Wagner, M. V. | bioRxiv Science

bioRxiv2022

Structures of human Malic Enzyme 3

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to capture the structures of ME3 in various ligand binding states. ME3 exists as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

molecular biology↗

bioRxiv2020

Precision genetic cellular models identify therapies protective against endoplasmic reticulum stress

Congenital disorders of glycosylation (CDG) and deglycosylation (CDDG) are a collection of rare pediatric disorders with symptoms that range from mild to life threatening. They typically affect multiple organ systems and usually present with neurological abnormalities including hypotonia, cognitive impairment, and intractable seizures. Several genes have been implicated in the thirty-six types of CDG, but currently NGLY1 is the only known CDDG gene. A common biological mechanism among CDG types and in CDDG is endoplasmic reticulum (ER) stress. Here, we develop two isogenic human cellular models of CDG (PMM2, the most prevalent type of CDG, and DPAGT1) and of the only CDDG (NGLY1) in an effort to identify drugs that can alleviate ER stress. Systematic phenotyping identified elevated ER stress and autophagy levels among other cellular and morphological phenotypes in each of the cellular models. We screened a complex drug library for compounds able to correct aberrant morphological phenotypes in each of the models using an agnostic phenotypic cell painting assay based on >300 cellular features. The image-based screen identified multiple candidate compounds able to correct aberrant morphology, and we show a subset of these are able to correct cellular and molecular defects in each of the models. These results provide new directions for the treatment of rare diseases of glycosylation and deglycosylation and a framework for new drug screening paradigms for more common neurodegenerative diseases characterized by ER stress. Summary sentenceNovel drug screening modality identifies compounds that correct aberrant molecular phenotypes in precision cellular models of glycosylation defects.

neuroscience↗