3-kinase Protein Kinase Research Articles

Clinical diagnostic value and potential regulatory mechanisms of lncRNA NOP14-AS1 in chronic kidney disease

In the early stages, chronic kidney disease (CKD) can be asymptomatic, marking diagnosis difficult. This study aimed to investigate the diagnostic role and potential regulatory mechanisms of nucleolar protein 14 (NOP14) -antisense RNA 1 (AS1) in patients with CKD. Herein, 68 patients with CKD, 65 patients with CKD undergoing peridialysis, and 80 healthy adults were included. The real-time reverse transcription-quantitative polymerase chain reaction was performed to assess NOP14-AS1 levels, and its diagnostic value was evaluated using receiver operating characteristic curves. Additionally, cell proliferation and apoptosis were assessed by Cell Counting Kit-8 assay. and flow cytometry, respectively. Oxidative stress levels were determined using superoxide dismutase and malondialdehyde MDA kits, and the dual-luciferase reporter assay was performed to determine the relationship between NOP14-AS1 and microRNA-326 (miR-326) target binding. Lastly, the potential mechanism underlying miR-326 target gene regulation in CKD progression were explored utilizing Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. Notably, patients with CKD exhibited decreasedNOP14-AS1 levels and upregulated miR-326 levels. NOP14-AS1 and miR-326 exhibited combined effects on cell proliferation, apoptosis, inflammatory factors, and oxidative stress levels. Furthermore, the target genes of miR-326 showed enrichment in CKD-associated rat sarcoma and phosphoinositide 3-kinase protein kinase B pathways. Altogether, the findings of this study show the potential of NOP14-AS1 as a diagnostic marker in CKD. Overall, NOP14-AS1 regulates the miR-326 expression, which, in turn, regulates various miR-326 target gene-associated signaling pathways, thereby affecting the occurrence and development of CKD.

Bipolar and schizophrenia risk gene AKAP11 encodes an autophagy receptor coupling the regulation of PKA kinase network homeostasis to synaptic transmission.

Human genomic studies have identified protein-truncating variants in AKAP11 associated with both bipolar disorder and schizophrenia, implicating a shared disease mechanism driven by loss-of-function. AKAP11, a protein kinase A (PKA) adaptor, plays a key role in degrading the PKA-RI complex through selective autophagy. However, the neuronal functions of AKAP11 and the impact of its loss-of-function remains largely uncharacterized. Through multi-omics approaches, cell biology, and electrophysiology analysis in mouse models and human induced neurons, we delineated a central role of AKAP11 in coupling PKA kinase network regulation to synaptic transmission. Loss of AKAP11 disrupted PKA activity and impaired cellular functions that significantly overlap with pathways associated with the psychiatric disease. Moreover, we identified interactions between AKAP11, the PKA-RI adaptor SPHKAP, and the ER-resident autophagy-related proteins VAPA/B, which co-adapt and mediate PKA-RI degradation. Notably, AKAP11 deficiency impaired neurotransmission and decreased presynaptic protein levels in neurons, providing key insights into the mechanism underlying AKAP11 -associated psychiatric diseases.

CaMKK2: bridging the gap between Ca2+ signaling and energy-sensing.

Calcium (Ca2+) ions are ubiquitous and indispensable signaling messengers that regulate virtually every cell function. The unique ability of Ca2+ to regulate so many different processes yet cause stimulus specific changes in cell function requires sensing and decoding of Ca2+ signals. Ca2+-sensing proteins, such as calmodulin, decode Ca2+ signals by binding and modifying the function of a diverse range of effector proteins. These effectors include the Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) enzyme, which is the core component of a signaling cascade that plays a key role in important physiological and pathophysiological processes, including brain function and cancer. In addition to its role as a Ca2+ signal decoder, CaMKK2 also serves as an important junction point that connects Ca2+ signaling with energy metabolism. By activating the metabolic regulator AMP-activated protein kinase (AMPK), CaMKK2 integrates Ca2+ signals with cellular energy status, enabling the synchronization of cellular activities regulated by Ca2+ with energy availability. Here, we review the structure, regulation, and function of CaMKK2 and discuss its potential as a treatment target for neurological disorders, metabolic disease, and cancer.

Blocking donor liver Pannexin 1 channels facilitates mitochondria protection during liver transplantation

Static cold storage (SCS) is the standard technique for organ preservation during transplantation, resulting in cold ischemic injury. Hypoxia can induce pannexin 1 (Panx1) channels to open, leading to release of adenosine triphosphate. However, it is unknown if Panx1 plays a role in SCS. In this study, livers from Panx1−/− mice exhibited reduced adenosine triphosphate release, resulting in hepatocyte protection during preservation. The donor liver damage was decreased during SCS when Panx1 activity was blocked. Transmission electron microscopy revealed a decrease in mitochondria-associated endoplasmic reticulum membranes and improved mitochondria morphology. Mechanistically, Panx1 blockade upregulated the phosphatidylinositol 3-kinase–protein kinase B pathway and increased B cell leukemia/lymphoma 2 levels to combat apoptosis during liver preservation. The data indicate that blocking Panx1 during preservation of the donor liver can effectively improve mitochondrial function and reduce cellular stress damage thereby decreasing cold ischemia and reperfusion-related injuries in liver transplantation.

Current Insights into Weak Seed Dormancy and Pre-Harvest Sprouting in Crop Species.

During the domestication of crops, seed dormancy has been reduced or eliminated to encourage faster and more consistent germination. This alteration makes cultivated crops particularly vulnerable to pre-harvest sprouting, which occurs when mature crops are subjected to adverse environmental conditions, such as excessive rainfall or high humidity. Consequently, some seeds may bypass the normal dormancy period and begin to germinate while still attached to the mother plant before harvest. Grains affected by pre-harvest sprouting are characterized by increased levels of α-amylase activity, resulting in poor processing quality and immediate grain downgrading. In the agriculture industry, pre-harvest sprouting causes annual economic losses exceeding USD 1 billion worldwide. This premature germination is influenced by a complex interplay of genetic, biochemical, and molecular factors closely linked to environmental conditions like rainfall. However, the exact mechanism behind this process is still unclear. Unlike pre-harvest sprouting, vivipary refers to the germination process and the activation of α-amylase during the soft dough stage, when the grains are still immature. Mature seeds with reduced levels of ABA or impaired ABA signaling (weak dormancy) are more susceptible to pre-harvest sprouting. While high seed dormancy can enhance resistance to pre-harvest sprouting, it can lead to undesirable outcomes for most crops, such as non-uniform seedling establishment after sowing. Thus, resistance to pre-harvest sprouting is crucial to ensuring productivity and sustainability and is an agronomically important trait affecting yield and grain quality. On the other hand, seed color is linked to sprouting resistance; however, the genetic relationship between both characteristics remains unresolved. The identification of mitogen-activated protein kinase kinase-3 (MKK3) as the gene responsible for pre-harvest sprouting-1 (Phs-1) represents a significant advancement in our understanding of how sprouting in wheat is controlled at the molecular and genetic levels. In seed maturation, Viviparous-1 (Vp-1) plays a crucial role in managing pre-harvest sprouting by regulating seed maturation and inhibiting germination through the suppression of α-amylase and proteases. Vp-1 is a key player in ABA signaling and is essential for the activation of the seed maturation program. Mutants of Vp-1 exhibit an unpigmented aleurone cell layer and exhibit precocious germination due to decreased sensitivity to ABA. Recent research has also revealed that TaSRO-1 interacts with TaVp-1, contributing to the regulation of seed dormancy and resistance to pre-harvest sprouting in wheat. The goal of this review is to emphasize the latest research on pre-harvest sprouting in crops and to suggest possible directions for future studies.

Circulating free heme induces cytokine storm and pulmonary hypertension through the MKK3/p38 axis.

Hemolysis is associated with pulmonary hypertension (PH), but the direct contribution of circulating free heme to the PH pathogenesis remains unclear. Here, we show that the elevated levels of circulating free heme are sufficient to induce PH and inflammatory response in mice and confirm the critical role of mitogen-activated protein kinase kinase-3 (MKK3)-mediated pathway in free heme signaling. Following the continuous infusion of heme for 2 wk, wild-type (WT) but not MKK3 knockout (KO) mice develop PH, as evidenced by a significantly elevated right ventricular (RV) systolic pressure, RV hypertrophy, and pulmonary vascular remodeling. The MKK3/p38 axis, markedly activated by heme infusion in WTs, results in upregulated proliferative/cytokine signaling targets Akt, ERK1/2, and STAT3, which were abrogated in MKK3 KO mice. Moreover, the MKK3 KOs were protected against heme-mediated endothelial barrier dysfunction by restoring the tight junction protein zonula occludens-1 expression and diminishing the inflammatory cell infiltration in the lungs. Plasma cytokine multiplex analysis revealed a severe cytokine storm already 24 h after initiation of heme infusion, with a significant increase of 19 cytokines, including IL-1b, IL-2, IL-6, IL-9, and TNF-a, in WT animals and complete attenuation of cytokine production in MKK3 KO mice. Together, these findings reveal a causative role of circulating free heme in PH through activating inflammatory and proliferative responses. The central role of MKK3 in orchestrating the heme-mediated pathogenic response supports MKK3 as an attractive therapeutic target for PH and other lung inflammatory diseases linked to hemolytic anemia.NEW & NOTEWORTHY This study demonstrates that elevated levels of circulating free heme can induce pulmonary hypertension (PH) and inflammation in mice. Continuous heme infusion activated the MKK3/p38 pathway, leading to increased right ventricular pressure, right ventricular hypertrophy, and vascular remodeling. This activation upregulated signaling cascades such as Akt, ERK1/2, and STAT3, whereas MKK3 knockout mice were protected against these changes and had reduced inflammatory responses, highlighting MKK3's potential as a therapeutic target for PH.

New target therapies in prostate cancer: from radioligand therapy, to PARP-inhibitors and immunotherapy.

Prostate cancer (PCa) remains a significant global health challenge, particularly in its advanced stages. Despite progress in early detection and treatment, PCa is the second most common cancer diagnosis among men. This review aims to provide an overview of current therapeutic approaches and innovations in PCa management, focusing on the latest advancements and ongoing challenges. We conducted a narrative review of clinical trials and research studies, focusing on PARP inhibitors (PARPis), phosphoinositide 3 kinase-protein kinase B inhibitors, immunotherapy, and radioligand therapies (RLTs). Data was sourced from major clinical trial databases and peer-reviewed journals. Androgen deprivation therapy and androgen-receptor pathway inhibitors remain foundational in managing castration-sensitive and early-stage castration-resistant PCa (CRPC). PARPi's, such as olaparib and rucaparib, have emerged as vital treatments for metastatic CRPC with homologous recombination repair gene mutations, highlighting the importance of personalized medicine. Immune checkpoint inhibitors (ICIs) have shown clinical benefit limited to specific subgroups of PCa, demonstrating significant improvement in efficacy in patients with microsatellite instability/mismatch repair or cyclin-dependent kinase 12 alteration, highlighting the importance of focusing ongoing research on identifying and characterizing these subgroups to maximize the clinical benefits of ICIs. RLTs have shown effectiveness in treating mCRPC. Different alpha emitters (like [225Ac]PSMA) and beta emitters compounds (like [177Lu]PSMA) impact treatment differently due to their energy transfer characteristics. Clinical trials like VISION and TheraP have demonstrated positive outcomes with RLT, particularly [177Lu]PSMA-617, leading to FDA approval. Ongoing trials and future perspectives explore the potential of [225Ac]PSMA, aiming to improve outcomes for patients with mCRPC. The landscape of PCa treatment is evolving, with significant advancements in both established and novel therapies. The combination of hormonal therapies, chemotherapy, PARPis, immunotherapy, and RLTs, guided by genetic and molecular insights, opens new possibilities for personalized treatment.

RAF and MEK Inhibitors in Non-Small Cell Lung Cancer.

Lung cancer, despite recent advancements in survival rates, represents a significant global health burden. Non-small cell lung cancer (NSCLC), the most prevalent type, is driven largely by activating mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and receptor tyrosine kinases (RTKs), and less in v-RAF murine sarcoma viral oncogene homolog B (BRAF) and mitogen-activated protein-kinase kinase (MEK), all key components of the RTK-RAS-mitogen-activated protein kinase (MAPK) pathway. Learning from melanoma, the identification of BRAFV600E substitution in NSCLC provided the rationale for the investigation of RAF and MEK inhibition as a therapeutic strategy. The regulatory approval of two RAF-MEK inhibitor combinations, dabrafenib-trametinib, in 2017, and encorafenib-binimetinib, in 2023, signifies a breakthrough for the management of BRAFV600E-mutant NSCLC patients. However, the almost universal emergence of acquired resistance limits their clinical benefit. New RAF and MEK inhibitors, with distinct biochemical characteristics, are in preclinical and clinical development. In this review, we aim to provide valuable insights into the current state of RAF and MEK inhibition in the management of NSCLC, fostering a deeper understanding of the potential impact on patient outcomes.

Mechanism of noise induced hidden hearing loss based on proteomics

Objective: To explore the mechanism of noise-induced hidden hearing loss by proteomics. Methods: In October 2022, 64 SPF male C57BL/6J mice were divided into control group and noise exposure group with 32 mice in each group according to random sampling method. The noise exposure group was exposed to 100 dB sound pressure level, 2000-16000 Hz broadband noise for 2 h, and the mouse hidden hearing loss model was established. Auditory brainstem response (ABR) was used to test the change of hearing threshold of mice on the 7th day after noise exposure, the damage of basal membrane hair cells was observed by immunofluorescence, and the differentially expressed proteins in the inner ear of mice in each group were identified and analyzed by 4D-Label-free quantitative proteomics, and verified by Western blotting. The results were statistically analyzed by ANOVA and t test. Results: On the 7th day after noise exposure, there was no significant difference in hearing threshold between the control group and the noise exposure group at click and 8000 Hz acoustic stimulation (P>0.05) . The hearing threshold in the noise exposure group was significantly higher than that in the control group under 16000 Hz acoustic stimulation (P<0.05) . Confocal immunofluorescence showed that the basal membrane hair cells of cochlear tissue in noise exposure group were arranged neatly, but the relative expression of C-terminal binding protein 2 antibody of presynaptic membrane in middle gyrus and basal gyrus was significantly lower than that in control group (P<0.05) . GO enrichment analysis showed that the functions of differentially expressed proteins were mainly concentrated in membrane potential regulation, ligand-gated channel activity, and ligand-gated ion channel activity. KEGG pathway enrichment analysis showed that differentially expressed proteins were significantly enriched in phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling pathway, NOD-like receptor signaling pathway, calcium signaling pathway, etc. Western blotting showed that the expression of inositol 1, 4, 5-trisphosphate receptor 3 (Itpr3) was increased and the expression of solute carrier family 38 member 2 (Slc38a2) was decreased in the noise exposure group (P<0.05) . Conclusion: Through proteomic analysis, screening and verification of the differential expression proteins Itpr3 and Slc38a2 in the constructed mouse noise-induced hidden hearing loss model, the glutaminergic synaptic related pathways represented by Itpr3 and Slc38a2 may be involved in the occurrence of hidden hearing loss.

Screening and identification of lncRNAs in preadipocyte differentiation in sheep

Studies of preadipocyte differentiation and fat deposition in sheep have mainly focused on functional genes, and with no emphasis placed on the role that long non-coding RNAs (lncRNAs) may have on the activity of those genes. Here, the expression profile of lncRNAs in ovine preadipocyte differentiation was investigated and the differentially expressed lncRNAs were screened on day 0 (D0), day 2(D2) and day 8(D8) of ovine preadipocyte differentiation, with their target genes being predicted. The competing endogenous RNA (ceRNA) regulatory network was constructed by GO and KEGG enrichment analysis for functional annotation, and some differentially expressed lncRNAs were randomly selected to verify the RNA-Seq results by RT-qPCR. In the study, a total of 2517 novel lncRNAs and 3943 known lncRNAs were identified from ovine preadipocytes at the three stages of differentiation, with the highest proportion being intergenic lncRNAs. A total of 3455 lncRNAs were expressed at all three stages of preadipocyte differentiation, while 214, 226 and 228 lncRNAs were uniquely expressed at day 0, day 2 and day 8, respectively. By comparing the expression of the lncRNAs between the three stages of differentiation stages, a total of 405, 272 and 359 differentially expressed lncRNAs were found in D0-vs-D2, D0-vs-D8, and D2-vs-D8, respectively. Functional analysis revealed that the differentially expressed lncRNAs were enriched in signaling pathways related to ovine preadipocyte differentiation, such as mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase protein kinase B (PI3K-Akt) pathway, and the transforming growth factor beta (TGF-β) pathway. In summary, lncRNAs from preadipocytes at different stages of differentiation in sheep were identified and screened using RNA-Seq technology, and the regulatory mechanisms of lncRNAs in preadipocyte differentiation and lipid deposition were explored. This study provides a theoretical reference for revealing the roles of lncRNAs in ovine preadipocyte differentiation and also offers a theoretical basis for further understanding the regulatory mechanisms of ovine preadipocyte differentiation.

Oxidative Stress Promotes Liver Cancer Metastasis via RNF25-Mediated E-Cadherin Protein Degradation.

Loss of E-cadherin (ECAD) is required in tumor metastasis. Protein degradation of ECAD in response to oxidative stress is found in metastasis of hepatocellular carcinoma (HCC) and is independent of transcriptional repression as usually known. Mechanistically, protein kinase A (PKA) senses oxidative stress by redox modification in its β catalytic subunit (PRKACB) at Cys200 and Cys344. The activation of PKA kinase activity subsequently induces RNF25 phosphorylation at Ser450 to initiate RNF25-catalyzed degradation of ECAD. Functionally, RNF25 repression induces ECAD protein expression and inhibits HCC metastasis in vitro and in vivo. Altogether, these results indicate that RNF25 is a critical regulator of ECAD protein turnover, and PKA is a necessary redox sensor to enable this process. This study provides some mechanistic insight into how oxidative stress-induced ECAD degradation promotes tumor metastasis of HCC.

MiRNA profiling of granulosa cell-derived exosomes reveals their role in promoting follicle development.

To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exoscould serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis.GC-Exoswere characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.

FGF4 protects the liver from immune-mediated injury by activating CaMKKβ-PINK1 signal pathway to inhibit hepatocellular apoptosis

Immune-mediated liver injury (ILI) is a condition where an aberrant immune response due to various triggers causes the destruction of hepatocytes. Fibroblast growth factor 4 (FGF4) was recently identified as a hepatoprotective cytokine; however, its role in ILI remains unclear. In patients with autoimmune hepatitis (type of ILI) and mouse models of concanavalin A (ConA)- or S-100-induced ILI, we observed a biphasic pattern in hepatic FGF4 expression, characterized by an initial increase followed by a return to basal levels. Hepatic FGF4 deficiency activated the mitochondria-associated intrinsic apoptotic pathway, aggravating hepatocellular apoptosis. This led to intrahepatic immune hyper-reactivity, inflammation accentuation, and subsequent liver injury in both ILI models. Conversely, administration of recombinant FGF4 reduced hepatocellular apoptosis and rectified immune imbalance, thereby mitigating liver damage. The beneficial effects of FGF4 were mediated by hepatocellular FGF receptor 4, which activated the Ca2+/calmodulin-dependent protein kinasekinase 2 (CaMKKβ) and its downstream phosphatase and tensin homologue-induced putative kinase 1 (PINK1)-dependent B-cell lymphoma 2-like protein 1-isoform L (Bcl-XL) signalling axis in the mitochondria. Hence, FGF4 serves as an early response factor and plays a protective role against ILI, suggesting a therapeutic potential of FGF4 and its analogue for treating clinical immune disorder-related liver injuries.

Single cell RNA sequencing reveals emergent notochord-derived cell subpopulations in the postnatal nucleus pulposus.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-betaand phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

Analysis of Chromatin Accessibility Changes Induced by BMMC Recognition of Foot-and-Mouth Disease Virus-like Particles through ATAC-seq

Mast cells can recognize foot-and-mouth disease virus-like particles (FMDV-VLPs) via mannose receptors (MRs) to produce differentially expressed cytokines. The regulatory role of chromatin accessibility in this process is unclear. Bone marrow-derived mast cells (BMMCs) were cultured, and an assay of transposase-accessible chromatin sequencing (ATAC-seq) was applied to demonstrate the regulation of chromatin accessibility in response to the BMMCs’ recognition of FMDV-VLPs. A pathway enrichment analysis showed that peaks associated with the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), and other signaling pathways, especially the NF-κB pathway, were involved in the BMMCs’ recognition of VLPs. Moreover, transcription factors including SP1, NRF1, AP1, GATA3, microphthalmia-associated transcription factor (MITF), and NF-κB-p65 may bind to the motifs with altered chromatin accessibility to regulate gene transcription. Furthermore, the expression of NF-κB, interleukin (IL)-9, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the BMMCs of the VLP group increased compared with that of the BMMCs in the control group, whereas the expression of IL-10 did not differ significantly between groups. After inhibiting the MRs, the expression of NF-κB, IL-9, TNF-α, and IFN-γ decreased significantly, whereas the expression of IL-10 increased. The expression of MAPK and IL-6 showed no significant change after MR inhibition. This study demonstrated that MRs expressed on BMMCs can affect the NF-κB pathway by changing chromatin accessibility to regulate the transcription of specific cytokines, ultimately leading to the differential expression of cytokines. These data provide a theoretical basis and new ideas for the development of a novel vaccine for FMD.

Cytochrome c regulates hyphal morphogenesis by interfering with cAMP-PKA signaling in Candida albicans

In the human fungal pathogen Candida albicans, invasive hyphal growth is a well-recognized virulence trait. We employed transposon-mediated genome-wide mutagenesis, revealing that inactivating CTM1 blocks hyphal growth. CTM1 encodes a lysine (K)methyltransferase, which trimethylates cytochrome c (Cyc1) at K79. Mutants lacking CTM1 or expressing cyc1K79A grow as yeast under hyphae-inducing conditions, indicating that unmethylated Cyc1 suppresses hyphal growth. Transcriptomic analyses detected increased levels of the hyphal repressor NRG1 and decreased levels of hyphae-specific genes in ctm1Δ/Δ and cyc1K79A mutants, suggesting cyclic AMP (cAMP)-protein kinase A (PKA) signaling suppression. Co-immunoprecipitation and invitro kinase assays demonstrated that unmethylated Cyc1 inhibits PKA kinase activity. Surprisingly, hyphae-defective ctm1Δ/Δ and cyc1K79A mutants remain virulent in mice due to accelerated proliferation. Our results unveil a critical role for cytochrome c in maintaining the virulence of C.albicans by orchestrating proliferation, growth mode, and metabolism. Importantly, this study identifies a biological function for lysine methylation on cytochrome c.

Anti-inflammatory activity of calmodulin-lysine N-methyltransferase through suppressing the caspase-11 non-canonical inflammasome

Calmodulin (CaM)-lysine N-methyltransferase (CAMKMT) is a novel methyltransferase that catalyzes lysine trimethylation in CaM. However, its specific roles in inflammatory responses and diseases remain unclear. In this study, we investigated the effects of CAMKMT on caspase-11 non-canonical inflammasomes. CAMKMT expression levels were significantly decreased during inflammatory responses activated by caspase-11 non-canonical inflammasome in macrophages. Moreover, CaM lysine trimethylation was markedly inhibited, but no change was observed in CaM expression during these inflammatory responses in macrophages. Activation of the CaM downstream effectors, CaM-dependent proteinkinase kinase 2 and CaM-dependent proteinkinase type IV, was also inhibited during inflammatory responses activated by caspase-11 non-canonical inflammasome in macrophages. Notably, forced expression of CAMKMT restrained caspase-11 non-canonical inflammasome activation via inhibiting proteolytic activation of caspase-11 and gasdermin D (GSDMD), which in turn suppressed pyroptosis and the release of interleukin (IL)-1β and IL-18 in macrophages. Finally, an in vivo study revealed that CAMKMT ameliorated lipopolysaccharide (LPS)-stimulated acute lethal sepsis in mice by increasing the survival rate and reducing the serum levels of IL-1 β. These findings suggest CAMKMT as a novel methyltransferase that plays an anti-inflammatory role through restraining caspase-11 non-canonical inflammasome in macrophages.

Maternal taurine as a modulator of Cl- homeostasis as well as of glycine/GABAA receptors for neocortical development.

During brain and spinal cord development, GABA and glycine, the inhibitory neurotransmitters, cause depolarization instead of hyperpolarization in adults. Since glycine and GABAA receptors (GABAARs) are chloride (Cl-) ion channel receptor, the conversion of GABA/glycine actions during development is influenced by changes in the transmembrane Cl- gradient, which is regulated by Cl- transporters, NKCC1 (absorption) and KCC2 (expulsion). In immature neurons, inhibitory neurotransmitters are released in a non-vesicular/non-synaptic manner, transitioning to vesicular/synaptic release as the neuron matures. In other word, in immature neurons, neurotransmitters generally act tonically. Thus, the glycine/GABA system is a developmentally multimodal system that is required for neurogenesis, differentiation, migration, and synaptogenesis. The endogenous agonists for these receptors are not fully understood, we address taurine. In this review, we will discuss about the properties and function of taurine during development of neocortex. Taurine cannot be synthesized by fetuses or neonates, and is transferred from maternal blood through the placenta or maternal milk ingestion. In developing neocortex, taurine level is higher than GABA level, and taurine tonically activates GABAARs to control radial migration as a stop signal. In the marginal zone (MZ) of the developing neocortex, endogenous taurine modulates the spread of excitatory synaptic transmission, activating glycine receptors (GlyRs) as an endogenous agonist. Thus, taurine affects information processing and crucial developmental processes such as axonal growth, cell migration, and lamination in the developing cerebral cortex. Additionally, we also refer to the possible mechanism of taurine-regulating Cl- homeostasis. External taurine is uptake by taurine transporter (TauT) and regulates NKCC1 and KCC2 mediated by intracellular signaling pathway, with-no-lysine kinase 1 (WNK1) and its subsequent kinases STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Through the regulation of NKCC1 and KCC2, mediated by the WNK-SPAK/OSR1 signaling pathway, taurine plays a role in maintaining Cl- homeostasis during normal brain development.

Activation of endoplasmic reticulum stress by harmine suppresses the growth of esophageal squamous cell carcinoma.

The worldwide overall 5-year survival rate of esophageal squamous cell carcinoma (ESCC) patients is less than 20%, and novel therapeutic strategies for these patients are urgently needed. Harmine is a natural β-carboline alkaloid, which received great interest in cancer research because of its biological and anti-tumor activities. The aim of this study is to examine the effects of harmine on ESCC and its mechanism. We investigated the effects of harmine on proliferation, cell cycle, apoptosis, and tumor growth in vivo. RNA sequencing (RNA-seq), real-time PCR, and western blotting were used to detect the mechanism. Harmine inhibited ESCC cell growth in vitro and tumor growth in vivo. Differentially expressed genes in harmine-treated ESCC cells were mainly involved in protein processing in the endoplasmic reticulum (ER). Real-time PCR and western blotting confirmed harmine-induced cellular ER stress. CRISPR-Cas9 knockout of C/EBP homologous protein (CHOP) abolished harmine-induced expression of death receptor 5 and apoptosis. Harmine also induced the expression of CHOP-mediated sestrin-2, which in turn contributes to autophagosome formation via suppressing the AMP-activated protein kinase-protein kinase B-mammalian target of rapamycin signaling pathway. In conclusion, our results demonstrate that harmine inhibits the growth of ESCC through its regulation of ER stress, suggesting that it is a promising candidate for ESCC treatment.

O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer's disease.

Aberrant activation of Extracellular Signal-Regulated Kinase (ERK) signaling is associated with Alzheimer's disease (AD) pathogenesis. For example, enhanced ERK signal activation mediated by Apolipoprotein E4 (APOE4), which is a critical genetic risk factor for AD, increases the transcription of amyloid precursor protein (APP). We hypothesize that O-linked N-acetylglucosamine (O-GlcNAc) regulates the phosphorylation and activation of ERK. O-GlcNAc is a single sugar post-translational modification that dynamically cycles on and off proteins in response to nutrient changes by the action of the enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, O-GlcNAc quickly returns to a baseline level after stimulus removal (called O-GlcNAc homeostasis). We did a serum reactivation time-course followed by western blot in SH-SY5Y neuroblastoma cells after long-term O-GlcNAcase (OGA) inhibition by Thiamet-G (TMG) treatment, O-GlcNAc transferase (OGT) knock-down (KD) and OGA KD. Brain tissues of C57BL6/J mice and 5XFAD Alzheimer's disease mice intra-peritoneally injected with TMG for 1 month and C57BL6/J mice intra-peritoneally injected with TMG for 6 months were also used for western blot. We found that ERK1/2 phosphorylation at Thr 202/Tyr204 and Thr183/Tyr185 (p-ERK) are amplified and hence ERK1/2 are activated after long-term OGA inhibition in SH-SY5Y cells. In addition to pharmacological treatment, genetic disruption of O-GlcNAc by OGT KD and OGA KD also increased p-ERK in SH-SY5Y cells suggesting O-GlcNAc homeostasis controls ERK signaling. To determine how O-GlcNAc regulates p-ERK, we probed the expression of phosphorylated mitogen-activated protein kinase-kinase (p-MEK) which phosphorylates and activates ERK and Dual specificity phosphatase-4 (DUSP4) which dephosphorylates and inactivates ERK in SH-SY5Y cells. p-MEK increases in TMG treated and OGT KD cells whereas total DUSP4 decreases in OGT KD and OGA KD cells with serum reactivation time course. Next, we probed the role of OGA inhibition in regulating ERK activation using mice brain-tissue samples. Interestingly, 6-month intra-peritoneal TMG injection in C57BL/6J mice showed an increase in amplitude of p-ERK and APP protein levels, indicating long-term OGA inhibition potentially contributes to AD progression. Furthermore, 1-month TMG injection was sufficient to increase the amplitude of p-ERK in 5XFAD AD mice brains suggesting AD phenotype contributes to the acceleration of ERK activation mediated by OGA inhibition. Together, these results indicate that disruptions to O-GlcNAc homeostasis amplify ERK signal activation in AD.