Proteomic Analysis Provides Insights Into PPIP5K2 Function and Its Impact on Corneal Energy Metabolism.

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.