RT Journal Article
SR Electronic
T1 Spatial and temporal alterations in protein structure by EGF regulate cryptic cysteine oxidation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 624304
DO 10.1101/624304
A1 Jessica B Behring
A1 Sjoerd van der Post
A1 Arshag D Mooradian
A1 Matthew J Egan
A1 Maxwell I Zimmerman
A1 Jenna L. Clements
A1 Gregory R Bowman
A1 Jason M Held
YR 2019
UL http://biorxiv.org/content/early/2019/04/30/624304.abstract
AB Stimulation of receptor tyrosine kinases (RTK) such as EGF locally increase reactive oxygen species (ROS) levels at the plasma membrane that oxidize cysteines in proteins to enhance downstream signaling. Spatial confinement of ROS is an important regulatory mechanism to redox signaling, but it remains unknown why stimulation of different receptor tyrosine kinases (RTKs) at the plasma membrane target distinct sets of downstream proteins. To uncover additional mechanisms specifying which cysteines are redox regulated by EGF stimulation, we performed time-resolved quantification of the oxidation of 4,200 cysteine sites subsequent to EGF stimulation in A431 cells. EGF induces three distinct spatiotemporal patterns of cysteine oxidation in functionally organized protein networks, consistent with the spatial confinement model. Unexpectedly, protein crystal structure analysis and molecular dynamic simulation indicate widespread redox regulation of cryptic cysteines that are only solvent exposed upon changes in protein conformation. Phosphorylation and increased flux of nucleotide substrates serve as two distinct modes by which EGF specifies which cryptic cysteines become solvent exposed and redox regulated. Since proteins structurally regulated by different RTKs or cellular perturbations are largely unique, solvent exposure and redox regulation of cryptic cysteines is an important mechanism contextually delineating redox signaling networks.Significance Statement Cellular redox processes are interconnected, but are not in equilibrium. Thus, understanding the redox biology of cells requires a systems-level, rather than reductionist, approach. Factors specifying which cysteines are redox regulated by a stimulus remain poorly characterized but are critical to understanding the fundamental properties of redox signaling networks. Here, we show that EGF stimulation induces oxidation of specific cysteines in 3 distinct spatiotemporal patterns. Redox regulated proteins include many proteins in the EGF pathway as well as many cysteines with known functional importance. Many redox regulated cysteines are cryptic and solvent exposed by changes in protein structure that were induced by EGF treatment. The novel finding that cryptic cysteines are redox regulated has important implications for how redox signaling networks are specified and regulated to minimize crosstalk. In addition, this time-resolved dataset of the redox kinetics of 4,200 cysteine sites is an important resource for others and is an important technological achievement towards systems-level understanding of cellular redox biology.DDAdata-dependent acquisitionDIAdata-independent acquisitionEGFEpidermal growth factorEGFREpidermal growth factor receptorERendoplasmic reticulumGOgene ontologyGSHglutathioneGSSGglutathione disulfideH2O2hydrogen peroxideHIFHypoxia-inducible factorIACiodoacetamideIPAIngenuity Pathway Analysis (Qiagen)LMW-PTPlow molecular weight protein tyrosine phosphatasem/zmass to charge ratioMSMass spectrometryNADPNicotinamide adenine dinucleotide diphosphateNADPHDihydronicotinamide adenine dinucleotide phosphateNEMN-ethylmaleimideOxRACOxidation analysis by resin-assisted capturePKMpyruvate kinasePTPsprotein tyrosine phosphatasesPRDXperoxyredoxinspYpan-phosphotyrosineROSreactive oxygen speciesRSArelative solvent accessibilityRTKreceptor tyrpsine kinaseSDSsodium dodecyl sulfateTCEPtris(2-carboxyethyl)phosphineUGDHUDP-glucose 6-dehydrogenaseVCPvalosin-containing protein