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1.
Int J Mol Sci ; 25(10)2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38791291

RESUMO

The src homology 2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are two proteins involved in intracellular signaling pathways and have been linked to the pathogenesis of several diseases. Both protein paralogs are well known for their involvement in the formation of various kinds of cancer. SHIP1, which is expressed predominantly in hematopoietic cells, has been implicated as a tumor suppressor in leukemogenesis especially in myeloid leukemia, whereas SHIP2, which is expressed ubiquitously, has been implicated as an oncogene in a wider variety of cancer types and is suggested to be involved in the process of metastasis of carcinoma cells. However, there are numerous other diseases, such as inflammatory diseases as well as allergic responses, Alzheimer's disease, and stroke, in which SHIP1 can play a role. Moreover, SHIP2 overexpression was shown to correlate with opsismodysplasia and Alzheimer's disease, as well as metabolic diseases. The SHIP1-inhibitor 3-α-aminocholestane (3AC), and SHIP1-activators, such as AQX-435 and AQX-1125, and SHIP2-inhibitors, such as K161 and AS1949490, have been developed and partly tested in clinical trials, which indicates the importance of the SHIP-paralogs as possible targets in the therapy of those diseases. The aim of this article is to provide an overview of the current knowledge about the involvement of SHIP proteins in the pathogenesis of cancer and other human diseases and to create awareness that SHIP1 and SHIP2 are more than just tumor suppressors and oncogenes.


Assuntos
Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Humanos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Domínios de Homologia de src , Transdução de Sinais , Inositol Polifosfato 5-Fosfatases/metabolismo , Inositol Polifosfato 5-Fosfatases/genética
2.
Structure ; 32(4): 453-466.e6, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38309262

RESUMO

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.


Assuntos
Domínios C2 , Monoéster Fosfórico Hidrolases , Inositol Polifosfato 5-Fosfatases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Humanos
3.
J Exp Clin Cancer Res ; 43(1): 17, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38200519

RESUMO

BACKGROUND: Prohibitin 2 (PHB2) exhibits opposite functions of promoting or inhibiting tumour across various cancer types. In this study, we aim to investigate its functions and underlying mechanisms in the context of gastric cancer (GC). METHODS: PHB2 protein expression levels in GC and normal tissues were examined using western blot and immunohistochemistry. PHB2 expression level associations with patient outcomes were examined through Kaplan-Meier plotter analysis utilizing GEO datasets (GSE14210 and GSE29272). The biological role of PHB2 and its subsequent regulatory mechanisms were elucidated in vitro and in vivo. GC cell viability and proliferation were assessed using MTT cell viability analysis, clonogenic assays, and BrdU incorporation assays, while the growth of GC xenografted tumours was measured via IHC staining of Ki67. The interaction among PHB2 and SHIP2, as well as between SHIP2 and NEDD4, was identified through co-immunoprecipitation, GST pull-down assays, and deletion-mapping experiments. SHIP2 ubiquitination and degradation were assessed using cycloheximide treatment, plasmid transfection and co-immunoprecipitation, followed by western blot analysis. RESULTS: Our analysis revealed a substantial increase in PHB2 expression in GC tissues compared to adjacent normal tissues. Notably, higher PHB2 levels correlated with poorer patient outcomes, suggesting its clinical relevance. Functionally, silencing PHB2 in GC cells significantly reduced cell proliferation and retarded GC tumour growth, whereas overexpression of PHB2 further enhanced GC cell proliferation. Mechanistically, PHB2 physically interacted with Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) in the cytoplasm of GC cells, thus leading to SHIP2 degradation via its novel E3 ligase NEDD4. It subsequently activated the PI3K/Akt signaling pathway and thus promoted GC cell proliferation. CONCLUSIONS: Our findings highlight the importance of PHB2 upregulation in driving GC progression and its association with adverse patient outcomes. Understanding the functional impact of PHB2 on GC growth contributes valuable insights into the molecular underpinnings of GC and may pave the way for the development of targeted therapies to improve patient outcomes.


Assuntos
Neoplasias Gástricas , Ubiquitina-Proteína Ligases , Humanos , Inositol Polifosfato 5-Fosfatases , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Neoplasias Gástricas/genética , Ubiquitinação
4.
Nat Cancer ; 5(3): 481-499, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38233483

RESUMO

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.


Assuntos
Melanoma , Humanos , Melanoma/tratamento farmacológico , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/uso terapêutico , Mutação , Transdução de Sinais , Inositol Polifosfato 5-Fosfatases/genética
5.
Eur J Immunol ; 53(12): e2350446, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37742135

RESUMO

Phosphoinositide-3-kinase/AKT (PI3K/AKT) signaling plays key roles in the regulation of cellular activity in both health and disease. In immune cells, this PI3K/AKT pathway is critically regulated by the phosphoinositide phosphatase SHIP1, which has been reported to modulate the function of most immune subsets. In this review, we summarize our current knowledge of SHIP1 with a focus on innate immune cells, where we reflect on the most pertinent aspects described in the current literature. We also present several small-molecule agonists and antagonists of SHIP1 developed over the last two decades, which have led to improved outcomes in several preclinical models of disease. We outline these promising findings and put them in relation to human diseases with unmet medical needs, where we discuss the most attractive targets for immune therapies based on SHIP1 modulation.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Imunoterapia , Imunidade Inata , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Inositol Polifosfato 5-Fosfatases/metabolismo
6.
J Biol Chem ; 299(8): 105022, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37423304

RESUMO

Signal transduction downstream of growth factor and immune receptor activation relies on the production of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) lipids by PI3K. Regulating the strength and duration of PI3K signaling in immune cells, Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) controls the dephosphorylation of PI(3,4,5)P3 to generate phosphatidylinositol-(3,4)-bisphosphate. Although SHIP1 has been shown to regulate neutrophil chemotaxis, B-cell signaling, and cortical oscillations in mast cells, the role that lipid and protein interactions serve in controlling SHIP1 membrane recruitment and activity remains unclear. Using single-molecule total internal reflection fluorescence microscopy, we directly visualized membrane recruitment and activation of SHIP1 on supported lipid bilayers and the cellular plasma membrane. We find that localization of the central catalytic domain of SHIP1 is insensitive to dynamic changes in PI(3,4,5)P3 and phosphatidylinositol-(3,4)-bisphosphate both in vitro and in vivo. Very transient SHIP1 membrane interactions were detected only when membranes contained a combination of phosphatidylserine and PI(3,4,5)P3 lipids. Molecular dissection reveals that SHIP1 is autoinhibited with the N-terminal Src homology 2 domain playing a critical role in suppressing phosphatase activity. Robust SHIP1 membrane localization and relief of autoinhibition can be achieved through interactions with immunoreceptor-derived phosphopeptides presented either in solution or conjugated to a membrane. Overall, this work provides new mechanistic details concerning the dynamic interplay between lipid-binding specificity, protein-protein interactions, and the activation of autoinhibited SHIP1.


Assuntos
Fosfatidilinositol 3-Quinases , Monoéster Fosfórico Hidrolases , Inositol Polifosfato 5-Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Domínios de Homologia de src , Fosfatidilinositóis , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo
7.
Iran Biomed J ; 26(6): 440-53, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36437782

RESUMO

Background: Background: Type I inositol polyphosphate-5-phosphatase A (INPP5A) is involved in different cellular events, including cell proliferation. Since INPP5A, HLAG1, IL-10, and matrix metalloproteinases (MMP)-21 genes play fundamental roles in esophageal squamous cell carcinoma (ESCC) tumorigenesis, we aimed in this study to clarify the possible interplay of these genes and explore the potential of these chemistries as a predictor marker for diagnosis in ESCC disease. Methods: Methods: Gene expression analysis of INPP5A, HLAG-1, IL-10, and MMP-21 was performed using relative comparative real-time PCR in 56 ESCCs compared to their margin normal tissues. Immunohistochemical staining was accomplished for INPP5A in ESCCs. Analysis of ROC curves and the AUC were applied to evaluate the diagnostic capability of the candidate genes. Results: Results: High levels of HLA-G1, MMP-21, and IL-10 were detected in nearly 23.2%, 62.5%, and 53.5% of ESCCs compared to the normal tissues, respectively, whereas INPP5A underexpression was detected in 19.6% of ESCCs, which all tested genes indicated significant correlations with each other. The protein expression level of INPP5A in ESCC tissues was significantly lower than that of the non-tumor esophageal tissues (p = 0.001). Interestingly, the concomitant expression of the INPP5A/HLA-G1, INPP5A/MMP-21, INPP5A/IL-10, HLA-G1/MMP-21, HLA-G1/IL-10, and MMP-21/IL-10 was significantly correlated with several clinicopathological variables. INPP5A, HLA-G1, MMP-21, and IL-10 showed to be the most appropriate candidates to discriminate tumor/non-tumor groups due to the total AUCs of all combinations (>60%). Conclusion: Conclusion: Our results represent a new regulatory axis containing INPP5A/HLAG-1/IL-10/MMP-21 markers in ESCC development and may provide novel insight into the mechanism of immune evasion mediated by the INPP5A/HLAG-1/IL-10/MMP-21 regulatory network in the disease.


Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Linhagem Celular Tumoral , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Inositol Polifosfato 5-Fosfatases/genética , Inositol Polifosfato 5-Fosfatases/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz Secretadas/metabolismo
8.
Int J Mol Sci ; 23(19)2022 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-36232599

RESUMO

SHIP2 is a multi-domain inositol 5-phosphatase binding to a variety of phosphotyrosine (pY)-containing proteins through its SH2 domain, so as to regulate various cell signaling pathways by modulating the phosphatidylinositol level in the plasma membrane. Unfavorably, Helicobacter pylori can hijack SHIP2 through the CagA protein to induce gastric cell carcinogenesis. To date, the interaction between SHIP2 and CagA was not analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Tyr-phosphorylated peptides from four EPIYA motifs (A/B/C/D) in CagA was studied using NMR spectroscopy. The results showed that EPIYA-C and -D bind to a similar interface of SHIP2-SH2, including a pY-binding pocket and a hydrophobic pocket, to achieve high affinity, while EPIYA-A and -B bind to a smaller interface of SHIP2-SH2 with weak affinity. By summarizing the interface and affinity of SHIP2-SH2 for CagA EPIYA-A/B/C/D, c-MET and FcgR2B ITIM, it was proposed that, potentially, SHIP2-SH2 has a selective preference for L > I > V for the aliphatic residues at the pY+3 position in its ligand. This study reveals the rule of the ligand sequence bound by SHIP2-SH2 and the mechanism by which CagA protein hijacks SHIP2, which will help design a peptide inhibitor against SHIP2-SH2.


Assuntos
Helicobacter pylori , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Carcinogênese , Helicobacter pylori/metabolismo , Humanos , Inositol Polifosfato 5-Fosfatases/metabolismo , Ligantes , Peptídeos/química , Fosfatidilinositóis/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Isoformas de Proteínas/metabolismo
9.
J Alzheimers Dis ; 89(1): 67-77, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35848021

RESUMO

BACKGROUND: Both INPP5D and INPP5F are members of INPP5 family. INPP5F rs117896735 variant was associated with Parkinson's disease (PD) risk, and INPP5D was an Alzheimer's disease (AD) risk gene. However, it remains unclear about the roles of INPP5F rs117896735 variant in AD. OBJECTIVE: We aim to investigate the roles of rs117896735 in AD. METHODS: First, we conducted a candidate variant study to evaluate the association of rs117896735 variant with AD risk using the large-scale AD GWAS dataset. Second, we conducted a gene expression analysis of INPP5F to investigate the expression difference of INPP5F in different human tissues using two large-scale gene expression datasets. Third, we conducted an expression quantitative trait loci analysis to evaluate whether rs117896735 variant regulate the expression of INPP5F. Fourth, we explore the potentially differential expression of INPP5F in AD and control using multiple AD-control gene expression datasets in human brain tissues and whole blood. RESULTS: We found that 1) rs117896735 A allele was associated with the increased risk of AD with OR = 1.15, 95% CI 1.005-1.315, p = 0.042; 2) rs117896735 A allele could increase INPP5F expression in multiple human tissues; 3) INPP5F showed different expression in different human tissues, especially in brain tissues; 4) INPP5F showed significant expression dysregulation in AD compared with controls in human brain tissues. CONCLUSION: Conclusion: We demonstrate that PD rs117896735 variant could regulate INPP5F expression in brain tissues and increase the risk of AD. These finding may provide important information about the role of rs117896735 in AD.


Assuntos
Doença de Alzheimer , Doença de Parkinson , Alelos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Inositol Polifosfato 5-Fosfatases , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Polimorfismo de Nucleotídeo Único/genética
10.
J Cell Biol ; 221(9)2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35878408

RESUMO

Upon antigen binding, the B cell receptor (BCR) undergoes clustering to form a signalosome that propagates downstream signaling required for normal B cell development and physiology. BCR clustering is dependent on remodeling of the cortical actin network, but the mechanisms that regulate actin remodeling in this context remain poorly defined. In this study, we identify the inositol 5-phosphatase INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells. INPP5B acts via dephosphorylation of the inositol lipid PI(4,5)P2 that in turn is necessary for actin disassembly, BCR mobilization, and cell spreading on immobilized surface antigen. These effects can be explained by increased actin severing by cofilin and loss of actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P2 hydrolysis. INPP5B is therefore a new player in BCR signaling and may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling.


Assuntos
Actinas , Inositol Polifosfato 5-Fosfatases , Receptores de Antígenos de Linfócitos B , Actinas/metabolismo , Linfócitos B , Humanos , Inositol Polifosfato 5-Fosfatases/genética , Inositol Polifosfato 5-Fosfatases/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases , Receptores de Antígenos de Linfócitos B/metabolismo
11.
J Exp Clin Cancer Res ; 41(1): 13, 2022 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-34996491

RESUMO

BACKGROUND: Increasing evidence has suggested inositol polyphosphate 5-phosphatase family contributes to tumorigenesis and tumor progression. However, the role of INPP5F in hepatocellular carcinoma (HCC) and its underlying mechanisms is unclear. METHODS: The expression of INPP5F in HCC was analyzed in public databases and our clinical specimens. The biological functions of INPP5F were investigated in vitro and vivo. The molecular mechanism of INPP5F in regulating tumor growth were studied by transcriptome-sequencing analysis, mass spectrometry analysis, immunoprecipitation assay and immunofluorescence assay. RESULTS: High expression of INPP5F was found in HCC tissues and was associated with poor prognosis in HCC patients. Overexpression of INPP5F promoted HCC cell proliferation, and vice versa. Knockdown of INPP5F suppressed tumor growth in vivo. Results from transcriptome-sequencing analysis showed INPP5F not only regulated a series of cell cycle related genes expression (c-MYC and cyclin E1), but also promoted many aerobic glycolysis related genes expression. Further studies confirmed that INPP5F could enhance lactate production and glucose consumption in HCC cell. Mechanistically, INPP5F activated Notch signaling pathway and upregulated c-MYC and cyclin E1 in HCC via interacting with ASPH. Interestingly, INPP5F was commonly nuclear-located in cells of adjacent non-tumor tissues, while in HCC, cytoplasm-located was more common. LMB (nuclear export inhibitor) treatment restricted INPP5F in nucleus and was associated with inhibition of Notch signaling and cell proliferation. Sequence of nuclear localization signals (NLSs) and nuclear export signals (NESs) in INPP5F aminoacidic sequence were then identified. Alteration of the NLSs or NESs influenced the localization of INPP5F and the expression of its downstream molecules. Furthermore, we found INPP5F interacted with both exportin and importin through NESs and NLSs, respectively, but the interaction with exportin was stronger, leading to cytoplasmic localization of INPP5F in HCC. CONCLUSION: These findings indicate that INPP5F functions as an oncogene in HCC via a translocation mechanism and activating ASPH-mediated Notch signaling pathway. INPP5F may serve as a potential therapeutic target for HCC patients.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Hepatocelular/genética , Inositol Polifosfato 5-Fosfatases/metabolismo , Neoplasias Hepáticas/genética , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Transdução de Sinais
12.
Nat Plants ; 7(5): 587-597, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34007035

RESUMO

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a low-abundance membrane lipid essential for plasma membrane function1,2. In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P2 production is involved in development, immunity and reproduction3-5. However, phospholipid synthesis is highly intricate6. It is thus likely that steady-state depletion of PI(4,5)P2 triggers confounding indirect effects. Furthermore, inducible tools available in plants allow PI(4,5)P2 to increase7-9 but not decrease, and no PIP5K inhibitors are available. Here, we introduce iDePP (inducible depletion of PI(4,5)P2 in plants), a system for the inducible and tunable depletion of PI(4,5)P2 in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P2 is critical for various aspects of plant development, including root growth, root-hair elongation and organ initiation. We show that PI(4,5)P2 is required to recruit various endocytic proteins, including AP2-µ, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we find that inducible PI(4,5)P2 perturbation impacts the dynamics of the actin cytoskeleton as well as microtubule anisotropy. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P2 in given cellular or developmental responses, and also to evaluate the importance of this lipid in protein localization.


Assuntos
Arabidopsis/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Inositol Polifosfato 5-Fosfatases/genética , Fosfatidilinositol 4,5-Difosfato/fisiologia , Fosfolipídeos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas
13.
Nat Commun ; 12(1): 2673, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976123

RESUMO

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fosfatos de Inositol/metabolismo , Lipídeos de Membrana/metabolismo , Animais , Transporte Biológico , Células COS , Chlorocebus aethiops , Colesterol/metabolismo , Endocitose , Células HEK293 , Células HeLa , Humanos , Inositol Polifosfato 5-Fosfatases/genética , Inositol Polifosfato 5-Fosfatases/metabolismo , Microscopia Confocal , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Triexosilceramidas/metabolismo
15.
Brain ; 144(8): 2427-2442, 2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-33792664

RESUMO

Marinesco-Sjögren syndrome is a rare human disorder caused by biallelic mutations in SIL1 characterized by cataracts in infancy, myopathy and ataxia, symptoms which are also associated with a novel disorder caused by mutations in INPP5K. While these phenotypic similarities may suggest commonalties at a molecular level, an overlapping pathomechanism has not been established yet. In this study, we present six new INPP5K patients and expand the current mutational and phenotypical spectrum of the disease showing the clinical overlap between Marinesco-Sjögren syndrome and the INPP5K phenotype. We applied unbiased proteomic profiling on cells derived from Marinesco-Sjögren syndrome and INPP5K patients and identified alterations in d-3-PHGDH as a common molecular feature. d-3-PHGDH modulates the production of l-serine and mutations in this enzyme were previously associated with a neurological phenotype, which clinically overlaps with Marinesco-Sjögren syndrome and INPP5K disease. As l-serine administration represents a promising therapeutic strategy for d-3-PHGDH patients, we tested the effect of l-serine in generated sil1, phgdh and inpp5k a+b zebrafish models, which showed an improvement in their neuronal phenotype. Thus, our study defines a core phenotypical feature underpinning a key common molecular mechanism in three rare diseases and reveals a common and novel therapeutic target for these patients.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Inositol Polifosfato 5-Fosfatases/genética , Mutação , Fenótipo , Fosfoglicerato Desidrogenase/genética , Degenerações Espinocerebelares/genética , Adolescente , Adulto , Animais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Proteômica , Degenerações Espinocerebelares/patologia , Peixe-Zebra
16.
Adv Med Sci ; 66(2): 231-236, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33798953

RESUMO

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is categorized among ten common aggressive malignancies, with a higher incidence and mortality rates in the developing than in developed countries. The inositol polyphosphate 5-phosphatase (INPP5A), as an intracellular-calcium mobilizer and modifier enzyme, facilitates cell responses to various stimuli. Epithelial-mesenchymal transition (EMT), a transformation procedure, has a vital role in cancer progression and metastasis when epithelial cells lose their traits in favor of obtaining mesenchymal features. In this study, we analyzed the correlation between the expression of INPP5A and the involved genes in EMT pathway through the progression and development of the ESCCs. MATERIALS AND METHODS: The gene expression analyses of INPP5A, TWIST1, MMP-2, and EGFR were performed using relative comparative real-time PCR in 58 ESCCs patients compared to corresponding margin-normal esophageal tissues. RESULTS: A significant inverse correlation between INPP5A and EGFR/MMP-2 mRNA expression was observed in tumor samples. Underexpression of INPP5A was significantly correlated with overexpression of TWIST1, MMP-2, and EGFR in different invasiveness and aggressiveness pathological features of the ESCCs (P â€‹< â€‹0.05). CONCLUSIONS: The results propose a tumor suppressor role for INPP5A and oncogenic function for concomitant expression of the other genes in ESCC invasion and metastasis. The current study is the first report elucidating the correlation between the downregulation of INPP5A and upregulation of TWIST1, MMP-2, and EGFR in ESCC and introduces this panel of the genes as a marker for poor prognosis of the disease.


Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células , Receptores ErbB/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Inositol Polifosfato 5-Fosfatases , Metaloproteinase 2 da Matriz/genética , Invasividade Neoplásica/genética , Proteínas Nucleares/genética , Prognóstico , Proteína 1 Relacionada a Twist/genética
17.
J Cell Sci ; 134(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33722976

RESUMO

Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent endomembrane fusion was necessary for the completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that the removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1, prevented macropinosome closure without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete the scission of macropinosomes or to prevent their back-fusion with the plasmalemma.


Assuntos
Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositóis , Inositol , Inositol Polifosfato 5-Fosfatases , Pinocitose
18.
Am J Med Genet A ; 185(6): 1841-1847, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33720513

RESUMO

Childhood-Onset Schizophrenia (COS) is a very rare and severe psychiatric disorder defined by adult schizophrenia symptoms occurring before the age of 13. We report a microduplication in the 10q26.3 region including part of the Inositol Polyphosphate-5-Phosphatase A (INPP5A) gene that segregates with Schizophrenia Spectrum Disorders (SSDs) in the family of a female patient affected by both COS and Autism Spectrum Disorder (ASD). Phenotyping and genotyping (including CGH-array) were performed for mother, healthy sister, and affected child according to the GenAuDiss protocol (NCT02565524). The duplication size is 324 kb and is present in a patient with COS and in her mother with SSD, but not in the patient's healthy sister. INPP5A encodes a membrane-associated 43 kDa type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase. This protein is found both in mouse and human brains and we found that its Drosophila homologue 5PtaseI is specifically expressed in the central nervous system. Hydrolyzed products from InsP3 5-phosphatases mobilize intracellular calcium, which is relevant for dendritic spine morphogenesis in neurons and altered in both schizophrenia and ASD. These may constitute arguments in favor of this gene alteration in the pathophysiology of COS.


Assuntos
Transtorno do Espectro Autista/genética , Transtornos Globais do Desenvolvimento Infantil/genética , Inositol Polifosfato 5-Fosfatases/genética , Esquizofrenia Infantil/genética , Adolescente , Adulto , Animais , Transtorno do Espectro Autista/complicações , Transtorno do Espectro Autista/patologia , Encéfalo/patologia , Criança , Transtornos Globais do Desenvolvimento Infantil/complicações , Transtornos Globais do Desenvolvimento Infantil/patologia , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Linhagem , Fenótipo , Esquizofrenia Infantil/complicações , Esquizofrenia Infantil/patologia , Irmãos , Adulto Jovem
19.
In Vitro Cell Dev Biol Anim ; 57(3): 342-349, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33537929

RESUMO

Hormesis describes a biphasic dose-response relationship generally characterized by a low-dose excitement and a high-dose inhibition. This phenomenon has been observed in the regulation of cell, organ, and organismic level. However, hormesis has not reported in oocytes. In this study, we observed, for the first time, hormetic responses of PIPP levels in oocytes by inhibitor of Akt1 or PKCδ. The expression of PIPP was detected by qPCR, immunofluorescent (IF), and Western Blot (WB). To observe the changes of PIPP levels, we used the inhibitors against pAkt1 (Ser473) or PKCδ, SH-6 or sotrastaurin with low and/or high-dose, treated GV oocytes and cultured for 4 h, respectively. The results showed that PIPP expression was significantly enhanced when oocytes were treated with SH-6 or sotrastaurin 10 µM, but decreased with SH-6 or sotrastaurin 100 µM. We also examined the changes of PIPP levels when GV oocytes were treated with exogenous PtdIns(3,4,5)P3 or LY294002 for 4 h. Our results showed that PIPP level was enhanced much higher under the treatment of 0.1 µM PtdIns(3,4,5)P3 than that of 1 µM PtdIns(3,4,5)P3, which is consistent with the changes of PIPP when oocytes were treated with inhibitors of pAkt1 (Ser473) or PKCδ. In addition, with PIPP siRNA, we detected that down-regulated PIPP may affect distributions of Akt, Cdc25, and pCdc2 (Tyr15). Taken together, these results show that the relationships between PIPP and Akt may follow the principle of hormesis and play a key role during release of diplotene arrest in mouse oocytes.


Assuntos
Hormese , Inositol Polifosfato 5-Fosfatases/metabolismo , Oócitos/metabolismo , Prolina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Forma Celular/efeitos dos fármacos , Cromonas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Hormese/efeitos dos fármacos , Prófase Meiótica I/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/metabolismo , Regulação para Cima/efeitos dos fármacos
20.
Biochem J ; 478(4): 895-910, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33635336

RESUMO

Thirty-six years after the publication of the important article by Busa and Nuccitelli on the variability of intracellular pH (pHi) and the interdependence of pHi and intracellular Ca2+ concentration ([Ca2+]i), little research has been carried out on pHi and calcium signaling. Moreover, the results appear to be contradictory. Some authors claim that the increase in [Ca2+]i is due to a reduction in pHi, others that it is caused by an increase in pHi. The reasons for these conflicting results have not yet been discussed and clarified in an exhaustive manner. The idea that variations in pHi are insignificant, because cellular buffers quickly stabilize the pHi, may be a limiting and fundamentally wrong concept. In fact, it has been shown that protons can move and react in the cell before they are neutralized. Variations in pHi have a remarkable impact on [Ca2+]i and hence on some of the basic biochemical mechanisms of calcium signaling. This paper focuses on the possible triggering role of protons during their short cellular cycle and it suggests a new hypothesis for an IP3 proton dependent mechanism of action.


Assuntos
Sinalização do Cálcio/fisiologia , Prótons , Animais , Cálcio/química , Retroalimentação Fisiológica , Humanos , Hidrogênio/química , Concentração de Íons de Hidrogênio , Inositol 1,4,5-Trifosfato/fisiologia , Inositol Polifosfato 5-Fosfatases/fisiologia , Modelos Químicos , Fosfolipases/fisiologia , Sistemas do Segundo Mensageiro/fisiologia
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