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Published on: September 27, 2016
Proteomic Analysis Provides Insights Into PPIP5K2 Function and Its Impact on Corneal Energy Metabolism
Theresa Akoto1, Caili Hao1, Zhong Chen1
1Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, Georgia, United States.
Purpose:
Variants in the Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) gene may contribute to familial keratoconus (KC) pathogenesis, although the underlying molecular mechanisms remain elusive. We aimed to determine the proteomic effect of PPIP5K2 loss-of-function in response to two KC-related factors (cyclic mechanical stretch [CMS] and TGFβ1 treatment) in primary human corneal fibroblasts (HCFs).
Methods:
PPIP5K2 knockdown HCFs (n = 4) were cultured and treated with 0, 5, and 10 ng/mL of TGFβ1 with or without 15% CMS (1 cycle/second, 24 hours) using a Flexcell Tension system. Cellular proteins (50 µg) were digested with trypsin and analyzed through label-free liquid chromatography-mass spectrometry. Differentially expressed proteins (DEPs) were determined using 2-way ANOVA by adjusting the effect of TGFβ1 and CMS with false discovery rate (FDR)-adjusted P values ≤ 0.1. Gene ontology (GO) and pathway analyses were performed with the PANTHER classification system. Metabolic profiles of the PPIP5K2 knockdown in HCFs were evaluated with Seahorse assays.
Results:
A total of 1549 proteins were identified across 48 samples. With FDR ≤ 0.1, the effect of PPIP5K2, TGFβ1 treatment, and CMS status revealed 19 DEPs in the PPIP5K2 knockdown HCFs. GO and pathway analyses revealed over-representation of proteins involved in energy metabolism pathways. Furthermore, a reduction in ATP levels was observed, corroborated by diminished glycolytic function following the loss of PPIP5K2 in HCFs.
Conclusions:
This study identified many proteins and energy-related pathways following the loss of PPIP5K2 in HCFs, suggesting a potential role of PPIP5K2 in regulating glycolysis in the cornea.
