Inhibiting SIRT-2 by AK-7 restrains airway inflammation and oxidative damage promoting lung resurgence through NF-kB and MAP kinase signaling pathway.

Introduction: Chronic obstructive pulmonary disease (COPD) is a major global cause of mortality with limited effective treatments. Sirtuins (SIRT) are histone deacetylases that are involved in the regulation of redox and inflammatory homeostasis. Hence, the present study aims to investigate the role of SIRT-2 in modulating inflammation in a murine model of COPD. Methods: COPD in mice was established by cigarette smoke (CS) exposure for 60 days, and AK-7 was used as the specific SIRT-2 inhibitor. AK-7 (100 µg/kg and 200 µg/kg body weight) was administered intranasally 1 h before CS exposure. Molecular docking was performed to analyze the binding affinity of different inflammatory proteins with AK-7. Results: Immune cell analysis showed a significantly increased number of macrophages (F4/80), neutrophils (Gr-1), and lymphocytes (CD4+, CD8+, and CD19+) in the COPD, group and their population was declined by AK-7 administration. Total reactive oxygen species, total inducible nitric oxide synthase, inflammatory mediators such as neutrophil elastase, C-reactive protein, histamine, and cytokines as IL4, IL-6, IL-17, and TNF-α were elevated in COPD and declined in the AK-7 group. However, IL-10 showed reverse results representing anti-inflammatory potency. AK-7 administration by inhibiting SIRT-2 decreased the expression of p-NF-κB, p-P38, p-Erk, and p-JNK and increased the expression of Nrf-2. Furthermore, AK-7 also declined the lung injury by inhibiting inflammation, parenchymal destruction, emphysema, collagen, club cells, and Kohn pores. AK-7 also showed good binding affinity with inflammatory proteins. Discussion: The current study reveals that SIRT-2 inhibition mitigates COPD severity and enhances pulmonary therapeutic interventions, suggesting AK-7 as a potential therapeutic molecule for COPD medication development.

The genetic risk factors for cerebral venous thrombosis: a case-control study in a Chinese national comprehensive hospital.

BACKGROUND: About 13-25% of cerebral venous thrombosis (CVT) cases lack clear etiology, which may be associated with underlying genetic factors. This study aims to investigate genetic factors in CVT patients using whole exome sequencing (WES). METHODS: Thirty-eight CVT patients hospitalized underwent WES. 977 subjects with WES data from a community cohort study --the Shunyi cohort were as the control group. Using bioinformatics analysis, differential genes with rare damaging variants between two groups were filtered (P < 0.05). KEGG enrichment analysis was performed on the screened genes to identify pathways associated with CVT. RESULTS: Through analysis of medical history, routine tests, and imaging examinations, the etiology of 38 patients: 8 cases of antiphospholipid syndrome, 6 cases with hematologic diseases, 3 cases of protein C deficiency, and 2 cases of protein S deficiency. Five cases occurred during pregnancy or puerperium, and 3 cases had a history of oral contraceptive use, and so on. The etiology was unknown in 12 cases (31.6%), and the etiology of 4 patients were further clarified through WES: F9 c.838 + 1_838 + 16del, Hemizygote: F9 EX1-EX7 Dup; CBS c.430G > A, CBS c.949 A > G; F2 c.1787G > A; SERPINC1 c.409-11G > T. Comparing the WES data of two groups, a total of 179 different genes with rare damaging variants were screened (P < 0.05), with 5 genes of interest (JAK2, C3, PROC, PROZ, SERPIND1). Enrichment analysis of the 179 different genes revealed the complement and coagulation pathway and the mitogen activated protein kinases (MAPK) pathway were associated with CVT. CONCLUSION: For CVT patients with unknown etiology, WES could help identify the cause of CVT early, which is of great significance for treatment decisions and prognosis. In addition to the complement and coagulation pathway, MAPK pathway is associated with CVT, potentially related to platelet regulation and inflammatory response.

Arrestins: A Small Family of Multi-Functional Proteins.

The first member of the arrestin family, visual arrestin-1, was discovered in the late 1970s. Later, the other three mammalian subtypes were identified and cloned. The first described function was regulation of G protein-coupled receptor (GPCR) signaling: arrestins bind active phosphorylated GPCRs, blocking their coupling to G proteins. It was later discovered that receptor-bound and free arrestins interact with numerous proteins, regulating GPCR trafficking and various signaling pathways, including those that determine cell fate. Arrestins have no enzymatic activity; they function by organizing multi-protein complexes and localizing their interaction partners to particular cellular compartments. Today we understand the molecular mechanism of arrestin interactions with GPCRs better than the mechanisms underlying other functions. However, even limited knowledge enabled the construction of signaling-biased arrestin mutants and extraction of biologically active monofunctional peptides from these multifunctional proteins. Manipulation of cellular signaling with arrestin-based tools has research and likely therapeutic potential: re-engineered proteins and their parts can produce effects that conventional small-molecule drugs cannot.

Mutational analyses of the interacting domains of Schizosaccharomyces pombe Byr2 with 14-3-3s.

The Byr2 kinase of fission yeast Schizosaccharomyces pombe is recruited to the membrane with the assistance of Ras1. Byr2 is also negatively regulated by 14-3-3 proteins encoded by rad24 and rad25. We conducted domain and mutational analysis of Byr2 to determine which region is critical for its binding to 14-3-3 proteins. Rad24 and Rad25 bound to both the Ras interaction domain in the N-terminus and to the C-terminal catalytic domain of Byr2. When amino acid residues S87 and T94 of the Ras-interacting domain of Byr2 were mutated to alanine, Rad24 could no longer bind to Byr2. S402, S566, S650, and S654 mutations in the C-terminal domain of Byr2 also abolished its interaction with Rad24 and Rad25. More than three mutations in the C-terminal domain were required to abolish completely its interaction with 14-3-3 protein, suggesting that multiple residues are involved in this interaction. Expression of the N-terminal domain of Byr2 in wild-type cells lowered the mating ratio, because it likely blocked the interaction of Byr2 with Ste4 and Ras1, whereas expression of the catalytic domain of Byr2 increased the mating ratio as a result of freeing from intramolecular regulation by the N-terminal domain of Byr2. The S87A and T94A mutations of Byr2 increased the mating ratio and attenuated inhibition of Byr2 by Rad24; therefore, these two amino acids are critical for its regulation by Rad24. S566 of Byr2 is critical for activity of Byr2 but not for its interaction with 14-3-3 proteins. In this study, we show that 14-3-3 proteins interact with two separate domains in Byr2 as negative regulators.

Role of Mitogen-activated protein kinase kinase kinase kinase 4 signaling in liver and metabolic diseases.

MAP4K4 is a serine/threonine protein kinase belonging to the germinal center kinase (GCK) sub-group of sterile 20 protein (Ste20p) family of kinases. MAP4K4 has been involved in regulating multiple biological processes and a plethora of pathologies, including systemic inflammation, cardiovascular diseases, cancers, metabolic and hepatic diseases. Recently, multiple reports have indicated the upregulation of MAP4K4 expression and signaling in hyperglycemia and liver diseases. This review summarizes our current understanding of MAP4K4 structure, expression, as well as its regulation and signaling, specifically in metabolic and hepatic diseases. Reviewing these promising studies will enrich our understanding of MAP4K4 signaling which will help us design innovative therapeutic interventions against metabolic and liver diseases using MAP4K4 as a target. Significance Statement Although most studies on the involvement of MAP4K4 in human pathologies are related to cancers, only recently its role in liver and other metabolic diseases is beginning to unravel. This mini review discusses recent advancements in MAP4K4 signaling and comprehensively characterizes MAP4K4 as a clinically relevant therapeutic target against liver diseases.

New therapies for unresectable or metastatic cutaneous eyelid and orbital melanoma.

PURPOSE: Newer treatment options offer the promise of improved outcomes for metastatic and unresectable melanoma. This investigation was performed to review these modalities for cutaneous eyelid and orbital disease. METHODS: A search for articles that were related to this subject was performed in the PubMed database, and the bibliographies of these manuscripts were reviewed to ensure capture of the appropriate literature. Data was abstracted and analyzed. RESULTS: Historically, patients who suffer from melanoma of the ocular adnexa have fared poorly. Approaches that employ BRAF and mitogen-associated protein kinase inhibitors, immunotherapy, and novel cellular therapies improve outcomes and survival rates, although the side effect profiles of these agents are problematic. Most of the existing strategies have not explored ocular adnexal disease specifically, and treatment plans are generally adapted from the general cutaneous oncology literature. CONCLUSIONS: Thanks to advances in our comprehension of the cellular biology of the disease, the management of unresectable and metastatic melanoma has evolved considerably over the past several years. Newer modalities will likely continue to improve survival and reduce adverse events.

The phosphorylation landscape of infection-related development by the rice blast fungus.

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

Electroacupuncture at the Dazhui and Baihui acupoints and different frequencies (10 and 50 Hz) protects against apoptosis by up-regulating ERK1/2-mediated signaling in rats after global cerebral ischemia.

Objectives: This study assessed the effects of electroacupuncture (EA) stimulation at different frequencies at the Dazhui and Baihui acupoints in the subacute phase after transient global cerebral ischemia (GCI). Materials and Methods: Rats were subjected to GCI for 25 min, followed by reperfusion for 7 days. EA at acupoints was performed at 10, 30, or 50 Hz, 1 day after reperfusion and then once daily for 6 consecutive days. Results: EA at acupoints at 10 and 50 Hz effectively down-regulated apoptosis in the hippocampal cornu ammonis 1(CA1) area and ameliorated memory deficits. Moreover, EA treatment at 10 and 50 Hz markedly increased phospho (p)-extracellular signal-regulated protein kinase 1/2 (ERK1/2), p-ERK1/2/neuronal nuclei (NeuN), p-cAMP response element-binding protein (CREB)/p-ERK1/2, B-cell lymphoma-2 (Bcl-2)/p-CREB, and X-linked inhibitor of apoptosis protein/NeuN expression levels and decreased Bcl-2 homologous antagonist/killer, second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI, cytochrome c, cleaved caspase-3, and apoptosis-inducing factor expression levels. Furthermore, 10-Hz EA treatment effectively increased p-p38 mitogen-activated protein kinase (MAPK), p-p38 MAPK/NeuN, and p-CREB/p-p38 MAPK expression levels. Pretreatment with U0126 (ERK1/2 inhibitor) completely abrogated the effects of 10- and 50-Hz EA treatments on the aforementioned protein expression levels. Similarly, pretreatment with SB203580 (p38 MAPK inhibitor) completely abrogated the effects of 10-Hz treatment on the aforementioned protein expression levels. Conclusion: The effects of 10- and 50-Hz EA treatments on mitochondria-related apoptosis can be attributed to the activation of ERK1/2/p38 MAPK/CREB/Bcl-2- and ERK1/2/CREB/Bcl-2-mediated signaling, respectively, in the hippocampal CA1 area at 7 days after transient GCI.

Studying RAS Interactions in Live Cells with BRET.

Bioluminescence resonance energy transfer (BRET) is a valuable technique for studying protein-protein interactions (PPIs) within live cells (Pfleger and Eidne, Nat Methods 3:165-174, 2006). Among the various BRET methodologies, a recent addition called NanoBRET has emerged, leveraging advancements in donor and acceptor technologies (Machleidt and Woodroofe, ACS Chem Biol 10:1797-1804, 2015). In this study, we present a developed methodology designed to measure PPIs involving the RAS protein family and their effectors and interactors at the plasma membrane. By utilizing the NanoLuc and HaloTag BRET pair, we provide evidence of a saturable interaction between KRAS4b-G12D and full-length RAF1. Conversely, the RAF1 R89L mutant, known to impede RAF1 binding to active RAS, exhibits nonspecific interactions. The assay exhibits remarkable signal-to-background ratios and is highly suitable for investigating the interactions of RAS with effectors, as well as for high-throughput screening assays.

Nucleolin lactylation contributes to intrahepatic cholangiocarcinoma pathogenesis via RNA splicing regulation of MADD.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a fatal malignancy of the biliary system. The lack of a detailed understanding of oncogenic signaling or global gene expression alterations has impeded clinical iCCA diagnosis and therapy. The role of protein lactylation, a newly unraveled post-translational modification that orchestrates gene expression, remains largely elusive in the pathogenesis of iCCA. METHODS: Proteomics analysis of clinical iCCA specimens and adjacent tissues was performed to screen for proteins aberrantly lactylated in iCCA. Mass spectrometry, macromolecule interaction and cell behavioral studies were employed to identify the specific lactylation sites on the candidate protein(s) and to decipher the downstream mechanisms responsible for iCCA development, which were subsequently validated using a xenograft tumor model and clinical samples. RESULTS: Nucleolin (NCL), the most abundant RNA-binding protein in the nucleolus, was identified as a functional lactylation target that correlates with iCCA occurrence and progression. NCL was lactylated predominantly at lysine 477 by the acyltransferase P300 in response to a hyperactivity of glycolysis, and promoted the proliferation and invasion of iCCA cells. Mechanistically, lactylated NCL bound to the primary transcript of MAP kinase-activating death domain protein (MADD) and led to efficient translation of MADD by circumventing alternative splicing that generates a premature termination codon. NCL lactylation, MADD translation and subsequent ERK activation promoted xenograft tumor growth and were associated with overall survival in patients with iCCA. CONCLUSION: NCL is lactylated to upregulate MADD through an RNA splicing-dependent mechanism, which potentiates iCCA pathogenesis via the MAPK pathway. Our findings reveal a novel link between metabolic reprogramming and canonical tumor-initiating events, and uncover biomarkers that can potentially be used for prognostic evaluation or targeted treatment of iCCA. IMPACT AND IMPLICATIONS: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive liver malignancy with largely uncharacterized pathogenetic mechanisms. Herein, we demonstrated that glycolysis promotes P300-catalyzed lactylation of nucleolin, which upregulates MAP kinase-activating death domain protein (MADD) through precise mRNA splicing and activates ERK signaling to drive iCCA development. These findings unravel a novel link between metabolic rewiring and canonical oncogenic pathways, and reveal new biomarkers for prognostic assessment and targeting of clinical iCCA.

BRAF V600E mutation mediates invasive and growth features in ameloblastoma.

OBJECTIVES: Ameloblastoma (AM), a locally aggressive tumor with extensive growth capacity, causes significant damage to the jaw and affects facial appearance. Although the high prevalence of BRAF V600E mutation in AM is known, its specific impacts on patients with AM remain unclear. Thus, the present study investigated the role of BRAF V600E mutation, thereby focusing on its impact on AM invasion and growth. MATERIALS AND METHODS: Immunohistochemical analysis was used to compare BRAF V600E, MMP2, MMP9, and Ki-67 expressions in AM (n = 49), normal oral mucosa (NOM) (n = 10), and odontogenic keratocyst (OKC) (n = 15) tissues. AM was further classified according to the presence or absence of BRAF V600E. The relationship between BRAF V600E and invasion as well as growth was evaluated. In addition, correlation analysis was performed using immunohistochemistry and confirmed via double-labeling immunofluorescence. Finally, comparative analyses using mass spectrometry, immunohistochemistry, and immunofluorescence were performed to explore and identify underlying mechanisms. RESULTS: AM exhibited a higher incidence of BRAF V600E mutation than NOM and OKC. BRAF V600E expression was positively correlated with the invasion-associated proteins MMP2 and MMP9 and the growth-related protein Ki-67. Proteomic data revealed that BRAF V600E primarily activates the MAPK signaling pathway in AM, particularly driving the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). CONCLUSIONS: In summary, the findings suggested that the BRAF V600E mutation enhances the invasion and growth abilities of AM via the MAPK/ERK signaling pathway. Thus, targeting BRAF V600E or the MAPK/ERK pathway may be a potential AM therapy.

Phosphorylated p38 MAP kinase expression by leucocytes is increased in allergic humans and associated with IgE responses.

Mitogen-activated protein kinases (MAPK) activate cascades that regulate cell proliferation, differentiation and death. Phosphorylated (phos-)p38 MAPK is a cell-signalling pathway associated with Th2 cytokine responses, which is required for immunoglobulin (Ig)E production. It is unknown whether MAPK are associated with IgE production. We examine the evidence linking p38 MAPK to inflammatory responses. Phos-p38, extracellular signal-related kinase (ERK) and c-JUN-n terminal (JNK) MAPK expression by blood leucocyte subsets and levels of serum Igs were measured in blood from adults with asthma and/or rhinoconjunctivitis (N = 28) and non-asthma (N = 10) (flow cytometry, microfluorenzymeimmunoassay). Peripheral blood mononuclear cells (PBMC) from allergic subjects were cultured for 10 days ± anti-CD40/recombinant IL-4 ± inhibitor of phos-P38. Culture supernatants were assayed for IgE (ELISA). Phos-p38 MAPK expression by all leucocyte subsets of allergic subjects was associated with serum IgE levels (p ≤ 0.01), after adjusting for cell counts, age, sex, race and smoking status (p ≤ 0.04). Leucocyte expression of phos-ERK and JNK did not correlate with IgE (p = 0.09-0.99). Instead, phos-ERK expression was associated with serum IgG. When PBMC from atopic subjects were cultured for 10 days with anti-CD40/rhIL-4, IgE levels were 26.2 ± 18 ng/mL. Inclusion of SB202190 (5-20 µg/mL), a specific inhibitor of phos-p38 MAPK, in culture suppressed IgE production in dose-dependent manner, with peak suppression obtained with SB202190 at 20 µg/mL (82.1% ± 11.8) (p = 0.0001), with virtually no cytotoxicity (<5%). Different MAPK pathways may be associated with IgE (p38) and IgG (ERK) responses. Phos-p38 MAPK can be a potential anti-allergy drug target.

The effects of Aurora Kinase inhibition on thyroid cancer growth and sensitivity to MAPK-directed therapies.

Thyroid cancer is one of the deadliest endocrine cancers, and its incidence has been increasing. While mutations in BRAF are common in thyroid cancer, advanced PTC patients currently lack therapeutic options targeting the MAPK pathway, and despite the approved combination of BRAF and MEK1/2 inhibition for BRAF-mutant ATC, resistance often occurs. Here, we assess growth and signaling responses to combined BRAF and MEK1/2 inhibition in a panel of BRAF-mutant thyroid cancer cell lines. We first showed that combined BRAF and MEK1/2 inhibition synergistically inhibits cell growth in four out of six of the -BRAF-mutant thyroid cancer cell lines tested. Western blotting showed that the MAPK pathway was robustly inhibited in all cell lines. Therefore, to identify potential mechanisms of resistance, we performed RNA-sequencing in cells sensitive or resistant to MEK1/2 inhibition. In response to MEK1/2 inhibition, we identified a downregulation of Aurora Kinase B (AURKB) in sensitive but not resistant cells. We further demonstrated that combined MEK1/2 and AURKB inhibition slowed cell growth, which was phenocopied by inhibiting AURKB and ERK1/2. Finally, we show that combined AURKB and ERK1/2 inhibition induces apoptosis in BRAF-mutant thyroid cancer cell lines, together suggesting a potential combination therapy for BRAF-mutant thyroid cancer patients.

Conformation selection by ATP-competitive inhibitors and allosteric communication in ERK2.

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Discovery of N-Benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine as a Potential Antifungal Agent against Sporidia Growth and Teliospore Germination of Sporisorium scitamineum.

The cultivation of sugar cane using perennial roots is the primary planting method, which is one of the reasons for the serious occurrence of sugar cane smut disease caused by the basidiomycetous fungus Sporisorium scitamineum in the sugar cane perennial root planting area. Consequently, it is crucial to eliminate pathogens from perennial sugar cane buds. In this study, we found that MAP kinase Hog1 is necessary for heat stress resistance. Subsequent investigations revealed a significant reduction in the expression of the heat shock protein 104-encoding gene, SsHSP104, in the ss1hog1Δ mutant. Additionally, the overexpression of SsHSP104 partially restored colony growth in the ss1hog1Δ strain following heat stress treatment, demonstrating the crucial role of SsHsp104 in SsHog1-mediated heat stress tolerance. Hence, we constructed the ss1hsp104:eGFP fusion strain in the wild type of S. scitamineum to identify small-molecule compounds that could inhibit the heat stress response, leading to the discovery of N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine as a potential compound that targets the SsHog1 mediation SsHsp104 pathway during heat treatment. Furthermore, the combination of N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine and warm water treatment (45 °C for 15 min) inhibits the growth of S. scitamineum and teliospore germination, thereby reducing the occurrence of sugar cane smut diseases and indicating its potential for eliminating pathogens from perennial sugar cane buds. In conclusion, these findings suggest that N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine is promising as a targeted compound for the SsHog1-mediated SsHsp104 pathway and may enable the reduction of hot water treatment duration and/or temperature, thereby limiting the occurrence of sugar cane smut diseases caused by S. scitamineum.

Amino acid metabolism and MAP kinase signaling pathway play opposite roles in the regulation of ethanol production during fermentation of sugarcane molasses in budding yeast.

Sugarcane molasses is one of the main raw materials for bioethanol production, and Saccharomyces cerevisiae is the major biofuel-producing organism. In this study, a batch fermentation model has been used to examine ethanol titers of deletion mutants for all yeast nonessential genes in this yeast genome. A total of 42 genes are identified to be involved in ethanol production during fermentation of sugarcane molasses. Deletion mutants of seventeen genes show increased ethanol titers, while deletion mutants for twenty-five genes exhibit reduced ethanol titers. Two MAP kinases Hog1 and Kss1 controlling the high osmolarity and glycerol (HOG) signaling and the filamentous growth, respectively, are negatively involved in the regulation of ethanol production. In addition, twelve genes involved in amino acid metabolism are crucial for ethanol production during fermentation. Our findings provide novel targets and strategies for genetically engineering industrial yeast strains to improve ethanol titer during fermentation of sugarcane molasses.

CRISPR-targeted mutagenesis of mitogen-activated protein kinase phosphatase 1 improves both immunity and yield in wheat.

Plants have evolved a sophisticated immunity system for specific detection of pathogens and rapid induction of measured defences. Over- or constitutive activation of defences would negatively affect plant growth and development. Hence, the plant immune system is under tight positive and negative regulation. MAP kinase phosphatase1 (MKP1) has been identified as a negative regulator of plant immunity in model plant Arabidopsis. However, the molecular mechanisms by which MKP1 regulates immune signalling in wheat (Triticum aestivum) are poorly understood. In this study, we investigated the role of TaMKP1 in wheat defence against two devastating fungal pathogens and determined its subcellular localization. We demonstrated that knock-down of TaMKP1 by CRISPR/Cas9 in wheat resulted in enhanced resistance to rust caused by Puccinia striiformis f. sp. tritici (Pst) and powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt), indicating that TaMKP1 negatively regulates disease resistance in wheat. Unexpectedly, while Tamkp1 mutant plants showed increased resistance to the two tested fungal pathogens they also had higher yield compared with wild-type control plants without infection. Our results suggested that TaMKP1 interacts directly with dephosphorylated and activated TaMPK3/4/6, and TaMPK4 interacts directly with TaPAL. Taken together, we demonstrated TaMKP1 exert negative modulating roles in the activation of TaMPK3/4/6, which are required for MAPK-mediated defence signalling. This facilitates our understanding of the important roles of MAP kinase phosphatases and MAPK cascades in plant immunity and production, and provides germplasm resources for breeding for high resistance and high yield.

Green leaf volatiles co-opt proteins involved in molecular pattern signalling in plant cells.

The green leaf volatiles (GLVs) Z-3-hexen-1-ol (Z3-HOL) and Z-3-hexenyl acetate (Z3-HAC) are airborne infochemicals released from damaged plant tissues that induce defenses and developmental responses in receiver plants, but little is known about their mechanism of action. We found that Z3-HOL and Z3-HAC induce similar but distinctive physiological and signaling responses in tomato seedlings and cell cultures. In seedlings, Z3-HAC showed a stronger root growth inhibition effect than Z3-HOL. In cell cultures, the two GLVs induced distinct changes in MAP kinase (MAPK) activity and proton fluxes as well as rapid and massive changes in the phosphorylation status of proteins within 5 min. Many of these phosphoproteins are involved in reprogramming the proteome from cellular homoeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteins and transcriptional regulators. These are well-known components of damage-associated molecular pattern (DAMP) signaling pathways. These rapid changes in the phosphoproteome may underly the activation of defense and developmental responses to GLVs. Our data provide further evidence that GLVs function like DAMPs and indicate that GLVs coopt DAMP signaling pathways.

Unveiling the role of host kinases at different steps of influenza A virus life cycle.

Influenza viruses remain a major public health concern causing contagious respiratory illnesses that result in around 290,000-650,000 global deaths every year. Their ability to constantly evolve through antigenic shifts and drifts leads to the emergence of newer strains and resistance to existing drugs and vaccines. To combat this, there is a critical need for novel antiviral drugs through the introduction of host-targeted therapeutics. Influenza viruses encode only 14 gene products that get extensively modified through phosphorylation by a diverse array of host kinases. Reversible phosphorylation at serine, threonine, or tyrosine residues dynamically regulates the structure, function, and subcellular localization of viral proteins at different stages of their life cycle. In addition, kinases influence a plethora of signaling pathways that also regulate virus propagation by modulating the host cell environment thus establishing a critical virus-host relationship that is indispensable for executing successful infection. This dependence on host kinases opens up exciting possibilities for developing kinase inhibitors as next-generation anti-influenza therapy. To fully capitalize on this potential, extensive mapping of the influenza virus-host kinase interaction network is essential. The key focus of this review is to outline the molecular mechanisms by which host kinases regulate different steps of the influenza A virus life cycle, starting from attachment-entry to assembly-budding. By assessing the contributions of different host kinases and their specific phosphorylation events during the virus life cycle, we aim to develop a holistic overview of the virus-host kinase interaction network that may shed light on potential targets for novel antiviral interventions.

Inactivation of ERK1/2 Signaling in Dopaminergic Neurons by Map Kinase Phosphatase MKP3 Regulates Dopamine Signaling and Motivation for Cocaine.

The mesolimbic dopamine system is a crucial component of reward and reinforcement processing, including the psychotropic effects of drugs of abuse such as cocaine. Drugs of abuse can activate intracellular signaling cascades that engender long-term molecular changes to brain reward circuitry, which can promote further drug use. However, gaps remain about how the activity of these signaling pathways, such as ERK1/2 signaling, can affect cocaine-induced neurochemical plasticity and cocaine-associated behaviors specifically within dopaminergic cells. To enable specific modulation of ERK1/2 signaling in dopaminergic neurons of the ventral tegmental area, we utilize a viral construct that Cre dependently expresses Map kinase phosphatase 3 (MKP3) to reduce the activity of ERK1/2, in combination with transgenic rats that express Cre in tyrosine hydroxylase (TH)-positive cells. Following viral transfection, we found an increase in the surface expression of the dopamine transporter (DAT), a protein associated with the regulation of dopamine signaling, dopamine transmission, and cocaine-associated behavior. We found that inactivation of ERK1/2 reduced post-translational phosphorylation of the DAT, attenuated the ability of cocaine to inhibit the DAT, and decreased motivation for cocaine without affecting associative learning as tested by conditioned place preference. Together, these results indicate that ERK1/2 signaling plays a critical role in shaping the dopamine response to cocaine and may provide additional insights into the function of dopaminergic neurons. Further, these findings lay important groundwork toward the assessment of how signaling pathways and their downstream effectors influence dopamine transmission and could ultimately provide therapeutic targets for treating cocaine use disorders.