Abstract
Purpose :
The major risk factor for primary open angle glaucoma (POAG), a leading cause of irreversible blindness, is elevated intraocular pressure (IOP) due to pathology in the conventional outflow pathway. IOP homeostasis requires the cells of the conventional outflow tract, which includes the trabecular meshwork (TM) and Schlemm’s canal (SC), to detect and respond to changes in IOP. We have identified caveolae as a mechanosensor and mechanoprotector in TM and SC cells. Importantly, sequence variants in the CAV1 gene, the primary component of caveolae, associate reproducibly with elevated IOP and POAG risk. We have previously found complementary and cell-intrinsic roles for CAV1 in TM and SC cells. However, the cell-type-specific molecular mechanisms in the TM and their interplay have yet to be fully understood. Therefore, the goal of the present study is to define the molecular machinery within TM cells with which caveolae transduce changes in IOP.
Methods :
To define the CAV1 interactome, we immunoprecipitated CAV1 and bound proteins from cultured human TM cells (n = 3 different strains). Successful immunoprecipitation was confirmed with western blotting for CAV1. After which, bound proteins were analyzed with quantitative mass spectrometry in the Mass Spectrometry/Molecular Biology Core Module at Duke University. CAV1 interactions were defined with the stringent criteria of 2 or more peptides, ≥ 2-fold enrichment, and an ANOVA result with p < 0.05.
Results :
We identified fifty-five CAV1-interacting proteins including known caveolae proteins like CAV2, PTRF/CAVIN1, PRKCDBP/CAVIN3, and EHD2; known lipid raft proteins like FLOT2 and STOM; and FLNA, a key protein linking caveolae to actin stress fibers. We also identified several novel interactions relevant to mechanobiology, including myosin phosphatase rho-interacting protein (MPRIP), desmosomal proteins (DSP and DSG1), and an extracellular matrix remodeling protein lysyl oxidase (LOX).
Conclusions :
These experiments have identified proteins that directly interact with CAV1 in caveolae in the TM. As caveolae are mechanosensors that are likely involved in TM mechanotransduction and IOP homeostasis, future studies will evaluate the response of the CAV1 interactome to mechanical stimulation with cyclic stretch.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.