Autophagy Modulators Profoundly Alter the Astrocyte Cellular Proteome.

Affan Ali Sher,Kevin M Coombs,Ang Gao

doi:10.3390/cells9040805

Affan Ali Sher, Kevin M Coombs + Show 1 more

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https://doi.org/10.3390/cells9040805

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Abstract

Autophagy is a key cellular process that involves constituent degradation and recycling during cellular development and homeostasis. Autophagy also plays key roles in antimicrobial host defense and numerous pathogenic organisms have developed strategies to take advantage of and/or modulate cellular autophagy. Several pharmacologic compounds, such as BafilomycinA1, an autophagy inducer, and Rapamycin, an autophagy inhibitor, have been used to modulate autophagy, and their effects upon notable autophagy markers, such as LC3 protein lipidation and Sequestosome-1/p62 alterations are well defined. We sought to understand whether such autophagy modulators have a more global effect upon host cells and used a recently developed aptamer-based proteomic platform (SOMAscan®) to examine 1305 U-251 astrocytic cell proteins after the cells were treated with each compound. These analyses, and complementary cytokine array analyses of culture supernatants after drug treatment, revealed substantial perturbations in the U-251 astrocyte cellular proteome. Several proteins, including cathepsins, which have a role in autophagy, were differentially dysregulated by the two drugs as might be expected. Many proteins, not previously known to be involved in autophagy, were significantly dysregulated by the compounds, and several, including lactadherin and granulins, were up-regulated by both drugs. These data indicate that these two compounds, routinely used to help dissect cellular autophagy, have much more profound effects upon cellular proteins.

Highlights

There is growing appreciation for the roles of apoptosis and autophagy in numerous areas of scientific research, including their roles in normal development and differentiation, and their potential roles in viral infections and in tumor cell development and function
Our results indicate that sub-toxic concentrations of both drugs impact proteins involved in autophagy, and dramatically impact numerous other cellular networks, pathways, and biologic functions, including development, cell morphology, cell movement, cell death and survival, lipid metabolism, the cardiovascular system, neurological and immunological diseases, and protein synthesis
To ensure that we treated our U-251 cells with sufficient concentrations of the autophagy modulating drugs BafA1 and Rapa, without inducing excessive cytotoxicity, we treated our cells with various concentrations of each drug for various periods of time and compared cell viability of the treated cells to non-treated cells

Summary

Introduction

There is growing appreciation for the roles of apoptosis and autophagy in numerous areas of scientific research, including their roles in normal development and differentiation, and their potential roles in viral infections and in tumor cell development and function. Autophagy is a complex cellular pathway that targets organelles, protein aggregates, and pathogen components for lysosomal degradation [1,2]. There are three general types of autophagic process: macroautophagy, microautophagy, and chaperone-mediated autophagy (reviewed in [3]). Microautophagy is a non-selective degradative process involving the direct engulfment of cytoplasmic material by the lysosomal membrane [5]. Chaperone-mediated autophagy recognizes and selects cytoplasmic components for degradation using Hsc chaperone protein [6,7,8]. This Hsc tags the substrate and brings it to the lysosomal membrane where it binds to LAMP-2A before being translocated inside the

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