Biochemistry Biochemistry最新文献

Yin-Yang 1 (YY1) is a CCHH-type classical zinc finger (ZF) protein that plays diverse roles in gene expression, acting as both a transcriptional activator and repressor, which is important for DNA repair, neuronal development, and oncogenesis. Classical ZFs adopt a ββα fold upon Zn(II) binding, and YY1 contains four CCHH-type domains. The two central domains (ZF2 and ZF3) are known to directly bind to DNA. Although ZFs have traditionally been viewed as just structural domains, emerging data shows that ZFs can be modified by the gaseous signaling molecule hydrogen sulfide, H₂S, to form persulfides. These data are principally from proteomics studies from which several classical ZFs, including YY1, were identified as persulfidated. Herein, we report how the classical ZF YY1 is persulfidated by H₂S and the effects of persulfidation on DNA binding using three ZF constructs containing the second domain (YY1-ZF2), third domain (YY1-ZF3), and both the second and third domains (YY1-ZF2-ZF3). Persulfidation of all three constructs was observed using an NBF-Cl/dimedone tag-switch method. Persulfidation required Zn(II) and O₂. Superoxide, as measured by hydroethidine and superoxide dismutase experiments, was also observed as an intermediate. YY1-ZF2-ZF3 was also shown to bind to the adeno-associated virus P5 initiator and IL-6 promoter DNA via a fluorescence anisotropy assay. This ZF/DNA binding was abrogated by H₂S; however, when DNA was bound to YY1-ZF2-ZF3, it was unreactive to H₂S modification suggesting a protective effect of the DNA macromolecule. In addition, H₂S disrupted the secondary structure of all three YY1 constructs as measured by circular dichroism.

Modulation of the nervous system by gut microbiota through metabolic pathways is a key mechanism of communication within the gut-brain axis. A critical factor determining whether gut microbial metabolites can exert functional effects in the brain is their ability to cross the blood-brain barrier (BBB). However, current methods for assessing BBB permeability lack systematic, standardized approaches and advanced predictive technologies. Traditional experimental techniques are often costly and time-consuming compared to computational methods. To address these limitations, we developed an automated molecular simulation workflow to generate a high-quality data set of gut microbial metabolites annotated with thermodynamic features related to BBB permeability. Based on this data set, we constructed an interpretable thermodynamic evaluation framework capable of accurately identifying key factors that influence transmembrane transport. The robustness and predictive power of our models were validated using two authoritative benchmark data sets, confirming their ability to reliably distinguish BBB-permeable from nonpermeable compounds. Furthermore, our findings highlight the substantial potential of gut microbiota metabolism to influence BBB permeability via metabolic pathways. Overall, this study provides a powerful tool for identifying gut microbiota-derived metabolites with potential biological activity in the brain and introduces a novel paradigm for the intelligent prediction of BBB permeability.

Thousands of recently discovered microproteins represent a new frontier in the search for functional and disease-causing genes. Though shorter than canonical proteins, some microproteins contain signal peptides and are predicted to produce secreted peptides. However, whether any of the microprotein-derived secreted peptides possess biological activity remains underexplored. Here, we screen a small library of secreted peptides from the microproteome by measuring signaling downstream from GPCRs. This approach identified several cAMP-stimulating peptides, including a secreted peptide from a "non-coding" HLA complex P5 RNA (HCP5). The HCP5-secreted peptide (HCP5-SP) is encoded by a small open reading frame embedded in the HCP5 mRNA. In vitro assays with synthetic HCP5-SP and HCP5-SP analogs validated its cAMP-stimulating activity and revealed the necessity for the wild-type C-terminal sequence for activity. Furthermore, HCP5-SP promotes the proliferation of HEK293T cells, providing an alternative mechanism that might explain some of the cancer biology associated with HCP5 mRNA. In summary, this work establishes a workflow for the preliminary identification of bioactive microproteins and demonstrates that the vast, largely untapped microproteome is a source of novel bioactive endogenous peptides.

Mycobacterium tuberculosis Mpa is an AAA+ ATPase that unfolds and translocates substrate proteins during proteasomal degradation. Mpa spontaneously assembles into a homohexamer driven by interdomain interactions, even in the absence of a nucleotide. To dissect the mechanisms underlying its oligomerization and deoligomerization, we perturbed the system using chemical and thermal denaturants and monitored the oligomeric states by far-UV CD, fluorescence, calorimetry, state-of-the-art single-particle mass photometry, and ATPase activity assays. Equilibrium chemical denaturation resulted in gradual changes in spectroscopic signals, whereas mass photometry revealed a direct transition from a hexamer to monomeric species, indicating a concerted but noncooperative pathway without any intermediate oligomeric or folded monomeric states. In contrast, thermal perturbation showed two sharp and distinct transitions, the first one corresponding to a concerted and cooperative transition from hexamer to unfolded state, which further aggregates, and the second transition to disaggregation at elevated temperatures. Both chemical and thermal unfolding processes were irreversible with respect to the reassembly of the functional oligomer. Using single-particle mass photometry complemented by spectroscopy and calorimetry, these findings establish that Mpa is an obligate oligomer and can provide insights into the oligomerization pathways of AAA+ enzymes that spontaneously hexamerize and have the potential to further illuminate evolutionary strategies underlying their assembly mechanisms.

CRISPR-Cas9 is natively present in the adaptive immune systems of a multitude of bacteria and has been adapted as an effective genome engineering tool. The Cas9 effector enzyme, which is composed of a single Cas9 protein and a single-guide RNA (sgRNA), identifies and cleaves double-stranded DNA targets through a series of conformational changes that require DNA distortion and unwinding. While most studies of Cas9 specificity have focused on the DNA sequence, the role of intrinsic DNA physical properties ("DNA shape") in modulating Cas9 activity remains insufficiently defined. We previously showed that with a 16-nucleotide (-nt) truncated guide, the intrinsic DNA duplex dissociation energy at the PAM+(17-20) segment beyond the RNA-DNA hybrid tunes Cas9 cleavage rates of linear substrates. Here, we examined the impact of DNA supercoiling on Cas9 cleavage with the 16-nt truncated guide. Enzyme kinetic analysis revealed that PAM+(17-20) DNA sequences beyond the RNA/DNA hybrid preserve their effects on Cas9 cleavage in the supercoiled state. Furthermore, combining a novel asymmetric hairpin construct with a parallel-sequential kinetics model, rates for first-step nicking and second-step cleavage by Cas9 were obtained for both supercoiled and linear substrates. With both topologies, it was found that first-step nicking is clearly impacted by PAM+(17-20) DNA sequences, and the effects can be correlated with DNA unwinding, which dictates R-loop dynamics. This work expands our understanding of DNA target recognition by Cas9, and the methods developed, in particular those for analyzing the progression of Cas9-induced nicks, will aid in further in-depth mechanistic investigation.

Glycine, the simplest amino acid with a single hydrogen atom as its side chain, plays a crucial role in protein folding and structural flexibility. In this study, we used copper/zinc superoxide dismutase (SOD1) as a model system to investigate how the substitution of glycine at position 93 with various amino acids affects the protein structure and stability. We engineered 19 G93 SOD1 mutants and evaluated their folding patterns and aggregation propensities using immunoblotting, fluorescence microscopy, and fluorescence loss in photobleaching (FLIP) assays. All mutants, regardless of the amino acid substitution, form protein aggregates with varying degrees of stability, demonstrating that position 93 exhibits extreme mutation sensitivity, with different substitutions producing distinct destabilization pathways. Our findings demonstrate that alteration of glycine's minimal side chain─consisting of a single hydrogen atom─ disrupts native protein structure. The physicochemical properties of the substituting amino acid, such as polarity, charge, and steric bulk, critically modulate the nature and extent of the resulting misfolding. Nonpolar residues promote aggregation primarily through hydrophobic interactions, while polar and charged residues drive aggregation via hydrogen bonding and electrostatic interactions. This study provides fundamental insights into glycine's unique structural contributions to protein architecture and presents a conceptual framework for understanding how side chain properties influence protein folding and stability. These findings also provide mechanistic implications for protein aggregation processes in neurodegenerative diseases.

Cyclic tetrapeptides (CTPs) are a diverse class of natural products with a broad range of biological activities. However, they are challenging to synthesize due to the ring strain associated with their small ring size. While chemical methods have been developed to access CTPs, they generally require the presence of certain amino acids, limiting their substrate scopes. Herein, we report the first bioinformatics-guided discovery of a thioesterase from a cryptic biosynthetic gene cluster for peptide cyclization. Specifically, we hypothesized that predicted penicillin-binding type thioesterases (PBP-TEs) from cryptic nonribosomal peptide synthetase (NRPS) gene clusters containing four adenylation domains would catalyze tetrapeptide cyclization. We found that one of the predicted PBP-TEs, WP516, efficiently cyclizes a wide variety of tetrapeptide substrates. To date, it is only the second stand-alone enzyme capable of efficiently cyclizing tetrapeptides, and its substrate scope greatly surpasses that of the only other reported tetrapeptide cyclase Ulm16. AlphaFold modeling, covalent docking, molecular dynamics, and mutational analyses were used to rationalize the broad substrate scope of WP516. Overall, the bioinformatics-guided workflow outlined in this paper and the discovery of WP516 represent promising tools for the biocatalytic production of head-to-tail CTPs, as well as a more general strategy for the discovery of enzymes for peptide cyclization.

Sweet proteins trigger sweet taste perception through interactions with the human T1R2/R3 sweet taste receptor. To date, relatively few proteins have been identified as causing sweet taste perception, and the four most studied proteins: monellin, brazzein, thaumatin, and honey truffle active component (HT-AC), have minimal sequence homology or structural similarities aside from positively charged surface sites. Sweet taste perception has also been found to be readily perturbed by minor changes in the protein structure, such as natural isoforms inherent to heterologous expression of the protein, and synthetic amino acid substitutions. This study uses ab initio rigid-body docking to predict the interactions of known sweet proteins and variants with a recently resolved cryo-EM structure of the T1R2/R3 sweet taste receptor, incorporating comparative analyses between apo-, holo-, and a potentially transient conformation of the receptor. HT-AC mediated activation of the sweet taste receptor is confirmed by in vitro cell-based assays, and results from in silico docking of various sweet proteins are used to derive additional insights regarding sweet taste perception. Perturbations of HT-AC due to naturally occurring post-translational modifications and synthetic modifications are evaluated using in vitro and in silico methods to determine robustness of the interaction between T1R2/R3 and sweet proteins with primary focuses on HT-AC.

Ubiquitination, a post-translational modification, regulates numerous cellular processes by attaching ubiquitin molecules to the target proteins, thereby altering their cellular levels and functions. A central aspect of ubiquitin-mediated signaling is the formation of different types of polyubiquitin (polyUb) chains, which can be either homotypic or heterotypic, generating a variety of cellular signals that activate distinct downstream pathways. Homotypic chains are linked via the same lysine on each molecule, whereas heterotypic chains are conjugated via multiple lysines. The synthesis of these diverse polyubiquitin chains is driven by interactions between substrate-conjugated ubiquitin molecules, ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). However, the molecular details of these interactions and how they govern the synthesis of different homotypic and heterotypic chains remain poorly understood. The E2 enzyme E2-25K preferentially extends K48-linked polyubiquitin chains on K63-linked template polyubiquitin chains, creating branched K48/K63 chains. In this study, we investigated the role of dynamic interactions between E2-25K and the K63-linked diubiquitin substrate to assess the molecular mechanism of branched-chain assembly. Our data reveal that E2-25K shows no preference for binding to the proximal or distal ubiquitin of a template chain. However, binding to the distal unit activates the complex; binding to the proximal unit does not. This study highlights the critical role of stereospecificity in enzyme/substrate interactions for branched ubiquitin chain synthesis and provides insights into the mechanisms of ubiquitin signaling.

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.