Human Ortholog of a Plant Salicylic Acid Receptor Found in SK-N-SH Cell Line
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Human Ortholog of a Plant Salicylic Acid Receptor Found in SK-N-SH Cell Line Hanna Skubatz • William N. Howald
Published online: 28 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Our previous studies have described the purification and characterization of a novel plant NAD(P)reductase like protein (RL) from the thermogenic appendix of the Sauromatum guttatum inflorescence. RL is mainly located in cytoplasm of thermogenic plants and it can act like a bistable switch. It adopts a compact conformation during heat-production and a more expanded conformation when heat is not generated. Addition of salicylic acid, a natural thermogenic inducer, at picomolar concentration to a solution of purified RL induced a discontinuous volume phase transition in which the volume of RL in the oligomeric form expanded and shrunk repeatedly every 4–5 min. In the present study using ESI–MS analysis we have demonstrated the existence of RL in the human SKN-SH cell line and in mouse brain tissue. The molecular mass of human RL is in the same range as of its plant counterpart, 34,140 ± 34 Da. The charge state distribution of the human RL is identical to its plant counterpart from the Sauromatum appendix during heat-production. Human RL was present in the compact state when it was purified from the SK-N-SH cell line When these cells were treated with salicylic acid (10 lM) a shift to a much more compact conformation was observed. It seems that the potential of RL to respond to salicylic acid was conserved. These
H. Skubatz (&) NeoPro Labs, 1124 Columbia St., Seattle, WA 98104, USA e-mail: [email protected] W. N. Howald Department of Medicinal Chemistry, School of Pharmacy Mass Spectrometry Center, University of Washington, Seattle, WA 98195, USA e-mail: [email protected]
results may reveal the existence of a thermoregulation system that is evolutionarily conserved and is operating by conformational changes. This discovery may also represent an opportunity for a better understanding of some of the diverse functions of salicylic acid and aspirin in plants and humans. Keywords Appendix ESI-MS Human neuroblastoma SK-N-SH cell line Mice NAD(P)reductase Salicylic acid Sauromatum guttatum Abbreviations ACN Acetonitrile ASA Aspirin CSD Charge-state distribution D-Day The day of inflorescence opening and heatproduction 2,6-DHBA 2,6-dihydroxybenzoic acid HHM Hidden Markov model IP Isopropanol ESI Electrospray ionization MaxEnt Maximum entropy deconvolution software of multiply charged electrospray spectra MS Mass spectrometry m/z Mass to charge ratio PBS Phosphate buffered saline PVP Polyvinylpyrrolidone RL NAD(P) reductase-like protein RP-HPLC Reversed-phase high-performance liquid chromatography SA Salicylic acid SDR Short-chain dehydrogenase/reductase enzymes TIC Total ion current TFA Trifluoroacetic acid
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1 Introduction The appendix (a 20 cm-long, slender organ) of the Sauromatum guutatum inflorescence generates heat on the day of the inflorescence opening. The appendix temperature can r
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