Covalent inhibition of pro-apoptotic BAX.

BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.

Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

Quantitative mass spectrometry-based multiplexing compares the abundance of 5000 S. cerevisiae proteins across 10 carbon sources.

UNLABELLED: The budding yeast Saccharomyces cerevisiae is a model system for investigating biological processes. Cellular events are known to be dysregulated due to shifts in carbon sources. However, the comprehensive proteomic alterations thereof have not been fully investigated. Here we examined proteomic alterations in S. cerevisiae due to the adaptation of yeast from glucose to nine different carbon sources - maltose, trehalose, fructose, sucrose, glycerol, acetate, pyruvate, lactic acid, and oleate. Isobaric tag-based mass spectrometry techniques are at the forefront of global proteomic investigations. As such, we used a TMT10-plex strategy to study multiple growth conditions in a single experiment. The SPS-MS3 method on an Orbitrap Fusion Lumos mass spectrometer enabled the quantification of over 5000 yeast proteins across ten carbon sources at a 1% protein-level FDR. On average, the proteomes of yeast cultured in fructose and sucrose deviated the least from those cultured in glucose. As expected, gene ontology classification revealed the major alteration in protein abundances occurred in metabolic pathways and mitochondrial proteins. Our protocol lays the groundwork for further investigation of carbon source-induced protein alterations. Additionally, these data offer a hypothesis-generating resource for future studies aiming to investigate both characterized and uncharacterized genes. BIOLOGICAL SIGNIFICANCE: We investigate the proteomic alterations in S. cerevisiae resulting from adaptation of yeast from glucose to nine different carbon sources - maltose, trehalose, fructose, sucrose, glycerol, acetate, pyruvate, lactic acid, and oleate. SPS-MS3 TMT10plex analysis is used for quantitative proteomic analysis. We showcase a technique that allows the quantification of over 5000 yeast proteins, the highest number to date in S. cerevisiae, across 10 growth conditions in a single experiment. As expected, gene ontology classification of proteins with the major alterations in abundances occurred in metabolic pathways and mitochondrial proteins, reflecting the degree of metabolic stress when cellular machinery shifts from growth on glucose to an alternative carbon source. Our protocol lays the groundwork for further investigation of carbon source-induced protein alterations. Improving depth of coverage - measuring abundance changes of over 5000 proteins - increases our understanding of difficult-to-study genes in the model system S. cerevisiae and by homology human cell biology. We submit this highly comprehensive dataset as a hypothesis generating resource for targeted studies on uncharacterized genes.