Regulation Of Kinase Activity Research Articles

Cancer-Associated Fibroblasts Regulate Kinase Activity in Mesothelioma Cell Lines via Paracrine Signaling and Thereby Dictate Cell Faith and Behavior.

Background: Malignant pleural mesothelioma (MPM) has an infaust prognosis due to resistance to systemic treatment with platin-analoga. MPM cells modulate the immune response to their benefit. They release proinflammatory cytokines, such as TGF-ß, awakening resting fibrocytes that switch their phenotype into activated fibroblasts. Signaling interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an integral part in tumor progression. This study aimed to investigate the role CAFs play in MPM progression, analyzing the impact this complex, symbiotic interaction has on kinase-related cell signaling in vitro. Methods: We simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052, MSTO-211H, and NCI-H2452 cell lines representing the three mayor MPM subtypes, while embryonal myofibroblast cell lines, IMR-90 and MRC-5, provide a CAFs-like phenotype. Subsequently, differences in proliferation rates, migratory behavior, apoptosis, necrosis, and viability were used as covariates for data analysis. Kinase activity of treated samples and corresponding controls were then analyzed using the PamStation12 platform (PamGene); Results: Treatment with myofibroblast-derived CM revealed significant changes in phosphorylation patterns in MPM cell lines. The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect was observed using CM derived from IMR-90 than MRC-5. The phosphorylation changes mainly affected the MAPK signaling pathway.; Conclusions: The factors secreted by myofibroblasts in fibroblasts CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in tested MPM cell lines. Our in vitro results indicate promising therapeutic effects by the use of MEK or ERK inhibitors and might have synergistic effects in combination with cisplatin-based treatment, improving clinical outcomes for MPM patients.

Agonist-Specific Regulation of G Protein-Coupled Receptors after Chronic Opioid Treatment.

Chronic treatment of animals with morphine results in a long lasting cellular tolerance in the locus coeruleus and alters the kinase dependent desensitization of opioid and nonopioid G protein-coupled receptors (GPCRs). This study examined the development of tolerance and altered regulation of kinase activity after chronic treatment of animals with clinically relevant opioids that differ in efficacy at the µ-opioid receptors (MOR). In slices from oxycodone treated animals, no tolerance to opioids was observed when measuring the MOR induced increase in potassium conductance, but the G protein receptor kinase 2/3 blocker, compound 101, no longer inhibited desensitization of somatostatin (SST) receptors. Chronic fentanyl treatment induced a rightward shift in the concentration response to [Met5]enkephalin, but there was no change in the kinase regulation of desensitization of the SST receptor. When total phosphorylation deficient MORs that block desensitization, internalization, and tolerance were virally expressed, chronic treatment with fentanyl resulted in the altered kinase regulation of SST receptors. The results suggest that sustained opioid receptor signaling initiates the process that results in altered kinase regulation of not only opioid receptors, but also other GPCRs. This study highlights two very distinct downstream adaptive processes that are specifically regulated by an agonist dependent mechanism. SIGNIFICANCE STATEMENT: Persistent signaling of MORs results in altered kinase regulation of nonopioid GPCRs after chronic treatment with morphine and oxycodone. Profound tolerance develops after chronic treatment with fentanyl without affecting kinase regulation. The homeostatic change in the kinase regulation of nonopioid GPCRs could account for the systems level in vivo development of tolerance that is seen with opioid agonists, such as morphine and oxycodone, that develop more rapidly than the tolerance induced by efficacious agonists, such as fentanyl and etorphine.

DUSP4 Inactivation Leads to Reduced Extracellular Signal‒Regulated Kinase Activity through Upregulation of DUSP6 in Melanoma Cells

A subset of dual-specificity phosphatases is a major negative regulator of MAPKs, and their involvement in tumorigenesis remains controversial. Among them, DUSP4 is reported to preferentially dephosphorylate extracellular signal‒regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase over p38. In this study, we aimed to identify a possible role of DUSP4 in melanoma genesis. An examination of large-scale public data on gene expression and dependency revealed a considerably high DUSP4 expression and dependency of the melanoma cell lines compared with those of other tumor cell lines, which was not apparent for the other 24 dual-specificity phosphatases genes encoded in the human genome. Using two melanoma lines, we confirmed that DUSP4 depletion impaired cell growth without notably inducing apoptosis. Interestingly, immunoblotting and kinase translocation reporter data revealed that DUSP4 depletion induces a decrease in ERK1/2 phosphorylation but barely affects c-Jun N-terminal kinase phosphorylation, suggesting that neither ERK nor c-Jun N-terminal kinase is a direct target of DUSP4 in our experimental setting. Notably, DUSP4 depletion led to an increase in DUSP6 level, possibly through a post-transcriptional process, and DUSP6 knockout almost eliminated the DUSP4-depletion effect on cell growth and ERK activity. Our findings suggest that DUSP4 plays a role in maintaining a high ERK1/2 activity by negatively regulating DUSP6 and thus contributes to the survival and growth of melanoma cells.

Mechanism of Huangjing Qianshi Decoction in treatment of prediabetes based on network pharmacology and molecular docking

This study analyzed the molecular mechanism of Huangjing Qianshi Decoction(HQD) in the treatment of prediabetes based on network pharmacology and molecular docking. The active components of HQD were identified and screened based on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP, http://Lsp.nwu.edu.cn/tcmsp.php) and then the targets of the components and the genes related to prediabetes were retrieved, followed by identifying the common targets of the decoction and the disease. The medicinal component-target network was constructed by Cytoscape to screen key components. The protein-protein interaction(PPI) network was established by STRING and hub genes were identified by Cytoscape-CytoNCA, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) of the hub genes with R-clusterProfi-ler. Thereby, the possible signaling pathways were predicted and the molecular mechanism was deduced. A total of 79 active components of HQD and 785 diabetes-related targets of the components were screened out. The hub genes mainly involved the GO terms of tricarboxylic acid cycle, peptide binding, amide binding, hydrolase activity, and kinase activity regulation, and the KEGG pathways of AGE-RAGE signaling pathway, TNF signaling pathway, AMPK signaling pathway, IL-17 signaling pathway, and insulin signaling pathway. Western blot result showed that HQD-containing serum significantly reduced the expression of AKT1, AGE, and RAGE proteins in insulin resistance model cells. HQD's treatment of prediabetes is characterized by multiple pathways, multiple targets, and multiple levels. The main mechanism is that the components zhonghualiaoine, baicalein, kaempferol, and luteolin act on AKT1 and inhibit the AGE-RAGE axis.

The potent inhibitory role of suppressing TBK1 in RIPK1 associated cerebral ischemia-reperfusion injury

The pathological mechanism of cell death features in cerebral ischemia-reperfusion injury (CIRI) was complicated. The occurrence of various cell death pathways during the progression of ischemia/reperfusion injury promoted complex further neuroinflammation. RIPK1, receptor interacting protein kinase 1, was convinced to be involved in both necroptosis and apoptosis, which is a special RIPK1-dependent apoptosis. More evidences indicated the physiological role of RIPK1 in necroptosis, apoptosis and also autophagy.In this study, we elucidated the RIPK1 exhibited characterization in various cell death pathways in time-course dependent feature. The necroptosis occupied dominant neuron death within 24 h after ischemia/reperfusion injury. However, the neuronal death feature seemed turned to apoptosis 24 h after reperfusion.In this study, it was also found that TBK1 (TANK binding kinase 1) played as suppressor in the regulation of kinase activity of RIPK1. This result might provide a potential approach in mediating the kinase activity of RIPK1 in clinic.

Abstract WMP110: Cerebral Ischemia-Reperfusion Alters Postsynaptic Kinase Activities Through Ubiquitination

Background: Aberrant activity of the postsynaptic density (PSD)-associated kinases CaMKII, PKC, Pyk2 and Cdk5 contributes to post-ischemic neuronal death. However, the mechanism(s) leading to altered activity after ischemia have not been discovered. We investigated the role of ubiquitination in post-ischemic PSD kinase activity regulation. Methods: Cerebral ischemia was induced in male C57BL6 mice by middle cerebral artery occlusion (MCAO), followed by 1-hour reperfusion. Ubiquitinated and phosphorylated proteins in the ischemic neocortex were identified by nanoLC-MS/MS and analyzed for GO enrichment. PSD isolation was carried out by detergent extraction. Kinase ubiquitination was confirmed by co-immunoprecipitation with ubiquitin. Protein phosphorylation was assessed with phospho-specific antibodies and kinase activities were measured by transfer of γ 32 P to peptide substrates. Removal of ubiquitin from PSD lysates was achieved by addition of USP2 deubiquitinase. Results: MS analysis revealed that MCAO increases ubiquitination of proteins associated with the PSD (p=5.14x10 -33 , n=60/group). This is also evidenced by biochemical detection of ubiquitin at the synapse and PSD but not cytosol of cortical MCAO extracts (p<0.001, n=6/group). Interestingly, a significant number of ubiquitinated PSD proteins exhibit kinase activity (p=1.02x10 -23 , n=60/group) and include the kinases CaMKII, PKC, Pyk2 and Cdk5, which were biochemically verified (n=3/group). Concurrent with ubiquitination, MCAO also changes the phosphorylation state of PSD-associated proteins (p=3.66x10 -13 , n=60/group). Pyk2 and Cdk5 target sites are hyper-phosphorylated in post-ischemic PSD extracts, while CaMKII and PKC targets show decreased phosphorylation (p<0.05, n=5/group). In line with this, post-ischemic CaMKII (-69±6% of sham, p<0.001, n=9/group) and PKC (-63±8% of sham, p<0.001, n=6/group) activities are severely suppressed at the PSD, which is restored by de-ubiquitination (CaMKII: 105.8±23% of sham, n=6/group; PKC: 101.4±11% of sham, n=4/group). Conclusions: Ubiquitination is a potent and reversible modulator of kinase activity at the PSD that could impact tissue outcome and, as such, may open new avenues for the treatment of ischemic stroke.

Cellular ATP Levels Determine the Stability of a Nucleotide Kinase.

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf 0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

A Pan-Cancer Analysis of Clinical Prognosis and Immune Infiltration of CKS1B in Human Tumors.

Although more and more evidence supports CDC28 protein kinase subunit 1B (CKS1B) is involved significantly in the development of human cancers, most of the researches have focused on a single disease, and pan-cancer studies conducted from a holistic perspective of different tumor sources have not been reported yet. Here, for the first time, we investigated the potential oncogenic and prognostic role of CKS1B across 33 tumors based on public databases and further verified it in a small scale by RNA sequencing or quantitative real-time PCR. CKS1B was generally highly expressed in a majority of tumors and had a notable correlation with the prognosis of patients, but its prognostic significance in different tumors was not exactly the same. In addition, CKS1B expression was also closely related to the infiltration of cancer-associated fibroblasts in tumors such as breast invasive carcinoma, kidney chromophobe, lung adenocarcinoma, and tumor-infiltrating lymphocytes in tumors such as glioblastoma multiforme, bladder urothelial carcinoma, and brain lower grade glioma. Moreover, reduced CKS1B methylation was observed in certain tumors, for example, adrenocortical carcinoma. Cell cycle and kinase activity regulation and PI3K-Akt signaling pathway were found to be involved in the functional mechanism of CKS1B. In conclusion, our first pan-cancer analysis of CKS1B contributes to a better overall understanding of CKS1B and may provide a new target for future cancer therapy.

Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation.

Statins represent the most prescribed class of drugs for the treatment of hypercholesterolemia. Effects that go beyond lipid-lowering actions have been suggested to contribute to their beneficial pharmacological properties. Whether and how statins act on macrophages has been a matter of debate. In the present study, we aimed at characterizing the impact of statins on macrophage polarization and comparing these to the effects of bempedoic acid, a recently registered drug for the treatment of hypercholesterolemia, which has been suggested to have a similar beneficial profile but fewer side effects. Treatment of primary murine macrophages with two different statins, i.e., simvastatin and cerivastatin, impaired phagocytotic activity and, concurrently, enhanced pro-inflammatory responses upon short-term lipopolysaccharide challenge, as characterized by an induction of tumor necrosis factor (TNF), interleukin (IL) 1β, and IL6. In contrast, no differences were observed under long-term inflammatory (M1) or anti-inflammatory (M2) conditions, and neither inducible NO synthase (iNOS) expression nor nitric oxide production was altered. Statin treatment led to extracellular-signal regulated kinase (ERK) activation, and the pro-inflammatory statin effects were abolished by ERK inhibition. Bempedoic acid only had a negligible impact on macrophage responses when compared with statins. Taken together, our data point toward an immunomodulatory effect of statins on macrophage polarization, which is absent upon bempedoic acid treatment.

Tyrosine Kinase Self-Phosphorylation Controls Exopolysaccharide Biosynthesis in Gluconacetobacter diazotrophicus Strain Pal5.

The biosynthesis of exopolysaccharides (EPSs) is essential for endophytic bacterial colonisation in plants bacause this exopolymer both protects bacterial cells against the defence and oxidative systems of plants and acts on the plant colonisation mechanism in Gluconacetobacter diazotrophicus. The pathway involved in the biosynthesis of bacterial EPS has not been fully elucidated, and several areas related to its molecular regulation mechanisms are still lacking. G. diazotrophicus relies heavily on EPS for survival indirectly by protecting plants from pathogen attack as well as for endophytic maintenance and adhesion in plant tissues. Here, we report that EPS from G. diazotrophicus strain Pal5 is a signal polymer that controls its own biosynthesis. EPS production depends on a bacterial tyrosine (BY) kinase (Wzc) that consists of a component that is able to phosphorylate a glycosyltranferase or to self-phosphorylate. EPS interacts with the extracellular domain of Wzc, which regulates kinase activity. In G. diazotrophicus strains that are deficient in EPS production, the Wzc is rendered inoperative by self-phosphorylation. The presence of EPS promotes the phosphorylation of a glycosyltransferase in the pathway, thus producing EPS. Wzc-mediated self-regulation is an attribute for the control of exopolysaccharide biosynthesis in G. diazotrophicus.

Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson’s disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC–COR–kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC’s homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment.

P2X7 Receptor Deficiency Ameliorates STZ-induced Cardiac Damage and Remodeling Through PKCβ and ERK.

Diabetic cardiomyopathy (DCM) is a complication of diabetes mellitus which result in cardiac remodeling and subsequent heart failure. However, the role of P2X7 receptor (P2X7R) in DCM has yet to be elucidated. The principal objective of this study was to investigate whether P2X7R participates in the pathogenesis of DCM. In this study, the C57BL/6 diabetic mouse model was treated with a P2X7R inhibitor (A438079). Cardiac dysfunction and remodeling were attenuated by the intraperitoneal injection of A438079 or P2X7R deficiency. In vitro, A438079 reduced high glucose (HG) induced cell damage in H9c2 cells and primary rat cardiomyocytes. Furthermore, HG/streptozotocin (STZ)-induced P2X7R activation mediated downstream protein kinase C-β (PKCβ) and extracellular regulated protein kinases (ERK) activation. This study provided evidence that P2X7R plays an important role in the pathogenesis of STZ-induced diabetic cardiac damage and remodeling through the PKCβ/ERK axis and suggested that P2X7R might be a potential target in the treatment of diabetic cardiomyopathy.

Integration of network pharmacology and molecular docking technology reveals the mechanism of the herbal pairing of Codonopsis Pilosula (Franch.) Nannf and Astragalus Membranaceus (Fisch.) Bge on chronic heart failure

The herbal pairing of Dangshen (DS) [Codonopsis pilosula (Franch.) Nannf.] and Huangqi (HQ) [Astragalus membranaceus (Fisch.) Bge.] (DHP) is a traditional Chinese herbal medicine that is frequently used to treat chronic heart failure (CHF) in China. However, the pharmacological mechanism of DHP has not been fully elucidated. This is the first study aimed to reveal the active mechanism of DHP in the treatment of CHF by using network pharmacology methods. The active ingredients of DHP were obtained from the TCMSP database, and the potential targets of DHP were predicted using the SwissTargetPrediction database. CHF-related targets were searched by the DisGeNET and GeneCards databases. The common targets between the disease and herbs were obtained using a Venn diagram. The STRING database was utilized to obtain the protein-protein interaction data. Next, we used Cytoscape 3.7.2 software to construct and analyze the herb-ingredient-potential targets-disease network. Topology analysis was used to identify the key ingredients and hub genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Metascape database to reveal the mechanism. Furthermore, molecular docking simulation was performed using AutoDock Vina software to assess the affinity of the key ingredients and hub genes. Five key ingredients and six hub genes were screened. The six hub genes were closely related to PI3K /AKT or ERK1/2 pathways. The KEGG pathways mainly involved the TNF signaling pathway, calcium signaling pathway, and cancer-related pathways. The GO enrichment analysis results showed that DHP might act on biological processes including positive regulation of kinase activity and cellular response to nitrogen compound via the three above-mentioned pathways in the treatment of CHF. Finally, the molecular docking results showed that the five key ingredients exhibited strong affinities to the six hub genes. This study revealed the molecular mechanism that the flavonoids in DHP may alleviate endothelial dysfunction and cardiac hypertrophy via regulation of the TNF pathway and its downstream PI3K/Akt or ERK1/2 signaling pathways, or improve excitation-contraction coupling by regulating calcium signaling pathway, thereby improving CHF. These results provide insights for further experimentation on its pharmacological effects.

MTOR Restricts Chromatin Access and Genomic Activity to Maintain Hematopoietic Stem Cell Quiescence and Engraftment

mTOR kinase is an anti-cancer drug target that integrates signals from nutrients, growth factors, and cellular energy status to control cell growth and proliferation, but its biological function in hematopoietic stem cells remains unclear. Numerous studies by using rapamycin or mTOR kinase inhibitors have indicated that mTOR inhibition suppresses chromatin access and gene expression and inhibits cell growth. Consistent with the notion that mTOR targeting suppresses gene activation, hyperactivation of mTOR by deletion of negative regulators of mTOR, TSC1/TSC2 or PTEN, causes a loss of quiescence and exhaustion of HSCs. To directly examine the function of mTOR and its kinase activity in HSC regulation, we used the conditional Mx-Cre;mTORflox/flox and the Mx-Cre;mTORflox/knockin mice that produce the mTOR-/- and the mTOR kinase-activity deficient D2338A mutant knockin genotype, respectively, upon poly I:C induced Cre recombination. We have determined the phenotypic, genomic and molecular effects of their HSCs. Our previous studies have found that depletion of mTOR drastically affected hematopoiesis, with a marked reduction in total BM cellularity and transient increase in the number of HSCs in bone marrow. Remarkably, mTOR-/- HSCs displayed a loss of quiescence, increased proliferation and a normal survival index (Guo et al., Haematologic a 98:1353). Our current study has found similar phenotypes of the HSCs in increased number and proliferation in the Mx-Cre;mTORflox/knockin mice where the mTOR kinase activity is abolished. Both mTOR-/- and mTORKI/- HSCs are defective in engraftment upon competitive transplant into recipient mice, with a clear molecular signature of drastically reduced downstream effectors p-S6K and p-4E-BP. ATAC-seq reveals that loss of mTOR or mTOR kinase activity results in selectively increased chromatin access and global gene activation, in contrast to the reported effects by mTOR inhibitors such as rapamycin or pp242/AZD2014. ChIP-seq of two active histone markers, H3K27Ac and H3K4Me3, yields consistent results that chromosome of the HSCs are activated upon the loss of mTOR or its kinase activity. In particular, increased H3K27Ac and H3K4Me3 ChIP-seq activities are observed in both the upstream and downstream region of c-myc locus, consistent with an activated c-myc expression. RNA-seq analysis reveals that upon mTOR KO or KI, thousands of genes exhibit differential expression. The up-regulated genes are particularly enriched in functions involved in ribosome biogenesis/protein translation, cell cycle progression, cell fate determination, and survival. Analysis using the STRING functional interaction network to search for the compensatory genes/pathways discovers a number of likely compensatorily upregulated genes, upon mTOR KO or KI, that share a significant number of interacting genes with mTOR, including c-myc, Fos, and Jun. These genes are enriched in pathways such as EGFR-MAPK pathway, PI3K-Akt pathway, Jak-Stat pathway, and NFkB pathway, which may compensate for mTOR loss. RT-PCR and Western blotting further verify that loss of mTOR or its kinase activity causes elevated mRNA and protein expression of selected compensatory genes, including c-myc, Fos, Jun, and Pik3cd. In addition, broad compensatory elevations in ribosome and translation machinery gene expression, activation of RNA pol-II, and activation of several interesting compensatory pathways are seen. The BET inhibitor, JQ1, that inhibits c-myc activity readily suppresses the hyper-proliferative phenotype of mTOR-/- and mTORKI/-HSCs. Thus, contrary to dogma established mostly by transient rapamycin or kinase inhibitor usages, our genetic and genomic studies demonstrate that mTOR, through its kinase activity, plays a restrictive role to maintain chromosomal access and genomic activity of HSCs. Loss of mTOR or its kinase activity unleashes a compensatory chromosomal and gene activation effects that involve master regulators of gene activities such as c-myc and Fos, protein translation and ribosomal proteins, and cell growth signaling pathways. These genomic changes may cause the HSC loss of quiescence and engraftment phenotypes seen upon mTOR loss, and need to be taken into consideration in the development of new generations of mTOR targeted therapy and combinatory therapy.Disclosures: No relevant conflicts of interest to declareASH Members: Yi Zheng DisclosuresNo relevant conflicts of interest to declare.

Regulatory Switching by Concerted Motions on the Microsecond Time Scale of the Oxygen Sensor Protein FixL.

Signal transduction proteins perceive external stimuli in their sensor module and regulate the biological activities of the effector module, allowing cellular adaptation in response to environmental changes. FixL is a dimeric heme protein kinase that senses the oxygen level in plant root nodules to regulate the transcription of nitrogen fixation genes via the phosphorylation of its cognate transcriptional activator. Dissociation of oxygen from the heme induces conformational changes in the protein, converting it from the inactive form for phosphorylation to the active form. However, how FixL undergoes conformational change to regulate kinase activity upon oxygen dissociation remains poorly understood. Here we report time-resolved ultraviolet resonance Raman spectra showing conformational changes for FixL from Sinorhizobium meliloti. We observed spectral changes with a time constant of about 3 μs, which were oxygen-specific. Furthermore, we found that the conformational changes in the sensor and kinase domains are coupled, enabling allosteric control of kinase activity. Our results demonstrate that concerted structural changes on the microsecond time scale serve as the regulatory switch in FixL.

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2.

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

MTOR kinase activity disrupts a phosphorylation signaling network in schizophrenia brain.

The AKT-mTOR signaling transduction pathway plays an important role in neurodevelopment and synaptic plasticity. mTOR is a serine/threonine kinase that modulates signals from multiple neurotransmitters and phosphorylates specific proteins to regulate protein synthesis and cytoskeletal organization. There is substantial evidence demonstrating abnormalities in AKT expression and activity in different schizophrenia (SZ) models. However, direct evidence for dysregulated mTOR kinase activity and its consequences on downstream effector proteins in SZ pathophysiology is lacking. Recently, we reported reduced phosphorylation of mTOR at an activating site and abnormal mTOR complex formation in the SZ dorsolateral prefrontal cortex (DLPFC). Here, we expand on our hypothesis of disrupted mTOR signaling in the SZ brain and studied the expression and activity of downstream effector proteins of mTOR complexes and the kinase activity profiles of SZ subjects. We found that S6RP phosphorylation, downstream of mTOR complex I, is reduced, whereas PKCα phosphorylation, downstream of mTOR complex II, is increased in SZ DLPFC. In rats chronically treated with haloperidol, we showed that S6RP phosphorylation is increased in the rat frontal cortex, suggesting a potential novel mechanism of action for antipsychotics. We also demonstrated key differences in kinase signaling networks between SZ and comparison subjects for both males and females using kinome peptide arrays. We further investigated the role of mTOR kinase activity by inhibiting it with rapamycin in postmortem tissue and compared the impact of mTOR inhibition in SZ and comparison subjects using kinome arrays. We found that SZ subjects are globally more sensitive to rapamycin treatment and AMP-activated protein kinase (AMPK) contributes to this differential kinase activity. Together, our findings provide new insights into the role of mTOR as a master regulator of kinase activity in SZ and suggest potential targets for therapeutic intervention.

Genetic association between major depressive disorder and type 2 diabetes mellitus: Shared pathways and protein networks

BackgroundMajor depressive disorder (MDD) and type 2 diabetes mellitus (T2DM) are common public health disorders that often co-occur. This study aims to determine whether gene expression profiles from individuals with MDD or T2DM overlap and if there are any functional interconnectivity between identified genes using protein-protein interaction (PPI). MethodsThe DNA microarray datasets were extracted from the Gene Expression Omnibus. Gene expression dataset GSE98793 from a case-control study of MDD (64 healthy control subjects, 128 patients) and dataset GSE15653 from a case-control study of T2DM (nine controls, nine individuals with T2DM) were used for this secondary and post-hoc analysis. GO enrichment analyses and Reactome pathway enrichment analysis were performed for functional enrichment analyses with the shared genes. PPI networks, PPI clusters and hub genes were performed to detect the potential relationships among differentially expressed genes (DEG) -encoding proteins in both MDD and T2DM. ResultsA total of 3640 DEGs were identified in the MDD group when compared to the control group, whereas 3700 DEGs were identified in the T2DM group when compared to the control groups, among which 244 DEGs were overlap genes. The identified DEGs were enriched for Interleukin-4 and Interleukin-13 signaling, neutrophil degranulation, as well as other select species of the innate immune system. The biological processes of neurofibrillary tangle assembly regulation, tau-protein kinase activity regulation, amyloid-beta clearance regulation, amyloid-beta formation regulation and neuron apoptotic processes were also identified. Molecular function analysis indicated that identified genes were mainly enriched for amyloid-beta binding. 925 out of 1006 protein-protein interactions and six sub-networks were identified reflecting the disparate biological domains of overlapping genes. Ten hub genes further highlight the putative importance of tau-protein kinase activity, inflammatory response and neuron apoptotic regulatory processes across MDD and T2DM. ConclusionsOur results indicate that an overlapping genetic architecture subserves MDD and T2DM. Genes relevant to the innate immune system, tau protein formation, and cellular aging were identified. Results indicate that the common, often comorbid, conditions of MDD and T2DM have a pathoetiologic nexus.

Insight into Liver lncRNA and mRNA Profiling at Four Developmental Stages in Ningxiang Pig.

Simple SummaryThe current study reveals the expression profiles and functional networks on messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the liver of Ningxiang piglets across four developmental stages (30, 90, 150, and 210 days after birth). Differentially expressed mRNAs (DEmRNAs) were upregulated at 30 days; however, most differentially expressed lncRNAs (DElncRNAs) were downregulated at 210 days. A complex interaction between mRNAs and lncRNAs was identified by Short Time-series Expression Miner (STEM) analysis and weighted gene co-expression network analysis (WGCNA), indicating that lncRNAs may be a critical regulatory element in mRNAs. STEM was used to identify significant temporal expression profiles and the genes associated with them and to compare the behavior of these genes across multiple conditions. WGCNA was used to study the biological networks based on pairwise correlations between variables. One particular mRNA profile 4 contained CAV1, PACSIN2, and CDC42, which are the target genes of lncRNAs in the same profile, suggesting the possible regulatory relationship between lncRNAs and mRNAs. Ningxiang pigs, a fat-type pig, are native to Ningxiang County in Hunan Province, with thousands of years of breeding history. This study aims to explore the expression profiles and functional networks on messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the liver. Liver tissue of Ningxiang piglets was collected at 30, 90, 150, and 210 days after birth (four development stages), and the mRNA and lncRNA expression was profiled. Compared to mRNA and lncRNA expression profiles, most differentially expressed mRNAs (DEmRNAs) were upregulated at 30 days; however, most DElncRNAs were downregulated at 210 days. Via Short Time-series Expression Miner (STEM) analysis and weighted gene co-expression network analysis (WGCNA), a complex interaction between mRNAs and lncRNAs was identified, indicating that lncRNAs may be a critical regulatory element for mRNAs. One module of genes in particular (module profile 4) was related to fibril organization, vasculogenesis, GTPase activator activity, and regulation of kinase activity. The mRNAs and lncRNAs in module profile 4 had a similar pattern of expression, indicating that they have functional and regulatory relationships. Only CAV1, PACSIN2, and CDC42 in the particular mRNA profile 4 were the target genes of lncRNAs in that profile, which shows the possible regulatory relationship between lncRNAs and mRNAs. The expression of these genes and lncRNAs in profile 4 was the highest at 30 days, and it is believed that these RNAs may play a critical role during the suckling period in order to meet the dietary requirements of piglets. In the lncRNA–mRNA co-expression network, the identified gene hubs and associated lncRNAs were shown to be involved in saccharide, lipid, and glucose metabolism, which may play an important role in the development and health of the liver. This result will lead to further investigation of liver lncRNA functions at various stages of development in Ningxiang pigs.

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.