Biology subjects

Biswas-Fiss, E. E.

Publications and source records attributed to Biswas-Fiss, E. E..

3 recordsLinked to original sources

bioRxiv2026

Pathogenic impact of ABCA4 missense variants in the structurally uncharacterized ECD1 region: implications for Stargardt disease.

Pathogenic mutations in the ABCA4 gene cause several inherited retinal diseases, particularly Stargardt disease (STGD1). However, many missense variants remain classified as variants of uncertain significance (VUS) due to inconclusive evidence regarding their pathogenic impact. The missense VUS span across all the domains of ABCA4, with the majority found in the larger extracellular domains (ECDs). The largest uncharacterized region of ABCA4 is located in ECD1, where limited structural information and inconsistent computational predictions hinder clinical interpretation of missense VUS in this region. Here, we integrated in silico analysis with in vitro functional assays to evaluate the pathogenicity of VUS in this region and improve their diagnostic classification. Missense VUS in the ECD1 uncharacterized region were curated from ClinVar. Six multiallelic sites were identified in the uncharacterized region and 13 missense VUS on these multiallelic sites were characterized using the integrated analysis. In the in silico platform, the pathogenicity of the VUS were predicted using multiple algorithms, and the structural effects of the variants were analyzed compared to the wild type. Recombinant variants were expressed in virus-like particles (VLPs), and protein expression, membrane localization, and ATPase activity were quantified relative to wild type to identify potential disease-causing variants. From the integrated analysis, variants with pronounced structural destabilization, impaired membrane trafficking, and reduced or absent N-retinylidene-phosphatidylethanolamine (NRPE) substrate stimulated ATPase activities were identified as potentially deleterious. Notably, VUS at p.H193P and p.I214N showed loss of function, with p.I214N reflecting selectively impaired membrane targeting and p.H193P reflecting combined expression and trafficking defects. Additionally, NRPE-stimulated ATPase activities were impaired in VUS, p.V195L, p.V195I, p.D197H, p.I214F and p.N269S. Overall structural destabilization interfered with the NRPE-stimulated ATPase activities of p.N269S, while the lack of NRPE-stimulated ATPase activities of p.D197H, p.V195L, p.V195I and p.I214F are thought to be due to impaired NRPE interactions with ABCA4. All the VUS at p.R140, p.H193Y, p.D197N and p.N269H showed both the basal and NRPE-stimulated ATPase activities but less than that of the wild type displaying a mild functional deficit. Together, these findings demonstrated that certain VUS within the unresolved ECD1 region disrupts ABCA4 stability and function, supporting their contribution to disease pathogenesis. This integrative approach highlights key residues likely to be pathogenic and advances the interpretation of VUS in inherited retinal disorders.

genetics↗

bioRxiv2026

De Novo Design and Computational Validation of a High-Affinity Peptide Inhibitor Targeting the HPV E1-E2 Interface

The oncogenic progression of high-risk Human Papillomavirus (HPV) strains relies on the cooperative interaction between the E1 replicative helicase and the E2 origin-binding protein to initiate viral DNA amplification. Disrupting this protein-protein interaction represents a promising, yet clinically unrealized, therapeutic paradigm for treating established HPV infections prior to malignant transformation. This study presents a comprehensive computational pipeline for the de novo design and evaluation of peptide inhibitors targeting the HPV E1-E2 interface, specifically a conserved arginine triad on the solvent-exposed surface of the E1 helicase. AlphaProteo was used for sequence discovery, and AlphaFold 3 for complex structural prediction, generating a candidate library that was subsequently subjected to dual-scale Molecular Dynamics (MD) simulations and MM/GBSA thermodynamic validation using GROMACS. Binder 8 emerged as the lead candidate, yielding a predicted binding free energy of -59.1 {+/-} 0.7 kcal/mol -- a statistically significant improvement over the native E1-E2 baseline (Welchs t-test, p = 8.14e-19; Cohens d = 2.21). As an implicit solvent method, MM/GBSA overestimates absolute affinities; reported values reflect effective binding enthalpy and should be interpreted as relative rankings. Per-residue energy decomposition confirms binding is anchored through multi-point interactions with the arginine triad. Physicochemical profiling via CSM-Toxin and AlgPred 2.0 confirms zero predicted toxicity and non-allergenic properties for Binder 8. Sequence alignment across 183 oncogenic Alpha-papillomavirus genotypes demonstrates near-universal conservation of the targeted triad, supporting Binder 8 as a candidate scaffold for broad-spectrum antiviral development. These findings provide a computationally validated blueprint for future in vitro validation via Bio-layer interferometry.

bioinformatics↗

bioRxiv2025

Design, Expression, and Purification of a Soluble Form of the Retina-Specific Membrane Transporter, ABCA4

The ATP-binding cassette transporter A-subfamily member, ABCA4, is highly expressed in rod and cone photoreceptors in the retina, where it transports cis- and trans-retinal and is indispensable for vision. Genetic mutations in the ABCA4 gene lead to a wide range of inherited retinal degenerative diseases, including Stargardt disease (STGD1) and autosomal recessive cone-rod dystrophy. It is an integral membrane protein with twelve transmembrane -helices that complicates studies with the full-length ABCA4 transporter. We have engineered the full-length ABCA4 by transforming its membrane helices, creating a soluble homolog (ABCA4s). Most hydrophobic residues in the membrane helices were substituted with structurally compatible but hydrophilic residues. The re-engineered ABCA4s was expressed in insect cells, and it was found in the cytosolic extract, which was purified by immunoaffinity chromatography. Purified ABCA4s was enzymatically active, all-trans-retinal stimulated its ATPase activity, and its activity remained stable. SignificanceABCA4 is a 12-pass transmembrane protein that plays essential roles in the human retina and multiple visual diseases. Historically, the purification of ABCA4 and other large membrane proteins has relied on detergent-based purification, which renders the proteins enzymatic activity highly unstable. We describe here the design of a truly soluble analog of ABCA4 with stable enzymatic activities. Its 12 transmembrane helices were transformed using selective amino acid substitution, and deleterious substitutions were carefully avoided. This soluble form will pave the way for mechanistic studies of the enzyme and its disease-causing genetic variants. The methodology described here should be widely applicable to other complex membrane proteins facilitating their studies without the need for reconstitution in lipids.

biochemistry↗