Biochemistry Biochemistry最新文献

Peptide nucleic acid (PNA) and DNAzymes have recently been used to develop an artificial DNA nuclease system named PNA-assisted double-stranded DNA nicking by DNAzymes (PANDA) for genetic engineering. Interestingly, the PANDA system demonstrated a higher sequence fidelity than CRISPR/Cas9, with the ability to discriminate single-nucleotide mismatches. To evaluate the source of PANDA’s sequence fidelity, we conducted kinetic experiments that separately examined the kinetics of PNA invasion and DNAzyme cleavage, each under rate-limiting conditions. Our results show that PNA serves as an initial mismatch “inspector,” while DNAzyme adds complementary specificity during the cleavage process. Notably, PNA and DNAzyme recognize mismatches at opposite ends of their binding regions, enabling cooperative discrimination of mismatches across the entire target site, including regions that are typically difficult to distinguish by other methods. This dual recognition mechanism enhances PANDA’s sequence fidelity, particularly in single-nucleotide mismatch discrimination. These findings establish PANDA as a promising molecular tool for precise, targeted DNA manipulation, offering a robust platform for applications that require stringent sequence specificity.

Use of noncanonical nucleotides for nucleic acid replication provides a critical way to understand how and why natural nucleotides were chosen by evolution and provides potential applications in biotechnology, diagnostics, therapeutics, and DNA computation. However, monitoring noncanonical nucleotide incorporation can be difficult, especially for high-throughput applications. Therefore, we have utilized a 6-letter genetic alphabet (G, A, T, C, Z, and P) for toehold-mediated DNA strand exchange assays. Toehold hybridization and branch migration were found to proceed readily through the formation of multiple consecutive non-natural base pairs between hydrogen bonding synthetic nucleotides dZ (6-amino-5-nitro-2(1H)-pyridone) and dP (2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one). Unlike other mismatches, a dZ:dG mis-pair was minimally disruptive to strand exchange. Overall, however, positional and numerary effects on mismatch tolerance could be leveraged to distinguish both dP and dZ mismatches. The success of the assay allowed a rapid assessment of the semisynthetic DNA replication by Escherichia coli DNA polymerases I, II, and IIIα and the identification of E. coli and Geobacillus stearothermophilus DNA Pol I mutants with improved fidelity. Given the difficulty of adapting organisms for expanded genetic alphabets, the ability to rapidly proof sequences and enzymes in vitro should lead to new parts and circuits that can be modularly introduced to improve the incorporation of more and more different noncanonical nucleotides.

Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of cellular signaling pathways, and its dysregulation is linked to diabetes, obesity, cancer, and immune dysfunction. While the catalytic mechanism of PTP1B is conserved across protein tyrosine phosphatases, its regulation by distal allosteric sites remains less understood. Here, we investigate how mutations at four allosteric sites (Y153, I275, M282, and E297) alter the PTP1B substrate specificity and enzymatic dynamics. Kinetic analyses with phosphotyrosine peptides and p-nitrophenylphosphate reveal that allosteric mutants display distinct changes in catalytic efficiency (k_cat/K_m), in some cases reversing substrate preference relative to the wild-type enzyme. Solution NMR spectroscopy and microsecond molecular dynamics simulations demonstrate that these mutations perturb long-range communication networks, disrupting coupling between helices α3 and α7 and altering acid-loop flexibility and active-site dynamics. Notably, the E297A mutation has the most pronounced effects, rigidifying the acid loop and weakening allosteric communication to the catalytic center. Community network analysis highlights the acid loop and helix α7 as central hubs linking distal sites to the active site. Together, these results establish that distal mutations can reshape PTP1B’s dynamic landscape, thereby modulating substrate specificity. This work expands our understanding of allosteric regulation in PTP1B and provides a framework for targeting dynamic networks to control phosphatase activity.

Collagen in the connective tissue plays a key role in the expression the aging phenotypes. While collagen production decreases with aging, collagenase expression increases, resulting in collagen breakdown. The purpose of this study is to investigate the change in the expression of proteins and genes related to the collagen signaling pathway, cell cycle, and aging phenotypes of cells with the collagen type 1 α (COL1A1) gene edited by the CRISPR/Cas9 system. The mutation of the COL1A1 gene was induced by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses, Sanger DNA sequencing analysis and Swiss protein modeling analysis were used to verify the induction of mutation. Aging phenotypes in the mutated cells were evaluated by collagen staining assay, SA-β-galactosidase staining assay, RT-PCR assay, Western blot analysis, gelatin zymography, and immunofluorescent staining assay. Sanger DNA sequencing analysis demonstrated that human fibrosarcoma cells with COL1A1 gene mutations were successfully established in this study. Swiss protein modeling analysis displayed the altered structure of COL1A1 in the edited cells. In addition, while collagen production was decreased, the SA-β-galactosidase staining level was increased in the edited cells. It was also found that the expression levels of CDC2, CDk2, and cyclin D were increased by down-regulating p53 and p21 levels through the increased expression of MDM2 in the edited cells. Moreover, the expression levels of MMP-1, MMP-2, MMP-9, AKT, and p-mTOR were reduced in the edited cells. These findings could provide a crucial clue in elucidating the close relationship between collagen production and senescence.

In this report, we examine the extensive research landscape of CRISPR with an emphasis on CRISPR therapeutics and showcase our results from an in-depth analysis of the most up-to-date scientific information consisting of more than 53,000 publications encompassing academic journal articles and patents, spanning nearly three decades, extracted from the CAS Content Collection. Our analysis indicates that cancer and infectious diseases are the most explored in the context of CRISPR. Identified gene targets associated with CRISPR-related publications are led by TP53, c-myc, and hemoglobin beta subunit (HBB). Among the many delivery methods, adeno-associated vectors (AAVs) appear to be highly explored. With >140 CRISPR-based therapeutics in the clinical development pipeline and billions of dollars in investment, the field of CRISPR continues to evolve rapidly. We also briefly discuss the ethical implications of CRISPR technology. While some fundamental challenges persist, the future of CRISPR is undoubtedly bright.

Pyrroloquinoline quinone (PQQ), an o-quinone-type nutrient, has been shown to exert diverse beneficial effects on the biochemical and physiological processes of mammals. However, the molecular mechanisms underlying these effects remain incompletely understood. Here, through screening of metabolite-sensing G protein-coupled receptors (GPCRs)─which respond to metabolites produced by gut microbiota or derived from nutrients─we found that PQQ selectively activates G-protein-coupled receptor 35 (GPR35) and characterized the molecular basis of its ligand recognition. Using a transforming growth factor α shedding assay, we demonstrated that PQQ selectively activated GPR35, a class A rhodopsin-like GPCR expressed in various tissues, including adipose tissue and the gastrointestinal tract. PQQ also promoted β-arrestin 1 recruitment to the plasma membrane, further supporting its role as a GPR35 agonist. Direct binding of PQQ to GPR35 was demonstrated using a clickable photoaffinity probe derived from PQQ. Molecular docking simulation and site-directed mutagenesis revealed that the 2-carboxylic acid moiety of PQQ forms critical hydrogen bonds with Arg100, Tyr101, and Arg151 of GPR35. Additionally, Phe163 appears to contribute to π–H interaction with PQQ. These findings indicate that PQQ functions as a food-derived agonist of GPR35 and provide new insights into the molecular mechanisms underlying the potential beneficial effects of PQQ.

Viruses are the most abundant biological entities on Earth and play central roles in shaping microbiomes and influencing ecosystem functions. Yet, most viral genes remain uncharacterized, comprising what is commonly referred to as “viral dark matter.” Metagenomic studies across diverse environments consistently show that 40–90% of viral genes lack known homologues or annotated functions. This persistent knowledge gap limits our ability to interpret viral sequence data, understand virus-host interactions, and assess the ecological or applied significance of viral genes. Among the most intriguing components of viral dark matter are auxiliary viral genes (AVGs), including auxiliary metabolic genes (AMGs), regulatory genes (AReGs), and host-physiology-modifying genes (APGs), which may alter host function during infection and contribute to microbial metabolism, stress tolerance, or resistance. In this Review, we explore recent advances in the discovery and functional characterization of viral dark matter. We highlight representative examples of novel viral proteins across diverse ecosystems, including human microbiomes, soil, oceans, and extreme environments, and discuss what is known and still unknown about their roles. We then examine the bioinformatic and experimental challenges that hinder functional characterization and present emerging strategies to overcome these barriers. Finally, we highlight both the fundamental and applied benefits that multidisciplinary efforts to characterize viral proteins can bring. By integrating computational predictions with experimental validation and fostering collaboration across disciplines, we emphasize that illuminating viral dark matter is both feasible and essential for advancing microbial ecology and unlocking new tools for biotechnology.

Escalating antimicrobial resistance presents an urgent need for novel antibiotics, particularly against Mycobacterium tuberculosis (M. tb), which claims 1.5 million lives annually. A promising avenue for antibiotic development is inhibiting DNA biosynthesis by targeting Thymidylate Synthase enzymes (TS) that produce 2′-deoxythymidine-5′-monophosphate (dTMP), an essential building block of DNA. Two forms of TS enzymes are known, and M. tb has been shown to rely on a unique flavin-dependent thymidylate synthase (FDTS), which is distinct from the human TSase enzyme. This work explores the mechanism and binding modes of inhibitors of the MtbFDTS enzyme by utilizing a nonproductive oxidase reaction (O₂ reduction to H₂O₂) catalyzed by the flavin cofactor. We discovered that inhibitors of MtbFDTS that bind competitively at the nucleotide-binding site potentiate the oxidase reaction. Conversely, inhibitors of the enzyme that bind at the folate’s binding site abate the oxidase reaction. We exploit this contrasting kinetic behavior to show that naphthoquinones inhibit FDTS by binding competitively at the nucleotide’s active site, activating the enzyme’s oxidase reaction and consequently producing substantial peroxide as a byproduct. We confirm our predicted inhibitor binding modes through direct measurement of folinic acid binding to the ternary enzyme–flavin–naphthoquinone complex, providing a revised kinetic mechanism for the FDTS-catalyzed reaction. Our findings show that naphthoquinones inhibit the native FDTS reaction and divert the enzyme’s activity to produce reduced oxygen species, which sheds light on their antimycobacterial activity and will be critical for future inhibitor development and high-throughput screening (HTS) methods.

Transthyretin (TTR) is a 56 kDa tetrameric protein complex that transports thyroxine and retinol but can misfold, causing amyloid diseases, such as senile systemic amyloidosis, familial amyloid cardiomyopathy, and familial amyloid polyneuropathy. Previous studies have found that TTR aggregation is initiated when tetramers disassemble into monomers, dimers, and trimers, which misfold and assemble into heterogeneous oligomers. These oligomers are thought to be cytotoxic, yet their formation and composition remain poorly understood. To investigate monomer misfolding, ion mobility-mass spectrometry (IM-MS) was applied to wild-type TTR (wtTTR) and the pathogenic L55P variant under varying pH conditions. IM-MS revealed that acidic pH promotes extended monomer conformations for both wtTTR and L55P. Additionally, L55P showed a higher abundance of extended conformations that are attributed to its increased amyloidogenicity. Orbitrap-based charge detection mass spectrometry is used via the direct mass technology (DMT) mode to evaluate oligomeric species, revealing that acidic pH and lower temperatures promote oligomerization and L55P formed oligomers more readily than wtTTR. Together, these results show that oligomerization and conformational changes depend on solution pH, temperature, and proteoform, supporting the role that changes in hydration play in TTR aggregation. More broadly, these findings demonstrate the complementary strengths of IM-MS and DMT for characterizing aggregation intermediates and provide new insights into TTR aggregation.

We report the results of experiments to test the hypothesis that binding energy from the adenosine diphosphate (ADP) fragment of the NAD⁺ cofactor is utilized to drive a protein conformational change that activates phosphite dehydrogenase (PTDH) for catalysis of hydride transfer from phosphite to NAD⁺. The ADP fragment of the NAD⁺ cofactor provides >8.5 kcal/mol stabilization of the transition state for PTDH-catalyzed hydride transfer. The ADP and AMP fragments of NAD⁺ activate PTDH for catalysis of hydride transfer from phosphite to nicotinamide riboside (NR). At a 1.0 M standard state these activators stabilize the hydride transfer transition state by 5.1 (ADP) and 2.7 (AMP) kcal/mol, so the activation is due to protein interactions with both the α- and β-ADP phosphates. There is no detectable stabilization of the transition state for PTDH-catalyzed hydride transfer to NR by the adenosine fragment of NAD⁺. Activation is proposed to result from stabilization of the closed form of PTDH by a cation–anion pair with the K76 side chain that bridges the α- and β-phosphates of NAD⁺. By comparison, the activation of formate dehydrogenase- and glycerol phosphate dehydrogenase-catalyzed hydride transfer by ADP is from enzyme interactions with the α-phosphate of ADP, with little or no contribution from the β-phosphate. These results show a diversity in the evolution of enzyme-activating conformational changes for dehydrogenase-catalyzed hydride transfer reactions.