Biology subjects

Sechrest, E.

Publications and source records attributed to Sechrest, E..

3 recordsLinked to original sources

bioRxiv2026

Mapping the chaperonin TRiC/CCT interactome in mouse photoreceptors reveals functional significance for energy metabolism

The eukaryotic chaperonin TRiC/CCT is essential for folding a diverse set of proteins, yet its interactome and functional roles in specialized neurons remain incompletely understood. To investigate TRiC-mediated folding in rod photoreceptors, we generated a transgenic mouse line expressing an epitope-tagged Tcp-1 subunit, enabling purification of intact TRiC complexes from retinal tissue. Mass spectrometry identified 226 TRiC-interacting proteins, including known TRiC substrates and co-chaperones as well as numerous novel candidates enriched in RNA processing, cytoskeletal organization, and cell-cycle regulation. Using a TRiC loss-of-function model in which expression of a short splice isoform of phosducin-like protein (PhLPs) competitively inhibits TRiC activity, we observed marked reductions in canonical TRiC substrates, including tubulins, transducin {beta} subunits, and triosephosphate isomerase, as well as secondary alterations in proteins involved in cytoskeletal stability, membrane trafficking, energy metabolism, and phototransduction. Quantitative metabolomic profiling revealed that TRiC deficiency induces a metabolic "energy crisis" characterized by reduced glycolytic- and tricarboxylic acid cycle intermediates, acylcarnitines, ATP, NAD, and NADH, implicating widespread impairment of glucose utilization, mitochondrial bioenergetics, and fatty acid oxidation. Integrative proteomic-metabolomic analysis identified a small subset of proteins, including Rab10 and Anxa1, as potential drivers of these metabolic disruptions, with defective Rab10-dependent GLUT4 trafficking emerging as a plausible mechanism underlying impaired glucose uptake in TRiC-deficient rods. Finally, experiments using a perpetually unfolded G{beta}1 mutant and G{gamma}1-knockout mice demonstrated that substrate overload sequesters TRiC and competitively displaces other clients, exacerbating proteostasis imbalance. Together, our study provides a comprehensive in vivo mapping of the TRiC interactome in mammalian rods, reveals a connection between TRiC-dependent proteostasis and energy metabolism in rods, and indicates a mechanism by which misfolded TRiC substrates exacerbate a proteostasis imbalance that ultimately results in neurodegeneration.

biochemistry↗

bioRxiv2022

Dual Palmitoylation of PRCD, a Photoreceptor-Specific Protein Linked to RP, Alters Protein Stability and Subcellular Localization

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (POS) disc-specific protein essential for maintaining outer segment (OS) structures, while also contributing to rhodopsin packaging densities and distribution in the disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation is found linked with retinitis pigmentosa (RP) that is crucial for protein stability and trafficking to POS. PRCD has several predicted structural domains with unknown significance, such as the polybasic region (PBR) where an Arg17Cys (R17C) mutation is linked with RP. In this study, we demonstrate that a mutation in the PBR augments additional palmitoyl lipid modification observed through acyl-RAC in the mutant cysteine (R17C). Immunolocalization of transiently expressed R17C protein in hRPE1 cells depicts similar characteristics to wild type (WT); however, a double mutant lacking endogenous palmitoylation at the Cys2 position is comparable to the C2Y protein as both are likely aggregated and mislocalized in the mitochondria. Subretinal injection of C2Y, R17C, and R17C/C2Y mutants followed by electroporation in murine retina exhibit mislocalization in the inner segment compared to WT PRCD. Our results in the R17C mutant show palmitoylation transpires at two different locations. Despite being dually palmitoylated and demonstrating membrane association, the mutation in the PBR affects protein stability and trafficking to the OS. Moreover, palmitoylation within the PBR alone does not compensate for protein stability or trafficking, revealing the PBR domain is indispensable and any defects likely lead to dysregulation of PRCD protein associated with blinding diseases.

biochemistry↗

bioRxiv2021

NMNAT1 facilitates energy metabolism and gene regulation in developing retinal neurons

Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, large-scale transcriptomics reveals dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

molecular biology↗