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Targeting and inactivation of endoplasmic reticulum chaperones by L‐homocysteine: a pathway to ER stress
Author(s) -
Glushchenko Alla V.,
Axhemi Armend A.,
AlBayati Hiam,
Austin Richard C.,
Jacobsen Donald W.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1002.1
Subject(s) - endoplasmic reticulum , unfolded protein response , homocysteine , kdel , protein disulfide isomerase , chemistry , glucose regulated protein , biochemistry , protein folding , recombinant dna , microsome , microbiology and biotechnology , in vitro , biology , golgi apparatus , gene
Homocysteine may disrupt protein folding within the endoplasmic reticulum (ER) causing ER stress and activating the unfolded protein response. We hypothesize that L‐homocysteine induces ER stress by interacting with and modulating the activity of 78‐kDa glucose‐regulated protein (GRP78) and protein disulfide isomerase (PDI). In vitro S ‐homocysteinylation of human recombinant GRP78 (hrGRP78) and rat recombinant PDI (rrPDI) was identified by SDS‐PAGE and phosphorimage analysis. We determined that 0.27 mol of 35 S ‐L‐homocysteine and 0.38 mol of 35 S ‐L‐homocysteine react per mol of hrGRP78 and rrPDI, respectively. Evidence for in situ S ‐homocysteinylation of GRP78 and PDI was obtained by incubating rat liver microsomes with 35 S ‐L‐homocysteine, immunoprecipitating with mouse anti‐KDEL monoclonal antibodies, phosphorimaging and LC/MS/MS. Using surface plasmon resonance, we observed weaker interactions between peptide pp38 and S ‐homocysteinylated‐hrGRP78, compared to native hrGRP78 protein. In contrast, L‐homocysteine stimulated the activity of PDI in the insulin precipitation assay. We conclude that L‐homocysteine directly targets and alters the function of the ER resident chaperones GRP78 and PDI. This pathway links homocysteine to ER stress, cardiovascular disease and other disorders associated with abnormal protein accumulation. (Supported by NIH, HL52234 (DWJ)).