms afford the price of daily biological rhythms, Mathematical biosciences; Systems biology; In silico biology

The airborne fungus Aspergillus fumigatus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine161, sumoylation of lysine242 and phosphorylation of threonine319. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of hapX caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in A. fumigatus.