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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.
Insights
Loss of Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) in corneal cells impacts energy metabolism and glycolysis. This finding suggests PPIP5K2 may regulate corneal glycolysis, offering insights into keratoconus pathogenesis.
Area of Science:
- Ophthalmology
- Molecular Biology
- Biochemistry
Background:
- Keratoconus (KC) is a corneal disease where genetic variants, including those in Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2), may play a role.
- The precise molecular mechanisms linking PPIP5K2 variants to KC pathogenesis are not fully understood.
Purpose of the Study:
- To investigate the proteomic and metabolic consequences of PPIP5K2 loss-of-function in human corneal fibroblasts (HCFs).
- To examine these effects under conditions mimicking KC-related factors: cyclic mechanical stretch (CMS) and TGFβ1 treatment.
Main Methods:
- Primary HCFs with reduced PPIP5K2 expression were treated with varying levels of TGFβ1 and subjected to CMS.
- Proteomic analysis was performed using liquid chromatography-mass spectrometry, followed by bioinformatics analysis (GO and pathway analysis).
- Cellular metabolic function was assessed using Seahorse assays to measure ATP levels and glycolytic rates.
Main Results:
- A total of 1549 proteins were identified, with 19 differentially expressed proteins (DEPs) found under PPIP5K2 knockdown conditions.
- Pathway analysis indicated significant enrichment of proteins involved in energy metabolism.
- Loss of PPIP5K2 led to reduced ATP levels and impaired glycolytic function in HCFs.
Conclusions:
- The study identified key proteins and energy metabolism pathways affected by PPIP5K2 loss in corneal fibroblasts.
- These findings suggest a novel role for PPIP5K2 in regulating corneal glycolysis.
- This implicates PPIP5K2 in the molecular mechanisms underlying keratoconus.
