Abstract
Compared with whole-cell proteomic analysis, subcellular proteomic analysis is advantageous not only for the increased coverage of low abundance proteins but also for generating organelle-specific data containing information regarding dynamic protein movement. In the present study, peroxisome-enriched fractions from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and subjected to quantitative proteomics analysis. We identified 311 proteins that were significantly changed by SeV infection. Among these altered proteins, 25 are immune response-related proteins. Further bioinformatic analysis indicated that SeV infection inhibits cell cycle-related proteins and membrane attack complex-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 candidate proteins. We identified LGALS3BP and CALU as potential negative regulators of the virus-induced activation of the type I interferons.
Highlights
One of the most significant evolutionary features in eukaryotes is the appearance of a membrane system to separate enzymatic reactions and to provide scaffolds for signal transduction
The Sendai virus (SeV)-infected sample was labeled with the “heavy” reagent, whereas the mock treated sample was labeled with the “light” reagent
Knockdown of CALU or LGALS3BP potentiated SeV-induced transcription of the IFNB1 gene and SeV-induced IFN- protein levels (Fig. 7A, 7B). These results collectively prove that CALU and LGALS3BP act as potential negative regulators in virus-triggered type I IFN signaling
Summary
One of the most significant evolutionary features in eukaryotes is the appearance of a membrane system to separate enzymatic reactions and to provide scaffolds for signal transduction. Subcellular organelles with extensive membrane systems include the endoplasmic reticulum (ER), mitochondria, endosomes and peroxisomes. Despite their well-recognized functions in cell metabolism, these organelles and their related membranes have been identified in recent years as important innate immune platforms [5]. To systematically analyze the role of peroxisomal-related proteins in innate immune responses, we used a modified two-step gradient centrifugation method to enrich the peroxisomes from cells with or without SeV infection, followed by a quantitative proteomic analysis. Luciferase reporter assays were performed to further screen for the significantly changed proteins that could affect SeV-induced activation of the type I IFN signaling pathway. Does our data provide new and unbiased protein-level information regarding viral infection processes, we provide direct evidence for the involvement of two proteins (CALU and LGALS3BP) as potential negative regulators in the virus-triggered induction of the type I IFNs
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