Biochemistry Biochemistry最新文献

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections, for which there are few treatment options. The quorum sensing (QS) pathway governs many pathogenic behaviors that allow for P. aeruginosa to stage infections. Within the QS pathway, there is a key protein–protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa, also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. Here, we performed a fluorescence polarization screen of an FDA-approved drug library to identify molecules that bind in the active site of PqsE. Three molecules were identified, two of which showed inhibitory activity consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in a cellular enzyme assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein–protein interaction via binding in the PqsE active site.

Amyloid beta (Aβ) oligomers are thought to play an important role during development and progression of Alzheimer’s disease (AD). Previously, we determined the Aβ oligomer concentrations in various AD mouse models and in human brain tissues of former AD patients. Here, we investigate which proteins are part of these Aβ oligomers, apart from Aβ itself. Because several oligomer-associated proteins have been implicated in mechanisms leading to AD pathology, identification of the Aβ oligomer proteome may provide insights into the formation of Aβ oligomers in vivo and may reveal novel targets for disease-modifying therapeutic approaches. Here, we separated different native Aβ assemblies in brain homogenates of transgenic (tg) AD mice and human AD post mortem samples by density gradient centrifugation, then isolated Aβ-containing assemblies by co-immunoprecipitation. Mass spectrometry of immunoprecipitated proteins with label-free quantification (LFQ) showed significant changes between the proteomes of Aβ oligomers from tg AD mice and wildtype (wt) mice, confirming some proteins that have been expected to bind Aβ species, like ApoE and Clusterin, but also indicating novel, so far unknown, protein content of Aβ oligomers, such as the RabGAP Tbc1d10b. Some of the hereby identified proteins, like, for example, Clusterin, were also found to be enriched in Aβ oligomers from human AD brain tissue derived homogenates as compared to brain tissue from non-demented controls (NC). Others, such as Netrin-1, were specifically enriched in Aβ oligomers in AD compared to NC samples, but not in mouse samples.

Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles released by all cells. EVs carry nucleic acids, proteins, lipids, and metabolites in intercellular communication. As potential liquid-biopsy biomarkers and drug-delivery vehicles for diseases, however, isolating specific EV subpopulations remains challenging owing to their high heterogeneity in size, density, and surface protein markers. We utilized chemical biology tools to develop EV membrane-curvature probes, including MARCKS-ED and other peptides. These short, membrane-anchoring peptides detect the fluid membrane of EVs and report membrane curvature. Here, we review the peptide selection principles, bioorthogonal reaction engineering mechanisms, and applications of these EV peptide probes and discuss future directions, such as stimulus-responsive or artificial intelligence-assisted peptide probe design for on-demand EV capture and subpopulation.

Human loss-of-function variants in 17βHSD13 have been associated with reduced risk and progression of metabolic and alcohol-related liver disease. Although in vitro experiments implicate 17βHSD13 in the processing of steroidal and lipogenic substrates, establishing an unambiguous mechanistic link to disease causation remains challenging, and the absence of robust pharmacodynamic biomarkers complicates clinical dose selection based on tissue target occupancy. To address this, we implemented a medium-throughput cellular thermal shift assay (CETSA) in humanized mouse liver homogenates to quantify the binding of BI-3231, an uncompetitive 17βHSD13 inhibitor, and compared results with functional cell assays. We systematically profiled isothermal dose–response fingerprints (ITDRF_CETSA) across 58–70.5 °C to account for temperature-induced relaxation of binding equilibria at elevated temperatures, which produced pronounced right-shifts in the apparent potency and confirmed NAD⁺-dependent binding. Complementary surface plasmon resonance (SPR) measurements across 5–42 °C defined the temperature dependence of NAD⁺ and BI-3231 binding. Extrapolating SPR affinities to the CETSA temperature range showed convergence with ITDRF_CETSA fingerprints, supporting the use of the SPR-derived K_D for BI-3231 (2.5 nM at 37 °C; pK_D = 8.60 ± 0.07, 95% CI) to estimate the occupancy also in liver homogenates. This work provides a generalizable approach to quantify target engagement for CETSA-responsive drug targets, while underscoring that occupancy estimates for uncompetitive inhibitors must incorporate cofactor saturation.

DNA polymerase β (Pol β) is an important polymerase that functions in DNA repair within the Base Excision Repair and Non-Homologous End-Joining pathways. It is estimated to function in the repair of up to 50,000 DNA lesions per cell per day, within the base excision repair pathway (BER). Given the significant role Pol β plays in repairing DNA, genetic variants of Pol β have the potential to perturb repair, resulting in mutation accumulation which can potentiate cancer formation. Here we identify an unstudied human germline variant of Pol β, the S180R variant (rs1585898410), which introduces a significant amino acid alteration within the dNTP binding pocket of the enzyme. We demonstrate that S180R is a low fidelity variant of Pol β due to its loss of the ability to discriminate correct nucleotides from incorrect nucleotides. We also show that this variant exhibits a much slower rate of nucleotide incorporation, which could further disrupt repair capacity in vivo. Structural data reveal that this variant not only has structural changes that may disrupt dNTP binding but also a loss of primer terminus positioning and dynamic flexibility of the fingers domain in the binary state, which likely are driving the low fidelity of S180R Pol β. This study highlights the importance of binary positioning and nucleotide coordinating residues for maintaining nucleotide selectivity, polymerase function, and fidelity. It also emphasizes the importance of further study of this human germline Pol β variant in vivo.

Antibiotics have revolutionized human health by significantly reducing morbidity and mortality associated with bacterial infections. Antibiotics exert bactericidal or bacteriostatic effects through inhibition of cell wall synthesis and disruption of cell membrane integrity, inhibition of protein, nucleic acid synthesis, and other metabolic pathways. Despite their remarkable success since the mid-20th century, antimicrobial resistance (AMR) has emerged as a major global health concern, undermining current treatments and complicating infection management. Key drivers of AMR include the overuse and misuse of antibiotics in clinical settings as well as bacterial adaptations such as genetic mutations and horizontal gene transfer. Mechanistically, these changes can lead to enzymatic inactivation of antibiotics, modification of drug targets, changes in permeability, and active efflux of antimicrobial agents. As resistance rises, antibiotic discovery and development have lagged, creating an urgent need for novel therapeutic strategies and chemical scaffolds. This review examines the antibiotic mechanisms and antibiotic evasion strategies, highlighting genetic and omics approaches used to identify high-priority targets for future drug discovery.

Peptide-derived macrocycles are an emerging class of therapeutics capable of modulating protein–protein interactions that remain inaccessible to small molecules. Genetically encoded library (GEL) platforms such as phage and mRNA display have accelerated macrocyclic ligand discovery by linking peptide sequence to genotype and enabling selections from libraries with up to 10¹³ members. Efforts to expand the chemical space of GELs have included incorporation of electrophiles, either to generate libraries of true covalent ligands or to enable intramolecular reactions such as peptide cyclization. In the latter case, the electrophile is consumed during library construction, producing transient covalent libraries that enhance stability and diversity but are not designed for direct covalent engagement with targets. By contrast, recent advances have established robust strategies for embedding persistent electrophilic warheads that remain intact during library preparation and selectively react with nucleophilic residues on proteins. These approaches have yielded both reversible and irreversible covalent inhibitors against diverse classes of proteins, while also highlighting challenges in balancing electrophile reactivity with library integrity. Complementary developments in DNA-encoded covalent libraries further underscore the breadth of discovery platforms, though genetically encoded approaches remain uniquely powerful for macrocyclic peptides. Together, these advances define the trajectory of covalent genetically encoded libraries (cGELs) and point toward new opportunities for discovering ligands to historically undruggable targets.

Technologies that rely on artificial intelligence are of increasing prominence in protein science. To a casual observer it may appear that human inputs are of diminishing importance in research. This Perspective emphasizes the essential contributions of human interactions and chance encounters to the discovery process. The topic is synthetic receptors for proteins. I summarize how a chance observation of N-terminal complexation led to multiple (unpredictable) protein−receptor cocrystal structures with diverse potential applications.

Microbial diversity encompasses vast genetic and functional capacities, with immense potential for biotechnological applications. Yet, most biotechnological advances have been confined to a narrow set of model organisms, leaving the broader repertoire of nonmodel microbes largely untapped due to species-specific barriers that hinder genetic manipulation. Over the past decade, the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has transformed microbial engineering by enabling precise, programmable, and scalable control of genomes and gene expression. Importantly, the relative independence of many CRISPR effectors from host cofactors has facilitated their use in microbes previously challenging to engineer, thus expanding opportunities to exploit their unique metabolic and biosynthetic traits. In this review, we summarize the major CRISPR-Cas toolkits and highlight recent innovations, with particular emphasis on translational applications in nonmodel organisms such as C1-gas-fixing acetogens, antibiotic-producing Streptomyces, and gut commensal Bacteroides. We emphasize three areas of emerging impact: engineering microbial cell factories for sustainable biomanufacturing, accelerating natural product discovery, and development of next-generation live biotherapeutics. Finally, we discuss current limitations and future opportunities, underscoring how the integration of genome editing, synthetic biology, and systems-level approaches is reshaping the landscape of microbial biotechnology.

Encapsulins are self-assembling protein nanocompartments widely distributed across prokaryotes that sequester diverse enzymes. While most encapsulin systems studied thus far are involved in nutrient storage or oxidative stress response, recent bioinformatic and experimental studies have also demonstrated their involvement in secondary metabolism, particularly terpenoid biosynthesis. In this perspective, we first present a comprehensive analysis of Family 2B encapsulin gene clusters likely involved in terpene or terpenoid biosynthetic pathways. We then highlight the structural features of Family 2B encapsulin shells, with a focus on their pore properties and putative ligand-binding domains. We review the mechanisms of enzyme cargo loading in Family 2B systems and examine known examples of terpenoid synthesis compartmentalized within Family 2B encapsulin shells. This is followed by a discussion of the molecular logic and potential functional advantages of enzyme encapsulation. Finally, we consider outstanding questions and future research directions aimed at elucidating the molecular details and physiological implications of encapsulin-mediated bacterial terpene biosynthesis.