Phosphorylation Status Of Proteins Research Articles

Bta-miR-181d and Bta-miR-196a mediated proliferation, differentiation, and apoptosis in Bovine Myogenic Cells.

Skeletal muscle is an important component of livestock and poultry organisms. The proliferation and differentiation of myoblasts are highly coordinated processes, which rely on the regulation of miRNA. MiRNAs are widely present in organisms and play roles in various biological processes, including cell proliferation, differentiation, and apoptosis. MiR-181d and miR-196a, identified as tumor suppressors, have been found to be involved in cell proliferation, apoptosis, directed differentiation, and cancer cell invasion. However, their role in beef cattle skeletal muscle metabolism remains unclear. In this study, we discovered that overexpression of bta-miR-181d and bta-miR-196a in Qinchuan cattle myoblasts inhibited proliferation and apoptosis while promoting myogenic differentiation through EDU staining, flow cytometry analysis, immunofluorescence staining, and Western blotting. RNA-seq analysis of differential gene expression revealed that after overexpression of bta-miR-181d and bta-miR-196a, the differentially expressed genes were mainly enriched in the PI3K-Akt and MAPK signaling pathways. Furthermore，the phosphorylation levels of key proteins p-AKT in the PI3K signaling pathway and p-MAPK in the MAPK signaling pathway were significantly decreased after overexpression of bta-miR-181d and bta-miR-196a. Overall，this study provides preliminary evidence that bta-miR-181d and bta-miR-196a may regulate proliferation，apoptosis，and differentiation processes in Qinchuan cattle myoblasts by affecting the phosphorylation status of key proteins in PI3K-Akt and MAPK-ERK signaling pathways.

Sex-specific cardiovascular remodeling leads to a divergent sex-dependent development of heart failure in aged hypertensive rats.

The prevalence of heart failure with preserved ejection fraction (HFpEF) is continuously rising and predominantly affects older women often hypertensive and/or obese or diabetic. Indeed, there is evidence on sex differences in the development of HF. Hence, we studied cardiovascular performance dependent on sex and age as well as pathomechanisms on a cellular and molecular level. We studied 15-week- and 1-year-old female and male hypertensive transgenic rats carrying the mouse Ren-2 renin gene (TG) and compared them to wild-type (WT) controls at the same age. We tracked blood pressure and cardiac function via echocardiography. After sacrificing the 1-year survivors we studied vascular smooth muscle and endothelial function. Isolated single skinned cardiomyocytes were used to determine passive stiffness and Ca2+-dependent force. In addition, Western blots were applied to analyse the phosphorylation status of sarcomeric regulatory proteins, titin and of protein kinases AMPK, PKG, CaMKII as well as their expression. Protein kinase activity assays were used to measure activities of CaMKII, PKG and angiotensin-converting enzyme (ACE). TG male rats showed significantly higher mortality at 1 year than females or WT male rats. Left ventricular (LV) ejection fraction was specifically reduced in male, but not in female TG rats, while LV diastolic dysfunction was evident in both TG sexes, but LV hypertrophy, increased LV ACE activity, and reduced AMPK activity as evident from AMPK hypophosphorylation were specific to male rats. Sex differences were also observed in vascular and cardiomyocyte function showing different response to acetylcholine and Ca2+-sensitivity of force production, respectively cardiomyocyte functional changes were associated with altered phosphorylation states of cardiac myosin binding protein C and cardiac troponin I phosphorylation in TG males only. Cardiomyocyte passive stiffness was increased in TG animals. On a molecular level titin phosphorylation pattern was altered, though alterations were sex-specific. Thus, also the reduction of PKG expression and activity was more pronounced in TG females. However, cardiomyocyte passive stiffness was restored by PKG and CaMKII treatments in both TG sexes. Here we demonstrated divergent sex-specific cardiovascular adaptation to the over-activation of the renin-angiotensin system in the rat. Higher mortality of male TG rats in contrast to female TG rats was observed as well as reduced LV systolic function, whereas females mainly developed HFpEF. Though both sexes developed increased myocardial stiffness to which an impaired titin function contributes to a sex-specific molecular mechanism. The functional derangements of titin are due to a sex-specific divergent regulation of PKG and CaMKII systems.

The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability.

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

Paternal Age Amplifies Cryopreservation-Induced Stress in Human Spermatozoa.

The global fall in male fertility is a complicated process driven by a variety of factors, including environmental exposure, lifestyle, obesity, stress, and aging. The availability of assisted reproductive technology (ART) has allowed older couples to conceive, increasing the average paternal age at first childbirth. Advanced paternal age (APA), most often considered male age ≥40, has been described to impact several aspects of male reproductive physiology. In this prospective cohort study including 200 normozoospermic patients, 105 of whom were ≤35 years (non-APA), and 95 of whom were ≥42 years (APA), we assessed the impact of paternal age on different endpoints representative of sperm quality and cryopreservation tolerance. Non-APA patients had superior fresh semen quality; DNA fragmentation was notably increased in APA as compared to non-APA individuals (21.7% vs. 15.4%). Cryopreservation further increased the DNA fragmentation index in APA (26.7%) but not in non-APA patients. Additionally, APA was associated with increased mtDNAcn in both fresh and frozen/thawed sperm, which is indicative of poorer mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, as indicated by reduced incidences of unreacted acrosome in relation to fresh counterparts in non-APA (from 71.5% to 57.7%) and APA patients (from 75% to 63%). Finally, cryopreservation significantly reduced the phosphorylation status of proteins containing tyrosine residues in sperm from young males. Therefore, the present findings shed light on the effects of paternal age and cryopreservation on sperm quality and serve as valuable new parameters to improve our understanding of the mechanisms underlying sperm developmental competence that are under threat in current ART practice.

The Role of Acyl-CoA Synthetase 1 in Bioactive Lipid Accumulation and the Development of Hepatic Insulin Resistance.

The liver plays a crucial role in glucose metabolism. Obesity and a diet rich in fats (HFD) contribute to the accumulation of intracellular lipids. The aim of the study was to explore the involvement of acyl-CoA synthetase 1 (ACSL1) in bioactive lipid accumulation and the induction of liver insulin resistance (InsR) in animals fed an HFD. The experiments were performed on male C57BL/6 mice divided into the following experimental groups: 1. Animals fed a control diet; 2. animals fed HFD; and 3. HFD-fed animals with the hepatic ACSL1 gene silenced through a hydrodynamic gene delivery technique. Long-chain acyl-CoAs, sphingolipids, and diacylglycerols were measured by LC/MS/MS. Glycogen was measured by means of a commercially available kit. The protein expression and phosphorylation state of the insulin pathway was estimated by Western blot. HFD-fed mice developed InsR, manifested as an increase in fasting blood glucose levels (202.5 mg/dL vs. 130.5 mg/dL in the control group) and inhibition of the insulin pathway, which resulted in an increase in the rate of gluconeogenesis (0.420 vs. 0.208 in the control group) and a decrease in the hepatic glycogen content (1.17 μg/mg vs. 2.32 μg/mg in the control group). Hepatic ACSL1 silencing resulted in decreased lipid content and improved insulin sensitivity, accounting for the decreased rate of gluconeogenesis (0.348 vs. 0.420 in HFD(+/+)) and the increased glycogen content (4.3 μg/mg vs. 1.17 μg/mg in HFD(+/+)). The elevation of gluconeogenesis and the decrease in glycogenesis in the hepatic tissue of HFD-fed mice resulted from cellular lipid accumulation. Inhibition of lipid synthesis through silencing ACSL1 alleviated HFD-induced hepatic InsR.

(2-Methylbutyryl)shikonin Naturally Occurring Shikonin Derivative Ameliorates the α-MSH-Induced Melanogenesis via ERK1/2 and p38 MAP Kinase-Mediated Down-Regulation of the MITF Transcription Factor.

Cutaneous pigmentation is an important phenotypic trait whose regulation, despite recent advances, has yet to be completely elucidated. Melanogenesis, a physiological process of melanin production, is imperative for organism survival as it provides protection against the environmental insults that majorly involve sunlight-induced skin photodamage. However, immoderate melanin synthesis can cause pigmentation disorders associated with a psychosocial impact. In this study, the hypopigmentation effect of (2-methylbutyryl)shikonin, a natural product present in the root extract of Lithospermum erythrorhizon, and the underlying mechanisms responsible for the inhibition of melanin synthesis in α-MSH-stimulated B16F10 cells and C57BL/6J mice was studied. Non-cytotoxic concentrations of (2-methylbutyryl)shikonin significantly repressed cellular tyrosinase activity and melanin synthesis in both in vitro and in vivo models (C57BL/6J mice). (2-Methylbutyryl)shikonin remarkably abolished the protein expression of MITF, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2, thereby blocking the production of pigment melanin via modulating the phosphorylation status of MAPK proteins, viz., ERK1/2 and p38. In addition, specific inhibition of ERK1/2 attenuated the inhibitory effects of (2-methylbutyryl)shikonin on melanin synthesis, whereas selective inhibition of p38 augmented the inhibitory effect of BSHK on melanin synthesis. Moreover, topical application of (2-methylbutyryl)shikonin on C57BL/6J mouse tails remarkably induced tail depigmentation. In conclusion, with these findings, we, for the first time, report the hypopigmentation effect of (2-methylbutyryl)shikonin via inhibition of cellular tyrosinase enzyme activity, subsequently ameliorating the melanin production, thereby indicating that (2-methylbutyryl)shikonin is a potential natural therapy for hyperpigmentation disorders.

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

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

Cultivation, Differentiation, and Lentiviral Transduction of Human-Induced Pluripotent Stem Cell (hiPSC)-Derived Glutamatergic Neurons for Studying Human Tau.

Tau pathology is a major hallmark of many neurodegenerative diseases summarized under the term tauopathies. In most of these disorders,such as Alzheimer's disease, the neuronal axonal microtubule-binding Tau protein becomes mislocalized to the somatodendritic compartment. In human disease, this missorting of Tau is accompanied by an abnormally high phosphorylation state of the Tau protein, and several downstream pathological consequences (e.g., loss of microtubules, degradation of postsynaptic spines, impaired synaptic transmission, neuronal death). While some mechanisms of Tau sorting, missorting, and associated pathologies have been addressed in rodent models, few studies have addressed human Tau in physiological disease-relevant human neurons. Thus, suitable human-derived in vitro models are necessary. This protocol provides a simple step-by-step protocol for generating homogeneous cultures of cortical glutamatergic neurons using an engineered Ngn2 transgene-carrying WTC11 iPSC line. We further demonstrate strategies to improve neuronal maturity, that is, synapse formation, Tau isoform expression, and neuronal activity by co-culturing hiPSC-derived glutamatergic neurons with mouse-derived astrocytes. Finally, we describe a simple protocol for high-efficiency lentiviral transduction of hiPSC-derived neurons at almost all stages of differentiation.

Phosphorylation Dynamics in a flg22-Induced, G Protein–Dependent Network Reveals the AtRGS1 Phosphatase

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2–dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein–coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

Differences in photoprotective strategy during winter in Eastern white pine and white spruce.

Conifers growing in temperate forests utilize sustained forms of thermal dissipation during winter to protect the photosynthetic apparatus from damage, which can be monitored via pronounced reductions in photochemical efficiency (Fv/Fm) during winter. Eastern white pine (Pinus strobus L.) and white spruce (Picea glauca (Moench) Voss) are known to recover from winter stress at different rates, with pine recovering more slowly than spruce, suggesting different mechanisms for sustained dissipation in these species. Our objectives were to monitor pine and spruce throughout spring recovery in order to provide insights into key mechanisms for sustained dissipation in both species. We measured chlorophyll fluorescence, pigments, and abundance and phosphorylation status of key photosynthetic proteins. We found that both species rely on two forms of sustained dissipation involving retention of high amounts of antheraxanthin (A)+zeaxanthin (Z), one that is very slowly reversible and temperature independent and one that is more dynamic and occurs only on subzero days. Differences in protein abundance suggest that spruce, but not pine, likely upregulates cyclic or alternative pathways of electron transport involving the cytochrome b6f complex and photosystem I (PSI). Both species show an increased sustained phosphorylation of the D1 protein on subzero days, and spruce additionally shows dramatic increases in the sustained phosphorylation of light-harvesting complex II (LHCII) and other PSII core proteins on subzero days only, suggesting that a mechanism of sustained dissipation that is temperature dependent requires sustained phosphorylation of photosynthetic proteins in spruce, possibly allowing for direct energy transfer from PSII to PSI as a mechanism of photoprotection. The data suggest differences in strategy among conifers in mechanisms of sustained thermal dissipation in response to winter stress. Additionally, the flexible induction of sustained A+Z and phosphorylation of photosynthetic proteins in response to subzero temperatures during spring recovery seem to be important in providing photoprotection during transitional periods with high temperature fluctuation.

Dynamics of ribosome composition and ribosomal protein phosphorylation in immune signaling in Arabidopsis thaliana.

In plants, the detection of microbe-associated molecular patterns (MAMPs) induces primary innate immunity by the activation of mitogen-activated protein kinases (MAPKs). We show here that the MAMP-activated MAPK MPK6 not only modulates defense through transcriptional regulation but also via the ribosomal protein translation machinery. To understand the effects of MPK6 on ribosomes and their constituent ribosomal proteins (RPs), polysomes, monosomes and the phosphorylation status of the RPs, MAMP-treated WT and mpk6 mutant plants were analysed. MAMP-activation induced rapid changes in RP composition of monosomes, polysomes and in the 60S ribosomal subunit in an MPK6-specific manner. Phosphoproteome analysis showed that MAMP-activation of MPK6 regulates the phosphorylation status of the P-stalk ribosomal proteins by phosphorylation of RPP0 and the concomitant dephosphorylation of RPP1 and RPP2. These events coincide with a significant decrease in the abundance of ribosome-bound RPP0s, RPP1s and RPP3s in polysomes. The P-stalk is essential in regulating protein translation by recruiting elongation factors. Accordingly, we found that RPP0C mutant plants are compromised in basal resistance to Pseudomonas syringae infection. These data suggest that MAMP-induced defense also involves MPK6-induced regulation of P-stalk proteins, highlighting a new role of ribosomal regulation in plant innate immunity.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Feasibility of mass cytometry proteomic characterisation of circulating tumour cells in head and neck squamous cell carcinoma for deep phenotyping

BackgroundCirculating tumour cells (CTCs) are a potential cancer biomarker, but current methods of CTC analysis at single-cell resolution are limited. Here, we describe high-dimensional single-cell mass cytometry proteomic analysis of CTCs in HNSCC.MethodsParsortix microfluidic-enriched CTCs from 14 treatment-naïve HNSCC patients were analysed by mass cytometry analysis using 41 antibodies. Immune cell lineage, epithelial-mesenchymal transition (EMT), stemness, proliferation and immune checkpoint expression was assessed alongside phosphorylation status of multiple signalling proteins. Patient-matched tumour gene expression and CTC EMT profiles were compared. Standard bulk CTC RNAseq was performed as a baseline comparator to assess mass cytometry data.ResultsCTCs were detected in 13/14 patients with CTC counts of 2–24 CTCs/ml blood. Unsupervised clustering separated CTCs into epithelial, early EMT and advanced EMT groups that differed in signalling pathway activation state. Patient-specific CTC cluster patterns separated into immune checkpoint low and high groups. Patient tumour and CTC EMT profiles differed. Mass cytometry outperformed bulk RNAseq to detect CTCs and characterise cell phenotype.DiscussionWe demonstrate mass cytometry allows high-plex proteomic characterisation of CTCs at single-cell resolution and identify common CTC sub-groups with potential for novel biomarker development and immune checkpoint inhibitor treatment stratification.

The effects of branched-chain amino acids on muscle protein synthesis, muscle protein breakdown and associated molecular signalling responses in humans: an update.

Branched-chain amino acids (BCAA: leucine, isoleucine and valine) are three of the nine indispensable amino acids, and are frequently consumed as a dietary supplement by athletes and recreationally active individuals alike. The popularity of BCAA supplements is largely predicated on the notion that they can stimulate rates of muscle protein synthesis (MPS) and suppress rates of muscle protein breakdown (MPB), the combination of which promotes a net anabolic response in skeletal muscle. To date, several studies have shown that BCAA (particularly leucine) increase the phosphorylation status of key proteins within the mechanistic target of rapamycin (mTOR) signalling pathway involved in the regulation of translation initiation in human muscle. Early research in humans demonstrated that BCAA provision reduced indices of whole-body protein breakdown and MPB; however, there was no stimulatory effect of BCAA on MPS. In contrast, recent work has demonstrated that BCAA intake can stimulate postprandial MPS rates at rest and can further increase MPS rates during recovery after a bout of resistance exercise. The purpose of this evidence-based narrative review is to critically appraise the available research pertaining to studies examining the effects of BCAA on MPS, MPB and associated molecular signalling responses in humans. Overall, BCAA can activate molecular pathways that regulate translation initiation, reduce indices of whole-body and MPB, and transiently stimulate MPS rates. However, the stimulatory effect of BCAA on MPS rates is less than the response observed following ingestion of a complete protein source providing the full complement of indispensable amino acids.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation.

The mitophagic degradation of mitochondrial matrix proteins in Saccharomyces cerevisiae was previously shown to be selective, reflecting a pre-engulfment sorting step within the mitochondrial network. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these same proteins also regulate the phosphorylation status and catalytic activity of the yeast pyruvate dehydrogenase complex, which is critical for mitochondrial metabolism. To understand the relationship between these two functions, we evaluated the role of the pyruvate dehydrogenase complex in mitophagic selectivity. Surprisingly, we identified a novel function of the complex in regulating mitophagic selectivity, which is independent of its enzymatic activity. Our data support a model in which the pyruvate dehydrogenase complex directly regulates the activity of its associated kinases and phosphatases. This regulatory interaction then determines the phosphorylation state of mitochondrial matrix proteins and their mitophagic fates.

Dexamethasone induces cancer mitigation and irreversible senescence in lung cancer cells via damaging cortical actin and sustained hyperphosphorylation of pRb

Activation of the glucocorticoid receptors by its cognate ligand, dexamethasone (DEX) is commonly used as an adjuvant treatment in solid tumors. However, its direct effect on cancerous phenotype is not fully understood. We explored the effect and molecular mechanisms of DEX action in lung cancer. In in vitro experiments, DEX treatment causes decrease in migration, invasion and colony formation ability of A549 cells even at lower doses. DEX also decreased adhesion of A549 cells by reducing the formation of cortical actin. Treatment with RU486, a GR antagonist, indicated that these effects are partially mediated through GR. Further; DEX induces G0/G1 arrest of A549 cells. Mechanistically, DEX induces expression of both CDK inhibitors (p21Cip1, p27Kip1) and cyclin-dependent kinases (CDK4, CDK6). Due to this compensatory activation of CDKs and CDKIs, DEX induces the hyper phosphorylation state of Rb protein (pRb) leading to irreversible senescence as confirmed by β-gal staining. Next, in clinical dataset of NSCLC (Non-small cell lung cancer), GR was lowly expressed in cancer patients as compared to the normal group, where higher expression of GR led to higher overall survival of NSCLC indicating for a protective role of GR. Interestingly, when combined with chemotherapeutic agents, DEX can modulate the drug-sensitivity of cells. Taken together, these data indicate that DEX through GR activation may suppress tumor growth by decreasing proliferation and inducing irreversible senescence and combination of standard chemotherapy and DEX can be a potential treatment for NSCLC.

H2O2 and Phosphorylated Peptide Dual-Responsive Nanochannel Device.

Oxidation and protein phosphorylation are critical mechanisms involved in regulating various cellular activities. Increasing research has suggested that oxidative stress could affect the activities of specific kinases or phosphatases, leading to alterations in the phosphorylation status of certain proteins. Ultimately, these alterations can affect cellular signaling pathways and gene expression patterns. However, the relationship between oxidation and protein phosphorylation remains complex and not yet fully understood. Therefore, the development of effective sensors capable of detecting both oxidation and protein phosphorylation simultaneously presents an ongoing challenge. To address this need, we introduce a proof-of-concept nanochannel device that is dual-responsive to both H2O2 and phosphorylated peptide (PP). Specifically, we design a peptide GGGCEG(GPGGA)4CEGRRRR, which contains an H2O2-sensitive unit CEG, an elastic peptide fragment (GPGGA)4, and a phosphorylation site recognition fragment RRRR. When the peptides are immobilized on the inner walls of conical nanochannels in a polyethylene terephthalate membrane, this peptide-modified nanochannel device exhibits a sensitive response to both H2O2 and PPs. The peptide chains undergo a random coil-to-α-helix transition in response to H2O2, which leads to a close-to-open transition of the nanochannel, accompanied with a remarkable increase in the transmembrane ionic current. In contrast, binding of the peptides with PPs shields the positive charge of the RRRR fragments, causing a decrease of the transmembrane ionic current. These unique features enable the sensitive detection of reactive oxygen species released by 3T3-L1 cells stimulated by platelet-derived growth factor (PDGF) as well as PDGF-induced change in the PP level. Real-time kinase activity monitoring further confirms the device's potential utility for kinase inhibitor screening.

Quantitative phosphoproteomics reveals novel roles of cAMP in plants.

3',5'-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3',5'-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular "sponge" that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.