title: Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance.
abstract: Green fluorescent protein (GFP) and other fluorescent proteins are essential tools for biological research. When fused to peptides or proteins as a reporter, GFP enables localization and quantitation of gene products in otherwise unmanipulated live cells or organisms. We previously reported that a sizable fraction of nascent GFP is post-translationally converted into a 20-kDa Triton X-100-insoluble proteasome substrate (Qian, S. B., Princiotta, M. F., Bennink, J. R., and Yewdell, J. W. (2006) J. Biol. Chem. 281, 392-400; Dolan, B. P., Li, L., Veltri, C. A., Ireland, C. M., Bennink, J. R., and Yewdell, J. W. (2011) J. Immunol. 186, 2065-2072). Here, we show that a similarly sized fragment is generated by all GFP and red fluorescent protein family members we examined. We demonstrate that fragmentation is a by-product of GFP chromophore rearrangement. A non-rearranging GFP mutant fails to fragment and generates diminished levels of K(b)-SIINFEKL complexes when SIINFEKL is genetically fused to either the C- or N-terminal domains of GFP fusion proteins. Instructively, another fragmenting GFP mutant that cannot create the functional chromophore but still generates fragments also demonstrates diminished K(b)-SIINFEKL generation. However, the mutant and wild-type fragments differ fundamentally in that wild-type fragments are rapidly liberated from the intact molecule and degraded quickly, accounting for increased K(b)-SIINFEKL generation. In the fragmenting mutant, the fragments are generated slowly and remain associated, likely in a native conformation based on their original structural description (Barondeau, D. P., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2006) J. Am. Chem. Soc. 128, 4685-4693). The wild-type GFP fragments represent the first biochemically defined natural defective ribosomal products to contribute peptides for immunosurveillance, enabling quantitation of peptide generation efficiency from this source of defective ribosomal products. More broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes.
authors.affiliation: From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Jiajie
authors.initials: J
authors.lastname: Wei
authors.affiliation: From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: James S
authors.initials: JS
authors.lastname: Gibbs
authors.affiliation: From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Heather D
authors.initials: HD
authors.lastname: Hickman
authors.affiliation: From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Cush
authors.affiliation: From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Jack R
authors.initials: JR
authors.lastname: Bennink
authors.affiliation: From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892 JYEWDELL@niaid.nih.gov.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Yewdell
mesh_terms: D017951:Antigen Presentation; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D015166:Monitoring, Immunologic; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D000097573:Red Fluorescent Protein
keywords: DRiPs; antigen processing; fluorescent protein; fragmentation; immunology; immunosurveillance; protein misfolding; protein processing; protein synthesis