Autophagy Modulators Profoundly Alter the Astrocyte Cellular Proteome

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.

Combination of IL-17A/F and TNF-α uniquely alters the bronchial epithelial cell proteome to enhance proteins that augment neutrophil migration

“…The SOMAmer ® V.2 protein arrays were used for profiling the abundance of 1322 protein targets in each sample. The arrays were processed and analyzed according to the manufacturer's recommended protocol (SOMALogic, Inc) and as detailed in previous studies [21,[71][72][73][74]. Protein abundance was quantified using the Agilent hybridization array scanner in relative fluorescence unit (RFU), as previously described [21,[71][72][73][74].…”

“…The arrays were processed and analyzed according to the manufacturer's recommended protocol (SOMALogic, Inc) and as detailed in previous studies [21,[71][72][73][74]. Protein abundance was quantified using the Agilent hybridization array scanner in relative fluorescence unit (RFU), as previously described [21,[71][72][73][74]. The RFU readout values were log2-transformed and used for pairwise differential analysis using an uncorrected Welch's T-test.…”

Uncovering the Depths of the Human Proteome: Antibody-based Technologies for Ultrasensitive Multiplexed Protein Detection and Quantification

“…In the recent years, the multiplexing ability of the SOMAscan assay evolved from measuring 1305 human proteins to quantifying up to 7000 analytes simultaneously in a volume of 55 μl serum, plasma, and other biological sample with ultrasensitivity (claimed LLOQ of 125 fg/ml) ( 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 ). An 8- to 10-log dynamic range is attained through three serial dilutions of every sample in efforts to detect high- and low-abundance proteins ( 118 ).…”

Impaired Mitophagy in Neurons and Glial Cells during Aging and Age-Related Disorders

“…According to proteomic analysis, the pharmacological modulators of autophagy rapamycin (an inducer of mitophagy) and bafilomycin (an inhibitor of mitophagy), induce changes in the expression of numerous glial molecular targets involved in the differentiation, development, and survival of astrocytes [ 76 ]. These targets include proteins involved in the activation of astrocytes during neuroinflammation and determine their pro-inflammatory or anti-inflammatory phenotype [ 77 ].…”