The Bcl-2 family of proteins governs mitochondrial outer membrane (MOM) permeabilization, a critical step in apoptosis that is dysfunctional in many cancers. Although cellular studies have long implicated direct interactions between the pore-forming apoptotic Bax protein and its opponent, the antiapoptotic Bcl-2 protein in apoptosis regulation, the underlying basic principles behind this control remained unresolved. To provide in-depth insight, we carried out a systematic biophysical study in which we utilized neutron reflectometry (NR) and ATR-FTIR to elucidate the molecular communication between those proteins in and around the mitochondrial membrane environment. The spatial and temporal changes across model MOM surfaces were resolved during the interaction of Bax with Bcl-2. The NR-derived membrane surface Bax distributions suggested that Bcl-2 mediated Bax sequestration through both Bcl-2/Bax heterodimerization and Bax/Bax oligomerization. Kinetic analysis revealed a two-step process: rapid formation of Bcl-2/Bax heterodimers, followed by slower Bax oligomerization on these complexes. Importantly, this sequestration mechanism was also observed in the presence of cardiolipin, a lipid known to promote the formation of an apoptotic pore by Bax in the absence of Bcl-2. These findings suggest a fundamental mechanism by which cancer cells may evade apoptosis by exploiting Bcl-2's ability to neutralize Bax through structural entrapment, even if excess Bax is present, either in response to treatment or natural death signals.

Type II aromatic polyketides represent a structurally diverse class of natural products with medicinally relevant properties, and their biosynthesis usually involves biosynthetic intermediates with terminal carboxyl groups. In certain instances, terminal decarboxylation occurs, which can significantly impact the structural complexity. However, the enzymes and their involved mechanisms of terminal decarboxylation in type II aromatic polyketide biosynthesis have rarely been studied. This study has now shown that MrqO5, a member of the antibiotic biosynthesis monooxygenase (ABM) family, unexpectedly functions as a terminal decarboxylase involved in the biosynthesis of murayaquinone. Furthermore, an in vitro biochemical study demonstrated that two homologous proteins of MrqO5 exhibited similar decarboxylase activity. Therefore, the functional assignment and mechanistic investigation of this polyketide terminal decarboxylase elucidated an overlooked step in type II polyketide biosynthesis. Also, the discovery of this new family of decarboxylases expands the functions of the ABM superfamily proteins. Our structural characterizations, combined with site-directed mutagenesis studies, have unveiled the key residues involved in the decarboxylation and allowed an enzymatic decarboxylation mechanism to be proposed. Our studies advance the currently incomplete understanding of type II aromatic polyketide biosynthesis and gain the insight necessary for future engineering of these enzymes.

Glycopeptide antibiotics (GPAs) are clinically important antibiotics characterized by a rigid, highly cross-linked structure. The cross-links in GPAs are installed by the activity of several cytochrome P450 (Oxy) enzymes, which are recruited to their peptide substrates by a unique domain, the X-domain. Given that this cross-linking cascade is the source of both the antibiotic activity and the synthetic complexity of GPAs, it remains a central point for exploring the tolerance of the Oxy enzymes for altered peptide substrates. In this study, we have investigated the ability of the Oxy enzymes to cross-link peptides with changes to their amide backbone, specifically a [Ψ[CH2NH]Tpg] methylene linkage that was inspired by synthetic efforts showing that such analogues can recover antibiotic activity toward resistant bacteria. Our results show that the Oxy enzymes are extremely sensitive to the presence of a methylene linkage in their peptide substrates, which suggests that these backbone carbonyl groups play a crucial role in maintaining the correct binding of peptide substrates to the P450 enzymes within the GPA cross-linking cascade.

Mincle, a member of the C-type lectin receptor (CLR) family, detects various glycolipids and glycerolipids such as trehalose dimycolate (TDM) from Mycobacterium tuberculosis, leading to the activation of the innate immune system. In this study, we developed new fluorescence-labeled molecular probes, TDE-Fluor-Ligand and TDE-Reactive-Probe, based on the structure of trehalose diester to elucidate the intracellular behavior of Mincle and its ligands. TDE-Fluor-Ligand was prepared for the ligand analysis, and TDE-Reactive-Probe was specifically designed to label Mincle by turn-on fluorescent affinity labeling. Live-cell imaging analysis using these probes revealed that TDE-Fluor-Ligand internalizes into the cell in a Mincle-dependent manner. Furthermore, imaging analysis using TDE-Reactive-Probe successfully detected Mincle in cells in a Mincle expression-dependent manner.

Point mutations in the exonuclease (ExoN) site of nonstructural protein 14 (NSP14) compromise the fitness of betacoronaviruses such as SARS-CoV-2, implicating NSP14 ExoN inhibition as an antiviral strategy. However, there are no advanced compounds that inhibit NSP14's ExoN activity. Building upon the reported crystal structures of two fragments bound to NSP14's ExoN site, we identified a series of 3,5-disubsituted pyrazoles that bound to and inhibited NSP14 ExoN. However, upon resynthesis, we discovered that these putative leads were false positives, perhaps due to contaminating divalent cations, which potently inhibit NSP14 ExoN. Our results provide a cautionary tale to the field about the sensitivity of NSP14 to divalent cations and illustrate the challenges associated with directly targeting the NSP14 ExoN site via fragment merging.

Hydrogen sulfide (H2S) is an important gasotransmitter that has shown many physiological effects, ranging from anti-inflammation to antioxidation. To advance research on H2S, donor compounds that can slowly release H2S in biological conditions while producing minimal bioactive byproducts are essential. Herein, we report the evaluation of thiosaccharides as hydrolysis-based H2S donors. These compounds were found to slowly produce H2S over days, in aqueous buffers and in cells. Their H2S release rates could be affected by the hydroxyl protection groups of thiosaccharides, with faster release by electron-donating groups and slower release by electron-withdrawing groups. We also demonstrated the vasodilatory effect of 1-thioglucose using arterial rings isolated from adult ewes, which is likely due to H2S release. Altogether, thiosaccharides might be suitable slow-releasing H2S donors for biological applications.

A mechanistic basis for luciferase bioluminescence provides a glimpse into its evolutionary role for organism survival, as it provides a blueprint to engineer luciferase enzymes for advanced technological applications. Gaussia Luciferase is among the brightest natural luciferases, but (1) the evolutionary development of its luminescence behavior remains unclear, (2) recent fundamental studies utilized Escherichia coli expression systems instead of eukaryotic expression systems, and (3) notable mutants have been discovered but not integrated into a comprehensive mechanistic analysis. We describe new mechanistic observations from GLuc by addressing these gaps. We monitored the fluorescent coelenterazine-to-coelenteramide conversion to study turnover kinetics of mammalian-derived GLuc; this assay characterized the positive cooperativity kinetics of GLuc. The nonluminescent mutants, R76A and R147A, still turn over the substrate with high efficiency, each demonstrating sustained positive cooperativity. Through mass spectrometry, mutational analysis, and analytical liquid chromatography, we demonstrate that GLuc undergoes methionine oxidation during substrate turnover and that this impacts the luciferase's flash-type luminescence; we did not observe indications of covalent attachment with the substrate, product, or their intermediates. Chromatography of luciferases derived from ancestral sequence reconstruction highlighted that the extent of methionine-induced surface changes was greater for earlier ancestral luciferases. Ancestral sequence reconstruction also revealed that earlier ancestral copepod luciferases produced less light when compared to GLuc.

Autophagy inhibition represents a promising therapeutic approach for the management of various cancers including nonsmall cell lung cancer (NSCLC). We previously reported SBP-7455, a dual inhibitor of unc-51-like kinase 1 (ULK1) and its homologue ULK2 and described its effects on triple-negative breast cancer (TNBC) cells. Herein we report the design, synthesis, and characterization of SBP-5147 and SBP-7501, two new dual ULK1/2 inhibitors that are cytotoxic against NSCLC cells, inhibit autophagic flux in A549 cells, and present greater oral exposure than SBP-7455 at a lower dose. In addition, SBP-5147 effectively modulates autophagy and increases the expression of major histocompatibility complex (MHC) class I in NSCLC cells, which may support the rationale for ULK1/2 inhibition as a strategy to overcome resistance to immunotherapy. Together these data support the use of ULK inhibitors as part of a cancer treatment strategy, either as a single agent or in combination with current therapies.

Fungal secondary metabolites have historically provided important applications in a variety of industries. Penicillium camemberti, a fungus with a role in cheese production, was domesticated to food use partly due to its metabolically depleted characteristic, minimizing the risk of toxic compound formation. However, antiSMASH analysis of the genome reveals that strains of the species do contain various cryptic biosynthetic gene clusters and, thus, have the potential capability of producing multiple secondary metabolites despite its limited compound production under normal laboratory conditions. Here, we genetically engineered Penicillium camemberti strain IMV00769, which is genetically similar to cheese-making isolates, by deleting negative global regulator, mcrA. This deletion resulted in the production of secondary metabolites not previously produced by this strain, including fumigermin, a compound patented for cosmetic applications for the reduction of skin wrinkles, enhancement of skin elasticity, and skin whitening. Our findings highlight the power of global regulator manipulation to activate cryptic biosynthetic pathways and expand the range of natural products accessible from domesticated fungal strains.