Plausible role of INPP4A dysregulation in idiopathic pulmonary fibrosis.

INPP4A has been shown to be involved in the regulation of cell proliferation and apoptosis of multiple cell types including fibroblasts. Previous reports from our group have demonstrated the role of inositol polyphosphate 4-phosphatase Type I A (INPP4A) in these functions. Though existing evidences suggest a critical role for INPP4A in the maintenance of lung homeostasis, its role in chronic lung diseases is relatively under explored. In the current study, we made an attempt to understand the regulation of INPP4A in idiopathic pulmonary fibrosis (IPF). Through integration of relevant INPP4A gene expression data from public repositories with our results from in vitro experiments and mouse models, we show that INPP4A is altered in IPF. Interestingly, the direction of the change is dependent both on the disease stage and the region of the lung used. INPP4A was found to be upregulated when analyzed in lung sample representative of the whole lung, but was downregulated in the fibrotic regions of the lung. Similarly, INPP4A was found to be high, compared to controls, only in the early stage of the disease. Though the observed increase in INPP4A was found to be negatively correlated to physiological indices, FVC, and DLCO, of lung function, treatment with anti-INPP4A antibody worsened the condition in bleomycin treated mice. These contrasting results taken together are suggestive of a nuanced regulation of INPP4A in IPF which is dependent on the disease stage, cellular state and extent of fibrosis in the lung region being analyzed.

Clinical features and genetic analysis of 15 Chinese children with dent disease.

OBJECTIVE:  The clinical characteristics, genetic mutation spectrum, treatment strategies and prognoses of 15 children with Dent disease were retrospectively analyzed to improve pediatricians' awareness of and attention to this disease. METHODS:  We analyzed the clinical and laboratory data of 15 Chinese children with Dent disease who were diagnosed and treated at our hospital between January 2017 and May 2023 and evaluated the expression of the CLCN5 and OCRL1 genes. RESULTS:  All 15 patients were male and complained of proteinuria, and the incidence of low-molecular-weight proteinuria (LMWP) was 100.0% in both Dent disease 1 (DD1) and Dent disease 2 (DD2) patients. The incidence of hypercalciuria was 58.3% (7/12) and 66.7% (2/3) in DD1 and DD2 patients, respectively. Nephrocalcinosis and nephrolithiasis were found in 16.7% (2/12) and 8.3% (1/12) of DD1 patients, respectively. Renal biopsy revealed focal segmental glomerulosclerosis (FSGS) in 1 patient, minimal change lesion in 5 patients, and small focal acute tubular injury in 1 patient. A total of 11 mutations in the CLCN5 gene were detected, including 3 missense mutations (25.0%, c.1756C > T, c.1166T > G, and c.1618G > A), 5 frameshift mutations (41.7%, c.407delT, c.1702_c.1703insC, c.137delC, c.665_666delGGinsC, and c.2200delG), and 3 nonsense mutations (25.0%, c.776G > A, c.1609C > T, and c.1152G > A). There was no significant difference in age or clinical phenotype among patients with different mutation types (p > 0.05). All three mutations in the OCRL1 gene were missense mutations (c.1477C > T, c.952C > T, and c.198A > G). CONCLUSION:  Pediatric Dent disease is often misdiagnosed. Protein electrophoresis and genetic testing can help to provide an early and correct diagnosis.

Identification of PTPN12 Phosphatase as a Novel Negative Regulator of Hippo Pathway Effectors YAP/TAZ in Breast Cancer.

The Hippo pathway plays crucial roles in governing various biological processes during tumorigenesis and metastasis. Within this pathway, upstream signaling stimuli activate a core kinase cascade, involving MST1/2 and LATS1/2, that subsequently phosphorylates and inhibits the transcriptional co-activators YAP and its paralog TAZ. This inhibition modulates the transcriptional regulation of downstream target genes, impacting cell proliferation, migration, and death. Despite the acknowledged significance of protein kinases in the Hippo pathway, the regulatory influence of protein phosphatases remains largely unexplored. In this study, we conducted the first gain-of-functional screen for protein tyrosine phosphatases (PTPs) regulating the Hippo pathway. Utilizing a LATS kinase biosensor (LATS-BS), a YAP/TAZ activity reporter (STBS-Luc), and a comprehensive PTP library, we identified numerous novel PTPs that play regulatory roles in the Hippo pathway. Subsequent experiments validated PTPN12, a master regulator of oncogenic receptor tyrosine kinases (RTKs), as a previously unrecognized negative regulator of the Hippo pathway effectors, oncogenic YAP/TAZ, influencing breast cancer cell proliferation and migration. In summary, our findings offer valuable insights into the roles of PTPs in the Hippo signaling pathway, significantly contributing to our understanding of breast cancer biology and potential therapeutic strategies.

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

Targeting Moonlighting Enzymes in Cancer.

Moonlighting enzymes are multifunctional proteins that perform multiple functions beyond their primary role as catalytic enzymes. Extensive research and clinical practice have demonstrated their pivotal roles in the development and progression of cancer, making them promising targets for drug development. This article delves into multiple notable moonlighting enzymes, including GSK-3, GAPDH, and ENO1, and with a particular emphasis on an enigmatic phosphatase, PTP4A3. We scrutinize their distinct roles in cancer and the mechanisms that dictate their ability to switch roles. Lastly, we discuss the potential of an innovative approach to develop drugs targeting these moonlighting enzymes: target protein degradation. This strategy holds promise for effectively tackling moonlighting enzymes in the context of cancer therapy.

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased ß-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

Exploring MTH1 inhibitory potential of Thymoquinone and Baicalin for therapeutic targeting of breast cancer.

Cancers frequently have increased ROS levels due to disrupted redox balance, leading to oxidative DNA and protein damage, mutations, and apoptosis. The MTH1 protein plays a crucial role by sanitizing the oxidized dNTP pools. Hence, cancer cells rely on MTH1 to prevent the integration of oxidized dNTPs into DNA, preventing DNA damage and allowing cancer cell proliferation. We have discovered Thymoquinone (TQ) and Baicalin (BC) as inhibitors of MTH1 using combined docking and MD simulation approaches complemented by experimental validations via assessing binding affinity and enzyme inhibition. Docking and MD simulations studies revealed an efficient binding of TQ and BC to the active site pocket of the MTH1, and the resultant complexes are appreciably stable. Fluorescence measurements estimated a strong binding affinity of TQ and BC with Ka 3.4 ×106 and 1.0 ×105, respectively. Treating breast cancer cells with TQ and BC significantly inhibited the growth and proliferation (IC50 values 28.3 µM and 34.8 µM) and induced apoptosis. TQ and BC increased the ROS production in MCF7 cells, imposing substantial oxidative stress on cancer cells and leading to cell death. Finally, TQ and BC are proven strong MTH1 inhibitors, offering promising prospects for anti-cancer therapy.

Boron nanosheets as a phosphatase mimicking nanozyme with ultrahigh catalytic activity for prodrug-based cancer therapy.

Boron nanosheets (BNSs) are reported as a new phosphatase mimicking nanozyme. Surprisingly, the catalytic rate of BNSs is up to 17 times those of known phosphatase mimicking nanozymes. By adding polyols and Lewis bases, the catalytic activity of BNSs was attributed to the Lewis acidity of the B centers of the BNSs. Theoretical investigation shows that the B centers are responsible for the catalytic hydrolysis of phosphoesters. Moreover, the biomimetic activity of the BNSs was further explored for enhancing anticancer therapy through nanozyme-catalyzed prodrug conversion.

Autoantibodies-Abzymes with Phosphatase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of MS (multiple sclerosis) evolution are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis simulating human MS) in C57BL/6 mice occurs due to the violation of bone marrow hematopoietic stem cell differentiation profiles, leading to the production of toxic for human autoantibody splitting MBP (myelin basic protein), MOG (mouse oligodendrocyte glycoprotein), five histones, DNA, and RNA. Here, we first analyzed the changes in the relative phosphatase activity of IgGs from C57BL/6 mice blood over time, corresponding to three stages of EAE: onset, acute, and remission. Antibodies have been shown to catalyze the hydrolysis of p-nitrophenyl phosphate at several optimal pH values, mainly in the range of 6.5-7.0 and 8.5-9.5. During the spontaneous development of EAE, the most optimal value is pH 6.5. At 50 days after the birth of mice, the phosphatase activity of IgGs at pH 8.8 is 1.6-fold higher than at pH 6.5. During spontaneous development of EAE from 50 to 100 days, an increase in phosphatase activity is observed at pH 6.5 but a decrease at pH 8.8. After mice were immunized with DNA-histone complex by 20 and 60 days, phosphatase activity increased respectively by 65.3 and 109.5 fold (pH 6.5) and 128.4 and 233.6 fold (pH 8.8). Treatment of mice with MOG at the acute phase of EAE development (20 days) leads to a maximal increase in the phosphatase activity of 117.6 fold (pH 6.5) and 494.7 fold (pH 8.8). The acceleration of EAE development after mice treatment with MOG and DNA-histone complex results in increased production of lymphocytes synthesizing antibodies with phosphatase activity. All data show that IgG phosphatase activity could be essential in EAE pathogenesis.

Distinct early role of PTEN regulation during HCMV infection of monocytes.

Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to ß1/ß3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.

Cancer-Related Mutations in the Sam Domains of EphA2 Receptor and Ship2 Lipid Phosphatase: A Computational Study.

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.

Autocrine phosphatase PDP2 inhibits ferroptosis by dephosphorylating ACSL4 in the Luminal A Breast Cancer.

Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p < 0.001), as indicated by data from flow cytometry detection of membrane-permeability cell death stained with 7-aminoactinomycin, lipid peroxidation, and Fe2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.

Phosphatase-degradable nanoparticles providing sustained drug release.

AIM: The study aimed to develop enzyme-degradable nanoparticles comprising polyphosphates and metal cations providing sustained release of the antibacterial drug ethacridine (ETH). METHODS: Calcium polyphosphate (Ca-PP), zinc polyphosphate (Zn-PP) and iron polyphosphate nanoparticles (Fe-PP NPs) were prepared by co-precipitation of sodium polyphosphate with cations and ETH. Developed nanocarriers were characterized regarding particle size, PDI, zeta potential, encapsulation efficiency and drug loading. Toxicological profile of nanocarriers was assessed via hemolysis assay and cell viability on human blood erythrocytes and HEK-293 cells, respectively. The enzymatic degradation of NPs was evaluated in presence of alkaline phosphatase (ALP) monitoring the release of monophosphate, shift in zeta potential and particle size as well as drug release. The antibacterial efficacy against Escherichia coli was determined via microdilution assay. RESULTS: NPs were obtained in a size range between 300 - 480 nm displaying negative zeta potential values. Encapsulation efficiency was in the range of 83.73 %- 95.99 %. Hemolysis assay underlined sufficient compatibility of NPs with blood cells, whereas drug and NPs showed a concentration dependent effect on HEK-293 cells viability. Ca- and Zn-PP NPs exhibited remarkable changes in zeta potential, particle size, monophosphate and drug release upon incubation with ALP, compared to Fe-PP NPs showing only minor differences. The released ETH from Ca- and Zn-PP nanocarriers retained the antibacterial activity against E. coli, whereas no antibacterial effect was observed with Fe-PP NPs. CONCLUSION: Polyphosphate nanoparticles cross-linked with divalent cations and ETH hold promise for sustained drug delivery triggered by ALP for parental administration.

INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.

BACKGROUND: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function. METHODS: The EGFP-2xP4MSidM, PHPLCδ1-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ. RESULTS: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP. CONCLUSIONS: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.

Differential effects of the lipidic and ionic microenvironment on NPP1's phosphohydrolase and phosphodiesterase activities.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by producing pyrophosphate (PPi) from adenosine triphosphate (ATP). Here, we have used liposomes bearing dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), and cholesterol (Chol) harboring NPP1 to mimic the composition of MV lipid rafts to investigate ionic and lipidic influence on NPP1 activity and mineral propagation. Atomic force microscopy (AFM) revealed that DPPC-liposomes had spherical and smooth surface. The presence of SM and Chol elicited rough and smooth surface, respectively. NPP1 insertion produced protrusions in all the liposome surface. Maximum phosphodiesterase activity emerged at 0.082 M ionic strength, whereas maximum phosphomonohydrolase activity arose at low ionic strength. Phosphoserine-Calcium Phosphate Complex (PS-CPLX) and amorphous calcium-phosphate (ACP) induced mineral propagation in DPPC- and DPPC:SM-liposomes and in DPPC:Chol-liposomes, respectively. Mineral characterization revealed the presence of bands assigned to HAp in the mineral propagated by NPP1 harbored in DPPC-liposomes without nucleators or in DPPC:Chol-liposomes with ACP nucleators. These data show that studying how the ionic and lipidic environment affects NPP1 properties is important, especially for HAp obtained under controlled conditions in vitro.

Deficiency of INPP4A promotes M2 macrophage polarization in eosinophilic chronic rhinosinusitis with nasal polyps.

OBJECTIVE: The treatment of eosinophilic chronic rhinosinusitis with nasal polyps (E-CRSwNP) remains a challenge due to its complex pathogenesis. Inositol polyphosphate-4-phosphatase type IA (INPP4A), a lipid phosphatase, has been implicated in allergic asthma. However, the expression and function of INPP4A in E-CRSwNP remain unclear. This study aims to investigate the role of INPP4A in macrophages in E-CRSwNP. METHODS: We assessed the expression of INPP4A in human and mouse nasal mucosal tissues via immunofluorescence staining. THP-1 cells were cultured and exposed to various cytokines to investigate the regulation of INPP4A expression and its functional role. Additionally, we established a murine nasal polyp (NP) model and administrated an INPP4A-overexpressing lentivirus evaluate its impact on NP. RESULTS: The percentage of INPP4A + CD68 + macrophages among total macrophages decreased in the E-CRSwNP group compared to the control and the non-eosinophilic CRSwNP (NE-CRSwNP) groups, exhibiting an inverse correlation with an increased percentage of CD206 + CD68 + M2 macrophages among total macrophages. Overexpression of INPP4A led to a reduced percentage of THP-1 cells polarizing towards the M2 phenotype, accompanied by decreased levels of associated chemotactic factors including CCL18, CCL22, CCL24, and CCL26. We also validated the involvement of the PI3K-AKT pathway in the function of INPP4A in vitro. Furthermore, INPP4A overexpression in the murine NP model resulted in the attenuation of eosinophilic inflammation in the nasal mucosa. CONCLUSIONS: INPP4A deficiency promotes macrophage polarization towards the M2 phenotype, leading to the secretion of chemokines that recruit eosinophils and Th2 cells, thereby amplifying eosinophilic inflammation in E-CRSwNP. INPP4A may exert a suppressive role in eosinophilic inflammation and could potentially serve as a novel therapeutic strategy.

PP4R1 promotes glycolysis and gallbladder cancer progression through facilitating ERK1/2 mediated PKM2 nuclear translocation.

Gallbladder cancer (GBC) is a common solid tumor of the biliary tract with a high mortality rate and limited curative benefits from surgical resection. Here, we aimed to elucidate the pathogenesis of GBC from the perspective of molecular mechanisms and determined that protein phosphatase 4 regulator subunit 1 (PP4R1) is overexpressed in GBC tissues and contributes to poor prognosis. Through a series of in vitro and in vivo experiments, we demonstrated that PP4R1 overexpression improved tumorigenesis in GBC cells. Further mechanistic exploration revealed that PP4R1 directly interacts with pyruvate kinase-M2 (PKM2), a key regulator of glycolysis. PP4R1 promotes the extracellular signal-related kinase 1 and 2 (ERK1/2)-mediated PKM2 nuclear translocation, thereby participating in the regulation of tumor glycolysis. Interestingly, we determined that PP4R1 strengthens the interaction between ERK1/2 and PKM2. Furthermore, PP4R1 enhanced the suppressive effects of the ERK inhibitor SCH772984 on GBC. In conclusion, our data showed that PP4R1 is a promising biomarker associated with GBC and confirmed that PP4R1 regulates PKM2-mediated tumor glycolysis, which provides a metabolic growth advantage to GBC cells, thereby promoting GBC tumor growth and metastasis1.

Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2.

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.