Catalytic Subunit Research Articles

PKA regulation of neuronal function requires the dissociation of catalytic subunits from regulatory subunits

Protein kinase A (PKA) plays essential roles in diverse cellular functions. However, the spatiotemporal dynamics of endogenous PKA upon activation remain debated. The classical model predicts that PKA catalytic subunits dissociate from regulatory subunits in the presence of cAMP, whereas a second model proposes that catalytic subunits remain associated with regulatory subunits following physiological activation. Here, we report that different PKA subtypes, as defined by the regulatory subunit, exhibit distinct subcellular localization at rest in CA1 neurons of cultured hippocampal slices. Nevertheless, when all tested PKA subtypes are activated by norepinephrine, presumably via the β-adrenergic receptor, catalytic subunits translocate to dendritic spines but regulatory subunits remain unmoved. These differential spatial dynamics between the subunits indicate that at least a significant fraction of PKA dissociates. Furthermore, PKA-dependent regulation of synaptic plasticity and transmission can be supported only by wildtype, dissociable PKA, but not by inseparable PKA. These results indicate that endogenous PKA regulatory and catalytic subunits dissociate to achieve PKA function in neurons.

METTL3 accelerates staphylococcal protein A (SpA)-induced osteomyelitis progression by regulating m6A methylation-modified miR-320a.

Osteomyelitis (OM) is an inflammatory disease of bone infection and destruction characterized by dysregulation of bone homeostasis. Staphylococcus aureus (SA) has been reported to be the most common pathogen causing infectious OM. Recent studies have demonstrated that N6-methyladenosine (m6A) regulators are associated with the development of OM. However, the molecular mechanism of m6A modifications in OM remains unclear. Here, we investigated the function of methyltransferase-like 3 (METTL3)-mediated m6A modification in OM development. In this study, human bone mesenchymal stem cells (hBMSCs) were treated with staphylococcal protein A (SpA), a vital virulence factor of SA, to construct cell models of OM. Firstly, we found that METTL3 was upregulated in OM patients and SpA-induced hBMSCs, and SpA treatment suppressed osteogenic differentiation and induced oxidative stress and inflammatory injury in hBMSCs. Functional experiments showed that METTL3 knockdown alleviated the inhibition of osteogenic differentiation and the promotion of oxidative stress and inflammation in SpA-treated hBMSCs. Furthermore, METTL3-mediated m6A modification upregulated miR-320a expression by promoting pri-miR-320a maturation, and the mitigating effects of METTL3 knockdown on SpA-mediated osteogenic differentiation, oxidative stress and inflammatory responses can be reversed by miR-320 mimic. In addition, we demonstrated that phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) was a downstream target of miR-320a, upregulation of PIK3CA alleviated miR-320a-induced inhibition of osteogenic differentiation, and upregulation of oxidative stress and inflammatory responses during SpA infection. Finally, we found that silencing METTL3 alleviated OM development by regulating the miR-320a/PIK3CA axis. Taken together, our data demonstrated that the METTL3/m6A/miR-320a/PIK3CA axis regulated SpA-mediated osteogenic differentiation, oxidative stress, and inflammatory responses in OM, which may provide a new therapeutic strategy for OM patients.

Overlapping Transcriptome Alterations Reveal the Mechanism of Interaction between Selenium and Zinc and Their Common Effect on Essential Nutrient Metabolism in Mung Bean (Vigna radiata L.).

Selenium (Se) and zinc (Zn) deficiencies have become serious global food security and public health problems. Biofortification through foliar fertilizer is a nonspecific, low-tech, and cost-effective strategy. Se and Zn have overlapping physiological roles and interacting relationships in plants. Mung bean is superior for Se enrichment and an excellent Zn carrier. However, the molecular mechanism underlying the interaction between Se and Zn in the mung bean remains unclear. Herein, Se and Zn accumulation, antioxidant activities, physiological determination, and transcriptomic analysis were performed under both Se and Zn treatments. Common essential roles of Se and Zn in mung bean were reflected by the comprehensively altered ten physiological indexes under both Se2 (24 g·ha-1) and Zn1 (1.2 kg·ha-1) treatments. Overlapping transcriptome changes and common DEGs in two compared groups revealed that the upregulated expression of sulfate transporters (SULTRs), phosphate transporters (PHTs), and Zinc-regulated/Iron-regulated-like protein (ZIP) family genes under Se and Zn treatments directly promoted both Se and Zn intakes. Furthermore, the altered Se/Sulfur, nitrogen, and carbohydrate metabolisms are closely interlinked with the uptake and assimilation of Se and Zn via the 20 key genes that we filtered through the protein-protein interaction (PPI) network analysis. Further analysis indicated that l-methionine γ-lyase (E 4.4.1.11) genes may play an important role in the transamination of selenomethionine and its derivatives; glutamine synthetase (GS), nitrate reductase (NR), and starch synthase (SS) genes may regulate the nitrogen assimilation and carbohydrate metabolism, which provide more carriers for Se and Zn; glutathione peroxidase (GPx), glutamate-cysteine ligase catalytic subunit (GCLC), and serine acetyltransferase (SAT) genes may accelerate the GSH-GSSH cycle and promote Se and Zn storages. This study provides new molecular insights into the comprehensive improvement of the nutritional quality of mung beans in Se and Zn biofortification productions.

The structures of protein kinase A in complex with CFTR: Mechanisms of phosphorylation and noncatalytic activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

Structural determinants of protein kinase A essential for CFTR channel activation

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, through phosphorylation of multiple serines in CFTR's regulatory (R) domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation, respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct hydrophobic patch (the "docking site") is responsible for noncatalytic CFTR activation, achieved by stabilizing the R domain in a "released" conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns, we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic, CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute to the exacerbation of lung disease.

ATP-Triggered Fe(CN)2CO Synthon Transfer from the Maturase HypCD to the Active Site of Apo-[NiFe]-Hydrogenase.

[NiFe]-hydrogenases catalyze the reversible activation of H2 using a unique NiFe(CN)2CO metal site, which is assembled by a sophisticated multiprotein machinery. The [4Fe-4S] cluster-containing HypCD complex, which possesses an ATPase activity with a hitherto unknown function, serves as the hub for the assembly of the Fe(CN)2CO subfragment. HypCD is also thought to be responsible for the subsequent transfer of the iron fragment to the apo-form of the catalytic hydrogenase subunit, but the underlying mechanism has remained unexplored. Here, we performed a thorough spectroscopic characterization of different HypCD preparations using infrared, Mössbauer, and NRVS spectroscopy, revealing molecular details of the coordination of the Fe(CN)2CO fragment. Moreover, biochemical assays in combination with spectroscopy, AlphaFold structure predictions, protein-ligand docking calculations, and crosslinking MS deciphered unexpected mechanistic aspects of the ATP requirement of HypCD, which we found to actually trigger the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase.

Human DCP1 is crucial for mRNA decapping and possesses paralog-specific gene regulating functions.

The mRNA 5'-cap structure removal by the decapping enzyme DCP2 is a critical step in gene regulation. While DCP2 is the catalytic subunit in the decapping complex, its activity is strongly enhanced by multiple factors, particularly DCP1, which is the major activator in yeast. However, the precise role of DCP1 in metazoans has yet to be fully elucidated. Moreover, in humans, the specific biological functions of the two DCP1 paralogs, DCP1a and DCP1b, remain largely unknown. To investigate the role of human DCP1, we generated cell lines that were deficient in DCP1a, DCP1b, or both to evaluate the importance of DCP1 in the decapping machinery. Our results highlight the importance of human DCP1 in decapping process and show that the EVH1 domain of DCP1 enhances the mRNA-binding affinity of DCP2. Transcriptome and metabolome analyses outline the distinct functions of DCP1a and DCP1b in human cells, regulating specific endogenous mRNA targets and biological processes. Overall, our findings provide insights into the molecular mechanism of human DCP1 in mRNA decapping and shed light on the distinct functions of its paralogs.

Daphnetin Protects Schwann Cells Against High-Glucose-Induced Oxidative Injury by Modulating the Nuclear Factor Erythroid 2-Related Factor 2/Glutamate–Cysteine Ligase Catalytic Subunit Signaling Pathway

Diabetic peripheral neuropathy (DPN), a common complication of diabetes mellitus, is primarily characterized by damage to Schwann cells caused by oxidative stress under hyperglycemic conditions. Recently, we demonstrated the ability of coumarin-rich Ficus formosana Maxim. to alleviate DPN in ovariectomized diabetic mice. However, the underlying mechanisms remain unclear. In this study, we established an in vitro DPN model using RSC96 Schwann cells exposed to high glucose levels. Daphnetin, a natural coumarin found abundantly in Ficus formosana Maxim., was co-incubated with Schwann cells in a high-glucose medium to investigate its protective effects against DPN. The free radical scavenging capacity of daphnetin was evaluated, along with assessments of cell viability, apoptosis, H2O2 levels, and the expression of proteins by the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutamate–cysteine ligase catalytic subunit (GCLC) pathway in RSC96 Schwann cells. The results showed that daphnetin was non-toxic within the tested concentration range of 6.25 μM to 50 μM in RSC96 Schwann cells. Moreover, daphnetin significantly improved cell viability, exhibited strong antioxidant activity, reduced H2O2 levels, and regulated the Nrf2/GCLC pathway protein expressions in RSC96 cells cultured in high-glucose medium. Additionally, daphnetin influenced apoptosis-related proteins by decreasing the expression levels of Bax and Caspase 3, while increasing the Bcl-2 expression level in high-glucose-treated RSC96 cells. These findings suggest that daphnetin may alleviate oxidative stress induced by high glucose levels through activation of the Nrf2/GCLC pathway and inhibition of Schwann cell apoptosis, underscoring its potential as a therapeutic agent for DPN.

Subunit-Specific Developmental Roles of PI3K in SF1-Expressing Cells.

Phosphatidylinositol 3-kinase (PI3K) regulates cellular development and energy homeostasis. However, the roles of its subunits in organ development remain largely unknown. We explored the roles of PI3K catalytic subunits in steroidogenic factor 1 (SF1)-expressing cells through knockout (KO) of the p110α and p110β subunits. We examined mice with a double KO of p110α and p110β in SF1-expressing cells (p110αβ KOSF1). Although these animals exhibited no significant changes in the development of the ventromedial hypothalamus, we noted pronounced hypotrophy in the adrenal cortex, testis, and ovary. Additionally, corticosterone and aldosterone levels were significantly reduced. The absence of these subunits also resulted in decreased body weight and survival rate, along with impaired glucose homeostasis, in p110αβ KOSF1 mice. The data demonstrate the specific roles of PI3K catalytic subunits in the development and function of SF1-expressing organs.

IGFBP6 Modulates Proteostasis by Activating ATF4 Targets and Reducing ER Retrotranslocon Expression.

Reduced expression of the IGFBP6 protein leads to an increase in the metastatic potential of breast cancer (BC) cells. The level of protein synthesis in tumor cells is increased, leading to a compensatory adjustment of proteostasis. One of the tools used to study proteostasis is protein toxins of the RIP-II family, which irreversibly inactivate ribosomes (particularly, viscumin). We investigated the effect of IGFBP6 gene knockdown on the proteostasis in the BC cell line MDA-MB-231. Ribosomes from MDA-MB-231IGFBP6 cells, knockdown for the IGFBP6 gene, are less efficiently modified by the toxin. This is probably due to the reduced transport of the viscumin catalytic subunit from the ER to the cytoplasm. MDA-MB-231IGFBP6 cells showed reduced expression of the retrotranslocon HRD1/Derlin subunit, which is a component of the ER-associated protein degradation system (ERAD). For ATF4 transcription factor, which is a part of the ER unfolded protein response (UPR) pathway, an increased expression of its targets was found.

Inavolisib-Based Therapy in PIK3CA-Mutated Advanced Breast Cancer

BackgroundInavolisib is a highly potent and selective inhibitor of the alpha isoform of the p110 catalytic subunit of the phosphatidylinositol 3-kinase complex (encoded by PIK3CA) that also promotes the degradation of mutated p110α. Inavolisib plus palbociclib–fulvestrant has shown synergistic activity in preclinical models and promising antitumor activity in early-phase trials.MethodsIn a phase 3, double-blind, randomized trial, we compared first-line inavolisib (at an oral dose of 9 mg once daily) plus palbociclib–fulvestrant (inavolisib group) with placebo plus palbociclib–fulvestrant (placebo group) in patients with PIK3CA-mutated, hormone receptor–positive, human epidermal growth factor receptor 2 (HER2)–negative locally advanced or metastatic breast cancer who had had relapse during or within 12 months after the completion of adjuvant endocrine therapy. The primary end point was progression-free survival as assessed by the investigator.ResultsA total of 161 patients were assigned to the inavolisib group and 164 to the placebo group; the median follow-up was 21.3 months and 21.5 months, respectively. The median progression-free survival was 15.0 months (95% confidence interval [CI], 11.3 to 20.5) in the inavolisib group and 7.3 months (95% CI, 5.6 to 9.3) in the placebo group (hazard ratio for disease progression or death, 0.43; 95% CI, 0.32 to 0.59; P<0.001). An objective response occurred in 58.4% of the patients in the inavolisib group and in 25.0% of those in the placebo group. The incidence of grade 3 or 4 neutropenia was 80.2% in the inavolisib group and 78.4% in the placebo group; grade 3 or 4 hyperglycemia, 5.6% and 0%, respectively; grade 3 or 4 stomatitis or mucosal inflammation, 5.6% and 0%; and grade 3 or 4 diarrhea, 3.7% and 0%. No grade 3 or 4 rash was observed. Discontinuation of any trial agent because of adverse events occurred in 6.8% of the patients in the inavolisib group and in 0.6% of those in the placebo group.ConclusionsIn patients with PIK3CA-mutated, hormone receptor–positive, HER2-negative locally advanced or metastatic breast cancer, inavolisib plus palbociclib–fulvestrant led to significantly longer progression-free survival than placebo plus palbociclib–fulvestrant, with a greater incidence of toxic effects. The percentage of patients who discontinued any trial agent because of adverse events was low. (Funded by F. Hoffmann–La Roche; INAVO120 ClinicalTrials.gov number, NCT04191499.)

The role of PIK3CA gene mutations in colorectal cancer and the selection of treatment strategies

PIK3CA gene encodes the p110α catalytic subunit of PI3K, which regulates the PI3K/AKT/mTOR signaling pathway. PIK3CA gene mutation is one of the most common mutations in colorectal cancer (CRC), affecting about 15%–20% of CRC patients. PIK3CA gene mutation leads to the persistent activation of the PI3K/AKT/mTOR signaling pathway, which promotes the proliferation, invasion, metastasis, and drug resistance of CRC. This article provides a summary of the key detection methods for PIK3CA gene mutation, and provides an introduction to the existing colorectal cancer treatments and their practical applications in the clinic. Besides, this article summarizes the role and mechanism of PIK3CA gene mutation in the occurrence and development of CRC. It also explores the relationship between PIK3CA gene mutation and the clinical features and prognosis of CRC. This article focuses on the influence and mechanism of PIK3CA gene mutation on the targeted therapy and immunotherapy of CRC, and discusses the potential value and future direction of PIK3CA gene mutation in the personalized therapy of CRC. We aim to provide new perspectives and ideas for the precise diagnosis and treatment of CRC.

Protein phosphatase 1 suppresses PKR/EIF2α signaling during human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world's population. Lytic HCMV replication in immunocompromised individuals or neonates can lead to severe disease in multiple organ systems and even death. The establishment of lytic replication is driven by the first viral proteins expressed upon infection, the immediate early proteins, which play a key role in creating an intracellular environment conducive to virus replication. Two immediate early proteins, the functional orthologs pTRS1 and pIRS1, stimulate immediate early gene expression by suppressing antiviral PKR/eIF2α signaling and enhance the translation of viral mRNAs independent of PKR antagonism. To better understand the molecular functions of pTRS1, we used proximity labeling proteomics to identify proteins that interact with pTRS1 in infected cells. Multiple novel host and viral interactors were identified, including the catalytic subunits of the protein phosphatase 1 (PP1) holoenzyme. Mutations to a PP1 catalytic subunit known to disrupt binding to PP1 regulatory subunits decreased binding to pTRS1. pTRS1 immune complexes contained phosphatase activity, and inhibition of phosphatase activity in transfected or infected cells reversed the ability of pTRS1 to inhibit the antiviral kinase PKR. Depletion of individual PP1 catalytic subunits decreased virus replication and increased the phosphorylation of the PKR substrate eIF2α. Taken together, our data suggest potential novel functions for pTRS1 and define a novel role for PP1 as an antagonist of the antiviral PKR/eIF2α signaling axis during HCMV infection.IMPORTANCEThe human cytomegalovirus (HCMV) pTRS1 and pIRS1 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining the molecular functions of pTRS1/pIRS1 is important for understanding the molecular events controlling HCMV replication and viral disease. These data provide new insights into potential pTRS1 functional roles, providing a starting point for others to understand new features of infected cell biology. Another important result of this study is the finding that specific protein phosphatase 1 (PP1) regulatory subunits are required to suppress PKR/eIF2α signaling, a critical cellular innate immune defense to viral infection. These data lay the groundwork for future efforts to discover therapeutics that disrupt pTRS1 interaction with PP1 allowing cellular defenses to limit HCMV replication and disease.

Prim-O-glucosylcimifugin alleviates influenza virus-induced pneumonia in mice by inhibiting the TGF-β1/PI3KCD/MSK2/RELA signalling pathway.

Prim-O-glucosylcimifugin (POG) is a chromone derived primarily from Saposhnikovia divaricata (Turcz) Schischk and Cimicifuga simplex. Previous research has shown that POG possesses antibacterial, anticancer, anti-inflammatory, antioxidant, anticonvulsant, antipyretic, and analgesic properties. However, the specific impact of POG on influenza-virus-induced pneumonia is not well understood. In this study, we investigated the protective effects and underlying mechanisms of POG in pneumonia caused by influenza A virus (IAV). In vitro, POG was found to have a protective effect against infections caused by the respiratory viruses respiratory syncytial virus (RSV), human coronavirus OC43, and influenza A virus. POG inhibited A/FM/1/1947(H1N1) infection with an EC50 ranging from 3.01 to 10.43 in vitro. Intraperitoneal infection of mice with POG at a dose of 5 or 10 mg/kg resulted in a reduction in IAV-induced pneumonia, as evidenced by decreased pulmonary edema, improved lung histopathology, and reduced inflammatory cell accumulation. At the higher dose (10 mg/kg), POG treatment significantly increased survival rates, decreased viral titres in the lungs, improved lung histology, and reduced lung inflammation in IAV-infected mice. POG also effectively alleviated pulmonary fibrosis by reducing the levels of fibrotic markers (hydroxyproline [Hyp] and transforming growth factor β1 [TGF-β1]) and suppressing the expression of alpha smooth muscle actin (α-SMA), p focal adhesion kinase (p-FAK), and TGF-β1 in lung tissues. In addition, POG inhibited the expression of the RELA proto-oncogene (RELA), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), and mitogen- and stress-activated protein kinase 2 (MSK2) in lung tissues. These results indicate that POG may have a protective effect against IAV-induced pneumonia by downregulating the TGF-β1/PI3KCD/MSK2/RELA signalling pathway in the lungs.

Evaluation of the Dietary Arginine Supplementation on Yellow Catfish: From a Low-Temperature Farming Perspective

The yellow catfish is an economically significant freshwater fish with increasing importance in aquaculture. However, the low temperature environments prevalent in certain regions pose challenges to its growth, development, and overall health. This study aimed to explore the impact of dietary arginine (Arg) addition on the growth, digestive capacity, and intestinal antioxidant response in fish under low temperature acclimation (18 °C). Total 720 fish were randomly distributed into six groups, each containing 120 fish. Over the course of eight weeks, each group was fed with diets about varying Arg concentrations (1.79–3.26 g/kg). The results indicated that Arg supplementation resulted in an increase in specific growth rate (SGR), feed intake (FI), feed efficiency (FE), as well as pancreatic enzyme activities in both pancreas and intestine. Conversely, malondialdehyde (MDA) and protein carbonyl (PC) contents initially decreased but increased with higher Arg concentrations. Glutathione peroxidase 1a (GPX1a) showed a positive correlation with nuclear factor-erythroid 2-related factor-2 (Nrf2), showing its role in antioxidative capacity. Furthermore, this study revealed that Arg significantly enhanced the activities of anti-superoxide anion, anti-hydroxyl radical, and anti-oxidative enzymes, along with the relative mRNA abundance of Copper-Zinc superoxide dismutase (CuZnSOD), catalase, GPX1a, glutamate-cysteine ligase catalytic subunit (GCLC), and Nrf2 in the intestine. It was determined that yellow catfish weighing between 61.0 g and 89.0 g require an intake of 26.8 g of Arg per kilogram of diet based on polynomial regression analysis of specific growth rate (SGR), which is equivalent to 37.0 g of dietary protein, under sub-low temperature conditions of 18 °C.

CIGB-300 internalizes and impairs viability of NSCLC cells lacking actionable targets by inhibiting casein kinase-2 signaling

Overall response rates in advanced Non-Small Cell Lung Cancer (NSCLC) remains low. Thus, novel molecular targets, tailored drugs and/or drug combinations are needed. Casein Kinase-2 (CK2) is a constitutively active and frequently over-expressed enzyme which fosters tumor survival, proliferation and metastasis. By using a clinical-grade and Cell Penetrating Peptide-based inhibitor coined as CIGB-300, we explore the anti-neoplastic effects caused by interruption of CK2 signaling in lung cancer cells lacking EGFR, ALK and ROS mutations. CIGB-300 penetrated and impaired viability and proliferation of Lung Adenocarcinoma (LUAD) (A549, NCI-H522) and Lung Squamous Carcinoma (LUSC) (NCI-H226 and SK-MES-1) cells in a dose-response manner. The differential activity could not be explained by overall peptide uptake or its subcellular distribution, as evidenced by flow cytometry and confocal microscopy. Upon internalization, CIGB-300 interacted with CK2 catalytic subunits (ɑ1/ɑ2) and CK2 substrates, thus impairing phosphorylation of enzyme substrates (CDC37s13, NPM1s125) and downstream proteins (RPS6s325/326). CK2 inhibition induced an early Reactive Oxygen Species (ROS) and mitochondrial membrane depolarization, which predates lung cancer cell death. Finally, intravenous injection of CIGB-300 in a cell line-based xenograft corroborated CIGB-300’s anti-tumor effects and suggested concurrent in situ reductions of CSNK2ɑ subunit and downstream RPS6s235/236 phosphorylation. Overall, CIGB-300 therapeutic hypothesis and antineoplastic effects demonstrated herein, further support the evaluation of this clinical-grade CK2 inhibitor in advanced NSCLC with limited therapeutic options.

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p&amp;lt;0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

Clinicopathogenomic analysis of PI3K/AKT/PTEN-altered luminal metastatic breast cancer in Japan.

This rapid communication highlights the correlation between protein kinase B alpha (AKT1)-phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)- phosphatase and tensin homolog (PTEN) alterations and clinicopathological factors in Japanese patients with metastatic recurrent breast cancer (mBC). This study analyzed 1967 patients with luminal-type breast cancer who underwent cancer gene panel testing. The results demonstrated that AKT pathway alterations, including PI3K/AKT/PTEN, occurred in 1038 (52.8%)cases. Patients with AKT pathway mutations were older (p = 0.002) and had a higher rate of invasive lobular carcinoma (ILC) histology (p = 0.001), progesterone receptor (PgR) positivity (p = 0.006), and bone metastases (p = 0.001), and a lower rate of germline BRCA2 (p < 0.001). Comprehensive genomic profile results demonstrated a higher tumor mutational burden (TMB) (< 0.001) and lower tumor BRCA1/2 expression (< 0.001) in patients with mutations in the AKT pathway. These results are crucial for characterizing candidates for AKT pathway-targeted molecular therapies and conceptualizing optimal treatment strategies. Clinical trial registration: This study is an observational study and is therefore not registered with the clinical trials registration.

Far-infrared irradiation inhibits proliferation of human upper airway epithelial cells via protein phosphatase 2A-promoted dephosphorylation of p70 S6 kinase.

Far-infrared (FIR) ray, an invisible electromagnetic radiation with a wavelength of 3‒1000μm, elicits various biological effects. Excessive proliferation of human upper airway epithelial cells (HUAEpCs) contributes to the development and exacerbation of nasal narrowing diseases, including nasal polyposis and chronic rhinosinusitis with nasal polyps (CRSwNP). Here, we investigated the molecular mechanisms through which FIR irradiation inhibits the proliferation of HUAEpCs. FIR irradiation significantly inhibited the proliferation of NCI-H292 cells without alteration in cell viability. The anti-proliferative effect of FIR radiation was accompanied by decreased phosphorylation of p70S6K at Thr389 (p-p70S6K-Thr389), without changes in the phosphorylation of mammalian target of rapamycin and adenosine monophosphate-activated protein kinase (AMPK). Overexpression of p70S6K-T389E mutant gene, not dominant negative-AMPKα1 gene, significantly reversed FIR irradiation-inhibited p-p70S6K-Thr389 and cell proliferation. Cotreatment with okadaic acid or knockdown of protein phosphatase 2A catalytic subunit (PP2Ac) gene expression significantly reversed FIR irradiation-decreased p-p70S6K-Thr389 and cell proliferation. FIR irradiation remarkably promoted the physical association of p70S6K and PP2Ac without change in total PP2Ac expression. Hyperthermal stimulus (39°C) did not alter p-p70S6K-Thr389 and cell proliferation. In line with NCI-H292 cell results, FIR irradiation, not hyperthermal stimulus, significantly decreased p-p70S6K-Thr389 and cell proliferation in primary human nasal turbinate and polyp epithelial cells. These results demonstrated that FIR irradiation decreased the proliferation of HUAEpCs through PP2A-mediated inhibition of p70S6K phosphorylation, independent of its hyperthermal effect. Our data suggest that FIR therapy can be used to treat upper airway narrowing epithelial hyperplastic diseases, including nasal polyposis and CRSwNP.

Autophagy and proteasomes in thymic epithelial cells: essential bulk protein degradation systems for immune homeostasis maintenance

The thymus is a central organ that controls T cell development. Thymic epithelial cells (TECs) create a unique microenvironment essential for the differentiation of major histocompatibility complex (MHC)-restricted and self-tolerant T cells. TECs present a complex of self-peptides and MHC molecules (self-pMHCs) to immature T cells and regulate their survival and differentiation based on their affinity for self-pMHCs. The processing of self-peptides in TECs depends on bulk protein degradation systems, specifically autophagy and proteasomes. Studies using autophagy- and proteasome-deficient mouse models have demonstrated that these degradation systems in TECs are indispensable for maintaining immune homeostasis. Although autophagy and proteasomes are ubiquitous in nearly all eukaryotic cells, TECs exhibit unique characteristics in their autophagy and proteasome functions. Autophagy in TECs is constitutively active and independent of stress responses, while TEC proteasomes contain specialized catalytic subunits. This review summarizes the distinctive characteristics of autophagy and proteasomes in TECs and their roles in immune system regulation.