Lower Phosphorylation Levels Research Articles

SRPK Inhibitors Reduce the Phosphorylation and Translocation of SR Protein Splicing Factors, thereby Correcting BIN1, MCL-1 and BCL2 Splicing Errors and Enabling Apoptosis of Cholangiocarcinoma Cells.

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium that is commonly found in the Thai population. CCA has poor prognosis and a low survival rate due to the lack of early diagnosis methods and the limited effectiveness of current treatments. A number of oncogenic spliced-transcripts resulting from mRNA splicing errors have been reported in CCA, and aberrant mRNA splicing is suspected to be a key driver of this cancer type. The hyperphosphorylation of serine/arginine rich-splicing factors (SRSFs) by serine/arginine protein kinases (SRPKs) causes them to translocate to the nucleus where they facilitate gene splicing errors that generate cancer-related mRNA/protein isoforms. The correlation between SRPK expression and the survival of CCA patients was analyzed using data from The Cancer Genome Atlas (TCGA) dataset. The effect of SRPK inhibitors (SRPIN340 and SPHINX31) on two CCA cell lines (KKU-213A and TFK-1) was also investigated. The induction of cell death was studied by Calcein-AM/PI staining, AnnexinV/7AAD staining, immunofluorescence (IF), and Western blotting (WB). The phosphorylation and nuclear translocation of SRSFs was tracked by WB and IF, and the repair of splicing errors was examined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). High levels of SRPK1 and SRPK2 transcripts, and in particular SRPK1, correlated with shorter survival in CCA patients. SRPIN340 and SPHINX31 increased the number of dead and apoptotic cells in a dose-dependent manner. CCA also showed diffuse expression of cytoplasmic cytochrome C and upregulation of cleaved caspase-3. Moreover, SRSFs showed low levels of phosphorylation, resulting in the accumulation of cytoplasmic SRSF1. To link these phenotypes with aberrant gene splicing, the apoptosis-associated genes Bridging Integrator 1 (BIN1), Myeloid cell leukemia factor 1 (MCL-1) and B-cell lymphoma 2 (BCL2) were selected for further investigation. Treatment with SRPIN340 and SPHINX31 decreased anti-apoptotic BIN1+12A and increased pro-apoptotic MCL-1S and BCL-xS. The SRPK inhibitors SRPIN340 and SPHINX31 can suppress the phosphorylation of SRSFs and their nuclear translocation, thereby producing BIN1, MCL-1 and BCL2 isoforms that favor apoptosis and facilitate CCA cell death.

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation

AbstractThe inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody–based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain–containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization.

YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors; differentiating between these roles may depend on the YAP1 phosphorylation pattern. The specific function of YAP1 in B cell acute lymphoblastic leukemia (B-ALL), however, is currently unclear. Thus, in the present study, the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets. The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting, quantitative real-time polymerase chain reaction, flow cytometry, immunostaining, and nude mouse subcutaneous tumorigenesis experiments. Gene expression levels of Hippo pathway-related molecules before and after verteporfin (VP) treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells. Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels. YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells; YAP1 was distributed in the nuclei in NALM6 cells. Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase. Before and after VP treatment, the expression of the upstream gene LATS1 was upregulated; its overexpression promoted YAP1-Ser127 phosphorylation. Further, YAP1 was distributed in the plasma. LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function; thus, VP, which targets this axis, may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

Unravelling transcriptomic complexity in breast cancer through modulation of DARPP-32 expression and signalling pathways

DARPP-32 is a key regulator of protein-phosphatase-1 (PP-1) and protein kinase A (PKA), with its function dependent upon its phosphorylation state. We previously identified DKK1 and GRB7 as genes with linked expression using Artificial Neural Network (ANN) analysis; here, we determine protein expression in a large cohort of early-stage breast cancer patients. Low levels of DARPP-32 Threonine-34 phosphorylation and DKK1 expression were significantly associated with poor patient prognosis, while low levels of GRB7 expression were linked to better survival outcomes. To gain insight into mechanisms underlying these associations, we analysed the transcriptome of T47D breast cancer cells following DARPP-32 knockdown. We identified 202 differentially expressed transcripts and observed that some overlapped with genes implicated in the ANN analysis, including PTK7, TRAF5, and KLK6, amongst others. Furthermore, we found that treatment of DARPP-32 knockdown cells with 17β-estradiol or PKA inhibitor fragment (6–22) amide led to the differential expression of 193 and 181 transcripts respectively. These results underscore the importance of DARPP-32, a central molecular switch, and its downstream targets, DKK1 and GRB7 in breast cancer. The discovery of common genes identified by a combined patient/cell line transcriptomic approach provides insights into the molecular mechanisms underlying differential breast cancer prognosis and highlights potential targets for therapeutic intervention.

Characterizing ATM Aberrations in Chronic Lymphocytic Leukemia (CLL): Prognostic Implications and Sensitivity to PARP Inhibition

Introduction ATM mutations (muts) frequently occur in CLL, yet their influence on ATM protein function and DNA break repair is poorly elucidated. Our prior study demonstrated functional hypomorphism of the rare germline variant L2307F in ATM and a higher occurrence of 11q deletions (del(11q)) with ATM germline variants in CLL (Lampson et al, JCO 2023). However, the role of other ATM germline variants in association with del(11q) or somatic muts remains unknown. Here, we analyzed the characteristics of CLL patients (pts) with ATM aberrancies and evaluated ATM function in primary CLL cells. Methods We retrospectively analyzed 278 CLL pts with ATM aberrancy, evaluated by clinical targeted sequencing from 2014 to 2019, as per our recent study (Lampson et al, JCO 2023). ATM, KAP1, and H2AX phosphorylation levels were examined after etoposide or irradiation (IR) exposure, by western blot or flow cytometry, in primary CLL cells with or without ATM aberrancy. We assessed the inhibitory effect of talazoparib, a PARP inhibitor, on CLL proliferation over 14 days using flow cytometry and Cell Trace Violet dye. Results We identified 294 ATM variants, 146 unique (germline N=74, somatic N=72). In CLL pts, 58 had dual ATM allele loss (biallelic group), including del(11q) together with only germline ATM variant(s) (N=29), only somatic mut(s) (N=18), or both (N= 11). 168 pts had ATM muts or variants without del(11q), including only germline (N=137), somatic (N=19), or both (N=12) ( ATM group), and 52 pts had del(11q) alone (11q group). Complex karyotype increased with ATM allele deficiency, particularly with 5+ abnormalities (complex karyotype 5+, biallelic vs 11q vs ATM: 22.6% vs 10.6% vs 5.8%, P=0.002). Unmutated IGHV was significantly higher in the groups with del(11q) versus ATM alone (biallelic vs 11q vs ATM: 85.1% vs 87.8% vs 53.9%, p<0.001). Interestingly, when we compared IGHV status in the ATM alone group with or without somatic ATM muts, unmutated IGHV significantly increased in those with somatic muts ( ATM-somatic vs ATM-germline alone, 81.8% vs 43.6%, p<0.001), and karyotypic complexity tended to be higher in the somatic ATM mut group (P=0.077). This might suggest that pts with ATM somatic muts have clinical characteristics and pathogenicity more similar to del(11q) as compared to those with germline muts. SF3B1 muts were more common in those with del(11q) (biallelic vs 11q vs ATM: 20.7% vs 26.9% vs 12.5%, P=0.036). For functional evaluation, we compared the five groups of primary CLL samples: del(11q) with somatic ATM mut with/without germline (11q+ ATM-S), del(11q) alone with no ATM muts (11q), somatic ATM muts without del(11q) with/without germline ( ATM-S), germline ATM muts alone ( ATM-G), and no ATM aberrancies (wild type, WT). Western blot demonstrated significantly lower phosphorylated protein levels (pATM, pKAP1, γH2AX) in response to DNA damage with etoposide in ATM aberrancy groups compared to WT (WT vs 11q, vs ATM-S, vs 11q+ ATM-S, all P<0.001 except for WT vs 11q in γH2AX, P<0.05) (Figure 1A). Flow cytometry also showed significantly lower phosphorylation levels of ATM after IR in 11q, ATM-S and 11q+ ATM-S groups compared to WT, while ATM-G group didn't show significance. To assess ATM deficient cell sensitivity to talazoparib, we compared proliferation inhibition across groups cocultured with talazoparib for 14 days. The 11q+ ATM-S group exhibited significantly greater sensitivity to talazoparib at lower dosage compared to the WT (talazoparib 0.1 μM, P<0.05; 0.5 μM, P=0.098) (Figure 1B). The monoallelic ATM deficient groups (del11q and ATM-G) tended to have lower proliferation, but not significantly. Higher concentrations of talazoparib (2.5μM) strongly inhibited proliferation of all CLLs including WT. Conclusions Pts with ATM somatic aberrancy including del11q had worse associated prognostic factors including unmutated IGHV and complex karyotype. Somatic events showed greater functional impact than some germline variants. CLL cells with ATM aberrancies had ATM pathway dysfunction, and those with biallelic ATM aberrancies were more sensitive to the PARP inhibitor, talazoparib, than WT. This work could potentially lead to additional avenues for treatment based on differential sensitivities of CLLs with ATM aberrancy. Further studies are needed to characterize specific ATM germline variants with or without somatic ATM events.

All-Trans-Retinoic Acid Induction Overcomes Immune Evasion through Downregulating Immune Checkpoint Molecules and Inducing a Cytokine Storm Triggered By Overexpression of S100A8/A9 Signaling

Background: More than 95% of acute promyelocytic leukemia (APL) patients can achieve complete remission by dual induction of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). However, early death is a treatment obstacle of APL. The main causes of early death in APL include hemorrhage, differentiation syndrome (DS) related to induction therapy, and infection. Currently, the mechanism of DS in APL largely remains unclear. This study aims to investigate the pathogenetic mechanism and treatment pattern for APL DS and find a suitable biomarker for DS using multiomics approaches. Methods: Eighty newly diagnosed APL patients were recruited through Jan 2019 to Jan 2021 from a clinical trial (NCT04446806). Matched RNA and plasma samples of APL at presentation and DS were studied by RNA-seq and Human Cytokine Array. Two matched peripheral blood samples at presentation and DS from 2 severe DS patients were selected for 10×scRNA-seq analysis, compared with 3 peripheral blood and 2 bone marrow controls. Results: The incidence of DS was 35.3% (29/80), including 21.3% (17/80) moderate DS and 15% (12/80) severe DS. Bulk RNA-seq suggested immune response and cytokines interaction might play important roles in the development of DS ( Figure 1A and B). Significant downregulation of immune checkpoint molecules was shown in the DS period, such as GAL-9, which plays a crucial role in regulating the immune response ( Figure 1C). The activated CD4 +T and CD8 +T cells were amplification at diagnosis period compared with the corresponding CR/CRi period, then declined dramatically at DS period ( Figure 1 D). 10×scRNA-seq analysis revealed the activation of neutrophils and monocytes through innate immune response and inflammatory response ( Figure 1 E). Consistent with bulk RNA-seq results, apparent elevated expression of multiple inflammatory storm-related cytokines/receptors are shown in the DS period in monocytes and neutrophils, which indicates they contribute to the inflammatory cytokine storm ( Figure 1 F). These results indicated that ATRA induction overcomes immune evasion and induces highly activated immune response through downregulating immune checkpoint molecules. Based on 10×scRNA-seq data, differentiated promyelocytic leukemic cell, neutrophils and monocytes are major cell populations and expressed significantly higher cytokine and chemokine in DS stage, indicating that these cells might be major sources of the cytokine storm ( data not shown). We found S100A8 and S100A9, which function as danger-associated molecular pattern (DAMP) molecules and activate innate immune responses via binding to PRRs, significantly upregulated among all known alarmins ( Figure 1G). S100A8/A9 can be induced by ATRA induction, and also activate ERK, JAK-STAT and NF-κB signaling pathways ( Figure 1H). S100A8/A9 expression can be repressed by ERK inhibitor ( Figure 1I). Knockdown of S100A9 induced lower phosphorylation level of STAT1 and P65 ( Figure 1J). These results indicated S100A8/A9 were regulated by the ERK signaling and controlled the JAK-STAT and NF-κB signaling pathway. As a result, JAK-STAT related cytokines could be regulated by S100A8/A9 signaling. Importantly, ROC analysis showed that S100A8/A9 displayed both high sensitivity and specificity as a biomarker for predicting APL DS ( Figure 1K). According to clinical and cytokine storm characteristics of APL DS patients, Dexamethasone (DEX) and Ruxolitinib (RUX) were applied in the treatment of APL DS patients. The percentage of severe DS reduced remarkably after DEX prevention ( Figure 1L, P<0.05). The early death rate of APL declined under the DEX prevention and RUX treatment, and no patient died due to DS ( Figure 1M). The expression of a series of inflammatory cytokines dramatically decreased, indicating that the cytokine storm was effectively suppressed by the treatment of DEX and/or RUX ( Figure 1N). Conclusion s : Our data indicate that ATRA induction could overcome immune evasion through downregulating immune checkpoint molecules. ATRA-induced upregulation of S100A8/A9 may contribute to the cytokine storm observed in the DS stage of APL patients by activating the JAK-STAT and NF-κB signaling pathway. S100A8/A9 is recommended as a biomarker for predicting APL DS. Dexamethasone and Ruxolitinib are appropriate prevention and treatment pattern for APL DS patients.

Effects of muscle-specific oxidative stress on protein phosphorylation and its relationship with mitochondrial dysfunction, muscle oxidation, and apoptosis.

This study aimed to investigate the effects of muscle-specific oxidative stress on phosphorylation and its relationship with mitochondrial dysfunction, muscle oxidation, and apoptosis of porcine PM (psoas major) and LL (longissimus lumborum) during the first 24h postmortem. The global phosphorylation level decreased and the mitochondrial dysfunction, oxidation level, and apoptosis increased significantly at 12h postmortem compared with 2h postmortem, suggesting that lower phosphorylation level was related to more mitochondrial dysfunction and apoptosis during the early postmortem, regardless of muscle type. PM exhibited a higher global phosphorylation level but showed greater mitochondrial dysfunction, oxidation level, and apoptosis than LL, regardless of aging time. The increased mitochondrial dysfunction and oxidative stress accelerated apoptosis, but their relationship with phosphorylation was different in various muscle types at different aging times. These findings provide insight regarding the roles of coordinated regulation of phosphorylation and apoptosis in development of quality of different muscles.

Phosphorylation promoted the glycolytic rate in postmortem meat by tightening the structure of glycogen phosphorylase

AbstractThe activity and structural variation of glycogen phosphorylase (GP) at different phosphorylation levels during incubation at 4 °C were explored in this study. The GP was assigned into four treatments to obtain high/low phosphorylation levels, which were (1) treated with glycogen phosphorylase kinase (Phk) to obtain high phosphorylation level, (2) treated with protein kinase A to obtain high phosphorylation level, (3) treated with alkaline phosphatase to obtain low phosphorylation level, and (4) control. Compared with the control group, the content of α-helix and β-sheet increased and the secondary structure of GP changed from disorder to order after phosphorylation. The activity of GP was increased and its structure was more tightly in the Phk group than that in the control group. The phosphorylation at Ser277, Ser430, Ser809, Thr304, Tyr298, and Tyr525 resulted in tighter spatial structure. In conclusion, phosphorylation of GP enhanced its catalytic activity by making the secondary and spatial structure more orderly, which is of great significance for controlling meat quality by regulating glycolysis.

Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome

The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is a key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following four different types ofexperience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3938 proteins and 2761 phosphoproteins in the sequential strategy covering a total of 4968 unique protein groups (at least two peptides including a unique peptide). On the phosphoproteins, we identified a total of 6188 unambiguous phosphosites (75%<site-localization probability). Strikingly, of the significantly IA-regulated phosphoproteins, a large fraction of these displayed an overall decrease in phosphorylation level. Bioinformatic analysis of proteins and phosphoproteins that were regulated by IA were annotated for involvement in the regulation of glutamate receptor functionality, RHO GTPase cycle, and synaptic plasticity. We also identified synaptic kinases, phosphatases, and their respective phosphosites regulated by IA training or immediate shock. Furthermore, we found that AMPA receptor surface expression was regulated by Mg2+/Mn2+ dependent protein phosphatase 1H (Ppm1h). Together, these results unravel the dynamic remodeling ofthe PSD upon IA learning or immediate shock and serve asa resource for elucidating the synaptic proteome dynamics induced by experience-dependent plasticity.

A highly efficient human cell-free translation system.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

Novel 5-aminopyrazoles endowed with anti-angiogenetic properties: Design, synthesis and biological evaluation

The promising anti-angiogenetic properties of previously synthesized pyrazolyl ureas provided the rationale for the synthesis of novel 5-aminopyrazoles 2–5, differently decorated on the pyrazole nucleus. All the derivatives were tested by MTT assays and proved to be non-cytotoxic against eight different tumor cell lines and normal fibroblasts. An EdU proliferation assay was carried out on human foreskin fibroblasts and VEGF stimulated human umbilical vein endothelial cells which confirmed the absence of cytotoxicity of the compounds on human cells up to 20 μM concentration. To evaluate the influence of the newly synthesized pyrazoles on MAPK and PI3K signaling pathways, the phosphorylation of ERK1/2 and Akt was analyzed by Western blots from HFF and HUVEC cell lysates stimulated with growth factors in the presence or absence of the compounds. Pyrazoles 3b and 3c showed a significant inhibition of Akt phosphorylation in both tested cell lines with lower phosphorylation levels than the reference compound GeGe-3 in HUVEC. Furthermore, derivatives 2 and 3 appeared to strongly affect the migration of HFF cells in a wound healing assay, confirming their potential ability to interfere with the angiogenesis process. The new pyrazole library extends the structure-activity relationships of the previously isolated compounds and highlights the attractiveness of this chemical class for pathological cell migration and angiogenesis.

Lipid kinase PIP5Kα contributes to Hippo pathway activation via interaction with Merlin and by mediating plasma membrane targeting of LATS1

BackgroundThe Hippo pathway plays a critical role in controlled cell proliferation. The tumor suppressor Merlin and large tumor suppressor kinase 1 (LATS1) mediate activation of Hippo pathway, consequently inhibiting the primary effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid present in the plasma membrane (PM), binds to and activates Merlin. Phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) is an enzyme responsible for PIP2 production. However, the functional role of PIP5Kα in regulation of Merlin and LATS1 under Hippo signaling conditions remains unclear.MethodsPIP5Kα, Merlin, or LATS1 knockout or knockdown cells and transfected cells with them were used. LATS1, YAP, and TAZ activities were measured using biochemical methods and PIP2 levels were evaluated using cell imaging. Low/high cell density and serum starvation/stimulation conditions were tested. Colocalization of PIP5Kα and PIP2 with Merlin and LATS1, and their protein interactions were examined using transfection, confocal imaging, immunoprecipitation, western blotting, and/or pull-down experiments. Colony formation and adipocyte differentiation assays were performed.ResultsWe found that PIP5Kα induced LATS1 activation and YAP/TAZ inhibition in a kinase activity-dependent manner. Consistent with these findings, PIP5Kα suppressed cell proliferation and enhanced adipocyte differentiation of mesenchymal stem cells. Moreover, PIP5Kα protein stability and PIP2 levels were elevated at high cell density compared with those at low cell density, and both PIP2 and YAP phosphorylation levels initially declined, then recovered upon serum stimulation. Under these conditions, YAP/TAZ activity was aberrantly regulated by PIP5Kα deficiency. Mechanistically, either Merlin deficiency or LATS1 deficiency abrogated PIP5Kα-mediated YAP/TAZ inactivation. Additionally, the catalytic domain of PIP5Kα directly interacted with the band 4.1/ezrin/radixin/moesin domain of Merlin, and this interaction reinforced interaction of Merlin with LATS1. In accordance with these findings, PIP5Kα and PIP2 colocalized with Merlin and LATS1 in the PM. In PIP5Kα-deficient cells, Merlin colocalization with PIP2 was reduced, and LATS1 solubility increased.ConclusionsCollectively, our results support that PIP5Kα serves as an activator of the Hippo pathway through interaction and colocalization with Merlin, which promotes PIP2-dependent Merlin activation and induces local recruitment of LATS1 to the PIP2-rich PM and its activation, thereby negatively regulating YAP/TAZ activity.11kBwjrDVBy7xy7exwWkZdVideo

The ERBB2 Exonic Variant Pro1170Ala Modulates Mitogen-Activated Protein Kinase Signaling Cascades and Associates with Allergic Asthma

Introduction: Previous studies have indicated the ERBB2 genetic variants in the 17q12 locus might be associated with asthma; however, the functional effects of these variants on asthma risk remain inconclusive. This study aimed to characterize the functional roles of asthma-associated ERBB2 single nucleotide polymorphisms (SNPs) in asthma pathogenesis by performing genetic association and functional analysis studies. Methods: This study belongs to a part of an ongoing Singapore/Malaysia cross-sectional genetics and epidemiological study (SMCSGES). Genotype-phenotype associations were assessed by performing a genotyping assay on n = 4,348 ethnic Chinese individuals from the SMCSGES cohort. The phosphorylation levels of receptors and signaling proteins in the MAPK signaling cascades, including ErbB2, EGFR, and ERK1/2, were compared across the genotypes of asthma-associated SNPs through in vitro and ex vivo approaches. Results: The ERBB2 tag-SNP rs1058808 was significantly associated with allergic asthma, with the allele “G” identified as protective against the disease (adjusted logistic p = 6.56 × 10⁻⁹, OR = 0.625, 95% CI: 0.544–0.718). The allele “G” of rs1058808 resulted in a Pro1170Ala mutation that results in lower phosphorylation levels of ErbB2 in HaCat cells (p < 0.001), whereas the overall ERBB2 mRNA expression and the phosphorylation levels of EGFR remained unaffected. In the SMCSGES cohort, individuals carrying the genotype “GG” of rs1058808 had lower phosphorylated ERK1/2 proteins in the MAPK signaling cascade. A lower phosphorylation level of ERK1/2 was also associated with reduced asthma risk. Conclusions: The present findings highlighted the involvement of a functional exonic variant of ERBB2 in asthma development via modulating the MAPK signaling cascade.

Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain.

Tenderness is an important characteristic of meat quality. Calpastatin and calpain play important roles in meat tenderization. However, it is not clear how phosphorylation affects the regulation of calpastatin on μ-calpain and, consequently, meat tenderness. Calpastatin with high and low phosphorylation levels were obtained in vitro corresponding to the treatments by protein kinase A (PKA) and alkaline phosphatase. Then, calpain was incubated with calpastatin with different phosphorylation levels, and the effect of calpastatin on calpain activity under different phosphorylation levels was analyzed. The results showed that PKA promoted the phosphorylation of calpastatin, and a high phosphorylation level was maintained during incubation. The degradation rate of μ-calpain in AP group was higher than that in the other groups, meaning there was lower inhibition of calpastatin on calpain activity. The degradation of calpastatin was lower and its structure was more stable after phosphorylation. One more serine 133 site of calpastatin was identified in PKA group compared with the other groups. Phosphorylation at serine 133 of calpastatin enhanced its inhibition on calpain activity by maintaining its structural stability, thus inhibiting the tenderization of meat.

Phosphatase regulatory subunit MYPT2 knockout partially compensates for the cardiac dysfunction in mice caused by lack of myosin light chain kinase 3

Cardiac contraction is modulated by the phosphorylation state of myosin regulatory light chain 2 (MLC-2v). The level of MLC-2v phosphorylation is dependent on the opposing activities of MLC kinases and phosphatases. The predominant MLC phosphatase found in cardiac myocytes contains Myosin Phosphatase Targeting Subunit 2 (MYPT2). Overexpression of MYPT2 in cardiac myocytes results in a decreased level of MLC phosphorylation, reduced left ventricular contraction, and induction of hypertrophy; however, the effect of knocking out MYPT2 on cardiac function is unknown. We obtained heterozygous mice containing a MYPT2 null allele from the Mutant Mouse Resource Center. These mice were produced in a C57BL/6N background which lack MLCK3, the main regulatory light chain kinase in cardiac myocytes. We found that mice null for MYPT2 were viable and had no obvious phenotypic abnormality when compared to WT mice. Additionally, we determined that WT C57BL/6N mice had a low basal level of MLC-2v phosphorylation, which was significantly increased when MYPT2 was absent. At 12-weeks, MYPT2 KO mice had smaller hearts and showed downregulation of genes involved in cardiac remodeling. Using cardiac echo, we found that 24-week-old male MYPT2 KO mice had decreased heart size with increased fractional shortening compared to their MYPT2 WT littermates. Collectively, these studies highlight the important role that MYPT2 plays in cardiac function invivo and demonstrate that its deletion can partially compensate for the lack of MLCK3.

Variations in protein levels of the apelinergic system in adipose tissue of hypertensive individuals with class 3 obesity

The aim of this study was to investigate the expression of apelin (APLN) and its receptor (APLNR) in visceral adipose tissue (VAT), and its effect on the downstream expression of endothelial nitric oxide synthase (eNOS) in individuals with class 3 obesity, with or without hypertension. Seventy-five unrelated individuals presenting obesity class 3 with or without hypertension were included. Gene expression of APLN, and APLNR were analyzed in VAT, by reverse transcription quantitative polymerase chain reaction. The APLN, APLNR and eNOS (total and phosphorylated) levels in VAT were evaluated by Western blot. Analysis of differences between groups of APLN, APLNR and eNOS were performed by a logistic regression adjusting by confounding factors. Forty-five individuals with hypertension formed the case group, and 30 individuals constituted the control group. The APLN mRNA and protein levels were higher in the group of individuals with hypertension versus individuals without hypertension (p = 0.027 and p = 0.036, respectively). Meanwhile, APLNR mRNA and protein levels in subjects with hypertension were lower versus the group of subjects without hypertension (p = 0.001 and p = 0.008, respectively). Further, the group with hypertension presented a lower level of phosphorylation of eNOS Ser1177, compared to the control group (p = 0.002). In conclusion, individuals with class 3 obesity and hypertension present a modified APLN/APLNR expression in visceral adipose tissue, which could be secondary to reduced eNOS phosphorylation.

Extracellular DEK Activates NRF2 Antioxidant Targets and Drives Expansion of Functional and ROS-Low Human Hematopoietic Stem Cells

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation (HCT), a life-saving treatment, due to lower incidence of graft versus host disease and its availability. CB HCT is limited by low numbers of HSCs, thus there is a strong need to increase numbers of engraftable HSCs, for instance by expanding functionally competent HSCs ex vivo. We have shown that extracellular DEK protein, an epigenetic regulator that is secreted in response to stress and inflammation, is a novel hematopoietic regulatory chemokine. Treatment with extracellular DEK in growth stimulating media significantly enhances the expansion of engraftable HSCs in mouse bone marrow (BM) and human CB (Capitano, et al. JCI 2019; 129(6):2555-2570). To examine mechanisms driving enhanced expansion of functional HSCs after DEK treatment, we performed RNA-sequencing on CB HSCs, multipotent progenitor cells (MPPs), common myeloid progenitors (CMPs), and granulocyte macrophage progenitors (GMPs) after overnight DEK treatment in expansion media. All cell populations upregulated gene programs related to antioxidant responses upon DEK treatment. Indeed, 7/10 genes upregulated by DEK in all HSC/HPC populations (NMRAL2P, PIR, NQO1, SELENOW, FTL, RIT1, and QPCT) promote antioxidant activity. Overnight DEK treatment significantly reduced mean fluorescence of the activated reactive oxygen species (ROS) indicator CM-H2DCFDA in HSCs, MPPs, CMPs, and GMPs. Thus, DEK may induce antioxidant activity. We next found that all 10 genes commonly upregulated in HSCs/HPCs are either direct transcriptional targets of NRF2 (NMRAL2P, LUCAT1,PIR, NQO1, FTL, and QPCT) or are directly affected by changes in NRF2 activation (SELENOW, EBI3, TNFRSF9, and RIT1). Further, several genes highly upregulated by DEK treatment in HSCs (i.e., CCL2, KYNU, and CXCL8) are NRF2 targets. To examine if DEK activates NRF2 transcriptional activity, we utilized a transgene reporter assay where expression of red fluorescent protein (RFP) is driven by activation of promoter antioxidant response elements (ARE), to which active NRF2 binds. DEK treatment of hematopoietic derived cell lines (mouse MLL-AF9 AML and human HL-60 APL) expressing the transgene significantly increased RFP signal, indicating that DEK could activate NRF2. We further examined whether DEK treatment affects mitochondrial metabolism, a source of ROS and tightly regulated process in hematopoiesis. Seahorse XFe analysis, which measures metabolic flux, showed DEK treatment of MLL-AF9 cells significantly reduced mitochondrial oxygen consumption rate, suggesting a reduced reliance on oxidative phosphorylation as an energy source in DEK treated cells. These data suggest that DEK treatment during expansion of primitive hematopoietic cells drives activation of NRF2 transcription, upregulation of antioxidant response genes, reduction of intracellular ROS levels, and may reduce the mitochondrial metabolic rate. These effects in a sense "mimic” the in vivo niche of HSCs, where they reside in low oxygen tensions, have low intracellular ROS, and exhibit low levels of oxidative phosphorylation. To test this hypothesis, we examined if DEK treatment has similar effects to maintaining HSCs in physiologic oxygen tensions (3%) compared to HSCs exposed to extraphysiologic ambient air (21%), which drives oxidative stress (Mantel, et al. Cell 2015; 161(7):1553-65). Isolating and maintaining mouse BM in physiologic oxygen tensions or treating mice in vivo with recombinant DEK preserved HSC numbers compared to exposing the BM cells to ambient air or treating with vehicle. However, combining in vivo DEK treatment with physiologic isolation of HSCs/HPCs had no additive or synergistic effect, suggesting that the overall effects of DEK treatment and maintaining low oxygen tensions may be similar. Thus, extracellular DEK treatment leads to expansion of functionally competent HSCs and reduction of oxidative stress. These data have important implications for the regulation of HSCs/HPCs in stress conditions, where DEK is most likely to be secreted. Further, DEK may present a novel way to mimic physiologic oxygen conditions for enhancement of functional HSC expansion, and the elucidated mechanisms may be exploited for more efficient targeting of molecular programs critical for functional HSC competency, potentially providing new ways to enhance HCT.

Feeding restriction unalters physiological responses to dietary lipid and carbohydrate levels in juvenile gibel carp (Carassius gibelio)

To investigate the physiological responses to different energy sources under feeding restriction in fish, gibel carp were fed a high lipid, low carbohydrate diet (HL diet, 12% lipid, 16% carbohydrate) or a high carbohydrate, low lipid diet (HC diet, 34% carbohydrate, 4% lipid) to satiation or 70% satiation (restriction) for 56 d; then, growth performance, nutrient metabolism, endoplasmic reticulum (ER) stress, autophagy, and apoptosis were determined. Fish that were fed the HL diet had higher final body weight, specific growth rate, and feed efficiency, lower phosphorylation levels of AKT and mTOR proteins and lower mRNA levels of gk than the HC group. When fed to 70% satiation, fish had lower feed intake, feeding rate, final body weight, and specific growth rate, and lower plasma glucose and nonesterified fatty acid levels. Feeding restriction induced the transcript level of gk and suppressed the mRNA level of acly, acc, fas. There were no significant differences in ER stress, autophagy and apoptosis between feeding levels or diets. Interactions between feeding level and diet were observed in plasma triglyceride level and the gene expression levels of fbpase and srebp1. Thus, gibel carp utilized HL diet better than HC diet, which was unalerted by feeding restriction.

Metabolic and epigenetic dysfunctions underlie the arrest of in vitro fertilized human embryos in a senescent-like state

Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3- to 8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a senescent-like state, marked by cell cycle arrest, the down-regulation of ribosomes and histones and down-regulation of MYC and p53 activity. The arrested embryos can be divided into 3 types. Type I embryos fail to complete the maternal-zygotic transition, and Type II/III embryos have low levels of glycolysis and either high (Type II) or low (Type III) levels of oxidative phosphorylation. Treatment with the SIRT agonist resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype, which is accompanied by changes in metabolic activity. Overall, our data suggests metabolic and epigenetic dysfunctions underlie the arrest of human embryos.