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1.
Commun Biol ; 6(1): 133, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36726025

RESUMO

Emerging studies indicate that extracellular vesicles (EVs) and their inner circular RNAs (circRNAs), play key roles in the gene regulatory network and cardiovascular repair. However, our understanding of EV-derived circRNAs in cardiac remodeling after myocardial infarction (MI) remains limited. Here we show that the level of circCEBPZOS is downregulated in serum EVs of patients with the adverse cardiac remodeling compared with those without post-MI remodeling or normal subjects. Loss-of-function approaches in vitro establish that circCEBPZOS robustly promote angiogenesis. Overexpression of circCEBPZOS in mice attenuates MI-induced left ventricular dysfunction, accompanied by a larger functional capillary network at the border zone. Further exploration of the downstream target gene indicates that circCEBPZOS acts as a competing endogenous RNA by directly binding to miR-1178-3p and thereby inducing transcription of its target gene phosphoinositide-dependent kinase-1 (PDPK1). Together, our results reveal that circCEBPZOS attenuates detrimental post-MI remodeling via the miR-1178-3p/PDPK1 axis, which facilitates revascularization, ultimately improving the cardiac function.


Assuntos
Vesículas Extracelulares , MicroRNAs , Infarto do Miocárdio , Camundongos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Remodelação Ventricular/genética , RNA Circular/genética , RNA Circular/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo
2.
Front Cell Infect Microbiol ; 12: 1048962, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36452305

RESUMO

Human babesiosis is a global emerging tick-borne disease caused by infection with intra-erythrocytic parasites of the genus Babesia. With the rise in human babesiosis cases, the discovery and development of new anti-Babesia drugs are essential. Phosphatidylinositol 4-kinase (PI4K) is a widely present eukaryotic enzyme that phosphorylates lipids to regulate intracellular signaling and trafficking. Previously, we have shown that MMV390048, an inhibitor of PI4K, showed potent inhibition against Babesia species, revealing PI4K as a druggable target for babesiosis. However, twice-administered, 7-day regimens failed to clear Babesia microti parasites from the immunocompromised host. Hence, in this study, we wanted to clarify whether targeting PI4K has the potential for the radical cure of babesiosis. In a B. microti-infected SCID mouse model, a 64-day-consecutive treatment with MMV390048 resulted in the clearance of parasites. Meanwhile, an atovaquone (ATO) resistant parasite line was isolated from the group treated with ATO plus azithromycin. A nonsynonymous variant in the Y272C of the cytochrome b gene was confirmed by sequencing. Likewise, MMV390048 showed potent inhibition against ATO-resistant parasites. These results provide evidence of PI4K as a viable drug target for the radical cure of babesiosis, which will contribute to designing new compounds that can eradicate parasites.


Assuntos
Babesia microti , Babesia , Babesiose , Gastrópodes , Camundongos , Humanos , Animais , Babesia microti/genética , Babesiose/tratamento farmacológico , Camundongos SCID , 1-Fosfatidilinositol 4-Quinase , Babesia/genética , Atovaquona , Hospedeiro Imunocomprometido
3.
Theranostics ; 12(16): 6972-6988, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36276647

RESUMO

Background: The emergence of chemoresistance in leukemia markedly impedes chemotherapeutic efficacy and dictates poor prognosis. Recent evidence has revealed that phosphatidylinositol 4 kinase-IIIα (PI4KA) plays a critical role in tumorigenesis. However, the molecular mechanisms of PI4KA-regulated chemoresistance and leukemogenesis remain largely unknown. Methods: Liquid chromatography-mass spectrometry (LC-MS), patient samples and leukemia xenograft mouse models were used to investigate whether PI4KA was an effective target to overcome chemoresistance in leukemia. Enzyme-linked immunosorbent assay (ELISA) and molecular mechanics/generalized born surface area (MM/GBSA) method were employed to identify cepharanthine (CEP) as a novel PI4KA inhibitor. Results: High expression of PI4KA was observed in drug-resistant leukemia cells or in relapsed leukemia patients, which was correlated with poor overall survival. Depletion of PI4KA sensitized drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo by regulating ERK/AMPK/OXPHOS axis. We also identified cepharanthine (CEP) as a novel PI4KA inhibitor, which could undermine the stability of the PI4KA/TTC7/FAM126 complex, enhancing the sensitivity of drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo. Conclusions: Our study underscored the potential of therapeutic targeting of PI4KA to overcome chemoresistance in leukemia. A combination of the PI4KA inhibitor with classic chemotherapeutic agents could represent a novel therapeutic strategy for the treatment of refractory leukemia.


Assuntos
1-Fosfatidilinositol 4-Quinase , Leucemia , Humanos , Camundongos , Animais , 1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas Quinases Ativadas por AMP , Leucemia/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral
4.
Sci Transl Med ; 14(667): eabo7219, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36260689

RESUMO

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.


Assuntos
Antimaláricos , Plasmodium , Animais , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum , Inibidores MTOR , 1-Fosfatidilinositol 4-Quinase , Guanosina Monofosfato , Estágios do Ciclo de Vida , Serina-Treonina Quinases TOR , Sirolimo , Mamíferos
5.
Bioorg Med Chem Lett ; 76: 129010, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36184029

RESUMO

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks.


Assuntos
1-Fosfatidilinositol 4-Quinase , Trifosfato de Adenosina , 1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/metabolismo , Ligantes , Trifosfato de Adenosina/metabolismo , Adenina , Relação Estrutura-Atividade , Desenho de Fármacos , Simulação de Acoplamento Molecular
6.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36169639

RESUMO

Melanosomes are pigment cell-specific lysosome-related organelles in which melanin pigments are synthesized and stored. Melanosome maturation requires delivery of melanogenic cargoes via tubular transport carriers that emanate from early endosomes and that require BLOC-1 for their formation. Here we show that phosphatidylinositol-4-phosphate (PtdIns4P) and the type II PtdIns-4-kinases (PI4KIIα and PI4KIIß) support BLOC-1-dependent tubule formation to regulate melanosome biogenesis. Depletion of either PI4KIIα or PI4KIIß with shRNAs in melanocytes reduced melanin content and misrouted BLOC-1-dependent cargoes to late endosomes/lysosomes. Genetic epistasis, cell fractionation, and quantitative live-cell imaging analyses show that PI4KIIα and PI4KIIß function sequentially and non-redundantly downstream of BLOC-1 during tubule elongation toward melanosomes by generating local pools of PtdIns4P. The data show that both type II PtdIns-4-kinases are necessary for efficient BLOC-1-dependent tubule elongation and subsequent melanosome contact and content delivery during melanosome biogenesis. The independent functions of PtdIns-4-kinases in tubule extension are downstream of likely redundant functions in BLOC-1-dependent tubule initiation.


Assuntos
1-Fosfatidilinositol 4-Quinase , Endossomos , Melaninas , Melanossomas , 1-Fosfatidilinositol 4-Quinase/metabolismo , Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melaninas/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico
7.
Biochem Biophys Res Commun ; 627: 111-121, 2022 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-36030652

RESUMO

Characterized by compensatory hyperplasia dependent on hepatocyte proliferation, the liver will initiate regeneration after partial hepatectomy (PH) and acute or chronic injuries. A variety of genes and noncoding RNAs play pivotal roles in these cell proliferation and growth processes. However, it is still unclear how competition endogenous RNAs (ceRNAs) modulate cellular activities during each phase of liver regeneration, and the specific mechanisms of posttranscriptional gene expression regulation in hepatocyte proliferation remain to be elucidated. To investigate the mechanism of liver regeneration through RNA-seq profiling and to determine the role of miR-34b-5p/PDK1 on hepatocyte proliferation, we established a 2/3 PH mouse model for whole transcriptome profiling based on high-throughput sequencing techniques. We subsequently constructed a lncRNA-miRNA-mRNA ceRNA regulatory network through integrative analyses of RNA interactions. Finally, plasmid transfection in NCTC 1469 cells, dual luciferase reporter gene assay, quantitative real-time PCR, western blotting, Cell Counting Kit-8, and EdU-DNA synthesis cell proliferation assay were used to demonstrate the role of the miR-34b-5p/PDK1 axis in hepatocyte proliferation in vitro. A total of 1443 mRNAs (962 up, 481 down), 48 miRNAs (35 up, 13 down), and 1955 lncRNAs (986 up, 969 down) were identified as significantly differentially expressed. We then successfully constructed a ceRNA regulatory network consisting of 7 lncRNAs, 15 miRNAs, and 347 mRNAs based on the predicted inverse interactions among ceRNAs. Additionally, miR-34b-5p/PDK1 was predicted to be closely related to hepatocyte proliferation. We further demonstrated that miR-34b-5p could bind specifically to the 3'-untranslated region (3'-UTR) of PDK1 using the dual luciferase reporter assay. Ectopic overexpression of miR-34b-5p significantly reduced the mRNA and protein expression of PDK1, while it markedly inhibited the proliferation of mouse NCTC 1469 cells in vitro. In contrast, knocking down miR-34b-5p exhibited the inverse effects on PDK1 expression and hepatocyte proliferation. Through analyzing the ceRNA network during mouse liver regeneration, this study reveals that miR-34b-5p can inhibit hepatocyte proliferation through negatively regulating PDK1 and may be a potential pharmacological intervention target.


Assuntos
MicroRNAs , RNA Longo não Codificante , 1-Fosfatidilinositol 4-Quinase/genética , Regiões 3' não Traduzidas , Animais , Proliferação de Células/genética , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Hiperplasia , Regeneração Hepática/genética , Camundongos , MicroRNAs/metabolismo , Fosfatidilinositóis , Proteínas Quinases/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA-Seq
8.
Antimicrob Agents Chemother ; 66(9): e0057422, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-35924942

RESUMO

The present study aimed to evaluate the anti-Babesia effect of MMV390048, a drug that inhibits Plasmodium by targeting the phosphatidylinositol 4-kinase (PI4K). The half inhibitory concentration (IC50) of MMV390048 against the in vitro growth of Babesia gibsoni was 6.9 ± 0.9 µM. In immunocompetent mice, oral treatment with MMV390048 at a concentration of 20 mg/kg effectively inhibited the growth of B. microti (Peabody mjr strain). The peak parasitemia in the control group was 30.5%, whereas the peak parasitemia in the MMV390048-treated group was 3.4%. Meanwhile, MMV390048 also showed inhibition on the growth of B. rodhaini (Australia strain), a highly pathogenic rodent Babesia species. All MMV390048-treated mice survived, whereas the mice in control group died within 10 days postinfection (DPI). The first 7-day administration of MMV390048 in B. microti-infected, severe combined immunodeficiency (SCID) mice delayed the rise of parasitemia by 26 days. Subsequently, a second 7-day administration was given upon recurrence. At 52 DPI, a parasite relapse (in 1 out of 5 mice) and a mutation in the B. microti PI4K L746S, a MMV390048 resistance-related gene, were detected. Although the radical cure of B. microti infection in immunocompromised host SCID mice was not achieved, results from this study showed that MMV390048 has excellent inhibitory effects on Babesia parasites, revealing a new treatment strategy for babesiosis: targeting the B. microti PI4K.


Assuntos
Antimaláricos , Babesia , Babesiose , 1-Fosfatidilinositol 4-Quinase , Aminopiridinas , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Babesiose/tratamento farmacológico , Babesiose/parasitologia , Camundongos , Camundongos SCID , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Sulfonas
9.
EMBO J ; 41(17): e110698, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35844135

RESUMO

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.


Assuntos
1-Fosfatidilinositol 4-Quinase , Fosfolipase D , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/metabolismo , Fosfolipase D/química , Fosfolipase D/genética , Fosfolipase D/metabolismo
10.
Commun Biol ; 5(1): 738, 2022 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-35871252

RESUMO

Human two-pore channels (TPCs) are endolysosomal cation channels and play an important role in NAADP-evoked Ca2+ release and endomembrane dynamics. We found that YM201636, a PIKfyve inhibitor, potently inhibits PI(3,5)P2-activated human TPC2 with an IC50 of 0.16 µM. YM201636 also effectively inhibits NAADP-activated TPC2 and a constitutively-open TPC2 L690A/L694A mutant channel; whereas it exerts little effect when applied in the channel's closed state. PI-103, a YM201636 analog and an inhibitor of PI3K and mTOR, also inhibits human TPC2 with an IC50 of 0.64 µM. With mutational, virtual docking, and molecular dynamic simulation analyses, we found that YM201636 and PI-103 directly block the TPC2's open-state channel pore at the bundle-cross pore-gate region where a nearby H699 residue is a key determinant for channel's sensitivity to the inhibitors. H699 likely interacts with the blockers around the pore entrance and facilitates their access to the pore. Substitution of a Phe for H699 largely accounts for the TPC1 channel's insensitivity to YM201636. These findings identify two potent TPC2 channel blockers, reveal a channel pore entrance blockade mechanism, and provide an ion channel target in interpreting the pharmacological effects of two commonly used phosphoinositide kinase inhibitors.


Assuntos
Canais de Cálcio , Fosfatidilinositóis , 1-Fosfatidilinositol 4-Quinase , Aminopiridinas , Cálcio/metabolismo , Furanos , Compostos Heterocíclicos com 3 Anéis , Histidina , Humanos , Piridinas , Pirimidinas
11.
Biosci Rep ; 42(7)2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35735144

RESUMO

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.


Assuntos
1-Fosfatidilinositol 4-Quinase , Insulina , 1-Fosfatidilinositol 4-Quinase/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Camundongos
12.
Cancer Res ; 82(14): 2625-2639, 2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35657206

RESUMO

Melanomas frequently harbor activating NRAS mutations. However, limited advance has been made in developing targeted therapy options for patients with NRAS mutant melanoma. MEK inhibitors (MEKi) show modest efficacy in the clinic and their actions need to be optimized. In this study, we performed a genome-wide CRISPR-Cas9-based screen and demonstrated that loss of phosphoinositide-dependent kinase-1 (PDPK1) enhances the efficacy of MEKi. The synergistic effects of PDPK1 loss and MEKi was validated in NRAS mutant melanoma cell lines using pharmacologic and molecular approaches. Combined PDPK1 inhibitors (PDPK1i) with MEKi suppressed NRAS mutant xenograft growth and induced gasdermin E-associated pyroptosis. In an immune-competent allograft model, PDPK1i+MEKi increased the ratio of intratumoral CD8+ T cells, delayed tumor growth, and prolonged survival; the combination treatment was less effective against tumors in immune-deficient mice. These data suggest PDPK1i+MEKi as an efficient immunostimulatory strategy against NRAS mutant melanoma. SIGNIFICANCE: Targeting PDPK1 stimulates antitumor immunity and sensitizes NRAS mutant melanoma to MEK inhibition, providing rationale for the clinical development of a combinatorial approach for treating patients with melanoma.


Assuntos
GTP Fosfo-Hidrolases , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Melanoma , 1-Fosfatidilinositol 4-Quinase/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética
13.
Cell Death Differ ; 29(10): 2060-2069, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35477991

RESUMO

Subcellular machinery of NLRP3 is essential for inflammasome assembly and activation. However, the stepwise process and mechanistic basis of NLRP3 engagement with organelles remain unclear. Herein, we demonstrated glycogen synthase kinase 3ß (GSK3ß) as a molecular determinant for the spatiotemporal dynamics of NLRP3 inflammasome activation. Using live cell multispectral time-lapse tracking acquisition, we observed that upon stimuli NLRP3 was transiently associated with mitochondria and subsequently recruited to the Golgi network (TGN) where it was retained for inflammasome assembly. This occurred in relation to the temporal contact of mitochondria to Golgi apparatus. NLRP3 stimuli initiate GSK3ß activation with subsequent binding to NLRP3, facilitating NLRP3 recruitment to mitochondria and transition to TGN. GSK3ß activation also phosphorylates phosphatidylinositol 4-kinase 2 Α (PI4k2A) in TGN to promote sustained NLRP3 oligomerization. Our study has identified the interplay between GSK3ß signaling and the organelles dynamics of NLRP3 required for inflammasome activation and opens new avenues for therapeutic intervention.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , 1-Fosfatidilinositol 4-Quinase , Glicogênio Sintase Quinase 3 beta , Complexo de Golgi/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
15.
Birth Defects Res ; 114(10): 487-498, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35416431

RESUMO

BACKGROUND: MMV390048 is an aminopyridine plasmodial PI4K inhibitor, selected as a Plasmodium blood-stage schizonticide for a next generation of malaria treatments to overcome resistance to current therapies. MMV390048 showed an acceptable preclinical safety profile and progressed up to Phase 2a clinical trials. However, embryofetal studies revealed adverse developmental toxicity signals, including diaphragmatic hernias and cardiovascular malformations in rats but not rabbits. METHODS: In vivo exposures of free plasma concentrations of compound in rats were assessed in relation to in vitro human kinase inhibition by MMV390048, using the ADP-Glo™ Kinase Assay. RESULTS: We demonstrate a potential link between the malformations seen in the embryofetal developmental (EFD) studies and inhibition of the mammalian PI4Kß paralogue, as well as inhibition of the off-target kinases MAP4K4 and MINK1. PI3Kγ may also play a role in the embryofetal toxicity as its in vitro inhibition is covered by in vivo exposure. The exposures in the rabbit embryofetal development studies did not reach concentrations likely to cause PI4K inhibition. Overall, we hypothesize that the in vivo malformations observed could be due to inhibition of the PI4K target in combination with the off-targets, MAP4K4 and MINK1. However, these relationships are by association and not mechanistically proven. CONCLUSIONS: Deciphering if the EFD effects are dependent on PI4K inhibition, and/or via inhibition of other off-target kinases will require the generation of novel, more potent, and more specific PI4K inhibitors.


Assuntos
Hérnia Diafragmática , Malária , Plasmodium , 1-Fosfatidilinositol 4-Quinase , Animais , Malária/tratamento farmacológico , Mamíferos , Coelhos , Ratos
16.
Artigo em Inglês | MEDLINE | ID: mdl-35427794

RESUMO

Reversible phosphorylation of phosphatidylinositol by phosphoinositide (PI) kinases and phosphatases generates seven distinct phosphoinositide phosphates, called phosphoinositides or PIPs. All seven PIPs are formed in the retina and photoreceptor cells. Around 50 genes in the mammalian genome encode PI kinases and PI phosphatases. There are no studies available on the distribution of these enzymes in the retina and photoreceptors. AIM: To employ Ribosomal Targeting Strategy and Nuclear Labeling to Analyze Phosphoinositide Signatures in rod-photoreceptor cells. METHODS: HA-tagging of ribosomal protein Rpl22 was induced with Cre-recombinase under the control of the rhodopsin promoter. Actively translating mRNAs associated with polyribosomes were isolated by immunoprecipitation with HA antibody, followed by RNA isolation and gene identification. We also isolated biotinylated-rod nuclei from NuTRAP mice under the control of the rhodopsin-Cre promoter and analyzed nuclear phosphoinositides. RESULTS: Our results indicate that the expression of class I and class III PI 3-kinase, PI4K IIIß, PI 5-kinase, PIKfyve, PI3-phosphatases, MTMR2, 4, 6, 7, 14, PI4-phosphatase, TMEM55A, PI 5-phosphatases, SYNJI, INPP5B, INPP5E, INPP5F, SKIP and other phosphatases with dual substrate specificity, PTPMT1, SCAM1, and FIG4 are highly enriched in rod photoreceptor cells compared with the retina and cone-like retina. Our analysis identified the presence of PI(4)P, PI(3,4)P2, PI(3,5)P2, and PI(4,5)P2 in the rod nuclei. CONCLUSIONS: Our studies for the first time demonstrate the expression of PI kinases, PI phosphatases, and nuclear PIPs in rod photoreceptor cells. The NuTRAP mice may be useful not only for epigenetic and transcriptomic studies but also for in vivo cell-specific lipidomics research.


Assuntos
Monoéster Fosfórico Hidrolases , Células Fotorreceptoras , Ribossomos , 1-Fosfatidilinositol 4-Quinase , Animais , Flavoproteínas , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatases de Fosfoinositídeos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Células Fotorreceptoras/metabolismo , Proteínas Tirosina Fosfatases não Receptoras , Rodopsina
17.
Bioengineered ; 13(4): 8277-8290, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35302432

RESUMO

Gastric cancer (GC) is one of the most common malignancies in the world, and effective therapeutic targets need to be identified for this type of cancer. In this study, circular RNA (circRNA) microarray analysis was utilized to screen differentially expressed circRNA in GC. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR), hsa_circ_0000081 (circRNA-0000081) expression was found to be up-regulated in tissues and cells and was negative correlated with patients' survival time. RNase R and Actinomycin D assays indicated that circRNA-0000081 was significantly more resistant to R enzyme and had a longer half-life than linear RNA. Moreover, the knockdown or overexpression of circRNA-000081 could influence the proliferation, migration, and invasion potential of GC. Finally, dual luciferase reporter, RNA immunoprecipitation, qRT-PCR, and western blotting assays were used to verify the targeting relationship between circRNA-000081 and miRNA-423-5p or miRNA-423-5p and 3-phosphoinositide-dependent kinase 1 (PDPK1). In conclusion, circRNA-0000081 promotes the function of GC through sponging hsa-miR-423-5p to influence PDPK1 expression, which has a promising therapeutic potential for treating patients with GC.


Assuntos
MicroRNAs , Neoplasias Gástricas , 1-Fosfatidilinositol 4-Quinase , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Fosfatidilinositóis , RNA Circular/genética , Neoplasias Gástricas/patologia
18.
Cell Rep ; 38(9): 110452, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35235793

RESUMO

Phosphatidylinositol 4-phosphate (PI4P) is a low abundant phospholipid with important roles in lipid transport and membrane trafficking. However, little is known of its metabolism and function in neurons. Here, we investigate its subcellular distribution and functional roles in dendrites of rodent hippocampal neurons during resting state and long-term synaptic potentiation (LTP). We show that neural activity causes dynamic reversible changes in PI4P metabolism in dendrites. Upon LTP induction, PI4KIIIα, a type III phosphatidylinositol 4-kinase, localizes to the dendritic plasma membrane (PM) in a calcium-dependent manner and causes substantial increase in the levels of PI4P. Acute inhibition of PI4KIIIα activity abolishes trafficking of the AMPA-type glutamate receptor to the PM during LTP induction, and silencing of PI4KIIIα expression in the hippocampal CA1 region causes severe impairment of LTP and long-term memory. Collectively, our results identify an essential role for PI4KIIIα-dependent PI4P synthesis in synaptic plasticity of central nervous system neurons.


Assuntos
1-Fosfatidilinositol 4-Quinase , Potenciação de Longa Duração , 1-Fosfatidilinositol 4-Quinase/metabolismo , Região CA1 Hipocampal/metabolismo , Hipocampo/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo
20.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34982154

RESUMO

Phosphatidylinositol(4,5)-bisphosphate (PtdInsP2) is an important modulator of many cellular processes, and its abundance in the plasma membrane is closely regulated. We examined the hypothesis that members of the Dishevelled scaffolding protein family can bind the lipid kinases phosphatidylinositol 4-kinase (PI4K) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K), facilitating synthesis of PtdInsP2 directly from phosphatidylinositol. We used several assays for PtdInsP2 to examine the cooperative function of phosphoinositide kinases and the Dishevelled protein Dvl3 in the context of two receptor signaling cascades. Simultaneous overexpression of PI4KIIIα (also known as PI4KA) and PIP5KIγ (also known as PIP5K1C) had a synergistic effect on PtdInsP2 synthesis that was recapitulated by overexpression of Dvl3. Increasing the activity of Dvl3 by overexpression increased resting plasma membrane PtdInsP2. Knockdown of Dvl3 reduced resting plasma membrane PtdInsP2 and slowed PtdInsP2 resynthesis following receptor activation. We confirm that Dvl3 promotes coupling of PI4KIIIα and PIP5KIγ and show that this interaction is essential for efficient resynthesis of PtdInsP2 following receptor activation.


Assuntos
1-Fosfatidilinositol 4-Quinase , Fosfatidilinositol 4,5-Difosfato , 1-Fosfatidilinositol 4-Quinase/metabolismo , Membrana Celular/metabolismo , Proteínas Desgrenhadas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)
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