Mapping wild-type and R345W fibulin-3 intracellular interactomes

John D. Hulleman, Joseph C. Genereux, Annie Nguyen

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate ‘heavy’ and ‘light’ isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.

Original languageEnglish (US)
Pages (from-to)165-169
Number of pages5
JournalExperimental Eye Research
Volume153
DOIs
StatePublished - Dec 1 2016

Fingerprint

Isotope Labeling
Retinal Degeneration
Retinal Pigment Epithelium
Protein Folding
Macular Degeneration
fibulin
Immunoprecipitation
Adenoviridae
Endoplasmic Reticulum
Disulfides
Proteolysis
Extracellular Matrix
Glycoproteins
Proteins
Cell Culture Techniques
Western Blotting
Technology
Light
Amino Acids
Mutation

Keywords

  • Endoplasmic reticulum
  • Fibulin-3
  • Malattia Leventinese
  • Mass spectrometry
  • MudPIT
  • SILAC

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Mapping wild-type and R345W fibulin-3 intracellular interactomes. / Hulleman, John D.; Genereux, Joseph C.; Nguyen, Annie.

In: Experimental Eye Research, Vol. 153, 01.12.2016, p. 165-169.

Research output: Contribution to journalArticle

Hulleman, John D. ; Genereux, Joseph C. ; Nguyen, Annie. / Mapping wild-type and R345W fibulin-3 intracellular interactomes. In: Experimental Eye Research. 2016 ; Vol. 153. pp. 165-169.
@article{f59022fab64a4823b789cad3e71a8b33,
title = "Mapping wild-type and R345W fibulin-3 intracellular interactomes",
abstract = "Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate ‘heavy’ and ‘light’ isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.",
keywords = "Endoplasmic reticulum, Fibulin-3, Malattia Leventinese, Mass spectrometry, MudPIT, SILAC",
author = "Hulleman, {John D.} and Genereux, {Joseph C.} and Annie Nguyen",
year = "2016",
month = "12",
day = "1",
doi = "10.1016/j.exer.2016.10.017",
language = "English (US)",
volume = "153",
pages = "165--169",
journal = "Experimental Eye Research",
issn = "0014-4835",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Mapping wild-type and R345W fibulin-3 intracellular interactomes

AU - Hulleman, John D.

AU - Genereux, Joseph C.

AU - Nguyen, Annie

PY - 2016/12/1

Y1 - 2016/12/1

N2 - Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate ‘heavy’ and ‘light’ isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.

AB - Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate ‘heavy’ and ‘light’ isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.

KW - Endoplasmic reticulum

KW - Fibulin-3

KW - Malattia Leventinese

KW - Mass spectrometry

KW - MudPIT

KW - SILAC

UR - http://www.scopus.com/inward/record.url?scp=84993978561&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84993978561&partnerID=8YFLogxK

U2 - 10.1016/j.exer.2016.10.017

DO - 10.1016/j.exer.2016.10.017

M3 - Article

C2 - 27777122

AN - SCOPUS:84993978561

VL - 153

SP - 165

EP - 169

JO - Experimental Eye Research

JF - Experimental Eye Research

SN - 0014-4835

ER -