Insight into PreImplantation Factor (PIF) mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP) through essential RIKP [corrected] binding site

• Published in: PloS one Vol. 9; no. 7; p. e100263
• Main Authors: Barnea, Eytan R, Lubman, David M, Liu, Yan-Hui, Absalon-Medina, Victor, Hayrabedyan, Soren, Todorova, Krassimira, Gilbert, Robert O, Guingab, Joy, Barder, Timothy J
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science (PLoS) 2014
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Computer Simulation; Embryo, Mammalian - metabolism; Embryonic Development; Horses - embryology; Mice; Models, Molecular; Oxidative Stress; Peptides - chemistry; Peptides - metabolism; Peptides - physiology; Protein Folding; Protein Structure, Tertiary; Thioredoxins - metabolism
• ISSN: 1932-6203
• DOI: 10.1371/journal.pone.0100263

Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.
FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis.
PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.
Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.