Subunit Of Kinase Research Articles

Mediator's Kinase Module: A Modular Regulator of Cell Fate.

Selective gene expression is crucial in maintaining the self-renewing and multipotent properties of stem cells. Mediator is a large, evolutionarily conserved, multi-subunit protein complex that modulates gene expression by relaying signals from cell type-specific transcription factors to RNA polymerase II. In humans, this complex consists of 30 subunits arranged in four modules. One critical module of the Mediator complex is the kinase module consisting of four subunits: MED12, MED13, CDK8, and CCNC. The kinase module exists in variable association with the 26-subunit Mediator core and affects transcription through phosphorylation of transcription factors and by controlling Mediator structure and function. Many studies have shown the kinase module to be a key player in the maintenance of stem cells that is distinct from a general role in transcription. Genetic studies have revealed that dysregulation of this kinase subunit contributes to the development of many human diseases. In this review, we discuss the importance of the Mediator kinase module by examining how this module functions with the more recently identified transcriptional super-enhancers, how changes in the kinase module and its activity can lead to the development of human disease, and the role of this unique module in directing and maintaining cell state. As we look to use stem cells to understand human development and treat human disease through both cell-based therapies and tissue engineering, we need to remain aware of the on-going research and address critical gaps in knowledge related to the molecular mechanisms that control cell fate.

NAIL: an evolutionarily conserved lncRNA essential for licensing coordinated activation of p38 and NFκB in colitis

ObjectiveNFκB is the key modulator in inflammatory disorders. However, the key regulators that activate, fine-tune or shut off NFκB activity in inflammatory conditions are poorly understood. In this study, we...

Abstract 14219: Oxidation of PKA-RIα Protects Against Ischemia-reperfusion Injury by Inhibiting Lysosomal-triggered Calcium Release

Introduction: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, the regulatory Iα subunit of Protein Kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulfide bonds within the holoenzyme complex. However, the impact of disulfide formation on kinase function, and its influence on PKA signaling in the context of heart disease remains unknown. Methods & Results: Myocardial ischemia-reperfusion (I/R) was found to be a potent inducer of PKARIα disulfide formation in vivo , both in mice and in humans. Using imaging modalities with high spatial and temporal resolution, we found that this conformation did not increase intrinsic PKA catalytic activity, but rather facilitated enhanced AKAP-dependent compartmentation of PKARIα in the adult mouse left ventricular (LV) myocyte, with preferential localization to the lysosome under oxidized conditions (n=38-41 myocytes, N=3 animals, p<0.01). Investigations in isolated LV myocytes revealed disulfide-modified PKARIα to be a significant regulator of lysosomal two pore channel (TPC)-dependent calcium-induced calcium release, with myocytes from ‘redox dead’ PKARIα mice (Cys17Ser) displaying spontaneous sarcoplasmic reticulum calcium release events and pronounced intracellular calcium oscillations. These events were prevented by ryanodine receptor blockade (1 mM tetracaine; n=14, p<0.01), acute depletion of lysosomal calcium stores (100 nM bafilomycin; n=7; p<0.01), or TPC inhibition (5 μM Ned-19; n=9; p<0.05). Absence of I/R-induced disulfide formation in “redox dead” PKARIα mouse hearts resulted in larger infarcts (2-fold increase, p<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, p<0.001), which could be fully prevented by administering the TPC inhibitor, Ned-19, at the time of reperfusion. Conclusions: Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKA oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics.

Preventing RIα Phase Separation Disrupts Signaling and Promotes Cancer

RIα, a regulatory subunit of protein kinase A, formed liquid droplets that concentrated cAMP.

The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.

A prion-like domain of Tpk2 catalytic subunit of protein kinase A modulates P-body formation in response to stress in budding yeast

Low complexity regions are involved in the assembly and disassembly of P-bodies (PBs). Saccharomyces cerevisiae contains three genes encoding the protein kinase A (PKA) catalytic subunit: TPK1, TPK2 and TPK3. Tpk2 and Tpk3 isoforms localize to PBs upon glucose starvation showing different mechanisms and kinetics of accumulation. In contrast to the other two isoforms, Tpk2 harbors a glutamine-rich prion-like domain (PrLD) at the N-terminus. Here we show that the appearance of Tpk2 foci in response to glucose starvation, heat stress or stationary phase was dependent on its PrLD. Moreover, the PrLD of Tpk2 was necessary for efficient PB and stress granule aggregation during stress conditions and in quiescent cells. Deletion of PrLD does not affect the in vitro and in vivo kinase activity of Tpk2 or its interaction with the regulatory subunit Bcy1. We present evidence that the PrLD of Tpk2 serves as a scaffold domain for PB assembly in a manner that is independent of Pat1 phosphorylation by PKA. In addition, a mutant strain where Tpk2 lacks PrLD showed a decrease of turnover of mRNA during glucose starvation. This work therefore provides new insight into the mechanism of stress-induced cytoplasmic mRNP assembly, and the role of isoform specific domains in the regulation of PKA catalytic subunit specificity and dynamic localization to cytoplasmic RNPs granules.

PRKAR2B-HIF-1α loop promotes aerobic glycolysis and tumour growth in prostate cancer.

ObjectivesReprogramming of cellular metabolism is profoundly implicated in tumorigenesis and can be exploited to cancer treatment. Cancer cells are known for their propensity to use glucose‐dependent glycolytic pathway instead of mitochondrial oxidative phosphorylation for energy generation even in the presence of oxygen, a phenomenon known as Warburg effect. The type II beta regulatory subunit of protein kinase A (PKA), PRKAR2B, is highly expressed in castration‐resistant prostate cancer (CRPC) and contributes to tumour growth and metastasis. However, whether PRKAR2B regulates glucose metabolism in prostate cancer remains largely unknown.Materials and methodsLoss‐of‐function and gain‐of‐function studies were used to investigate the regulatory role of PRKAR2B in aerobic glycolysis. Real‐time qPCR, Western blotting, luciferase reporter assay and chromatin immunoprecipitation were employed to determine the underlying mechanisms.ResultsPRKAR2B was sufficient to enhance the Warburg effect as demonstrated by glucose consumption, lactate production and extracellular acidification rate. Mechanistically, loss‐of‐function and gain‐of‐function studies showed that PRKAR2B was critically involved in the tumour growth of prostate cancer. PRKAR2B was able to increase the expression level of hypoxia‐inducible factor 1α (HIF‐1α), which is a key mediator of the Warburg effect. Moreover, we uncovered that HIF‐1α is a key transcription factor responsible for inducing PRKAR2B expression in prostate cancer. Importantly, inhibition of glycolysis by the glycolytic inhibitor 2‐deoxy‐d‐glucose (2‐DG) or replacement of glucose in the culture medium with galactose (which has a much lower rate than glucose entry into glycolysis) largely compromised PRKAR2B‐mediated tumour‐promoting effect. Similar phenomenon was noticed by genetic silencing of HIF‐1α.ConclusionsOur study identified that PRKAR2B‐HIF‐1α loop enhances the Warburg effect to enable growth advantage in prostate cancer.

Budlein A methylacrylate demonstrates potent activity against triple-negative breast cancer by targeting IκBα kinase and exportin-1

Triple-negative breast cancer (TNBC) remains the most challenging breast cancer subtype to treat because there are no targeted therapies. Currently, chemotherapy is the only clinical option for TNBC despite development of resistance. New therapeutic agents with unique mechanisms of action are urgently needed; therefore, this study investigated the potential anti-TNBC effects of budlein A methylacrylate (BAM), a natural sesquiterpene lactone isolated from plants of the Helianthus genus. We discovered that BAM selectively suppressed and induced apoptosis TNBC cell growth versus other breast cancer or normal mammary epithelial cells. Mechanistically, BAM co-inhibited inhibitor of nuclear factor κBα (IκBα) kinase subunit β (IKKβ) and exportin-1 (XPO-1; chromosome region maintenance 1, CRM1), which are two dysregulated onco-related proteins in TNBC cells, by covalently modifying key functional cysteine residues (Cys179 of IKKβ, Cys528 of XPO-1). Dual inhibition led to the stabilization and nuclear retention of IκBα, impairment of NF-κB transcriptional activity, and consequent induction of TNBC cell apoptosis. In conclusion, this study provides evidence that co-inhibition of IKKβ and XPO-1 by BAM was effective against TNBC, demonstrating it as a representative new generation inhibitor with potential for TNBC treatment.

Astaxanthin Inhibits p70 S6 Kinase 1 Activity to Sensitize Insulin Signaling.

Astaxanthin (AST) is a carotenoid with therapeutic values on hyperglycemia and diabetic complications. The mechanisms of action of AST remain incompletely understood. p70 S6 kinase 1 (S6K1) is a serine/threonine kinase that phosphorylates insulin receptor substrate 1 (IRS-1)S1101 and desensitizes the insulin receptor (IR). Our present study aims to determine if AST improves glucose metabolisms by targeting S6K1. Western blot analysis revealed that AST inhibited the phosphorylation of two S6K1 substrates, S6S235/236 and IRS-1S1101, but enhanced the phosphorylation of AKTT308, AKTS473, and S6K1T389 by feedback activation of the phosphatidylinositol-3 (PI-3) kinase in 3T3-L1 adipocytes and L6 myotubes. In vitro kinase assays revealed that AST inhibited S6K1 activity with an IC50 value of approximately 13.8 μM. AST increased insulin-induced IR tyrosine phosphorylation and IRS-1 binding to the p85 subunit of PI-3 kinase. Confocal microscopy revealed that AST increased the translocation of the glucose transporter 4 (GLUT4) to the plasma membrane in L6 cells. Glucose uptake assays using a fluorescent dye, 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), revealed that AST increased glucose uptake in 3T3-L1 adipocytes and L6 myotubes under insulin resistance conditions. Our study identifies S6K1 as a previously unrecognized molecular target of AST and provides novel insights into the mechanisms of action of AST on IR sensitization.

Discovering functional evolutionary dependencies in human cancers.

Cancer cells retain genomic alterations that provide a selective advantage. The prediction and validation of advantageous alterations are major challenges in cancer genomics. Moreover, it is crucial to understand how the coexistence of specific alterations alters response to genetic and therapeutic perturbations. In the present study, we inferred functional alterations and preferentially selected combinations of events in >9,000 human tumors. Using a Bayesian inference framework, we validated computational predictions with high-throughput readouts from genetic and pharmacological screenings on 2,000 cancer cell lines. Mutually exclusive and co-occurring cancer alterations reflected, respectively, functional redundancies able to rescue the phenotype of individual target inhibition, or synergistic interactions, increasing oncogene addiction. Among the top scoring dependencies, co-alteration of the phosphoinositide 3-kinase (PI3K) subunit PIK3CA and the nuclear factor NFE2L2 was a synergistic evolutionary trajectory in squamous cell carcinomas. By integrating computational, experimental and clinical evidence, we provide a framework to study the combinatorial functional effects of cancer genomic alterations.

CKS1B as Drug Resistance-Inducing Gene-A Potential Target to Improve Cancer Therapy.

Cancer is a threat to human health and life. Although previously centered on chemical drug treatments, cancer treatment has entered an era of precision targeted therapy. Targeted therapy entails precise guidance, allowing the selective killing of cancer cells and thereby reducing damage to healthy tissues. Therefore, the need to explore potential targets for tumor treatment is vital. Cyclin-dependent kinase regulatory subunit 1B (CKS1B), a member of the conserved cyclin kinase subunit 1 (CKS1) protein family, plays an essential role in cell cycling. A large number of studies have shown that CKS1B is associated with the pathogenesis of many human cancers and closely related to drug resistance. Here, we describe the current understanding of the cellular functions of CKS1B and its underlying mechanisms, summarize a recent study of CKS1B as a target for cancer treatment and discuss the potential of CKS1B as a therapeutic target.

In silico drug discovery of IKK-β inhibitors from 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl) pyridine derivatives based on QSAR, docking, molecular dynamics and drug-likeness evaluation studies

The Inhibitor of IKK-β (nuclear factor kappa B kinase subunit beta), a specific modulator of NF-κB (nuclear factor-κB), is considered a valid target to discover new active compounds for various cancers and rheumatoid arthritis treatment. In this study a series of thirty 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl) pyridine derivatives was involved for a quantitative structure activity relationship model (QSAR) elaboration which allows the prediction of the pIC50 values of new designed compounds. The model can be used to predict the activity of new compounds within its applicability domain. Then a molecular docking study was carried out to identify the interactions between the compounds and the amino acids of the active site. After that, golden triangle, Veber’s rule, and Lipinski’s rule properties were calculated to identify the drug-likeness properties of the investigated compounds. Finally, in-silico-toxicity studies were performed to predict the toxicity of the new designed compounds. The analysis of the results of QSAR model and molecular docking succeeded to screen 21 interesting compounds with better inhibitory concentration having a good affinity to IKK-β. All compounds were within the range set by Veber’s rule and Lipinski’s rule. the analysis of golden triangle showed that the thirty 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl) pyridine derivatives would not have clearance and cell membrane permeability problems except comp6 comp12,comp20, comp21, and comp26.As for the new designed compounds, their properties may have these problems, except two compounds which are: A8m, A8p. The A1m, A1p, A3p and A11m compounds were predicted to be nontoxic. These findings indicate that the novel potent candidate drugs have promising potential to IKK-β enzyme inhibition and should motivate future experimental investigations. Communicated by Ramaswamy H. Sarma

Protective effects of HY1702 on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice

Acute respiratory distress syndrome is an inflammatory disease with no effective pharmacological treatment. We investigated the therapeutic effect of HY1702, a new small molecule diterpene obtained from the processing and modification of Glaucocalyxin A and may exhibit anti-inflammatory activity. Specifically, we studied the anti-inflammatory effects of HY1702 on lipopolysaccharide-induced inflammatory responses in RAW264.7 and THP-1 cells in vitro and its protective efficacy on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice.Our results showed that HY1702 significantly decreased lipopolysaccharide-induced inflammatory cytokine expression in RAW264.7 and THP-1 cells and attenuated the secretion of nitric oxide and prostaglandin E2 by down-regulating the expression of inducible nitric oxide synthase and cyclooxygenase 2 in RAW264.7 cells. In mice with lipopolysaccharide-induced mild acute respiratory distress syndrome, HY1702 alleviated histological alterations in the lungs and reduced the alveolar cavity protein leakage and inflammatory cytokine expression in murine bronchial alveolar lavage fluid. HY1702 decreased the myeloperoxidase activity and lung wet to dry weight ratio. In our mechanism studies in lipopolysaccharide-exposed RAW264.7 cells, HY1702 suppressed the inflammation stimulated by lipopolysaccharide through inhibiting phosphorylation of inhibitor of nuclear factor κB kinase subunit α/β (IKKα/β) and inhibitor of nuclear factor κB subunit α (IκBα), further affecting the nuclear transfer of phosphorylated p65. Meanwhile, phosphorylation of p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) was inhibited. These data suggest that HY1702 can reduce inflammation on lipopolysaccharide-stimulated macrophages and attenuate the symptoms of mild acute respiratory distress syndrome in a murine model by regulating the nuclear factor κB and MAP kinase signalling pathways.

IKKγ/NEMO Is Required to Confer Antimicrobial Innate Immune Responses in the Yellow Mealworm, Tenebrio Molitor.

IKKγ/NEMO is the regulatory subunit of the IκB kinase (IKK) complex, which regulates the NF-κB signaling pathway. Within the IKK complex, IKKγ/NEMO is the non-catalytic subunit, whereas IKKα and IKKβ are the structurally related catalytic subunits. In this study, TmIKKγ was screened from the Tenebrio molitor RNA-Seq database and functionally characterized using RNAi screening for its role in regulating T. molitor antimicrobial peptide (AMP) genes after microbial challenges. The TmIKKγ transcript is 1521 bp that putatively encodes a polypeptide of 506 amino acid residues. TmIKKγ contains a NF-κB essential modulator (NEMO) and a leucine zipper domain of coiled coil region 2 (LZCC2). A phylogenetic analysis confirmed its homology to the red flour beetle, Tribolium castaneum IKKγ (TcIKKγ). The expression of TmIKKγ mRNA showed that it might function in diverse tissues of the insect, with a higher expression in the hemocytes and the fat body of the late-instar larvae. TmIKKγ mRNA expression was induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenges in the whole larvae and in tissues such as the hemocytes, gut and fat body. The knockdown of TmIKKγ mRNA significantly reduced the survival of the larvae after microbial challenges. Furthermore, we investigated the tissue-specific induction patterns of fourteen T. molitor AMP genes in TmIKKγ mRNA-silenced individuals after microbial challenges. In general, the mRNA expression of TmTenecin1, -2, and -4; TmDefensin1 and -2; TmColeoptericin1 and 2; and TmAttacin1a, 1b, and 2 were found to be downregulated in the hemocytes, gut, and fat body tissues in the TmIKKγ-silenced individuals after microbial challenges. Under similar conditions, TmRelish (NF-κB transcription factor) mRNA was also found to be downregulated. Thus, TmIKKγ is an important factor in the antimicrobial innate immune response of T. molitor.

Fish IKKα from Japanese eel (Anguilla japonica) can activate NF-κB, AP1, and type I IFN signaling pathways

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays a pivotal role in the activation of nuclear factor kappa-B (NF-κB) pathway in response to pathogens infections in mammals, but the information about IKKα in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an IKKα homologue, AjIKKα, was cloned by 5′ and 3′ SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and A. hydrophila infection were investigated both in vivo and in vitro using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjIKKα GFP fusion protein and the induction of AjIKKα in the activation of NF-κB, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjIKKα has typical conserved domains, including an N-terminal kinase domain, an ubiquitin-like domain, a scaffold dimerization domain, and a C-terminal NEMO-binding domain. The predicted three-dimensional structure of AjIKKα is similar to that of human IKKα. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjIKKα in a wide range of tissues, with the highest expression in the liver, followed by the intestine, gills, and spleen, and with a lower expression in the muscle and heart. The AjIKKα expressions in the liver and kidney were significantly induced following injection with the viral mimic poly I:C and Aeromonas hydrophila infection, whereas the bacterial mimic LPS down-regulated the expression of AjIKKα in the spleen. In vitro, the AjIKKα transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of different concentration of Aeromonas hydrophila (1 × 106 cfu/mL, 1 × 107 cfu/mL, and 1 × 108 cfu/mL). Luciferase assays demonstrated that AjIKKα expression could significantly induce NF-κB, AP-1 and type I IFN promoter activation in a dose-dependent manner. Additionally, subcellular localization studies showed that AjIKKα was evenly distributed in the cytoplasm in the natural state, but AjIKKα was found to aggregate into spots in the cytoplasm after the stimulation of LPS and poly I:C. These results collectively indicated that AjIKKα plays an important role in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB, AP1and type I IFN signaling pathway.

Desulfatiglans anilini Initiates Degradation of Aniline With the Production of Phenylphosphoamidate and 4-Aminobenzoate as Intermediates Through Synthases and Carboxylases From Different Gene Clusters.

The anaerobic degradation of aniline was studied in the sulfate-reducing bacterium Desulfatiglans anilini. Our aim was to identify the genes and their proteins that are required for the initial activation of aniline as well as to characterize intermediates of this reaction. Aniline-induced genes were revealed by comparison of the proteomes of D. anilini grown with different substrates (aniline, 4-aminobenzoate, phenol, and benzoate). Most genes encoding proteins that were highly abundant in aniline- or 4-aminobenzoate-grown D. anilini cells but not in phenol- or benzoate-grown cells were located in the putative gene clusters ani (aniline degradation), hcr (4-hydroxybenzoyl-CoA reductase) and phe (phenol degradation). Of these putative gene clusters, only the phe gene cluster has been studied previously. Based on the differential proteome analysis, four candidate genes coding for kinase subunits and carboxylase subunits were suspected to be responsible for the initial conversion of aniline to 4-aminobenzoate. These genes were cloned and overproduced in E. coli. The recombinant proteins were obtained in inclusion bodies but could be refolded successfully. Two subunits of phenylphosphoamidate synthase and two carboxylase subunits converted aniline to 4-aminobenzoate with phenylphosphoamidate as intermediate under consumption of ATP. Only when both carboxylase subunits, one from gene cluster ani and the other from gene cluster phe, were combined, phenylphosphoamidate was converted to 4-aminobenzoate in vitro, with Mn2+, K+, and FMN as co-factors. Thus, aniline is degraded by the anaerobic bacterium D. anilini only by recruiting genes for the enzymatic machinery from different gene clusters. We conclude, that D. anilini carboxylates aniline to 4-aminobenzoate via phenylphosphoamidate as an energy rich intermediate analogous to the degradation of phenol to 4-hydroxybenzoate via phenylphosphate.

Combined culture experiment of mouse bone marrow mesenchymal stem cells and bioceramic scaffolds.

Articular cartilage injuries are common orthopedic conditions that severely affect the quality of life of patients. Tissue engineering can facilitate cartilage repair and the key points involve scaffolding and seed cell selection. Pre-experiments found a range of microstructures of bioceramic scaffolds suitable for chondrocyte adhesion and proliferation, and maintaining chondrocyte phenotype. Three-dimensional cultures of bone marrow mesenchymal stem cell (BMSC) scaffolds were implanted into mice. According to the shape of the bioceramic scaffolds and the implantation time in vivo, RNA sequencing was performed on the removed scaffolds to explore the molecular mechanism. The in vitro bone plate culture can induce differentiation of chondrocytes, making culture different to that produced in vitro. Implantation of scaffolds in vivo increases the expression of bone-related genes. The ceramic rod-like material was found to be superior to the disc shape, and the bone repair effect was more marked with longer implantation times. Gene Ontology analysis revealed that ‘cell chemotaxis’, ‘negative regulation of ossification’ and ‘bone development’ pathways were involved in recovery. It was further confirmed that BMSCs were suitable as seed cells for cartilage tissue engineering, and that the β-tricalcium phosphate scaffold maybe ideal as cartilage tissue engineering scaffold material. The present research provided new insights into the molecular mechanism of cartilage repair by BMSCs and bioceramic scaffolds. Bioinformatics analysis revealed that AMMECR1L-like protein, tumor necrosis factor-induced protein 2, inhibitor of nuclear factor-B kinase subunit and protein kinase C type and ‘negative regulation of ossification’ and ‘bone development’ pathways may be involved in osteoblast maturation and bone regeneration.

Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

FTY720 induces ferroptosis and autophagy via PP2A/AMPK pathway in multiple myeloma cells

AimsMultiple myeloma (MM) is the second hematological plasma cell malignany and sensitive to fingolimod (FTY720), a novel immunosuppressant. Previous study shows FTY720-induced apoptosis and autophagy can cause cell death in MM cells, however, the high death rate cannot fully be explained. The study aims to investigate further mechanism of how FTY720 kills MM cells. Materials and methodsExperiments are performed on 25 human primary cell samples and two MM cell lines by flow cytometry, fluorescence microscopy, and transmission electron microscopy. Expressions of relative factors are tested by qRT-PCR or western blot. Key findingsFerroptosis-specific inhibitors, deferoxamine mesylate (DFOM) and ferropstatin-1 (Fer-1), reverse FTY720-induced cell death in MM cells. Glutathione peroxidase 4 (GPX4) and soluble carrier family 7 member 11 (SLC7A11), key regulators of ferroptosis, are highly expressed in primary MM cells and can be decreased by FTY720 at the mRNA and protein level in MM cells. In addition, FTY720 induces other characteristic changes of ferroptosis. Furthermore, FTY720 can dephosphorylate AMP-activated protein kinase subunit ɑ (AMPKɑ) at the Thr172 site by activating protein phosphatase 2A (PP2A) and reduce the expression of phosphorylated eukaryotic elongation factor 2 (eEF2), finally cause MM cell death. Using LB-100, a PP2A inhibitor, AICAR, an agonist of AMPK, and bafilomycin A1 (Baf-A1), an autophagy inhibitor, we discover that FTY720 induces ferroptosis and autophagy through the PP2A/AMPK pathway, and ferroptosis and autophagy can reinforce each other. SignificanceThese results provide a new perspective on the treatment of MM.

SNW1 interacts with IKKγ to positively regulate antiviral innate immune responses against influenza A virus infection

The Ski-interacting protein (SNW1) acts as a transcriptional co-regulator associated with mRNA splicing and transcription, cell cycle progression, acute and chronic inflammatory responses, however, its role involved in host antiviral innate immune responses remains to be explored. Here, for the first time, we demonstrated that SNW1 positively regulates the expression of pro-inflammatory cytokines and interferon (IFN) responses induced by influenza A virus (IAV) infection, and further inhibits virus replication by performing SNW1 depletion or overexpression approaches. Furthermore, we showed that reduced interferon beta (IFN-β) expression caused by interfering SNW1 impairs the activation of JAK-STAT pathway in response to IAV or poly I:C. Importantly, by interacting with IKKγ, the regulatory subunit of IκB kinase (IKK) complex, SNW1 promotes IAV-induced activation of NF-κB and phosphorylation of TBK1 kinase, leading to the increase of antiviral effectors interleukin 6 (IL-6), C-X-C motif chemokine 10 (CXCL10), IFN-β and myxovirus resistance protein 1 (MX1). Taken together, our study revealed that SNW1 is an important mediator of host defenses against IAV through the induction of pro-inflammatory factors and IFN signaling, providing novel insights in modulating innate immune responses to protect host from IAV infection.