RESUMO
Aims: AGR2 is a tissue-restricted member of the protein disulfide isomerase family that has attracted interest because it is highly expressed in a number of cancers, including gastroesophageal adenocarcinoma. The behavior of AGR2 was analyzed under oxidizing conditions, and an alkylation trapping and immunoprecipitation approach were developed to identify novel AGR2 interacting proteins. Results: The data show that AGR2 is induced in esophageal adenocarcinoma, where it participates in redox-responsive, disulfide-dependent complexes. AGR2 preferentially engages with MUC-5 as a primary client and is coexpressed with the acidic mucin in Barrett's esophagus and esophageal adenocarcinoma tissue. Innovation: New partner chaperones for AGR2 have been identified, including peroxiredoxin IV, ERp44, P5, ERp29, and Ero1α. AGR2 interacts with unexpected metabolic enzymes, including aldehyde dehydrogenase (ALDH)3A1, and engages in an alkylation-sensitive association with the autophagy receptor SQSTM1, suggesting a potential mechanism for the postendoplasmic reticulum targeting of AGR2 to mucin granules. Disulfide-driven AGR2 complex formation provides a framework for a limited number of client proteins to interact, rather than for the recruitment of multiple novel clients. Conclusion: The extended AGR2 interactome will facilitate the development of therapeutics to target AGR2/mucin pathways in esophageal cancer and other conditions, including chronic obstructive pulmonary disease.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Mucina-5AC/metabolismo , Mucinas/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismo , Adenocarcinoma/genética , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Neoplasias Esofágicas/genética , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Espectrometria de Massas , Microscopia Confocal , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mucina-5AC/genética , Mucoproteínas/genética , Proteínas Oncogênicas/genética , Oxirredução , Ligação Proteica , Proteômica , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismoRESUMO
Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition.
