Novel cytosolic form of human HSPA5 involved specifically in sustained responses to infection

CRISTIAN JORGE ALEJANDRO ASENSIO; RODOLFO C GARCÍA

Porto Alegre

Congreso; IX Cell Stress Society International Workshop on the Molecular Biology of Stress Responses; 2012

Cell Stress Society International

INTRODUCTION: Many infectious/inflammatory diseases are latent or chronic. Most human diseases have also some inflammatory component due to exogenous /endogenous receptor ligands. On the other hand, some infectious agents induce some level of UPR, stressing the ER. Also different diseases are influenced by the UPR. There is however a clear paucity of knowledge concerning the cellular and molecular regulation of the chronic adaptation of the stress signaling pathways occurring in the cytoplasm during late-phase inflammatory / infectious events. By using a combination of novel proteomic and biochemical methods we searched for novel cytoplasmic proteome responses of innate immune cells during infection, in order to dissect the late-phase signaling pathways involved. We found a novel form of an ?ER chaperone? having a novel PTM. OBJETIVES: To develop novel methods to study how cells mount sustained proteome responses to infection by focusing in the alterations occurring in the cytoplasm/cytosol. Collaterally, to study the role (if any) of the UPR during infection or sustained ligation of innate immune receptors. METHODOLOGY: We developed novel ways to label cytoplasmic proteins after cell harvest and fractionation, without disturbing the in vivosignaling pathways, in order to quantitatively and sensitively monitor some interesting proteins, that otherwise cannot be studied. We employed a combination of proteomics and protein biochemistry approaches using in vitro assays, SDS-PAGE electrophoretic separation of proteins and autoradiography. We applied these methods to the study of infected human cells. RESULTS: Among other proteins, we discovered a novel, low-abundant post-translationally generated, cytosolic form of human HSPA5. This novel form is not regulated or influenced by different chemical stressors of the ER. This is the first cytosolic form of HSPA5 isolated from the UPR. The novel form is specifically involved in responses to infection/ innate immune receptor ligation. It contains a novel kind of reproducible PTM not reported before in other forms of HSPA5 or any HSP. This form is not a consequence of alternative splicing. It is a PTM variant. This HSPA5 form is specifically involved in the sustained sensing of microbial molecules through a receptor. It is always reproducibly upregulated regardless of the microbe type. It has a half-life of many days. We have developed the only method able to monitor this novel form in a sensitive, reproducible and quantitative way (not possible to quantify it with the known methods). CONCLUSION: It is now evident that some cytosolic HSPA5 forms might have specific roles in the cytosol and also that there is at least one cytosolic form not influenced by the UPR. SUPPORT: ICGEB.