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Glutamine metabolism via glutaminase 1 in autosomal-dominant polycystic kidney disease

Soomro, Irfana; Sun, Ying; Li, Zhai; Diggs, Lonnette; Hatzivassiliou, Georgia; Thomas, Ajit G; Rais, Rana; Parker, Seth J; Slusher, Barbara S; Kimmelman, Alec C; Somlo, Stefan; Skolnik, Edward Y
PMID: 31329939
ISSN: 1460-2385
CID: 4637312

Discoidin Domain Receptor 1 (DDR1) tyrosine kinase is upregulated in PKD kidneys but does not play a role in the pathogenesis of polycystic kidney disease

Soomro, Irfana; Hong, Aram; Li, Zhai; Duncan, James S; Skolnik, Edward Y
Tolvaptan is the only drug approved to slow cyst growth and preserve kidney function in patients with autosomal dominant polycystic kidney disease (ADPKD). However, its limited efficacy combined with significant side effects underscores the need to identify new and safe therapeutic drug targets to slow progression to end stage kidney disease. We identified Discoidin Domain Receptor 1 (DDR1) as receptor tyrosine kinase upregulated in vivo in 3 mouse models of ADPKD using a novel mass spectrometry approach to identify kinases upregulated in ADPKD. Previous studies demonstrating critical roles for DDR1 to cancer progression, its potential role in the pathogenesis of a variety of other kidney disease, along with the possibility that DDR1 could provide new insight into how extracellular matrix impacts cyst growth led us to study the role of DDR1 in ADPKD pathogenesis. However, genetic deletion of DDR1 using CRISPR/Cas9 failed to slow cyst growth or preserve kidney function in both a rapid and slow mouse model of ADPKD demonstrating that DDR1 does not play a role in PKD pathogenesis and is thus a not viable drug target. In spite of the negative results, our studies will be of interest to the nephrology community as it will prevent others from potentially conducting similar experiments on DDR1 and reinforces the potential of performing unbiased screens coupled with in vivo gene editing using CRISPR/Cas9 to rapidly identify and confirm new potential drug targets for ADPKD.
PMID: 31260458
ISSN: 1932-6203
CID: 3967862

Glutamine metabolism via glutaminase 1 in autosomal-dominant polycystic kidney disease

Soomro, Irfana; Sun, Ying; Li, Zhai; Diggs, Lonnette; Hatzivassiliou, Georgia; Thomas, Ajit G; Rais, Rana; Slusher, Barbara S; Somlo, Stefan; Skolnik, Edward Y
Background/UNASSIGNED:Metabolism of glutamine by glutaminase 1 (GLS1) plays a key role in tumor cell proliferation via the generation of ATP and intermediates required for macromolecular synthesis. We hypothesized that glutamine metabolism also plays a role in proliferation of autosomal-dominant polycystic kidney disease (ADPKD) cells and that inhibiting GLS1 could slow cyst growth in animal models of ADPKD. Methods/UNASSIGNED:Primary normal human kidney and ADPKD human cyst-lining epithelial cells were cultured in the presence or absence of two pharmacologic inhibitors of GLS1, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) and CB-839, and the effect on proliferation, cyst growth in collagen and activation of downstream signaling pathways were assessed. We then determined if inhibiting GLS1 in vivo with CB-839 in the Aqp2-Cre; Pkd1fl/fl and Pkhd1-Cre; Pkd1fl/fl mouse models of ADPKD slowed cyst growth. Results/UNASSIGNED:We found that an isoform of GLS1 (GLS1-GAC) is upregulated in cyst-lining epithelia in human ADPKD kidneys and in mouse models of ADPKD. Both BPTES and CB-839 blocked forskolin-induced cyst formation in vitro. Inhibiting GLS1 in vivo with CB-839 led to variable outcomes in two mouse models of ADPKD. CB-839 slowed cyst growth in Aqp2-Cre; Pkd1fl/fl mice, but not in Pkhd1-Cre; Pkd1fl/fl mice. While CB-839 inhibited mammalian target of rapamycin (mTOR) and MEK activation in Aqp2-Cre; Pkd1fl/fl, it did not in Pkhd1-Cre; Pkd1fl/fl mice. Conclusion/UNASSIGNED:These findings provide support that alteration in glutamine metabolism may play a role in cyst growth. However, testing in other models of PKD and identification of the compensatory metabolic changes that bypass GLS1 inhibition will be critical to validate GLS1 as a drug target either alone or when combined with inhibitors of other metabolic pathways.
PMID: 29420817
ISSN: 1460-2385
CID: 2948292

Regulation of KATPChannel Trafficking in Pancreatic β Cells by Protein Histidine Phosphorylation

Srivastava, Shekhar; Li, Zhai; Soomro, Irfana; Sun, Ying; Wang, Jianhui; Bao, Li; Coetzee, William A; Stanley, Charles A; Li, Chonghong; Skolnik, Edward Y
Protein histidine phosphatase 1 (PHPT-1) is an evolutionarily conserved 14 kDa protein that dephosphorylates phosphohistidine.PHPT-1
PMID: 29440278
ISSN: 1939-327x
CID: 2958302

Novel screen to identify kinase drug targets for autosomal dominant polycystic kidney disease [Meeting Abstract]

Soomro, I H; Hong, A; Li, Z; Skolnik, E Y
Background: Activation of kinases and the downstream signaling pathways they activate is central to the pathogenesis of cyst growth in ADPKD. However, while the human kinome consists of more than 500 kinases, only a fraction of these kinases have been tested to determine if they play a role in ADPKD pathogenesis. As a result, there are likely many kinases that are more active in ADPKD kidneys that play prominent roles in disease that are yet-to-be discovered and may be good therapeutic targets. We have now adapted a novel approach to broadly screen PKD kidneys in an unbiased manner for kinases that are more active in PKD kidneys compared with wild type kidneys.
Method(s): Active kinases were affinity captured by passing wild type and PKD kidney lysates through columns containing multiplexed kinase inhibitor beads. Bound kinases were then identified by LC separation followed by tandem mass spectrometry. Increase in kinase expression and/or activity was validated by Western Blot and the specific kidney cells expressing the kinase was determined by Immunohistochemistry. The relevance of a kinase to cyst growth in vivo was assessed by treating PKD mutant mice with specific kinase inhibitors and/or genetically by generating kinase knockouts using CRISPR/Cas9.
Result(s): We identified a number of both known and unknown kinases specifically upregulated or downregulated in mouse PKD kidneys. Focal adhesion Kinase (FAK) is one of the promising kinase identified in the screen. We found that FAK and phospho-FAK expression was upregulated in cyst lining epithelium in PKD kidneys. Consistent with FAK playing an important role in cyst growth, treatment of Pkhd1-Cre;Pkd1fl/fl mice with the FAK inhibitor VS-4718 slowed cyst growth, preserved renal function, and prolonged survival. VS-4718 treatment led to the inhibition of multiple signaling pathways that could account for the therapeutic benefit including paxillin, p130cas, AKT and Stat3.
Conclusion(s): This is the first time PKD kidneys have been probed proteomically in an unbiased manner to identify the full range of kinases that are more active in PKD kidneys with the goal of identifying new therapeutic targets. So far we have identified FAK as a potential drug target that can slow cyst growth and preserve renal function and are currently in the process of working up several other promising kinases
ISSN: 1533-3450
CID: 4758032

PLCepsilon1 regulates SDF-1alpha-induced lymphocyte adhesion and migration to sites of inflammation

Strazza, Marianne; Azoulay-Alfaguter, Inbar; Peled, Michael; Smrcka, Alan V; Skolnik, Edward Y; Srivastava, Shekhar; Mor, Adam
Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cepsilon 1 (PLCepsilon1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1alpha)-induced Rap1 activation. We have shown that SDF-1alpha-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCepsilon1 was necessary for SDF-1alpha-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCepsilon1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCepsilon1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1alpha induces T-cell adhesion through activation of PLCepsilon1, suggesting that PLCepsilon1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.
PMID: 28213494
ISSN: 1091-6490
CID: 2449442

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure

Abu-Taha, Issam H; Heijman, Jordi; Hippe, Hans-Jorg; Wolf, Nadine M; El-Armouche, Ali; Nikolaev, Viacheslav O; Schafer, Marina; Wurtz, Christina; Neef, Stefan; Voigt, Niels; Baczko, Istvan; Varro, Andras; Muller, Marion; Meder, Benjamin; Katus, Hugo A; Spiger, Katharina; Vettel, Christiane; Lehmann, Lorenz H; Backs, Johannes; Skolnik, Edward Y; Lutz, Susanne; Dobrev, Dobromir; Wieland, Thomas
BACKGROUND: -Chronic heart failure (HF) is associated with altered signal transduction via beta-adrenoceptors and G proteins, and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of end-stage HF patients, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. METHODS: -Real-time PCR, (Far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined using immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). RESULTS: -NDPK-C was essential for the formation of a NDPK-B/G proteins complex. Protein and mRNA levels of NDPK-C were up-regulated in end-stage human HF, in rats following chronic isoprenaline (ISO) stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with ISO. ISO also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to ISO-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression, but showed contractile dysfunction and exacerbated cardiac remodeling during chronic ISO stimulation. In human end-stage HF, the complex formation between NDPK-C and Galphai2 was increased, whereas NDPK-C/Galphas interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP-reduction in HF. CONCLUSIONS: -Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and with G proteins. NDPK-C is a novel critical regulator of beta-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Galphas to Galphai2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.
PMID: 27927712
ISSN: 1524-4539
CID: 2354352

Phosphatidlyinositol-3-kinase C2 beta (PI3KC2beta) is a potential new target to treat IgE mediated disease

Srivastava, Shekhar; Li, Zhai; Skolnik, Edward Y
Cross linking of the IgE receptor (FcepsilonRI) on mast cells plays a critical role in IgE-dependent allergy including allergic rhinitis, asthma, anaphylaxis, and delayed type hypersensitivity reactions. The Ca2+ activated K+ channel, KCa3.1, plays a critical role in IgE-stimulated Ca2+ entry and degranulation in mast cells by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca2+ influx. Of the 3 classes of PI3K, the class II PI3Ks are the least studied and little is known about the roles for class II PI3Ks in vivo in the context of the whole organism under normal and pathological conditions. Studying bone marrow derived mast cells (BMMC) isolated from PI3KC2beta-/- mice, we now show that the class II PI3KC2beta is critical for FcepsilonRI stimulated KCa3.1 channel activation and the subsequent activation of mast cells. We found FcepsilonRI-stimulated Ca2+ entry, cytokine production, and degranulation are decreased in BMMC isolated from PI3KC2beta-/- mice. In addition, PI3KC2beta-/- mice are markedly resistant to both passive cutaneous and passive systemic anaphylaxis. These findings identify PI3KC2beta as a new pharmacologic target to treat IgE-mediated disease.
PMID: 28820911
ISSN: 1932-6203
CID: 2670662

Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y
KCa2.1, KCa2.2, KCa2.3, and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NPDK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site.
PMID: 27542194
ISSN: 2050-084x
CID: 2219562

Identification of PGAM5 as a Mammalian Protein Histidine Phosphatase that Plays a Central Role to Negatively Regulate CD4+ T Cells

Panda, Saswati; Srivastava, Shekhar; Li, Zhai; Vaeth, Martin; Fuhs, Stephen R; Hunter, Tony; Skolnik, Edward Y
Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4+ T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K+ channel KCa3.1, which is required for TCR-stimulated Ca2+ influx and cytokine production. Using recently developed monoclonal antibodies that specifically recognize phosphorylation of nitrogens at the N1 (1-pHis) or N3 (3-pHis) positions of the imidazole ring, we detect for the first time phosphoisoform-specific regulation of histidine-phosphorylated proteins in vivo, and we link these modifications to TCR signaling. These results represent an important step forward in studying the role of histidine phosphorylation in mammalian biology and disease.
PMID: 27453048
ISSN: 1097-4164
CID: 2191412