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RNA silencing mediated by 19-to-24 nucleotide-long small RNAs is a central mechanism regulating gene expression. In plants, the two major types of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are derived from long hairpin RNA precursors while siRNAs are derived from long double-stranded RNAs. In plants, the majority of miRNAs and certain siRNAs associate with the ARGONAUTE1 (AGO1) protein to exert their regulatory effects by forming an RNA-induced silencing complex (RISC). Even though many studies have been done to characterize Arabidopsis AGO1 and AGO1 activities, little is known about the mechanisms that regulate AGO1 RISC formation. In my dissertation research, two mechanisms that regulate AGO1 RISC formation were identified and reported in chapters two and three. In the second chapter, we found that the long structurally unresolved N-terminal extension (NTE) is essential for the functions of AGO1, as an AGO1 mutant lacking this region is unable to rescue the developmental and molecular phenotypes of an ago1 null allele. RNA sequencing of total small RNAs and AGO1-associated small RNAs showed that amino acids (a.a.) 91-189 of the NTE are required for RISC formation, thus crucial for the activities of AGO1 in gene silencing. Furthermore, a.a. 1-90 and a.a. 91-189 regions of the NTE redundantly promote the activities of AGO1 in the biogenesis of trans-acting siRNAs (ta-siRNAs). This work provides a new understanding of the AGO1 NTE region in RISC assembly and ta-siRNA biogenesis. In the third chapter, through immunoprecipitation-mass spectrometry, we found that AGO1 is phosphorylated in vivo. Analysis of AGO1-associated small RNAs in vivo and RISC formation assays in vitro showed that phospho-mimetic AGO1 is compromised in the loading of both miRNA and siRNA duplexes and in passenger strand ejection of siRNA but not miRNA duplexes. Non-phosphorylatable AGO1 is compromised in target RNA repression. This work revealed a regulatory role of phosphorylation and dephosphorylation in the small RNA association and target repression activities of AGO1. Taken together, my dissertation research provided new insights into mechanisms regulating the activities of plant AGO1 and these insights have implications beyond plant AGO1.

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.

It has been nearly three decades since the discovery of the first bacterial serine/threonine protein kinase (STPK). Since then, a blend of technological advances has led to the characterization of a multitude of STPKs and phosphorylation substrates in several bacterial species that finely regulate intricate signaling cascades. Years of intense research from several laboratories have demonstrated unexpected roles for serine/threonine phosphorylation, regulating not only bacterial growth and cell division but also antibiotic persistence, virulence and infection, metabolism, chromosomal biology, and cellular differentiation. This review aims to provide an account of the most recent and significant developments in this up and growing field in microbiology.

Nuclear factor κB (NF-κB) is a ubiquitous regulator of the signalome and is indispensable for various biological cell functions. NF-κB consists of five transcription factors that execute both cytoplasmic and nuclear signaling processes in cells. NF-κB is the only signaling molecule that governs both pro- and anti-apoptotic, and pro- and anti-inflammatory responses. This is due to the canonical and non-canonical components of the NF-κB signaling pathway. Together, these pathways orchestrate cancer-related inflammation, hyperplasia, neoplasia, and metastasis. Non-canonical NF-κB pathways are particularly involved in the chemoresistance of cancer cells. In view of its pivotal role in cancer progression, NF-κB represents a potentially significant therapeutic target for modifying tumor cell behavior. Several phytochemicals are known to modulate NF-κB pathways through the stabilization of its inhibitor, IκB by inhibiting the phosphorylation and ubiquitination thereof. Several natural pharmacophores are known to inhibit the nuclear translocation of NF-κB and associated pro-inflammatory responses and cell survival pathways. In view of this, and the high degree of specificity exhibited by various phytochemicals for the NF-κB component, we herein present an in-depth overview of these phytochemicals and discuss their mode of interaction with the NF-κB signaling pathways for controlling fate of tumor cells for cancer-directed interventions.

Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities. However, genetic control of C. acetobutylicum biofilm has not been dissected so far. Here, to identify potential genes controlling C. acetobutylicum biofilm formation, over 40 gene candidates associated with extracellular matrix, cell surface, cell signaling or gene transcription, were tried to be disrupted to examine their individual impact. A total of 25 disruptants were finally obtained over years of attempts, for which biofilm and relevant phenotypes were characterized. Most of these disruptants formed robust biofilm still, or suffered both growth and biofilm defect. Only a strain with a disrupted histidine kinase gene (CA_C2730, designated bfcK in this study) abolished biofilm formation without impairing cell growth or solvent production. Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels. The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase (encoded by CA_C0404) that might negatively regulate biofilm formation. Based on these findings, possible bfcK-mediated mechanisms for biofilm formation were proposed. This is a big step toward understanding the biofilm formation in C. acetobutylicum and will help further engineering of its biofilm-based industrial processes.

The number of acetylated proteins identified from bacteria to mammals has grown exponentially in the last ten years, and it is now accepted that acetylation is a key component in most eukaryotic signaling pathways and is as important as phosphorylation. The enzymes involved in this process are well described in mammals; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids, several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this review article, we present an update of the most important known acetylated proteins in trypanosomatids, analyzing the acetylomes available. Also, we summarize the information available regarding acetyltransferases and deacetylases in trypanosomes and their potential use as chemotherapeutic targets.

During development, neural progenitors are in proliferative and immature states; however, the molecular machinery that cooperatively controls both states remains elusive. Here, we report that cyclin D1 (CCND1) directly regulates both proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates S309 of ATOH1, which inhibits additional phosphorylation at S328 and consequently prevents S328 phosphorylation‐dependent ATOH1 degradation. Additionally, PROX1 downregulates Ccnd1 expression by histone deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. Moreover, WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT‐PROX1‐CCND1‐ATOH1 signaling cascade cooperatively controls proliferative and immature states of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which are suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.

Protein kinases are enzymes that transfer the γ-phosphate of ATP to acceptor residues, most often serine, threonine or tyrosine, in proteins. Phosphorylation by protein kinases are integral to the signaling processes that control virtually all aspects of cellular physiology and phosphorylation is the most abundant post-translational modification in signal transduction networks. Covalent attachment of a phosphate group to a protein can cause a conformational change, introduce a new epitope that enhances or prevents interaction with another protein, increase or decrease the rate of turnover, and/or affect subcellular localization. In these ways, phosphorylation-dependent events can influence cell metabolism, morphology, and differentiation. Target of rapamycin (TOR) complex 2 (TORC2) is an evolutionarily conserved multi-subunit protein kinase and indispensable regulator of plasma membrane homeostasis. Genetic studies in yeast have demonstrated that the sole essential downstream target and effector of TORC2 is the protein kinase Ypk1 (and its paralog Ypk2), whose mammalian ortholog is SGK1. In humans, mutations in the TORC2 signaling network have been implicated in inflammatory disorders, metabolic diseases, and multiple cancers. In this doctoral dissertation, I describe the studies I undertook to explore novel aspects of TORC2-Ypk1 signaling. Because a Rab5-specific guanine nucleotide exchange factor (GEF), Muk1, was identified in a prior global screen for candidate Ypk1 targets, I investigated the role of Rab5-type GTPases in the TORC2 signaling network. I confirmed first that Muk1 is a substrate of Ypk1 and demonstrated that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Second, and strikingly, I found that yeast lacking its two Rab5 GEFs (Muk1 and Vps9), or its three Rab5 paralogs (Vps21/Ypt51, Ypt52 and Ypt53), or overexpressing Msb3 (a Rab5-directed GTPase-activating protein), all exhibited pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Finally, I showed that Vps21 co-immunoprecipitates with TORC2, and furthermore, that purified GTP-bound Vps21 is able to stimulate in vitro the activity of TORC2 immuno-enriched from Rab5-deficient cells. My findings suggest that TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling

Background: Cycle-regulating and transcriptional cyclin-dependent kinases (CDKs) are attractive targets in cancer drug development. Several CDK inhibitors have already been obtained or are close to regulatory approval for clinical applications. Objective: Phenylazopyrazole CAN508 has been described as the first selective CDK9 inhibitor with an IC50 of 350 nM. Since the azo-moiety is not a suitable functionality for drugs due to pharmacological reasons, the preparation of carbo-analogues of CAN508 with similar biological activities is desirable. The present work is focused on the synthesis of carbo-analogues similar to CAN508 and their CDK inhibition activity. Methods: Herein, the synthesis of 21 novel carbo analogues of CAN508 and their intermediates is reported. Subsequently, target compounds 8a - 8u were evaluated for protein kinase inhibition (CDK2/cyclin E, CDK4/cyclin D, CDK9/cyclin T) and antiproliferative activities in cell lines (K562, MCF-7, MV4-11). Moreover, the binding mode of derivative 8s in the active site of CDK9 was modelled. Results: Compounds 8a - 8u were obtained from key intermediate 7, which was prepared by linear synthesis involving Vilsmeier-Haack, Knoevenagel, Hunsdiecker, and Suzuki-Miyaura reactions. Styrylpyrazoles 8t and 8u were the most potent CDK9 inhibitors with IC50 values of approximately 1 μM. Molecular modelling suggested binding in the active site of CDK9. The flow cytometric analysis of MV4-11 cells treated with the most active styrylpyrazoles showed a significant G1-arrest. Conclusion: The prepared styrylpyrazoles showed inhibition activity towards CDKs and can provide a novel chemotype of kinase inhibitors.

Memory storage is a conserved survivability feature, present in virtually any complex species . During the last few decades, much effort has been devoted to understanding how memories are formed and which molecular switches define whether a memory should be stored for a short or a long period of time. Among these, de novo protein synthesis is known to be required for the conversion of short- to long-term memory. There are a number translational control pathways involved in synaptic plasticity and memory consolidation, including the phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α), which has emerged as a critical molecular switch for long-term memory consolidation. In this review, we discuss findings pertaining to the requirement of de novo protein synthesis to memory formation, how local dendritic and axonal translation is regulated in neurons, and how these can influence memory consolidation. We also highlight the importance of eIF2α-dependent translation initiation to synaptic plasticity and memory formation. Finally, we contextualize how aberrant phosphorylation of eIF2α contributes to Alzheimer’s disease (AD) pathology and how preventing disruption of eIF2-dependent translation may be a therapeutic avenue for preventing and/or restoring memory loss in AD.

Manganese (Mn) is an essential micronutrient for all living organisms. However, excess supply of Mn as occurring in acid or waterlogged soils has toxic effects on plant physiology and development. Although a variety of Mn transporter families has been characterized, our understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants remains rudimentary. Here, we identify two calcineurin-B-like proteins, CBL2/3, and their interacting kinases, CIPK3/9/26, as key regulators for plant Mn homeostasis. We found that Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkable high-Mn tolerance. Intriguingly, CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron (Fe) transporter MTP8 primarily at Ser35, which is conserved among MTP8 proteins from various species. Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole. Moreover, we report that sequential phosphorylation of MTP8 initially at Ser31/32 by the calcium-dependent protein kinases CPK5 and subsequently by CIPK26 at Ser35 provides an activation/deactivation fine-tuning mechanism for differential Mn transport regulation. Collectively, our studies define a two-tiered calcium-controlled mechanism for dynamically orchestrating Mn homeostasis under conditions of fluctuating Mn supply.

The relationship between the checkpoint kinase Chk1 and the STAT3 pathway was examined in multiple myeloma cells. Gene expression profiling of U266 cells exposed to low (nmol/L) Chk1 inhibitor [PF-477736 (PF)] concentrations revealed STAT3 pathway-related gene downregulation (e.g., BCL-XL, MCL-1, c-Myc), findings confirmed by RT-PCR. This was associated with marked inhibition of STAT3 Tyr705 (but not Ser727) phosphorylation, dimerization, nuclear localization, DNA binding, STAT3 promoter activity by chromatin immunoprecipitation assay, and downregulation of STAT-3-dependent proteins. Similar findings were obtained in other multiple myeloma cells and with alternative Chk1 inhibitors (e.g., prexasertib, CEP3891). While PF did not reduce GP130 expression or modify SOCS or PRL-3 phosphorylation, the phosphatase inhibitor pervanadate antagonized PF-mediated Tyr705 dephosphorylation. Significantly, PF attenuated Chk1-mediated STAT3 phosphorylation in in vitro assays. Surface plasmon resonance analysis suggested Chk1/STAT3 interactions and PF reduced Chk1/STAT3 co-immunoprecipitation. Chk1 CRISPR knockout or short hairpin RNA knockdown cells also displayed STAT3 inactivation and STAT3-dependent protein downregulation. Constitutively active STAT3 diminished PF-mediated STAT3 inactivation and downregulate STAT3-dependent proteins while significantly reducing PF-induced DNA damage (γH2A.X formation) and apoptosis. Exposure of cells with low basal phospho-STAT3 expression to IL6 or human stromal cell conditioned medium activated STAT3, an event attenuated by Chk1 inhibitors. PF also inactivated STAT3 in primary human CD138+ multiple myeloma cells and tumors extracted from an NSG multiple myeloma xenograft model while inhibiting tumor growth. Implications: These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.

Airway smooth muscle thickening, a key characteristic of chronic asthma, is largely attributed to increased smooth muscle cell proliferation and reduced smooth muscle apoptosis. Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that participates in the pathogenesis of airway smooth muscle remodeling. Although the role of Plk1 in cell proliferation and migration is recognized, its function in smooth muscle apoptosis has not been previously investigated. Caspase-9 (Casp9) is a key enzyme that participates in the execution of apoptosis. Casp9 phosphorylation at Ser-196 and Thr-125 is implicated in regulating its activity in cancer cells and epithelial cells. Here, exposure of human airway smooth muscle (HASM) cells to platelet-derived growth factorfor 24 hours enhanced the expression of Plk1 and Casp9 phosphorylation at Ser-196, but not Thr-125. Overexpression of Plk1 in HASM cells increased Casp9 phosphorylation at Ser-196. Moreover, the expression of Plk1 increased the levels of pro-Casp9 and pro-Casp3 and inhibited apoptosis, demonstrating a role of Plk1 in inhibiting apoptosis. Knockdown of Plk1 reduced Casp9 phosphorylation at Ser-196, reduced pro-Casp9/3 expression, and increased apoptosis. Furthermore, Casp9 phosphorylation at Ser-196 was upregulated in asthmatic HASM cells, which was associated with increased Plk1 expression. Knockdown of Plk1 in asthmatic HASM cells decreased Casp9 phosphorylation at Ser-196 and enhanced apoptosis. Together, these studies disclose a previously unknown mechanism that the Plk1-Casp9/3 pathway participates in the controlling of smooth muscle apoptosis.

There are over 7 million people worldwide suffering from Parkinson's disease, and this number will double in the next decade. Causative mutations and risk variants in >20 genes that predominantly act at synapses have been linked to Parkinson's disease. Synaptic defects precede neuronal death. However, we are only now beginning to understand which molecular mechanisms contribute to this synaptic dysfunction. In this review, we discuss recent data demonstrating that Parkinson proteins act centrally to various protein quality control pathways at the synapse, and we argue that disturbed synaptic proteostasis is an early driver of neurodegeneration in Parkinson's disease. ispartof: CURRENT OPINION IN NEUROBIOLOGY vol:72 pages:72-79 ispartof: location:England status: published

Graphical abstract Schematic illustration of the miR-4521-FAM129A axis in HCC malignancy. FAM129A negatively correlates with miR-4521, leading to suppressed migration and invasion by the TIMP-1/MMP9/MMP2 and EMT pathways. miR-4521-FAM129A axial regulation reduces proliferation and induced apoptosis via the p-FAK/p-AKT/MDM2/P53 and p-FAK/p-AKT /BCL-2/BAX/Cytochrome-C/Caspase-3/Caspase-9 pathways, respectively., Highlights • MiR-4521 deficiency reversely correlates with FAM129A upregulation in HCC. • MiR-4521 suppresses the in vitro migration and invasion capacities of HCC cells. • FAM129A is a direct and efficient target of miR-4521 in HCC cell invasiveness. • MiR-4521-FAM129A axis regulates HCC progression and metastasis via TIMP-1/MMP2/MMP9 and p-FAK/p-AKT pathway., Introduction Globally, hepatocellular carcinoma (HCC) is the sixth most common malignancy and it has the fourth highest mortality. MicroRNAs play a significant part in biological processes in cell formation and advancement by targeting genes in many cancers including HCC. Objective In the present study we examine the involvement of miR-4521 and FAM129A correlations in HCC occurrence and progression. Methods Expression levels of miR-4521 and FAM129A in HCC tissues and cells were detected. Immunohistochemistry was carried out to detect expression of FAM129A, MMP9 and TIMP-1 in HCC tissues. Western blot assays were used to examine expression levels of different genes involve in signaling pathways. Transwell chamber, MTT and wound healing assays were performed to check cell migration, invasion and proliferation rates. Results Overexpression of FAM129A positively correlated with upregulation of MMP9 and negatively correlated with TIMP-1 in HCC patient samples, which encouraged progression and metastasis of HCC. An antagonistic relation between miR-4521 and FAM129A was detected in current study, down-regulation of miR-4521 and up-regulation of FAM129A was demonstrated in HCC tissues and cell lines as compare to normal tissue samples and the normal cell line LO2. Overexpressing miR-4521 and silencing FAM129A impaired HCC cell migratory and invasive properties and suppressed cell proliferation. Mutually, miR-4521-FAM129A axial regulation inhibited in vitro proliferation of cells by promoting apoptosis through the p-FAK/p-AKT/MDM2/P53 and p-FAK/p-AKT/BCL-2/BAX/Cytochrome-C/Caspase-3/Caspase-9 pathways, respectively, and suppressed the migration and invasion capabilities of HCCLM3 and HepG2 cells via the TIMP-1/MMP9/MMP2 and p-FAK/p-AKT pathway. Conclusion Our work found the axial regulation mechanism of miR-4521-FAM129A in HCC. Deficiency of miR-4521 and abundance of FAM129A synergistically enhanced cancer progression by increasing cell proliferation and malignant invasion and by inhibiting apoptosis. These discoveries suggest that miR-4521/FAM129A might play a vital role in hepatic cancer progression and could be a candidate for its therapy.

Some previous studies in tissue fibrosis have suggested a profibrotic contribution from elevated expression of a protein termed either Regulator of Cell Cycle (RGCC) or Response Gene to Complement 32 Protein (RGC-32). Our analysis of public gene expression datasets, by contrast, revealed a consistent decrease in RGCC mRNA levels in association with pulmonary fibrosis. Consistent with this observation, we found that stimulating primary adult human lung fibroblasts with TGF-β in cell culture elevated collagen expression and simultaneously attenuated RGCC mRNA and protein levels. Moreover, overexpression of RGCC in cultured lung fibroblasts attenuated the stimulating effect of TGF-β on collagen levels. Similar to humans with pulmonary fibrosis, the levels of RGCC were also decreased in vivo in lung tissues of wild-type mice challenged with bleomycin in both acute and chronic models. Mice with constitutive RGCC gene deletion accumulated more collagen in their lungs in response to chronic bleomycin challenge than did wild-type mice. RNA-Seq analyses of lung fibroblasts revealed that RGCC overexpression alone had a modest transcriptomic effect, but in combination with TGF-β stimulation, induced notable transcriptomic changes that negated the effects of TGF-β, including on extracellular matrix-related genes. At the level of intracellular signaling, RGCC overexpression delayed early TGF-β-induced Smad2/3 phosphorylation, elevated the expression of total and phosphorylated antifibrotic mediator STAT1, and attenuated the expression of a profibrotic mediator STAT3. We conclude that RGCC plays a protective role in pulmonary fibrosis and that its decline permits collagen accumulation. Restoration of RGCC expression may have therapeutic potential in pulmonary fibrosis.

Background: The sarcoplasmic reticulum (SR) Ca 2+ -ATPase 2 (SERCA2) mediates Ca 2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca 2+ release from SR and triggers contraction. Ca 2+ /CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca 2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. Methods: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. Results: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca 2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca 2+ reuptake by SERCA2 and Ca 2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca 2+ -frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. Conclusions: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Alzheimer’s disease (AD) is characterized by progressive cognitive decline, and the number of affected individuals has increased worldwide. However, there are no effective treatments for AD. Therefore, it is important to prevent the onset of dementia. Oxidative stress and endoplasmic reticulum (ER) stress are increased in the brains of AD patients, and are postulated to induce neuronal cell death and cognitive dysfunction. In this study, Centella asiatica, a traditional Indian medicinal herb, were fractionated and compared for their protective effects against glutamate and tunicamycin damage. Araliadiol was identified as a component from the fraction with the highest activity. Further, murine hippocampal cells (HT22) were damaged by glutamate, an oxidative stress inducer. Centella asiatica and araliadiol suppressed cell death and reactive oxygen species production. HT22 cells were also injured by tunicamycin, an ER stress inducer. Centella asiatica and araliadiol prevented cell death by mainly inhibiting PERK phosphorylation; additionally, Centella asiatica also suppressed the expression levels of GRP94 and BiP. In Y-maze test, oral administration of araliadiol (10 mg/kg/day) for 7 days ameliorated the arm alternation ratio in mice with scopolamine-induced cognitive impairment. These results suggest that Centella asiatica and its active component, araliadiol, have neuroprotective effects, which may prevent cognitive dysfunction.

Protein phosphorylation is a crucial regulator of protein and cellular function, yet, despite identifying an enormous number of phosphorylation sites, the role of most is still unclear. Each phosphoform, the particular combination of phosphorylations, of a protein has distinct and diverse biological consequences. Aberrant phosphorylation is implicated in the development of many diseases. To investigate their function, access to defined protein phosphoforms is essential. Materials obtained from cells often are complex mixtures. Recombinant methods can provide access to defined phosphoforms if site-specifically acting kinases are known, but the methods fail to provide homogenous material when several amino acid side chains compete for phosphorylation. Chemical and chemoenzymatic synthesis has provided an invaluable toolbox to enable access to previously unreachable phosphoforms of proteins. In this review, we selected important tools that enable access to homogeneously phosphorylated protein and discuss examples that demonstrate how they can be applied. Firstly, we discuss the synthesis of phosphopeptides and proteins through chemical and enzymatic means and their advantages and limitations. Secondly, we showcase illustrative examples that applied these tools to answer biological questions pertaining to proteins involved in signal transduction, control of transcription, neurodegenerative diseases and aggregation, apoptosis and autophagy, and transmembrane proteins. We discuss the opportunities and challenges in the field.

Hepatic ischemia/reperfusion injury (HIRI) is a serious complication that occurs following shock and/or liver surgery. Gut microbiota and their metabolites are key upstream modulators of development of liver injury. Herein, we investigated the potential contribution of gut microbes to HIRI. Ischemia/reperfusion surgery was performed to establish a murine model of HIRI. 16S rRNA gene sequencing and metabolomics were used for microbial analysis. Transcriptomics and proteomics analysis were employed to study the host cell responses. Our results establish HIRI was significantly increased when surgery occurred in the evening (ZT12, 20:00) when compared with the morning (ZT0, 08:00); however, antibiotic pretreatment reduced this diurnal variation. The abundance of a microbial metabolite 3,4-dihydroxyphenylpropionic acid was significantly higher in ZT0 when compared with ZT12 in the gut and this compound significantly protected mice against HIRI. Furthermore, 3,4-dihydroxyphenylpropionic acid suppressed the macrophage pro-inflammatory response in vivo and in vitro. This metabolite inhibits histone deacetylase activity by reducing its phosphorylation. Histone deacetylase inhibition suppressed macrophage pro-inflammatory activation and diminished the diurnal variation of HIRI. Our findings revealed a novel protective microbial metabolite against HIRI in mice. The potential underlying mechanism was at least in part, via 3,4-dihydroxyphenylpropionic acid-dependent immune regulation and histone deacetylase (HDAC) inhibition in macrophages.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The uspS35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The uspS35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

Background & Aims Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. Methods We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. Results The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2–hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. Conclusions The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans., Graphical abstract

Atherosclerosis is a chronic inflammatory vascular disease, and inflammation plays a critical role in its formation and progression. Elevated serum homocysteine (Hcy) is an independent risk factor for atherosclerosis. Previous studies have shown that fatty acid binding protein 4 (FABP4) plays an important role in macrophage inflammation and lipid metabolism in atherosclerosis induced by Hcy. However, the underlying molecular mechanism of FABP4 in Hcy-induced macrophage inflammation remains unknown. In this study, we found that FABP4 activated the Janus kinase 2/signal transducer and activator of transcription 2 (JAK2/STAT2) pathway in macrophage inflammation induced by Hcy. Of note, we further observed that ras-related protein Rap-1a (Rap1a) induced the Tyr416 phosphorylation and membrane translocation of non-receptor tyrosine kinase (c-Src) to activate the JAK2/STAT2 pathway. In addition, the suppressor of cytokine signaling 1 (SOCS1)—a transcriptional target of signal transducer and activator of transcription (STATs) inhibited the JAK2/STAT2 pathway and Rap1a expression via a negative feedback loop. In summary, these results demonstrated that FABP4 promotes c-Src phosphorylation and membrane translocation via Rap1a to activate the JAK2/STAT2 pathway, contributing to Hcy-accelerated macrophage inflammation in ApoE−/− mice. A proposed regulatory model of FABP4 in Hcy-induced macrophage inflammation in atherosclerosis of ApoE−/− mice. FABP4 activates the JAK2/STAT2 pathway via Rap1a in Hcy-induced macrophage inflammatory response and atherosclerosis in ApoE−/− mice, which is attributed to Rap1a-dependent promotion of the c-Src phosphorylation at Tyr416 and membrane translocation. SOCS1 has a negative regulatory role in FABP4-dependent activation of the JAK2/STAT2 pathway and Rap1a in macrophage inflammatory response induced by Hcy.

It is well known that lectin-like oxidized low-density lipoprotein (ox-LDL) and its receptor LOX-1, angiotensin II (AngII) and its type 1 receptor (AT1-R) play an important role in the development of cardiac hypertrophy. However, the molecular mechanism is not clear. In this study, we found that ox-LDL-induced cardiac hypertrophy was suppressed by inhibition of LOX-1 or AT1-R but not by AngII inhibition. These results suggest that the receptors LOX-1 and AT1-R, rather than AngII, play a key role in the role of ox-LDL. The same results were obtained in mice lacking endogenous AngII and their isolated cardiomyocytes. Ox-LDL but not AngII could induce the binding of LOX-1 and AT1-R; inhibition of LOX-1 or AT1-R but not AngII could abolish the binding of these two receptors. Overexpression of wild type LOX-1 with AT1-R enhanced ox-LDL-induced binding of two receptors and phosphorylation of ERKs, however, transfection of LOX-1 dominant negative mutant (lys266ala / lys267ala) or an AT1-R mutant (glu257ala) not only reduced the binding of two receptors but also inhibited the ERKs phosphorylation. Phosphorylation of ERKs induced by ox-LDL in LOX-1 and AT1-R-overexpression cells was abrogated by an inhibitor of Gq protein rather than Jak2, Rac1 or RhoA. Genetically, an AT1-R mutant lacking Gq protein coupling ability inhibited ox-LDL induced ERKs phosphorylation. Furthermore, through bimolecular fluorescence complementation analysis, we confirmed that ox-LDL rather than AngII stimulation induced the direct binding of LOX-1 and AT1-R. We conclude that direct binding of LOX-1 and AT1-R and the activation of downstream Gq protein are important mechanisms of ox-LDL-induced cardiomyocyte hypertrophy.

Radiation therapy, a mainstay of treatment for head and neck cancer, is not always curative due to the development of treatment resistance; additionally, multi-institutional trials have questioned the efficacy of concurrent radiation with cetuximab, the epidermal growth factor receptor (EGFR) inhibitor. We unraveled a mechanism for radiation resistance; that is, radiation induces EGFR, which phosphorylates TRIP13 (thyroid hormone receptor interactor 13) on tyrosine 56. Phosphorylated (phospho-)TRIP13 promotes non-homologous end joining (NHEJ) repair to induce radiation resistance. NHEJ is the main repair pathway for radiation-induced DNA damage. Tumors expressing high TRIP13 do not respond to radiation but are sensitive to cetuximab or cetuximab combined with radiation. Suppression of phosphorylation of TRIP13 at Y56 abrogates these effects. These findings show that EGFR-mediated phosphorylation of TRIP13 at Y56 is a vital mechanism of radiation resistance. Notably, TRIP13-pY56 could be used to predict the response to radiation or cetuximab and could be explored as an actionable target.

Although the DNA damage response (DDR) is associated with the radioresistance characteristics of lung cancer cells, the specific regulators and underlying mechanisms of the DDR are unclear. Here, we identified the serine proteinase inhibitor clade E member 2 (SERPINE2) as a modulator of radiosensitivity and the DDR in lung cancer. Cells exhibiting radioresistance after ionizing radiation show upregulation of SERPINE2, and SERPINE2 knockdown improves tumor radiosensitivity in vitro and in vivo. Functionally, SERPINE2 deficiency causes a reduction in homologous recombination repair, rapid recovery of cell cycle checkpoints, and suppression of migration and invasion. Mechanistically, SERPINE2 knockdown inhibits the accumulation of p-ATM and the downstream repair protein RAD51 during DNA repair, and RAD51 can restore DNA damage and radioresistance phenotypes in lung cancer cells. Furthermore, SERPINE2 can directly interact with MRE11 and ATM to facilitate its phosphorylation in HR-mediated DSB repair. In addition, high SERPINE2 expression correlates with dismal prognosis in lung adenocarcinoma patients, and a high serum SERPINE2 concentration predicts a poor response to radiotherapy in non-small cell lung cancer patients. In summary, these findings indicate a novel regulatory mechanism by which SERPINE2 modulates the DDR and radioresistance in lung cancer.

The ameliorative effect of Dendrobium fimbriatum polysaccharide (cDFPW1) on ulcerative colitis (UC) was investigated using dextran-sodium-sulfate-induced (DSS-induced) mouse model in the present study. Results showed that cDFPW1 effectively improved colitis mice by ameliorating weight loss, disease activity index (DAI) and colonic pathological damage, and protecting intestinal barrier function integrity. Moreover, cDFPW1 modulated the composition and metabolism of intestinal microbiota through enhancing Romboutsia, Lactobacillus and Odoribacter, and reducing Parasutterella, Burkholderia-Caballeronia-Paraburkholderia and Acinetobacter in colitis mice. Notably, cDFPW1 significantly restored the homeostasis of Th17/regulatory T (Treg) cells and the expression of specific cytokines. Western blot of colon tissues showed that cDFPW1 markedly up-regulated the expression of Nrf2 and inhibited the phosphorylation of NF-κB signaling. These results indicated that cDFPW1 possesses the potential of improving UC and its effect on palliating colitis may be connected with the regulation of Nrf2/NF-κB signaling.