Biology subjects

Jastrzebska, B.

Publications and source records attributed to Jastrzebska, B..

4 recordsLinked to original sources

bioRxiv2022

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent apoptosis

We identified cyto-protective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in the mouse models of Stargardt disease and macular degeneration. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only MEFs. M109S also inhibited apoptosis induced by staurosporine (mouse embryonic fibroblasts), etoposide (Neuro2a cells), and obatoclax (ARPE19 cells). M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a new research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway. (128words)

cell biology↗

bioRxiv2022

Chromenone derivatives as novel pharmacological chaperones for retinitis pigmentosa-linked rod opsin mutants

The correct expression of folded, functional rhodopsin (Rho) is critical for visual perception. However, this seven-transmembrane helical G protein-coupled receptor (GPCR) is prone to mutations with pathological consequences of retinal degeneration in retinitis pigmentosa (RP) due to Rho misfolding. Pharmacological chaperones that stabilize the inherited Rho variants by assisting their folding and membrane targeting could slow the progression of RP. In this study, we employed virtual screening of synthetic compounds with natural product scaffold in conjunction with in vitro and in vivo evaluations to discover a novel chromenone-containing small molecule with favorable pharmacological properties that stabilizes rod opsin. This compound reversibly binds to unliganded bovine rod opsin with an EC50 value comparable to the 9-cis-retinal chromophore analog and partially rescued membrane trafficking of multiple RP-related rod opsin variants in vitro. Importantly, this novel ligand of rod opsin was effective in vivo in murine models, protecting photoreceptors from deterioration caused either by bright light or genetic insult. Together, our current study suggests potential broad therapeutic implications of the new chromenone-containing non-retinoid small molecule against retinal diseases associated with photoreceptor degeneration.

pharmacology and toxicology↗

bioRxiv2022

Molecular Basis for Variations in the Sensitivity of Pathogenic Rhodopsin Variants to 9-cis-Retinal

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. 67 of these apparent class II variants exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts in relation to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants ({Delta}N73, S131P, and R135G) and show that two retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

biochemistry↗

bioRxiv2021

Systematic Profiling of Temperature- and Retinal-Sensitive Rhodopsin Variants by Deep Mutational Scanning

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of "temperature-sensitive" variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and an investigational corrector (9-cis-retinal) on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures is correlated with the response to retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants within this helix appear to disrupt a network of hydrogen bonds that stabilizes a native helical kink. By comparison, mutants that alter a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest this insensitivity primarily arises from an abundance of mutations that enhance its membrane integration, stabilize its native conformation, and/ or perturb the retinal binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings elucidate various factors that mediate the sensitivity of genetic variants to temperature and to small molecule correctors.

biophysics↗