Biology subjects

Lieberman, R. L.

Publications and source records attributed to Lieberman, R. L..

7 recordsLinked to original sources

bioRxiv2025

GOLF: A Generative AI Framework for Pathogenicity Prediction of Myocilin OLF Variants

Missense mutations in the MYOC gene, particularly those affecting the olfactomedin (OLF) domain of the myocilin protein, can be causal for open-angle glaucoma--a leading cause of irre-versible blindness. However, predicting the pathogenicity of these mutations remains challenging due to the complex effects of toxic gain-of-function variants and the scarcity of labeled clinical data. Herein, we present GOLF, a generative AI framework for assessing and explaining the pathogenicity of OLF domain variants. GOLF collects and curates a comprehensive dataset of OLF homologs and trains generative models that predict the effect of monoallelic missense mutations. While these models exhibit diverse predictive behaviors, they collectively achieve accurate classification of known pathogenic and benign variants. To interpret their decision mechanisms, GOLF uses a sparse autoencoder (SAE) that reveals the underlying biochemical features exploited by the generative models to predict variant effects. GOLF enables accurate evaluation of disease-causing mutations, supporting early genetic risk stratification for glaucoma and facilitating interpretable investigations into the molecular basis of pathogenic variants.

bioinformatics↗

bioRxiv2025

Monitoring mutant myocilin secretion and localization in trabecular meshwork cell cultures using a protein complementation-based luminescence assay

Approximately 2-4% of adult onset and 10% of juvenile onset cases of primary open angle glaucoma can be attributed to non-synonymous coding mutations in MYOC. One of the key characteristics of a pathogenic MYOC mutant is the inability of the resulting protein to be secreted from trabecular meshwork cells. Instead, pathogenic myocilin variants accumulate in the endoplasmic reticulum. Typically, localization of MYOC mutants is compared to wild-type myocilin in cellular secretion assays that use immunoblot to detect myocilin in extracellular media, alongside intracellular soluble and insoluble (aggregated) fractions. Here, we implement a new method that utilizes a complement-based luminescence method in which an 11-residue HiBiT tag is appended to myocilin and complements a truncated nanoluciferase. The method allows for highly sensitive luminescence detection and does not require immunoblot. We tested non-synonymous coding variants T377R, D384G, D395ins, C433Y, T455K, and L486F, in an established immortalized trabecular meshwork cell line. Secretion was tested in 96-well plate format, revealing poor secretion for these mutants compared to wild-type myocilin. For assays conducted in 6-well plates, myocilin mutants were accumulated in intracellular fractions. HiBiT luminescence signals correlated well with immunofluorescence as well as immunoblot but is more sensitive than the latter. Overall, our study demonstrates that complement-based detection of mutant myocilin using luminescence allows for facile and sensitive detection of myocilin localization and has confirmed secretion defects for seven variants. HighlightsO_LIMutations in myocilin are causal for early onset open angle glaucoma C_LIO_LIIn the lab pathogenic myocilin mutations are characterized by secretion defects C_LIO_LIWe validate a complement luminescence assay for detection of myocilin localization C_LIO_LIThe luminescence-based assay is more sensitive than traditional immunoblot C_LIO_LIWe confirm secretion defects for several mutants not previously characterized C_LI TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/640780v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1647a7aorg.highwire.dtl.DTLVardef@1a4e56dorg.highwire.dtl.DTLVardef@d29165org.highwire.dtl.DTLVardef@477e4f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Structural basis for anomalous cellular trafficking behavior of glaucoma-associated A427T mutant myocilin

Familial mutations in myocilin cause vision loss in glaucoma due to misfolding and a toxic gain of function in a senescent cell type in the anterior eye. Here we characterize the cellular behavior and structure of the myocilin (myocilin A427T) mutant, of uncertain pathogenicity. Our characterization of A427T demonstrates that even mutations that minimally perturb myocilin structure and stability can present challenges for protein quality control clearance pathways. Namely, when expressed in an inducible immortalized trabecular meshwork cell line, inhibition of the proteasome reroutes wild-type myocilin, but not myocilin A427T, from endoplasmic reticulum associated degradation to lysosomal degradation. Yet, the crystal structure of the A427T myocilin olfactomedin domain shows modest perturbations largely confined to the mutation site. The previously unappreciated range of mutant myocilin behavior correlating with variable stability and structure provides a rationale for why it is challenging to predict causal pathogenicity of a given myocilin mutation, even in the presence of clinical data for members of an affected family. Comprehending the continuum of mutant myocilin behavior in the laboratory supports emerging efforts to use genetics to assess glaucoma risk in the clinic. In addition, the study supports a therapeutic strategy aimed at enhancing autophagic clearance of mutant myocilin. Significance statementO_LIRare familial mutations cause early onset glaucoma C_LIO_LIA427T is a case of uncertain pathogenicity C_LIO_LIA427T is structurally similar to wild-type but is not efficiently degraded C_LI

biochemistry↗

bioRxiv2024

Evidence for S331-G-S-L within the amyloid core of myocilin olfactomedin domain fibrils based on low-resolution 3D solid-state NMR spectra

Myocilin-associated glaucoma is a protein-conformational disorder associated with formation of a toxic amyloid-like aggregate. Numerous destabilizing single point variants, distributed across the myocilin olfactomedin {beta}-propeller (OLF, myocilin residues 245-504, 30 kDa) are associated with accelerated disease progression. In vitro, wild type (WT) OLF can be promoted to form thioflavin T (ThT)-positive fibrils under mildly destabilizing (37{degrees}C, pH 7.2) conditions. Consistent with the notion that only a small number of residues within a protein are responsible for amyloid formation, 3D 13C-13C solid-state NMR spectra show that OLF fibrils are likely to be composed of only about one third of the overall sequence. Here, we probe the residue composition of fibrils formed de novo from purified full-length OLF. We were able to make sequential assignments consistent with the sequence S331-G-S-L334. This sequence appears once within a previously identified amyloid-prone region (P1, G326AVVYSGSLYFQ) internal to OLF. Since nearly half of the pairs of adjacent residues (di-peptides) in OLF occur only once in the primary structure and almost all the 3-residue sequences (tri-peptides) are unique, remarkably few sequential assignments are necessary to uniquely identify specific regions of the amyloid core. This assignment approach could be applied to other systems to expand our molecular comprehension of how folded proteins undergo fibrillization. TOC GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/606901v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@ff42feorg.highwire.dtl.DTLVardef@875722org.highwire.dtl.DTLVardef@169b1deorg.highwire.dtl.DTLVardef@1ca311_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Mainly on the Plane: Deep Subsurface Bacterial Proteins Bind and Alter Clathrate Structure

Gas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a low-pressure analog for structure II gas clathrate. When grown in the presence of CbpAs, THF clathrate was polycrystalline and plate-like instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a plate-like structure during gas clathrate transportation. Table of Contents Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2019

Progranulin deficiency leads to reduced glucocerebrosidase activity

Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GBA), and is essential for proper GBA activity. GBA activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.

neuroscience↗

bioRxiv2017

Structure and misfolding of the flexible tripartite coiled coil domain of glaucoma-associated myocilin

Glaucoma-associated myocilin is a member of the olfactomedins, a protein family broadly involved in neuronal development and human disease. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a disulfide-linked, parallel dimer-of-dimers Y-shaped molecule, with distinct tetramer and dimer regions. The structure of the C-terminal 7-heptad dimer elucidates an unexpected repeat pattern involving electrostatic inter-strand stabilization. Molecular dynamics simulations reveal an alternate conformation in which the terminal inter-strand disulfide bond limits the extent of unfolding and results in a kinked configuration. Taken together, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of extracellular coiled coils for protein design and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.\n\nHighlightsO_LIGlaucoma-causing extracellular protein associated with amyloid forming propensity\nC_LIO_LIStructural studies confirm tripartite parallel dimer-of-dimers coiled coil\nC_LIO_LILeucine zipper exhibits non-canonical heptad repeat pattern with disulfide cap\nC_LIO_LIGlaucoma-associated variants in tetramer alter structure but not stability\nC_LI\n\neTOC blurbHill et al describe the structure of the coiled-coil region of myocilin, the extracellular olfactomedin family member closely associated with the ocular disorder glaucoma. Myocilins coiled coil adopts a unique Y-shaped parallel dimer-of-timers employing an unusual heptad repeat pattern. Selected disease variants alter quaternary structure.

biochemistry↗