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Science > Publications

Publications

Fluorescence-based biochemical high throughput primary assay to identify inhibitors of phospholipase C isozymes (PLCβ). 

National Center for Biotechnology Information. PubChem BioAssay Database; AID=720700, https://pubchem.ncbi.nlm.nih.gov/bioassay/720704.

+ Read abstract
Extracellular stimuli including hormones, growth factors, and neurotransmitters promote activation of phospholipase C (PLC) isozymes and cleavage of the membrane lipid phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P2) into the classical second messengers, diacylglycerol and inositol 1,4,5- trisphosphate (IP3) [1]. These second messengers coordinately control numerous signaling cascades through the mobilization of intracellular Ca2+ stores and the activation of protein kinase C. Aberrant regulation of PLCs contribute to diverse human diseases including cancer [2-4], cardiovascular diseases [5-6], and neuropathic pain [7], as well as schizophrenia and epilepsy [5, 8-9]. Consequently, small molecule PLC inhibitors will be valuable pharmacological tools to dissect the roles of PLCs in development and disease, and could potentially serve as candidates for drug development. Read the full article here

Fluorescence-based biochemical high throughput primary assay to identify inhibitors of phospholipase C isozymes (PLCγ1).

National Center for Biotechnology Information. PubChem BioAssay Database; AID=720700, https://pubchem.ncbi.nlm.nih.gov/bioassay/720700.

+ Read abstract
Extracellular stimuli including hormones, growth factors, and neurotransmitters promote activation of phospholipase C (PLC) isozymes and cleavage of the membrane lipid phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P2) into the classical second messengers, diacylglycerol and inositol 1,4,5- trisphosphate (IP3) [1]. These second messengers coordinately control numerous signaling cascades through the mobilization of intracellular Ca2+ stores and the activation of protein kinase C. Aberrant regulation of PLCs contribute to diverse human diseases including cancer [2-4], cardiovascular diseases [5-6], and neuropathic pain [7], as well as schizophrenia and epilepsy [5, 8-9]. Consequently, small molecule PLC inhibitors will be valuable pharmacological tools to dissect the roles of PLCs in development and disease, and could potentially serve as candidates for drug development. Read the full article here

A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes

Huang, W., Hicks, S. N., Sondek, J., & Zhang, Q. (2011). A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes. ACS Chemical Biology, 6(3), 223–228. http://doi.org/10.1021/cb100308n

+ Read abstract
Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer’s disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable Km and Vmax with the endogenous PIP2. This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high throughput screening of PLC inhibitors. Read the full article here

Small molecule inhibitors of phospholipase C from a novel high-throughput screen

Huang, W., Barrett, M., Hajicek, N., Hicks, S., Harden, T. K., Sondek, J., & Zhang, Q. (2013). Small molecule inhibitors of phospholipase C from a novel high-throughput screen. The Journal of Biological Chemistry, 288(8), 5840–5848.  http://doi.org/10.1074/jbc.M112.422501

+ Read abstract
Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity. Read the full article here

Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C

Jong Bae Seo, Seung-Ryoung Jung, Weigang Huang, Qisheng Zhang, Duk-Su Koh, PLoS One,11;10(12):e0144432 (2015), doi.org/10.1371/journal.pone.0144432

+ Read abstract
Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically. 

Interactions Between Autotrophic and Heterotrophic Strains Improve CO2 Fixing Efficiency of Non-photosynthetic Microbial Communities

Hu J, Wang L, Zhang S, Xi X, Le Y, Fu X, Tsang Y, Gao M., Appl Biochem Biotechnol 176(5):1459-71 (2015), doi: 10.1007/s12010-015-1657-4.

+ Read abstract
Five autotrophic strains isolated from non-photosynthetic microbial communities (NPMCs), which were screened from oceans with high CO2 fixing capability, were identified as Ochrobactrum sp. WH-2, Stenotrophomonas sp. WH-11, Ochrobactrum sp. WH-13, Castellaniella sp. WH-14, and Sinomicrobium oceani WH-15. The CO2 fixation pathways of all these strains were Calvin-Benson-Bassham pathway. These strains could metabolize multifarious organic compounds, which allowed switching them to autotrophic culture after enrichment in heterotrophic culture. The central composite response surface method indicated that these strains possessed many interactive effects, which increased the CO2 fixing efficiency of a combined community composed of these strains by 56 %, when compared with that of the single strain. Furthermore, another combined community composed of these autotrophic strains and NPMC had richer interactive relationships, with CO2 fixing efficiency being 894 % higher than that of the single strain and 148 % higher than the theoretical sum of the CO2 fixing efficiency of each of its microbial components. The interaction between strictly heterotrophic bacteria in NPMC and isolated autotrophic strains played a crucial role in improving the CO2 fixing efficiency, which not only eliminated self-restraint of organic compounds generated during the growth of autotrophic bacteria but also promoted its autotrophic pathway. 

A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes

Weigang Huang, Xiaoyang Wang, Stuart Endo-Streeter, Matthew Barrett§, Jarod Waybright, Christian Wohlfeld, Nicole Hajicek, T. Kendall Harden, John Sondek and Qisheng Zhang, JBC Papers in Press (2017) Latest version at http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000926, doi: 10.1074/jbc.RA117.000926

+ Read abstract
A diverse group of cell‒surface receptors, including many G protein‒coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the wide-spread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-β3 by Gαq. Therefore, XY-69 can replace radioactive PIP2 used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution. 

Membrane-induced Allosteric Control of Phospholipase C- Isozymes

Thomas H. Charpentier, Gary L. Waldo, Matthew O. Barrett, Weigang Huang, Qisheng Zhang, T. Kendall Harden and John Sondek, J Biol Chem 289(43):29545–29557 (2014), doi: 10.1074/jbc.M114.586784.

+ Read abstract
All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β(PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gαq, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gβ1γ2 did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes. 


LIM and cysteine-rich domains 1 is required for thrombin-induced smooth muscle cell proliferation and promotes atherogenesis

Janjanam J, Zhang B, Mani AM, Singh NK, Traylor JG Jr., Orr AW, Rao GN, J. Biol. Chem293(9): 3088–3103 (2018), doi: 10.1074/jbc.RA117.000866.

+ Read abstract
Restenosis arises after vascular injury and is characterized by arterial wall thickening and decreased arterial lumen space. Vascular injury induces the production of thrombin, which in addition to its role in blood clotting acts as a mitogenic and chemotactic factor. In exploring the molecular mechanisms underlying restenosis, here we identified LMCD1 (LIM and cysteine-rich domains 1) as a gene highly responsive to thrombin in human aortic smooth muscle cells (HASMCs). Of note, LMCD1 depletion inhibited proliferation of human but not murine vascular smooth muscle cells. We also found that by physically interacting with E2F transcription factor 1, LMCD1 mediates thrombin-induced expression of the CDC6 (cell division cycle 6) gene in the stimulation of HASMC proliferation. Thrombin-induced LMCD1 and CDC6 expression exhibited a requirement for protease-activated receptor 1-mediated Gαq/11-dependent activation of phospholipase C β3. Moreover, the expression of LMCD1 was highly induced in smooth muscle cells located at human atherosclerotic lesions and correlated with CDC6 expression and that of the proliferation marker Ki67. Furthermore, the LMCD1- and SMCα actin-positive cells had higher cholesterol levels in the atherosclerotic lesions. In conclusion, these findings indicate that by acting as a co-activator with E2F transcription factor 1 in CDC6 expression, LMCD1 stimulates HASMC proliferation and thereby promotes human atherogenesis, suggesting an involvement of LMCD1 in restenosis. 


PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration

Jagadeesh Janjanam, Giri Kumar Chandaka, Sivareddy Kotla, and Gadiparthi N. Rao, Mol Biol Cell, 26 (2015), doi: 10.1091/mbc.E15-08-0570

+ Read abstract
Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein–coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδactivation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδactivation, cortactin phosphorylation, cortactin–WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration. 


Pharmacologic inhibition of phospholipase C in the brain attenuates early memory formation in the honeybee (Apis mellifera L.)

Suenami S, Iino S, Kubo T, Bio Open 7(1), bio028191. doi:10.1242/bio.028191 (2018), doi: 10.1242/bio.028191.

+ Read abstract
Although the molecular mechanisms involved in learning and memory in insects have been studied intensively, the intracellular signaling mechanisms involved in early memory formation are not fully understood. We previously demonstrated that phospholipase C epsilon (PLCe), whose product is involved in calcium signaling, is almost selectively expressed in the mushroom bodies, a brain structure important for learning and memory in the honeybee. Here, we pharmacologically examined the role of phospholipase C (PLC) in learning and memory in the honeybee. First, we identified four genes for PLC subtypes in the honeybee genome database. Quantitative reverse transcription-polymerase chain reaction revealed that, among these four genes, three, including PLCe, were expressed higher in the brain than in sensory organs in worker honeybees, suggesting their main roles in the brain. Edelfosine and neomycin, pan-PLC inhibitors, significantly decreased PLC activities in homogenates of the brain tissues. These drugs injected into the head of foragers significantly attenuated memory acquisition in comparison with the control groups, whereas memory retention was not affected. These findings suggest that PLC in the brain is involved in early memory formation in the honeybee. To our knowledge, this is the first report of a role for PLC in learning and memory in an insect. 


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