Icon Legend

This session is not in your schedule.

This session is in your schedule. Click again to remove it.

Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

U8 - Intrinsically Disordered Regions of Rab Escort Proteins as Potential Regulators of Activity and Therapeutic Targets for Choroideremia

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Bhavya Boddu

Undergraduate Researcher
University of Virginia, United States

Co-Author(s)

Bhavya Boddu

Undergraduate Researcher
University of Virginia, United States
Nicholas Johnstone

Undergraduate Researcher
University of Virginia
Park City, Utah, United States
EB

Emily Baum

PhD student
University of Virginia, United States
JS

Jagathpala Shetty

Research Scientist
University of Virginia, United States
RB

Rishi Bhogaraju

Undergraduate Researcher
University of Virginia, United States
Ally O'Shea

Undergraduate Researcher
University of Virginia
Glen Allen, Virginia, United States
Grace Burns

Undergraduate Researcher
University of Virginia
Aiken, South Carolina, United States
TB

Thomas H. Barker, PhD

Professor
University of Virginia
Charlottesville, Virginia, United States

Primary Investigator(s)

TB

Thomas H. Barker, PhD

Professor
University of Virginia
Charlottesville, Virginia, United States

Introduction: : Intrinsically disordered regions (IDRs) are segments of proteins that challenge the traditional sequence-structure paradigm. Instead, IDRs lack a stable tertiary structure under physiological conditions and exist as dynamic ensembles of rapidly interconverting conformations (1). This structural plasticity enables IDRs to engage in multivalent interactions, participate in key regulatory and signaling pathways, undergo post-translational modifications (PTMs), and facilitate liquid-liquid phase separation (LLPS), often contributing to the formation of membraneless organelles such as P-bodies and stress granules (2). In this study, we investigate the IDRs of Rab escort proteins 1 and 2 (REP-1 and REP-2), which are essential for Rab prenylation. Complete loss of REP1 function is implicated in the pathogenesis of Choroideremia, a progressive retinal degenerative disease that causes progressive blindness in young male patients (3). Although REP-2 is 75% sequence identical and 90% sequence similar to REP-1, it does not fully compensate for REP-1 loss in Choroideremia. Differences in REP-1 and REP-2 sequences lie largely within their two IDRs, which have not yet been thoroughly studied. This suggests that the differences in the IDRs may be involved in the lack of REP-2s ability to compensate for REP-1, and thus were chosen to be the target regions of this study (4).

Materials and

Methods: : Several computational tools were used to collect information about the REP structures, IDR sequences, and phase separation properties. Full-length amino acid sequences of REP-1 and REP-2 were obtained from the AlphaFold Protein Structure Database. Disordered regions were identified using a combined approach: AlphaFold-derived low-confidence regions (pLDDT < 70) and AIUPred disorder scores (threshold > 0.5) for robust identification of IDRs. To further elucidate the structure of the IDR regions, STARLING was utilized to generate possible conformational ensembles of the IDR sequences. These structures will be used in future molecular dynamics simulations to model IDRs. Lastly, the phase separation potentials of REP-1 and REP-2 were assessed using both catGRANULE 2.0 ROBOT and FuzDrop, which score sequences for LLPS propensity based on physicochemical and disorder features.
Experimentally,immunofluorescence (IF) staining protocols were developed and implemented to visualize REP-1 and REP-2 location in retinal pigment epithelial (ARPE-19) cells. These protocols will be applied to both wild-type and CHM knockout ARPE-19 cells under normal and stress conditions, such as heat shock or oxidative stress. Imaging will be performed on a Keyence fluorescence microscope and puncta will be analyzed using Fiji. In addition, a temperature-dependent turbidity assay protocol was adapted for REP-1 and REP-2 and will be used to construct concentration-temperature phase diagrams of the proteins. Finally, we will integrate proteomics data from ARPE-19 cells with curated droplet-driver databases to identify candidate co-phase separation partners for the REPs, which will be experimentally tested for co-LLPS.

Results, Conclusions, and Discussions:: Disorder prediction tools identified two IDRs in both REP-1 and REP-2, located in corresponding positions across the two proteins.
Phase separation propensities of REP-1 and REP-2 were assessed using both FuzDrop and catGRANULE 2.0 ROBOT, which yielded consistent predictions. FuzDrop assigned a higher probability of spontaneous LLPS for REP-1 (pLLPS = 0.4837) than REP-2 (pLLPS = 0.2406). However, both pLLPS were below the threshold of 0.6, suggesting that neither proteins undergo homotypic phase separation in cellular conditions nor are likely to be droplet-drivers. While droplet-driver proteins spontaneously undergo liquid-liquid phase separation, droplet-client proteins require interactions with partners or specific cellular conditions to form the droplet state (5). FuzDrop also identified droplet-promoting regions and aggregation hotspots in both REP-1 and REP-2. The droplet-promoting regions for both proteins were aligned with their respective IDR regions. However, of the two, the tool identified a higher percentage of the droplet-promoting regions as aggregation hotspots in REP-2 than REP-1. While LLPS may increase efficiency of cellular processes through reversible increased localization of involved proteins, liquid-solid phase transitions (LSPT) promote the irreversible formation of aggregates that decreases the protein’s accessibility.
catGranule assigned a higher total score to REP-2 (0.695) than to REP-1 (0.575). The score for REP-2 suggests that it falls under the DropletAmyloid class, which suggests that REP-2 may undergo LLPS or LSPT under different conditions. Databases such as DrLLPS have also classified REP-2 as a droplet-regulator in the formation of stress granules. Through further testing, we hope to identify the components of stress granules and droplets under basal and stress conditions.
Collectively, this work provides a multifaceted characterization of REP-1/2 IDRs, integrating sequence-based prediction, evolutionary context, structural modeling, and preliminary experimental validation. Our findings lay the groundwork for future studies into the role of REP IDR-mediated phase separation in retinal cell biology and offer potential insights for novel therapeutic strategies for choroideremia.

Acknowledgements and/or References (Optional): : (1) https://doi.org/10.1146/annurev-biochem-072711-164947
(2) https://doi.org/10.1038/s41580-023-00673-0
(3) https://doi.org/10.1016/j.ophtha.2016.06.051
(4) https://www.jbc.org/article/S0021-9258(17)42142-9/fulltext
(5) https://doi.org/10.1093/nar/gkac386