title: TULP1 Missense Mutations Induces the Endoplasmic Reticulum Unfolded Protein Response Stress Complex (ER-UPR).
abstract: Mutations in the TULP1 gene are associated with early-onset retinitis pigmentosa (RP); however, the molecular mechanisms related to the deleterious effects of TULP1 mutations remains unknown. Several studies have shown that misfolded proteins secondary to genetic mutations can accumulate within the endoplasmic reticulum (ER), causing activation of the unfolded protein response (UPR) complex followed by cellular apoptosis. We hypothesize that TULP1 mutations produce misfolded protein products that accumulate in the ER and induce cellular apoptosis via the UPR. To test our hypothesis, we first performed three in-silico analyses of TULP1 missense mutations (I459K, R420P and F491L), which predicted misfolded protein products. Subsequently, the three mutant TULP1-GFP constructs and wild-type (wt) TULP1-GFP were transiently transfected into hTERT-RPE-1 cells. Staining of cells using ER tracker followed by confocal microscopy showed wt-TULP1 localized predominantly to the cytoplasm and plasma membrane. In contrast, all three mutant TULP1 proteins revealed cytoplasmic punctate staining which co-localized with the ER. Furthermore, western blot analysis of cells expressing mutant TULP1 proteins revealed induction of downstream targets of the ER-UPR complex, including BiP/GPR-78, phosphorylated-PERK (Thr980) and CHOP. Our in-vitro analyses suggest that mutant TULP1 proteins are misfolded and accumulate within the ER leading to induction of the UPR stress response complex.
authors.affiliation: Department of Ophthalmic Research-i31, Cole Eye Institute, Cleveland Clinic, 9500 Euclid Avenue, 44195, Cleveland, OH, USA. hagstrs@ccf.org.
authors.forename: Glenn P
authors.initials: GP
authors.lastname: Lobo
authors.affiliation: Department of Ophthalmic Research-i31, Cole Eye Institute, Cleveland Clinic, 9500 Euclid Avenue, 44195, Cleveland, OH, USA. ebkel@ccf.org.
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Ebke
authors.affiliation: Department of Ophthalmic Research-i31, Cole Eye Institute, Cleveland Clinic, 9500 Euclid Avenue, 44195, Cleveland, OH, USA. aca14@case.edu.
authors.forename: Adrian
authors.initials: A
authors.lastname: Au
authors.affiliation: Department of Ophthalmic Research-i31, Cole Eye Institute, Cleveland Clinic, 9500 Euclid Avenue, 44195, Cleveland, OH, USA.|Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 44195, Cleveland, OH, USA.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Hagstrom
mesh_terms: D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000235:genetics; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D020125:Mutation, Missense; D010766:Phosphorylation / Q000235:genetics; D017510:Protein Folding; D051742:Transcription Factor CHOP / Q000378:metabolism; D056811:Unfolded Protein Response / Q000235:genetics*; D019892:eIF-2 Kinase / Q000378:metabolism
chemical_list: C497249:DDIT3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D005136:Eye Proteins; D006360:Heat-Shock Proteins; C105443:TULP1 protein, human; D051742:Transcription Factor CHOP; C460640:EIF2AK3 protein, human; D019892:eIF-2 Kinase
keywords: Endoplasmic reticulum; Photoreceptor; Retinal degeneration; Tulp1; Unfolded protein response