Biochemistry Biochemistry最新文献

RNA-binding proteins (RBPs) are essential regulators of posttranscriptional gene expression, influencing mRNA processing, translation, and stability. Defining their binding sites on RNA is key to understanding how they assemble into functional ribonucleoprotein (RNP) complexes, but existing footprinting and cross-linking approaches often yield low signal-to-noise, variable efficiency, or require highly purified complexes. To address these limitations, we developed Tethered Micrococcal Nuclease Mapping (TM-map), a sequencing-based strategy that determines the three-dimensional binding sites of RBPs on RNA in vitro. In TM-map, the RBP is fused to micrococcal nuclease (MNase), which upon Ca²⁺ activation cleaves proximal RNA regions, producing fragments whose 3' termini report the spatial proximity of the fusion. We first validated TM-map using the bacteriophage MS2 coat protein bound to its cognate RNA stem-loop engineered into the Escherichia coli ribosome. Cleavage sites mapped proximal to the engineered stem-loop, confirming that tethered MNase reliably reports local protein-RNA proximity on the ribosome surface. We then applied TM-map to the Drosophila Fragile X Mental Retardation Protein (FMRP), a translational regulator with an unresolved ribosome-binding site. Both N- and C-terminal MNase-FMRP fusions produced reproducible cleavage clusters on the 18S rRNA localized to the body and head of the 40S subunit. The similar profiles suggest that FMRP's termini are conformationally flexible and sample multiple orientations relative to the ribosome, consistent with a dynamic interaction rather than a fixed binding mode. TM-map thus provides a simple, proximity-based, and generalizable in vitro approach for visualizing RBP-RNA interactions within native RNP assemblies.

The Ras superfamily of small GTPases are challenging targets for therapeutic inhibition, partially due to a lack of pockets amenable to small molecule inhibition. Our previous work identified high-affinity cyclized peptide binders of Cdc42, a member of the Rho family of small GTPases, capable of inhibiting activity. To further optimize these Cdc42 inhibitors, we have engineered modifications to the best sequence available from the original maturation and screened the ability of these third-generation peptides to compete with Cdc42-effector interactions. Improvements in affinity were achieved by single amino acid substitutions at several residue positions. We present the structure of one of these nanomolar affinity, cyclized peptides in complex with Cdc42. The structure reveals that the peptide binds in a β-hairpin conformation to create an extension of the β-sheet of the GTPase Rossman fold, acting as a structural mimic of native Cdc42 effectors. We additionally elucidate the NMR structures of four unbound C-terminal alanine variants and employ both the bound and unbound structures to inform the rational design of substituted peptide inhibitors. Overall, this study expands our understanding of how Ras GTPases can be targeted, by demonstrating a rare example of an inhibitor binding contiguously with a surface of β-strand of the small G protein, which illustrates an innovative avenue for noncovalent therapeutic design.

Reader proteins that bind histone trimethyllysine (Kme3) have become active therapeutic targets in recent years. Binding of Kme3 in the conserved aromatic cages of these readers via cation-π interactions is a key contributor to these protein–protein interactions. We explored whether the replacement of one methyl group of Kme3 with an electron-withdrawing group (EWG) could improve binding by increasing the electrostatic component of the cation-π interaction. We examined these Kme3 analogs in binding studies with histone reader proteins, nuclear magnetic resonance (NMR) studies in a β-hairpin peptide model system, and computational studies to gain mechanistic insight into their binding. Kme3 analogs with both alkyl and benzyl substitution with EWGs were examined. We found that EWGs on the ligand differentially affected binding to a series of Kme3 readers but did not improve binding. This is likely due to sterics of the different analogs that preclude optimal cation-π interactions. The unique selectivity patterns of these ligands nonetheless offer promise toward selective inhibitor development within this protein class. NMR studies of the alkyl-substituted analogs in the β-hairpin model system show that EWGs strengthen the cation-π interaction enthalpically but with an entropic penalty. NMR studies demonstrate that the benzyl group makes direct contacts with Trp in the β-hairpin model, providing aromatic interactions in addition to cation-π interactions. Computational energy decomposition analysis confirms the more favorable electrostatic interactions compared to Kme3, but other factors counter the enhanced electrostatic component. This mechanistic investigation demonstrates how EWG-containing Kme3 analogs contribute to cation-π interactions and provide unique selectivity patterns relative to Kme3.

Quantum mechanical tunneling (QMT) is now recognized as a significant contributor to some enzyme catalyzed hydrogen transfer reactions. In this perspective, we examine recent theoretical and experimental advances that investigate when, and how, QMT contributes to enzyme catalysis. We highlight progress and challenges in computing the rate constants of reactions involving tunneling, including developments in semiclassical approaches and in nuclear-electronic orbital density functional theory. Case studies on flavoenzymes, ribonucleotide reductase, catechol O-methyl transferase and Morita–Baylis–Hillmanase illustrate how protein dynamics, vibrational gating and electrostatic effects apparently modulate barrier width and sustain tunneling-derived rate enhancements. We expect that continued integration of improved theoretical methods and dynamics-sensitive experiments will be essential to move QMT from a mechanistic phenomenon to a tunable design parameter in future enzyme engineering and rational catalyst development.

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.