title: 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.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Research & Development, SIEP The Society for the Investigation of Early Pregnancy, Cherry Hill, New Jersey, United States of America; Research & Development, BioIncept, LLC, Cherry Hill, New Jersey, United States of America; Department of Obstetrics, Gynecology and Reproduction, UMDNJ-Robert Wood Johnson Medical School, Camden, New Jersey, United States of America.
authors.forename: Eytan R
authors.initials: ER
authors.lastname: Barnea
authors.affiliation: Department Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, United States of America.
authors.forename: David M
authors.initials: DM
authors.lastname: Lubman
authors.affiliation: Department Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, United States of America.
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Liu
authors.affiliation: Reproductive Medicine, Cornell University, College of Veterinary Medicine, Ithaca, New York, United States of America.
authors.forename: Victor
authors.initials: V
authors.lastname: Absalon-Medina
authors.affiliation: Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors.forename: Soren
authors.initials: S
authors.lastname: Hayrabedyan
authors.affiliation: Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors.forename: Krassimira
authors.initials: K
authors.lastname: Todorova
authors.affiliation: Reproductive Medicine, Cornell University, College of Veterinary Medicine, Ithaca, New York, United States of America.
authors.forename: Robert O
authors.initials: RO
authors.lastname: Gilbert
authors.affiliation: Chemical Biology and Proteomics, Banyan Biomarkers, Alachua, Florida, United States of America.
authors.forename: Joy
authors.initials: J
authors.lastname: Guingab
authors.affiliation: Research & Development, Eprogen, Downers Grove, Illinois, United States of America.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Barder
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D004622:Embryo, Mammalian / Q000378:metabolism*; D047108:Embryonic Development; D006736:Horses / Q000196:embryology; D051379:Mice; D008958:Models, Molecular; D018384:Oxidative Stress; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D010455:Peptides; C561617:preimplantation factor, mouse; D013879:Thioredoxins