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Abstract: Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.

Neuronal communication crucially relies on exocytosis of neurotransmitters from synaptic vesicles (SVs) which are clustered at synapses. To ensure reliable neurotransmitter release, synapses need to maintain an adequate pool of SVs at all times. Decades of research have established that SVs are clustered by synapsin 1, an abundant SV-associated phosphoprotein. The classical view postulates that SVs are crosslinked in a scaffold of protein-protein interactions between synapsins and their binding partners. Recent studies have shown that synapsins cluster SVs via liquid-liquid phase separation (LLPS), thus providing a new framework for the organization of the synapse. We discuss the evidence for phase separation of SVs, emphasizing emerging questions related to its regulation, specificity, and reversibility.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.

Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%–4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development ( kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 μM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 μM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 μM), and 100, 250 and 500 μM PRO, but decreased with 1000 μM PRO (p 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 μM: 77.8 ± 10.6%, 100 μM: 63.7 ± 11.7%, 250 μM: 52.1 ± 12.9%, 500 μM: 37.7 ± 11.6%, 1000 μM: 33.1 ± 14.4%; p 0.05), decreased head and entire tail phosphorylated cells (0 μM: 34.6%, 1000 μM: 5.1% p 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 μM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs.

Leucine and insulin-like growth factor-1 (IGF-1) are important regulators of protein synthesis in skeletal muscle. The mechanistic target of rapamycin complex 1 (mTORC1) is of particular importance in their mechanism of action. In the present study, pathways through which leucine and IGF-1 converge to mediate activation of mTORC1 were examined in L6 myoblasts that were deprived of leucine and serum followed by readdition of either leucine or IGF-1. Compared with leucine- and serum-deprived myoblasts, IGF-1, but not leucine, promoted phosphorylation of protein kinase B (AKT), tuberous sclerosis complex 2 (TSC2), and the autophosphorylation site on mTOR (S2481) and also stimulated mTOR kinase activity in mTOR immunoprecipitated samples. Both leucine and IGF-1 promoted phosphorylation of mTOR on S2448. The association of mTOR with the regulatory-associated protein of mTOR (Raptor) was altered by IGF-1 treatment and trended ( P = 0.065) to be altered by leucine treatment. Alterations in the association of mTOR with Raptor were proportional to changes in phosphorylation of the mTOR substrates, eIF4E-binding protein 1 (4E-BP1), and ribosomal protein S6 Kinase-β1 (p70S6K1). Surprisingly, leucine, but not IGF-1, stimulated protein synthesis suggesting a unique role for nutrients in regulating protein synthesis. Overall, the results are consistent with a model whereby IGF-1 stimulates phosphorylation of 4E-BP1 and p70S6K1 in L6 myoblasts through an AKT-TSC2-mTORC1 signaling pathway that also involves changes in the interaction between mTOR and Raptor. In contrast, leucine signaling to mTOR results in alterations in certain mTOR phosphorylation sites and the interaction between mTOR and Raptor and stimulates protein synthesis.

Bone metastasis remains a challenge in cancer treatment. Here we show enzymatic responsive rigid-rod aromatics acting as the substrates of "undruggable" phosphatases to kill cancer cells in a mimetic bone microenvironment. By phosphorylation and conjugating nitrobenzoxadiazole (NBD) to hydroxybiphenylcarboxylate (BP), we obtained pBP-NBD (

BackgroundWe established a novel diethylnitrosamine (DEN) -induced mouse model that reflected the progression of cholangiocarcinoma (CCA) from atypical cystic hyperplasia.MethodsBALB/c mice were administered DEN by oral gavage. Cells isolated from livers were analyzed for expression of CSNK2A1, MAX and MAX-interacting proteins. Human CCA cell lines (MzChA-1, HuCCT1), normal human cholangiocyte (H69), human hepatic stellate cells (LX-2), macrophages (RAW 264.7), and primary hepatic cells were used for cellular and molecular biology assays.ResultsExpression of MAX, CSNK2A1, C-MYC, β-catenin, HMGB1, and IL-6 was upregulated in hepatic cells from CCA liver tissue. The half-life of MAX is higher in CCA cells, and this favors their proliferation. Overexpression of MAX increased growth, migration, and invasion of MzChA-1, whereas silencing of MAX had the opposite effect. MAX positively regulated IL-6 and HMGB1 through paracrine signaling in HepG2, LX2, and RAW cells and autocrine signaling in MzChA-1 cells. CSNK2A1-mediated MAX phosphorylation shifts MAX-MAX homodimer to C-MYC-MAX and β-catenin-MAX heterodimers and increases the HMGB1 and IL-6 promoter activities. Increase of MAX phosphorylation promotes cell proliferation, migration, invasion, and cholangiocarcinogenesis. The casein kinase 2 inhibitor CX-4945 induces cell cycle arrest and inhibits cell proliferation, migration, invasion, and carcinogenesis in MzChA-1 cells through the downregulation of CSNK2A1, MAX, and MAX-interaction proteins.ConclusionC-MYC-MAX and β-catenin-MAX binding to E-box site or β-catenin-MAX bound to TCFs/LEF1 enhanced HMGB1 or IL-6 promoter activities, respectively. IL-6 and HMGB1 secreted by hepatocytes, HSCs, and KCs exert paracrine effects on cholangiocytes to promote cell growth, migration, and invasion and lead to the progression of cholangiocarcinogenesis. CX-4945 provides perspectives on therapeutic strategies to attenuate progression from atypical cystic hyperplasia to cholangiocarcinogenesis.

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.

The aim of the current study was to evaluate the influence of HFD on the functionality of LepR by quantifying phosphorylated levels of 705Tyr-STAT3 in hippocampus astrocytes from mice that consumed an HFD either during the juvenile or the adult period.Five- and eight-week-old male mice, fed during 8 weeks with either control chow or HFD, received a single dose of leptin and their brains were prepared for immunofluorescence to identify double-positive GFAP/p705Tyr-STAT3 cells.HFD intake led to increased pSTAT3 immunoreactivity in GFAP+ cells in the CA1/CA3 hippocampus areas. The effect was observed both in adolescent and adult mice. Leptin increased pSTAT3 immunoreactivity in control animals but was devoid of effect in HFD mice. HFD itself has no effect on the number of GFAP+ cells.Our data show that regular intake of HFD enhances STAT3 signaling in CA1/CA3 astrocytes, an effect that could be linked to the increase of leptin triggered by HFD. The increase of pSTAT3 might be integral to homeostatic mechanisms aimed at maintaining hippocampus function.

TP53 gene abnormalities represent the most important biomarker in chronic lymphocytic leukemia (CLL). Altered protein modifications could also influence p53 function, even in the wild-type protein. We assessed the impact of p53 protein phosphorylations on p53 functions as an alternative inactivation mechanism. We studied p53 phospho-profiles induced by DNA-damaging agents (fludarabine, doxorubicin) in 71 TP53-intact primary CLL samples. Doxorubicin induced two distinct phospho-profiles: profile I (heavily phosphorylated) and profile II (hypophosphorylated). Profile II samples were less capable of activating p53 target genes upon doxorubicin exposure, resembling TP53-mutant samples at the transcriptomic level, whereas standard p53 signaling was triggered in profile I. ATM locus defects were more common in profile II. The samples also differed in the basal activity of the hypoxia pathway: the highest level was detected in TP53-mutant samples, followed by profile II and profile I. Our study suggests that wild-type TP53 CLL cells with less phosphorylated p53 show TP53-mutant-like behavior after DNA damage. p53 hypophosphorylation and the related lower ability to respond to DNA damage are linked to ATM locus defects and the higher basal activity of the hypoxia pathway.

d-Allulose is a desirable sucrose substitute with potential applications in food and health care. d-Allulose can be synthesized using d-glucose as a substrate through coupling glucose isomerase with d-allulose 3-epimerase (DAEase); however, the product yield is typically less than 20% at reaction equilibrium and thus limits its use in industrial applications. Here, a 3R-ketose phosphorylation pathway coupled with an adenosine triphosphate (ATP) regeneration system was developed for the efficient synthesis of d-allulose in

c-Jun N-terminal Kinases (JNKs) have been identified as key disease drivers in a number of pathophysiological settings and central oncogenic signaling nodes in various cancers. Their roles in driving primary tumor growth, positively regulating cancer stem cell populations, promoting invasion and facilitating metastatic outgrowth have led JNKs to be considered attractive targets for anti-cancer therapies. However, the homeostatic, apoptotic and tumor-suppressive activities of JNK proteins limit the use of direct JNK inhibitors in a clinical setting. In this review, we will provide an overview of the different JNK targeting strategies developed to date, which include various ATP-competitive, non-kinase and substrate-competitive inhibitors. We aim to summarize their distinct mechanisms of action, review some of the insights they have provided regarding JNK-targeting in cancer, and outline the limitations as well as challenges of all strategies that target JNKs directly. Furthermore, we will highlight alternate drug targets within JNK signaling complexes, including recently identified scaffold proteins, and discuss how these findings may open up novel therapeutic options for targeting discrete oncogenic JNK signaling complexes in specific cancer settings.

In mammals, nemo-like kinase 2 (NLK2) is a conservative protein kinase involved in Wnt/β-catenin signaling pathway and immune response. However, the role of NLK2 in immune response in teleost remain unclear. In this study, we identified an ortholog of mammalian NLK from grass carp (Ctenopharyngodon idellus) named CiNLK2. CiNLK2 shares a high level of homology with the counterparts, especially with that of Cyprinus carpio. CiNLK2 was ubiquitously expressed in all tested tissues (liver, brain, spleen, gill, kidney and eye) and its expression was up-regulated under the treatment with poly I:C or GCRV. Overexpression of CiNLK2 suppressed the production of IFN I in CIK cells whether or not treated with poly I:C. However, knockdown of CiNLK2 increased the expression level of IFN I. The analysis of subcellular localization showed that CiNLK2 protein was scattered throughout the cytoplasm and nucleus. In terms of mechanism, CiNLK2 can directly interact with MAVS and inhibit MAVS-induced IFN I response. Moreover, CiNLK2 increased the phosphorylation level of MAVS, which led to the degradation of MAVS protein. On the other hand, CiNLK2 suppressed the phosphorylation and nuclear translocation of IRF3. In general, CiNLK2 served as an inhibitor for IFN I response by targeting MAVS-IRF3 signal axis.

Protein-protein interaction networks - interactomes - are charted with the hope to understand how phenotypes emerge and how they are altered in disease states. Early efforts to map interactomes have focused on the assembly of context agnostic, reference networks. However, recent studies have mapped interactomes across different cell lines and tissues, finding highly variable interactomes due to the rewiring of protein-protein interactions in different contexts. Increasing evidence points to significant links between protein structure and interactome diversity seen across cell types and tissues. We discuss how recent findings support the key role of alternative splicing and phosphorylation, two well-established regulators of protein structural and functional diversity, in defining cell type- and tissue-specific interactomes. Moreover, we show that intrinsically disordered protein regions are most favorably equipped to support interactome rewiring by acting as hubs of protein structure and function regulation.

Phosphorylation is the most common post-translational modification (PTM) in eukaryotes, occurring particularly frequently in intrinsically disordered proteins (IDPs). These proteins are highly flexible and dynamic by nature. Thus, it is intriguing that the addition of a single phosphoryl group to a disordered chain can impact its function so dramatically. Furthermore, as many IDPs carry multiple phosphorylation sites, the number of possible states increases, enabling larger complexities and novel mechanisms. Although a chemically simple and well-understood process, the impact of phosphorylation on the conformational ensemble and molecular function of IDPs, not to mention biological output, is highly complex and diverse. Since the discovery of the first phosphorylation site in proteins 75 years ago, we have come to a much better understanding of how this PTM works, but with the diversity of IDPs and their capacity for carrying multiple phosphoryl groups, the complexity grows. In this Essay, we highlight some of the basic effects of IDP phosphorylation, allowing it to serve as starting point when embarking on studies into this topic. We further describe how recent complex cases of multisite phosphorylation of IDPs have been instrumental in widening our view on the effect of protein phosphorylation. Finally, we put forward perspectives on the phosphorylation of IDPs, both in relation to disease and in context of other PTMs; areas where deep insight remains to be uncovered. Phosphorylation is the most common post-translational modification (PTM) in eukaryotes, occurring particularly frequently in intrinsically disordered proteins (IDPs). These proteins are highly flexible and dynamic by nature. Thus, it is intriguing that the addition of a single phosphoryl group to a disordered chain can impact its function so dramatically. Furthermore, as many IDPs carry multiple phosphorylation sites, the number of possible states increases, enabling larger complexities and novel mechanisms. Although a chemically simple and well-understood process, the impact of phosphorylation on the conformational ensemble and molecular function of IDPs, not to mention biological output, is highly complex and diverse. Since the discovery of the first phosphorylation site in proteins 75 years ago, we have come to a much better understanding of how this PTM works, but with the diversity of IDPs and their capacity for carrying multiple phosphoryl groups, the complexity grows. In this Essay, we highlight some of the basic effects of IDP phosphorylation, allowing it to serve as starting point when embarking on studies into this topic. We further describe how recent complex cases of multisite phosphorylation of IDPs have been instrumental in widening our view on the effect of protein phosphorylation. Finally, we put forward perspectives on the phosphorylation of IDPs, both in relation to disease and in context of other PTMs; areas where deep insight remains to be uncovered.

Bruton's tyrosine kinase (BTK) is a critical enzyme which is involved in multiple signaling pathways that regulate cellular survival, activation, and proliferation, making it a major cancer therapeutic target. We applied the novel integrated structure-based pharmacophore modeling, machine learning, and other in silico studies to screen the Korean chemical database (KCB) to identify the potential BTK inhibitors (BTKi). Further evaluation of these inhibitors on three different human cancer cell lines showed significant cell growth inhibitory activity. Among the 13 compounds shortlisted, four demonstrated consistent cell inhibition activity among breast, gastric, and lung cancer cells (IC50 below 3 μM). The selected compounds also showed significant kinase inhibition activity (IC50 below 5 μM). The current study suggests the potential of these inhibitors for targeting BTK malignant tumors.

Although several studies have focused on cancer diagnosis and therapy, prostate cancer (PC) remains an intractable disease. Androgen deprivation therapy (ADT), which is used to treat early stage PC can lead to the development of castration-resistant prostate cancer (CRPC), which is highly associated with androgen receptor (AR) mutations. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a chaperone that shuttles between the nucleus and the cytoplasm. Studies suggest that NOLC1 regulates PC progression; however, the underlying mechanisms remain unclear. Herein, we showed that NOLC1 knockdown suppresses PC cell proliferation by altering the signaling pathways and the expression of various proteins involved in DNA replication, amino acid metabolism, and RNA processing. Mechanistically, NOLC1 knockdown suppressed cell cycle progression by inhibiting AKT phosphorylation and β-catenin accumulation. Finally, we showed that NOLC1 expression is higher in human PC than in human hyperplastic prostate tissues. Altogether, we demonstrated that NOLC1 knockdown suppresses the progression of both AR-positive and AR-negative PC cells by inducing changes in the expression of several genes leading to cell cycle arrest. Thus, NOLC1 might be a novel and promising therapeutic target for PC.

In plants, transitory starch is synthetized during the day and degraded at night to provide the continuous carbon needed for growth and development. Starch metabolism is highly coordinated, as the starch degradation rate must be coupled to the amount of starch synthetized during the day. Maltose is one of the chloroplastic products obtained from starch degradation, and maltose is exported to the cytosol where disproportionating enzyme-2 (DPE2) is responsible for its metabolism. The amount of DPE2 remained unchanged throughout the day, but its activity notably increased at the end of the day (7 p.m.), suggesting that posttranslational modification drives the mechanism underlying the regulatory activity of this enzyme. Sucrose nonfermenting-related kinase-1 (SnRK1), a protein kinase that controls the activity of several metabolic enzymes, was able to interact and phosphorylate DPE2 at three different residues localized in the α-glucanotransferase domain. This phosphorylation acts as a positive regulator of DPE2, increasing its activity. Complementation of dpe2-deficient mutants with the wild-type (WT) and S786A forms of DPE2 showed that the nonphosphorylated form of DPE2 only partially restored starch degradation, suggesting that phosphorylation at S786 is involved in enzyme regulation.

Dysregulation of microRNAs has been implicated in diverse diseases, including Alzheimer's disease (AD). MiR-191-5p in plasma/serum has been identified as a novel and promising noninvasive diagnostic biomarker for AD. However, whether miR-191-5p is involved in AD pathogenesis is largely unknown, and its levels in human AD brains are undetermined. Herein, we demonstrated that miR-191-5p downregulated tau phosphorylation at multiple AD-related sites and promoted neurite outgrowth using immunoblotting, immunofluorescence, and neurite outgrowth assays. Moreover, immunoblotting and enzyme-linked immunosorbent assays indicated that miR-191-5p decreased amyloid precursor protein phosphorylation levels and beta-amyloid (Aβ) generation. Furthermore, miR-191-5p reduced ceramide-induced neuronal cell death analyzed by trypan blue staining, the in situ cell death detection kit, and Annexin V-FITC/PI flow cytometry. Next, we verified that death-associated protein kinase 1 (DAPK1) was a direct target of miR-191-5p through the dual luciferase reporter assay and confirmed that the effects of miR-191-5p were antagonized by restoration of DAPK1 expression. Finally, the hippocampal miR-191-5p level was found to be decreased in humans with AD compared with controls and was inversely correlated with the DAPK1 expression level. Collectively, these findings suggest that miR-191-5p might exert inhibitory effects on tau phosphorylation, Aβ secretion, and neuronal cell death by directly targeting DAPK1, providing an attractive therapeutic option for AD.

The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-β by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-β and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-β signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1−/− mice produced less serum IFN-β and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-β production, thus revealing a novel target for controlling antiviral innate immune responses.

Two-component systems (TCSs) are modular signaling circuits that regulate diverse aspects of microbial physiology in response to environmental cues. These molecular circuits comprise a sensor histidine kinase (HK) protein that contains a conserved histidine residue, and an effector response regulator (RR) protein with a conserved aspartate residue. HKs play a major role in bacterial signaling, since they perceive specific stimuli, transmit the message across the cytoplasmic membrane, and catalyze their own phosphorylation, and the trans-phosphorylation and dephosphorylation of their cognate response regulator. The molecular mechanisms by which HKs co-ordinate these functions have been extensively analyzed by genetic, biochemical, and structural approaches. Here, we describe the most common modular architectures found in bacterial HKs, and address the operation mode of the individual functional domains. Finally, we discuss the use of these signaling proteins as drug targets or as sensing devices in whole-cell biosensors with medical and biotechnological applications.

p38α is a key serine/threonine kinase that can enable atypical auto-activation through Zap70 phosphorylation and initiate T cell receptor signaling. The auto-activation plays an important role in autoimmune diseases. Although the classical activation mechanism of p38α has been studied in-depth, the atypical activation mechanism of Y323 phosphorylation-induced p38α auto-activation remains largely unexplained, especially the regulatory effects of phosphorylation on different sites (Y323 vs T180). From the X-ray experimental data, we identified the inactive and active states of p38α using principal component analysis. To understand the auto-activation process and the internal driving mechanism, a computational paradigm that couples the targeted molecular dynamics simulations, the String Method, and the umbrella sampling strategy were employed to generate the conformational landscape of p38α, including p38α T180-Y323, p38α T180-pY323, and p38α pT180-pY323 systems (pT180/pY323: phosphorylated T180/Y323). We explored that pY323 could change the conformational distribution and promote the conformational transition of p38α from the inactive state to the active state. Auto-activation of p38α is regulated by pY323 through destabilization of the hydrophobic core structure and aided by R173. This study will further explain the conformational transition of p38α induced by Y323 phosphorylation and provide insights into the universal molecular auto-activation mechanism of the p38 subfamily at the atomic level.

A casual decision made one evening in 1976, in a bar near the Biochemistry Department at the University of Dundee, led me to start my personal research journey by following up a paper that suggested that acetyl-CoA carboxylase (ACC) (believed to be a key regulatory enzyme of fatty acid synthesis) was inactivated by phosphorylation by what appeared to be a novel, cyclic AMP-independent protein kinase. This led me to define and name the AMP-activated protein kinase (AMPK) signalling pathway, on which I am still working 46 years later. ACC was the first known downstream target for AMPK, but at least 100 others have now been identified. This article contains some personal reminiscences of that research journey, focussing on: (i) the early days when we were defining the kinase and developing the key tools required to study it; (ii) the late 1990s and early 2000s, an exciting time when we and others were identifying the upstream kinases; (iii) recent times when we have been studying the complex role of AMPK in cancer. The article is published in conjunction with the Sir Philip Randle Lecture of the Biochemical Society, which I gave in September 2022 at the European Workshop on AMPK and AMPK-related kinases in Clydebank, Scotland. During the early years of my research career, Sir Philip acted as a role model, due to his pioneering work on insulin signalling and the regulation of pyruvate dehydrogenase.

Signal transduction pathways are responsible for maintaining cellular functions, including proliferation, differentiation, apoptosis, and cell cycle progression. These pathways are maintained through the propagation of phosphorylation signals by protein kinases, as well as the removal of phosphorylation signals by protein phosphatases. Depending on the context, post-translational modification could have either a positive or negative effect on a signaling pathway. Intricate networks of positive and negative regulators offer a challenging target for the dissection of cell signaling mechanisms, particularly regarding the more subtle dampening of signal transduction through phosphatases. We report the development of two complimentary methods for the optical control of a complex phosphatase: SH2 domain-containing protein tyrosine phosphatase-2 (SHP2). We investigated controlling the catalytic function of SHP2 through (1) site-specific incorporation of a caged tyrosine for light activation of catalytic activity for the control of an essential substrate binding residue and (2) site-specific incorporation of a caged lysine at a conserved residue within an allosteric pocket for the control of SHP2 binding partner docking sites. These methods are generalizable to proteins bearing either a protein tyrosine phosphatase (PTP) catalytic domain or an SH2 domain, including SHP1, PTP family phosphatases, and a diverse range of SH2 domain-containing proteins.

Background: Alzheimer’s disease (AD) was the main cause of dementia in an aging society; unfortunately, there is no effective treatment for AD now. Meditation has been reported to thicken the cerebral cortex, and gamma wave at a frequency of 40 hertz (Hz) was recorded during the meditation process from the brain. Previous study showed that non-invasive scintillation gamma frequency oscillation increased the space in recognition and memory of auditory cortex hippocampal gyrus in AD mice model. However, the AD-related molecular change by exposure of 40 Hz gamma frequency in brain cells was still unclear. Objective: We investigated the AD-related molecular change by exposure of 40 Hz gamma frequency in SH-SY5Y cells. Methods: We designed the light and sound generators at 40 Hz gamma frequency for this study. SH-SY5Y cells were exposed to sound or light of 40 Hz gamma frequency, respectively. The concentrations of amyloid-β40 (Aβ40) and amyloid-β42 (Aβ42) were quantified by enzyme-linked immunosorbent assay. The protein levels were examined by Western blotting. The aggregation of Aβ42 was examined by thioflavin T assay. Results: Our results showed that the secretion of Aβ, phosphorylation of AKT, mTOR, and tau, and aggregation of Aβ42 were significantly inhibited by 40 Hz gamma frequency in SH-SY5Y cells. The phosphorylation of 4E-BP1, downstream of mTOR, was induced by 40 Hz gamma frequency in SH-SY5Y cells. Conclusion: Our study showed 40 Hz gamma frequency involved in the inhibition of secretion and aggregation of Aβ and inhibition of p-Tau protein expression through the mTOR/4E-BP1/Tau signaling pathway.

Sevoflurane (Sev) might cause neurotoxicity in elderly rats. However, the role of Lin28A in Sev-induced neurotoxicity remains unclear in elderly rats. In this study, elderly rats were used to construct an Sev-induced nerve injury model. Learning and memory abilities were assessed by Morris water maze (MWM) trainings; pathological alterations in hippocampal region were assessed by HE staining; neuronal apoptosis was assessed by TUNEL; related protein expression was analyzed by immunofluorescence, immunohistochemistry, and Western blotting. Results of this study showed that Sev treatment caused nerve injury and cognitive dysfunction in elderly rats, with increased neuronal apoptosis and decreased Lin28A levels. Pathological impairment and learning and memory abilities of elderly rats were significantly improved after forced overexpression of Lin28A using AAV, accompanied by decreased expression of CD68, Iba-1, and GFAP. TUNEL analysis showed that Lin28A overexpression significantly reversed Sev-induced neuronal apoptosis. Further mechanistic analysis showed that Lin28A significantly promoted SIRT1 expression, which further reversed Sev-induced Tau acetylation at lysine 280 and 686 and Tau hyperphosphorylation, thereby alleviating nerve injury and cognitive dysfunction in elderly rats. The introduction of SIRT1 inhibitor EX527 further confirmed the involvement of SIRT1 in the regulation of Lin28A in elderly rats. In conclusion, our findings demonstrated that Lin28A reduced sevoflurane-induced nerve injury and cognitive dysfunction by inhibiting Tau acetylation and phosphorylation via activating SIRT1 in elderly rats.

Atherosclerosis (AS) is the pathological basis of many cardiovascular and cerebrovascular diseases, in which macrophage-derived foam cells are the critical step and a typical pathological feature of early atherosclerosis. We previously confirmed that low-molecular-weight fucoidan (LMWF) had a good anti-AS effect, but the mechanism is still unclear. Here with aim to investigate the inhibitory effect of LMWF on foam cells and its molecular mechanism. Oil red O staining showed that LMWF effectively alleviated lipid accumulation and the formation of foam cells. Flow cytometry detection showed that LMWF promoted foam cells apoptosis. In addition, immunofluorescence showed that LMWF inhibited macrophage scavenger receptor A1 (SR-A1)-mediated lipid uptake and promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol outflow. Western blot showed that LMWF downregulated SR-A1 protein expression and upregulated ABCA1 protein expression by inhibiting p38 mitogen activated protein kinase (p38MAPK) phosphorylation. Moreover, the mRNA transcriptions of Stat1, Elk-1, and Myc were downregulated when treated with LMWF. It concluded that, LMWF achieved bidirectional regulation of SR-A1 and ABCA1, then prevented the formation of foam cells, finally ameliorated the development of AS.

The adaptor protein GAREM has two subtypes. Each is involved in Erk activation signaling downstream of the cell growth factor receptor in cultured cells. Regarding their role in individual animals, we have previously reported that mice deficient in GAREM2, which is highly expressed in the brain, exhibit emotional changes. In this paper, we report an amino acid substitution mutation (K291R) in GAREM1, in a patient with idiopathic short stature, which indicates that the mutant exhibits dominant-negative properties. The GAREM K291R mutant did not promote Erk activation in EGF-stimulated cultured cells. Similar features were also observed in cells in which GAREM1 expression was suppressed by genome editing; along with Erk, phosphorylation of S6 kinase and 4EBP1, whose activation is necessary for cell proliferation and biological growth, were inhibited Furthermore, we generated mice deficient in GAREM1 and showed that the mutant mice are lighter in weight. Overall, the results of this paper suggest that GAREM1 is required for normal growth and for maintaing average body size in humans and mice.

To evaluate the phosphorylation of estrogen receptor α at serine-118 (phospho-ERα S118) in the endometrium, ovarian endometrioma, and deep infiltrating endometriosis (DIE).Experimental study.University-affiliated hospital and academic research laboratory.Twenty-five patients underwent a hysterectomy, 18 patients underwent surgical removal of ovarian endometrioma, and 6 patients underwent DIE.Tissue samples were obtained from patients who underwent surgical procedures.Immunostaining for phospho-ERα S118, ERα, or phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK) was performed to evaluate the endometrium with or without endometriosis, ovarian endometrioma, and DIE. For in vitro analysis, endometrial epithelial cells (Ishikawa cells) were stimulated with estradiol (E2) or tumor necrosis factor alpha (TNFα), and the expression levels of phospho-ERα S118 and phospho-p44/42 MAPK were evaluated via Western blotting.First, phospho-ERα S118 level was significantly higher in the glands and stroma of ovarian endometriosis samples than in those of endometrial and DIE samples. Second, colocalization of phospho-p44/42 MAPK and phospho-ERα S118 was observed in the glands of ovarian endometrioma. The proportions of cells strongly expressing phospho-p44/42 and phospho-ERα were 87% in phosphor-p44/42 MAPK-positive cells and 79% in phosphor-ERα-positive cells. Third, E2 stimulation significantly enhanced phospho-ERα S118 after 15 and 30 minutes in in vitro analysis using endometrial epithelial cells. Fourth, TNFα stimulation modestly but significantly enhanced phospho-ERα S118 after 15 and 30 minutes. Fifth, in Ishikawa cells, treatment with a p44/42 inhibitor (PD98059) significantly reduced phospho-ERα S118 by TNFα but not by E2.ERα-S118 phosphorylation was increased in ovarian endometriosis. Our findings may provide a new perspective for understanding the mechanism of increased ERα action in the pathophysiology of endometriosis.

Our previous studies illustrated that 2% Hsub2/subinhalation can protect against sepsis-associated encephalopathy (SAE) which is characterized by high mortality and has no effective treatment. To investigate the underlying role of protein phosphorylation in SAE and Hsub2/subtreatment, a mouse model of sepsis was constructed by caecal ligation and puncture (CLP), then treated with Hsub2/sub(CLP + Hsub2/sub). Brain tissues of the mice were collected to be analysed with tandem mass tag-based quantitative proteomics coupled with IMAC enrichment of phosphopeptides and LC-MS/MS analysis. In proteomics and phosphoproteomics analysis, 268 differentially phosphorylated proteins (DPPs) showed a change in the phosphorylated form in the CLP + Hsub2/subgroup (p lt; 0.05). Gene ontology analysis revealed that these DPPs were enriched in multiple cellular components, biological processes, and molecular functions. KEGG pathway analysis revealed that they were enriched in glutamatergic synapses, tight junctions, the PI3K-Akt signalling pathway, the HIF-1 signalling pathway, the cGMP-PKG signalling pathway, the Rap1 signalling pathway, and the vascular smooth muscle contraction. The phosphorylated forms of six DPPs, including ribosomal protein S6 (Rps6), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (Ywhag/14-3-3), phosphatase and tensin homologue deleted on chromosome ten (Pten), membrane-associated guanylate kinase 1 (Magi1), mTOR, and protein kinase N2 (Pkn2), were upregulated and participated in the PI3K-Akt signalling pathway. The WB results showed that the phosphorylation levels of Rps6, Ywhag, Pten, Magi1, mTOR, and Pkn2 were increased. The DPPs and phosphorylation-mediated molecular network alterations in Hsub2/sub-treated CLP mice may elucidate the biological roles of protein phosphorylation in the therapeutic mechanism of Hsub2/subtreatment against SAE.

Mammalian cells immediately inhibit transcription upon exposure to genotoxic stress to avoid fatal collision between ongoing transcription and newly recruited DNA repair machineries to protect genomic integrity. However, mechanisms of this early transcriptional inhibition are poorly understood. In this study, we decipher a novel role of human EAF1, a positive regulator of ELL-dependent RNA Polymerase II-mediated transcription in vitro, in regulation of temporal inhibition of transcription during genotoxic stress. Our results show that, besides Super Elongation Complex (SEC) and Little Elongation Complex (LEC), human ELL (aka ELL1) also forms a complex with EAF1 alone. Interestingly, contrary to the in vitro studies, EAF1 inhibits ELL-dependent RNA polymerase II-mediated transcription of diverse target genes. Mechanistically, we show that intrinsic self-association property of ELL leads to its reduced interaction with other SEC components. EAF1 enhances ELL self-association and thus reduces its interaction with other SEC components leading to transcriptional inhibition. Physiologically, we show that upon exposure to genotoxic stress, ATM-mediated ELL phosphorylation-dependent enhanced EAF1 association results in reduced ELL interaction with other SEC components that lead to global transcriptional inhibition. Thus, we describe an important mechanism of dynamic transcriptional regulation during genotoxic stress involving post-translational modification of a key elongation factor.

G protein-coupled receptors (GPCRs), the largest family of signaling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK), a master regulator of cell proliferation and survival(1). However, the precise mechanisms underlying GPCR-mediated ERK activation are not clearly understood(2–4). Here we investigated how spatially organized β(2)-adrenergic receptor (β(2)AR) signaling controls ERK. Using subcellularly targeted ERK activity biosensors(5), we show that β(2)AR signaling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gα(s) and requires ligand-stimulated β(2)AR endocytosis. We further identify an endosomally localized non-canonical signaling axis consisting of Gα(s), Raf and mitogen-activated protein kinase kinase (MEK), resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal β(2)AR and Gα(s) signaling blunted nuclear ERK activity, c-Myc gene expression and cell proliferation. These results uncover a non-canonical mechanism for the spatial regulation of ERK via GPCR signaling and identify a functionally important endosomal signaling axis.

Increasing evidence has demonstrated that STAT3 phosphorylation at Tyr

Major depressive disorder (MDD) is a severe disease of unknown pathogenesis with a lifetime prevalence of ~10%. Therapy requires prolonged treatment that often fails. We have previously demonstrated that ceramide levels in the blood plasma of patients and in mice with experimental MDD are increased. Neutralization of blood plasma ceramide prevented experimental MDD in mice. Mechanistically, we demonstrated that blood plasma ceramide accumulated in endothelial cells of the hippocampus, inhibited phospholipase D (PLD) and thereby decreased phosphatidic acid in the hippocampus. Here, we demonstrate that phosphatidic acid binds to and controls the activity of phosphotyrosine phosphatase (PTP1B) in the hippocampus and thus determines tyrosine phosphorylation of a variety of cellular proteins including TrkB. Injection of PLD, phosphatidic acid, or inhibition of PTP1B abrogated MDD and normalized cellular tyrosine phosphorylation, including phosphorylation of TrkB and neurogenesis in the hippocampus. Most importantly, these treatments also rapidly normalized behavior of mice with experimental MDD. Since phosphatidic acid binds to and inhibits PTP1B, the lack of phosphatidic acid results in increased activity of PTP1B and thereby in reduced tyrosine phosphorylation of TrkB and other cellular proteins. Thus, our data indicate a novel pathogenetic mechanism of and a rapidly acting targeted treatment for MDD. (Figure presented.).

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1–10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11–16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17–20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

Liquid–liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for cell function. We investigated the underlying mechanism of LLPS in virus infected cells, where virus inclusion bodies are formed by an RNA-binding phosphoprotein (NS2) of Bluetongue virus to serve as sites for subviral particle assembly and virus maturation. We show that NS2 undergoes LLPS that is dependent on protein phosphorylation and RNA-binding and that LLPS occurrence is accompanied by a change in protein secondary structure. Site-directed mutagenesis identified two critical arginine residues in NS2 responsible for specific RNA binding and thus for NS2–RNA complex driven LLPS. Reverse genetics identified the same residues as essential for VIB assembly in infected cells and virus viability. Our findings suggest that a specific arginine–RNA interaction in the context of a phosphorylated state drives LLPS in this, and possibly other, virus infections.

The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition

Hydroxyapatite scaffolds with different morphologies have been widely used in bone tissue engineering. Moreover, microRNAs (miRNAs) have been proven to be extensively involved in regulating bone regeneration. We developed grooved porous hydroxyapatite (HAG) scaffolds with good osteogenic efficiency. However, little is known about the role of miRNAs in HAG scaffold-mediated promotion of bone regeneration. The objective of this study was to reveal the mechanism from the perspective of differential miRNA expression.Scanning electron microscopy (SEM) was used to perform the coculture of cells and scaffolds. The miRNA profiles were generated by a microarray assay. A synthetic miR-129-5p mimic and inhibitor were used for overexpression or inhibition. The expression of osteogenic marker mRNAs and proteins was detected by quantitative real-time PCR (qRT-PCR), Western blotting, and immunofluorescence. An ALP activity kit and alizarin red staining (ARS) were used to measure ALP activity and mineral deposition formation. Cell migration ability was examined by wound healing and transwell assays. Protein kinase A (PKA) activity was measured by enzyme-linked immunosorbent assay (ELISA) after miR-129-5p transfection. Target genes were identified by a dual-luciferase reporter assay. H89 preculture evaluated the cross talk between miR-129-5p and PKA activity. Heterotopic implantation models, hematoxylin-eosin (HE), immunohistochemistry staining, and micro-CT were used to evaluate miR-129-5p osteogenesis in vivo.miRNAs were differentially expressed during osteogenic differentiation induced by HAG in vitro and in vivo. miR-129-5p was the only highly expressed miRNA both in vitro and in vivo. miR-129-5p overexpression promoted osteoblast differentiation and cell migration, while its inhibition weakened the effect of HAG. Moreover, miR-129-5p activated PKA to regulate the phosphorylation of β-catenin and cAMP-response element binding protein (CREB) by inhibiting cAMP-dependent protein kinase inhibitor alpha (Pkia). H89 prevented the effects of miR-129-5p on osteogenic differentiation and cell migration. HE, immunohistochemistry staining and micro-CT results showed that miR-129-5p promoted in vivo osteogenesis of the HAG scaffold.The HAG scaffold activates Pka by upregulating miR-129-5p and inhibiting Pkia, resulting in CREB-dependent transcriptional activation and accumulation of β-catenin and promoting osteogenic marker expression.

LIM domain kinases 1 and 2 (LIMK1 and LIMK2) regulate actin dynamics and subsequently key cellular functions such as proliferation and migration. LIMK1 and LIMK2 phosphorylate and inactivate cofilin leading to increased actin polymerization. As a result, LIMK inhibitors are emerging as a promising treatment strategy for certain cancers and neurological disorders. High-quality chemical probes are required if the role of these kinases in health and disease is to be understood. To that end, we report the results of a comparative assessment of 17 reported LIMK1/2 inhibitors in a variety of

Cancer progression is associated with metabolic reprogramming and causes significant intracellular stress; however, the mechanisms that link cellular stress and growth signalling are not fully understood. Here, we identified a mechanism that couples the mitochondrial stress response (MSR) with tumour progression. We demonstrated that the MSR is activated in a significant proportion of human thyroid cancers via the upregulation of heat shock protein D family members and the mitokine, growth differentiation factor 15. Our study also revealed that MSR triggered AKT/S6K signalling by activating mTORC2 via activating transcription factor 4/sestrin 2 activation whilst promoting leucine transporter and nutrient-induced mTORC1 activation. Importantly, we found that an increase insupmt/supDNA played an essential role in MSR-induced mTOR activation and that crosstalk between MYC and MSR potentiated mTOR activation. Together, these findings suggest that the MSR could be a predictive marker for aggressive human thyroid cancer as well as a useful therapeutic target.

Background: Histone-like proteins are small molecular weight DNA-binding proteins that are widely distributed in prokaryotes. These proteins have multiple functions in cellular structures and processes, including the morphological stability of the nucleoid, DNA compactness, DNA replication, and DNA repair. Deinococcus radiodurans, an extremophilic microorganism, has extraordinary DNA repair capability and encodes an essential histone-like protein, DrHU. Objective: We aim to investigate the phosphorylation regulation role of a histone-like HU protein from Deinococcus radiodurans. Methods: LC-MS/MS analysis was used to determine the phosphorylation site of endogenous DrHU. The predicted structure of DrHU-DNA was obtained from homology modeling (Swissmodel) using Staphylococcus aureus HU-DNA structure (PDB ID: 4QJU) as the starting model. Two types of mutant proteins T37E and T37A were generated to explore their DNA binding affinity. Complemented-knockout strategy was used to generate the ΔDrHU/pk-T37A and ΔDrHU/pk-T37E strains for growth curves and phenotypical analyses. Results and Discussion: The phosphorylation site Thr37, which is present in most bacterial HU proteins, is located at the putative protein-DNA interaction interface of DrHU. Compared to the wild-type protein, one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E) showed enhanced double-stranded DNA binding but a weakened protective effect against hydroxyl radical cleavage. Complementation of T37E in a DrHU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress. Conclusions: Phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from D. radiodurans, which contributes to the environmental adaptation of this organism.

We previously showed that the adaptive response of BRAFV600-mutated melanoma cells to BRAF inhibition emerges from a subpopulation of cells expressing an intermittent lower level of the mRNA-binding protein HuR. In this study, following initial overexpression experiments in which we confirm our previous results, we use wild-type and mutants HuR full-length mRNA constructs and in vivo-interacting assays and demonstrate that a highly conserved interferon-γ-activated inhibitor of translation (GAIT)-like motif located upstream of the GU-rich elements of HuR major polyadenylation site (PAS2), interacts with constituents of the GAIT complex and affects HuR post-transcriptional expression regulation. Knockdown of the ribosomal protein L13a or the inhibition of the DAPK1-ZIPK axis involved in L13a phosphorylation, reduces the proportion of HuR

A growing body of research is contributing to the development of three-dimensional (3D) tissue models to close the gap between two-dimensional (2D) cell culture and animal models. Here, we report fundamental studies to confirm the modification of vascular endothelial (VE)-cadherin by a tumor microenvironment using 2D and 3D in vitro models of triple-negative breast cancer cells co-cultured with endothelial cells.Breast cancer cells were cultivated as a monolayer (2D) on plates for 5 days or as microtumor spheroids (3D) with endothelial cells for up to 6 days. Phosphotyrosine-containing protein panels were analyzed in both cell types and upon co-culture. Microtumor spheroid size was evaluated via phase contrast microscopy. The content of VE-cadherin and phospho-VE-cadherin was determined. The effect of microtumor spheroid on the capillary network formed by endothelial cells was quantified by ImageJ Angiogenesis Analyzer. Sunitinib was used to determine drug efficacy in this model.The activity of signaling pathways in endothelial cells, including phosphorylation of YIn summary, we established 2D and 3D breast cancer-endothelial cell test systems compatible for detection of minimally tyrosine-phosphorylated proteins including VE-cadherin. The systems are capable of quantifying the effect of drugs on a tissue model of angiogenesis. This is a step towards developing tools for drug-efficacy testing that do not rely on live animals.

Cellulose is an interesting biopolymer offering numerous functionalization possibilities for various applications. Yet, cellulose functionalization usually involves expensive chemicals and complex processes. Here, we aim to utilize inexpensive fertilizer-grade phosphate for cellulose functionalization. Cellulose microfibers (CMF) were isolated from Giant Reed (GR) and were then phosphorylated using either a reagent-grade or a fertilizer-grade diammonium hydrogen phosphate (DAP) in the presence of urea following a water-based protocol. The effect of DAP on the phosphorylation reaction was mainly studied by conductometric titration, ICP-OES and FTIR, while further characterization was performed by SEM/EDX, TGA and XRD to investigate the morphology, composition, charge content, structure, and thermal degradation of the phosphorylated materials. It was found that cellulose phosphorylation using DAP fertilizer gave materials with the same charge content as that registered when using the reagent-grade DAP. Optimizing the reaction conditions with respect to the amount of fertilizer-grade DAP used for the phosphorylation gave high charge content (7000 mmol·g

Triacylglycerols (TGs) serve as reservoirs for diacylglycerols and fatty acids, which play important roles in synthesizing energy and membrane lipids that are required for cell cycle progression. In the yeast, Saccharomyces cerevisiae, Tgl4, the functional ortholog of murine adipose triacylglycerol lipase (ATGL), is activated by Cdk1/Cdc28-mediated phosphorylation and facilitates the G1/S transition. However, little is known about how Tgl4 is inactivated during the cell cycle. To monitor the phosphorylation status and the stability of endogenous Tgl4, we raised a specific antibody against Tgl4. We found that in contrast to the previous suggestion, Tgl4 was a stable protein throughout the cell cycle. We also showed that Tgl4 was dephosphorylated upon entry into G1 phase. These results suggest that Tgl4 is a stable protein and is inactivated during G1 phase by dephosphorylation.

Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 μg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.

For proper intracellular vesicle transport, it is essential for transport vesicle membranes to fuse with the appropriate target membranes. Ykt6 is a SNARE protein with functions in diverse vesicle transport pathways, including secretory, endocytotic and autophagic pathways. To exert these functions, the association of Ykt6 with vesicle membranes and the change of its conformation from closed to open play key roles. Recent studies have revealed regulatory mechanisms involved in Ykt6 membrane association and conformation change. When in the cytosol, the vicinal cysteine residues within the C-terminal CCAIM sequence of Ykt6 undergo diprenylation (farnesylation of the distal cysteine residues by farnesyltransferase; this is followed by geranylgeranylation of the proximal cysteine residue by geranylgeranyltransferase-III). Phosphorylation of a serine residue within the SNARE domain triggers the conversion of the Ykt6 conformation from closed to open, allowing Ykt6 membrane association. In this commentary, I briefly summarize and discuss the recently revealed regulatory mechanisms of Ykt6 function by diprenylation and phosphorylation.

Background: Urokinase-type plasminogen activator (uPA) is a serine proteinase found in excitatory synapses located in the II/III and V cortical layers. The synaptic release of uPA promotes the formation of synaptic contacts and the repair of synapses damaged by various forms of injury, and its abundance is decreased in the synapse of Alzheimer’s disease (AD) patients. Inactivation of the Wingless/Int1 (Wnt)-β-catenin pathway plays a central role in the pathogenesis of AD. Soluble amyloid-β (Aβ) prevents the phosphorylation of the low-density lipoprotein receptor-related protein-6 (LRP6), and the resultant inactivation of the Wnt-β-catenin pathway prompts the amyloidogenic processing of the amyloid-β protein precursor (AβPP) and causes synaptic loss. Objective: To study the role of neuronal uPA in the pathogenesis of AD. Methods: We used in vitro cultures of murine cerebral cortical neurons, a murine neuroblastoma cell line transfected with the APP-695 Swedish mutation (N2asw), and mice deficient on either plasminogen, or uPA, or its receptor (uPAR). Results: We show that uPA activates the Wnt-β-catenin pathway in cerebral cortical neurons by triggering the phosphorylation of LRP6 via a plasmin-independent mechanism that does not require binding of Wnt ligands (Wnts). Our data indicate that uPA-induced activation of the Wnt-β-catenin pathway protects the synapse from the harmful effects of soluble Aβ and prevents the amyloidogenic processing of AβPP by inhibiting the expression of β-secretase 1 (BACE1) and the ensuing generation of Aβ40 and Aβ42 peptides. Conclusion: uPA protects the synapse and antagonizes the inhibitory effect of soluble Aβ on the Wnt-β-catenin pathway by providing an alternative pathway for LRP6 phosphorylation and β-catenin stabilization.