Biochemistry Biochemistry最新文献

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.

The unfolding of telomeric G-quadruplexes (G4s) is a key step in telomere elongation and regulation. Within cells, the highly crowded intracellular milieu significantly influences the structural stability and dynamics of G4s; however, the molecular mechanism governing their unfolding under such conditions remains poorly understood. In this study, we have investigated the thermal unfolding of various human telomeric G4 sequences in KCl, both in the absence and presence of molecular crowders, using temperature-dependent circular dichroism (CD) spectroscopy combined with singular value decomposition, multivariate curve resolution alternating least-squares (MCR-ALS), and well-tempered metadynamics simulations. In KCl alone, telomeric G4s exhibit a two-state unfolding mechanism, where the hybrid-type topology directly converts into the unfolded random-coil state. In contrast, under crowded conditions, particularly in the presence of hydrophobic crowders, the unfolding follows a three-state pathway involving a distinct intermediate. The hybrid structure initially transitions to a parallel-type topology at elevated temperatures before fully unfolding. This stabilization of the parallel topology arises from preferential interactions between hydrophobic crowders and the exposed loop nucleobases of the parallel G4 form. On the other hand, hydrophilic crowders exert minimal influence on the unfolding pathway, which remains similar to that observed in KCl solution. Overall, these findings provide molecular-level insights into the unfolding process of telomeric G4 DNA in crowded cell-like environments and may be useful in understanding the complex telomere elongation process.

Mandelate racemase (MR) catalyzes the Mg²⁺-dependent interconversion of (R)- and (S)-mandelate and has been employed as a model enzyme to demonstrate that an enzyme catalyzing the deprotonation of a carbon acid substrate may be inhibited by boronic acids. We report a detailed structure–activity-based study of the ability of various boronic acid derivatives to competitively inhibit MR. 2-Naphthylboronic acid (K_i = 0.32 ± 0.01 μM), furan-3-boronic acid (K_i = 10 ± 1 μM), and thiophene-3-boronic acid (K_i = 1.27 ± 0.06 μM) were potent inhibitors of MR, while 1-naphthylboronic acid (K_i = 28 ± 3 μM) and nitrogen-heterocycles (e.g., isoxazole, indole, 1H-indazole, pyridine, and pyrimidine) bearing boronic acid groups were generally weaker inhibitors. A chlorine substituent on the pyridine (i.e., 2-chloro-pyridine-5-boronic or 2-chloro-pyridine-4-boronic acids) or pyrimidine (i.e., 2-chloro-pyrimidine-5-boronic acid) ring enhanced the binding affinity by 3- to 27-fold. Surprisingly, benzoxaboroles, including the antifungal agent tavaborole (i.e., 5-fluorobenzoxaborole, K_i = 1.06 ± 0.09 μM), were also potent competitive inhibitors of MR. The pH-dependence of the inhibition by benzoxaborole suggested that the species with the tetrahedral, sp³-hybridized boron atom was the more potent inhibitor. Interestingly, ¹¹B NMR spectroscopy and X-ray crystallography revealed that aryl boronic acids and benzoxaboroles interact with MR via different binding modes. Unlike phenylboronic acid, which forms an N^ε2–B bond with His 297 at the active site, the 1.8-Å resolution structure of the MR-tavaborole adduct revealed the presence of an N^ζ–B bond between the bound tavaborole and Lys 166 at the active site.

Gene transcription in eukaryotes is orchestrated by intricate regulatory architectures that depend on chromatin context and a diverse repertoire of transcription factors. Consequently, eukaryotic promoters are thought to be incompatible with the streamlined transcriptional machinery of bacteria. Here, we challenge this assumption by demonstrating that a minimal 90-bp fragment of the human TMIGD1 promoter functions as an active, constitutive promoter in Escherichia coli. This compact human sequence drives reproducible reporter expression at levels comparable to established moderate-strength bacterial promoters, revealing an unexpected convergence between human and bacterial transcriptional recognition mechanisms. The ability of a human promoter to operate across domains of life suggests that fundamental features of promoter architecture may be more deeply conserved─or evolutionarily constrained─than previously appreciated. Our findings expand the boundaries of promoter compatibility and open new opportunities for designing hybrid regulatory systems that leverage cross-kingdom transcriptional activity.

UBQLN2 is a member of the UBL-UBA domain protein family that functions as extrinsic substrate receptors for the 26S proteasome. UBQLN2 has been shown to undergo phase separation in vitro. In cells, UBQLN2 forms condensates that may be of importance for tuning protein degradation via the ubiquitin-proteasome system and potentially of relevance for UBQLN2-linked amyotrophic lateral sclerosis (ALS). Here we show that UBQLN2 is ubiquitylated on lysine residues in the N-terminal UBL domain. The C-terminal region of UBQLN2 is lysine-depleted, and we show that introducing lysine residues in this region leads to its E6AP-dependent degradation. The UBL domain critically stabilizes UBQLN2 and protects it from proteasomal degradation. Fusion of ubiquitin to the UBQLN2 N-terminus stabilizes UBQLN2 and increases its propensity for locating in puncta, indicating that ubiquitylation of the UBQLN2 UBL domain regulates abundance and localization.

Zika virus infections in humans were identified in several African countries in the 1950s and spread globally in the mid-2010s. Cases began to surge in South America around 2016, coinciding with a rise in severe developmental disorders in babies born to infected mothers. Gaining a deeper understanding of ways to treat infection is crucial. To this end, we sought to investigate the dynamics of Zika Virus Protease NS2B-NS3, a drug target that will enable effective treatment of infection and may be invaluable in treating more lethal variants that may eventually emerge. Specifically, we employed hydrogen–deuterium exchange mass spectrometry on NS2B-NS3 to observe which regions of the protein are quick or slow to exchange, and how these exchange patterns change in the presence of an allosteric inhibitor. From these studies, we observe that Zika Virus Protease populates the open conformation when it is unbound or bound to the allosteric inhibitor MH1. We further identified a single substitution in NS3, A125C, that directly blocks allosteric inhibition. The observed deuterium uptake patterns provide a detailed view of Zika Virus Protease dynamics in unbound and inhibitor-bound states, allowing us to visualize how allosteric binding at NS3 prevents closure of NS2B and propagates structural perturbations that together result in protease inhibition. Importantly, our studies predict that interactions between the NS2B cofactor and the NS3 core determine the potency of this class of inhibitors across the flaviviral proteases. Pan-flaviviral inhibitors would provide invaluable antiviral modalities, and insights from these studies should aid in their development.

Methylglyoxal (MGO) and glyoxal (GO) are reactive carbonyl species (RCS) generated as side products in glycolysis and carbohydrate, protein, and fat catabolism, which are enriched in most cancer cells. MGO/GO-induced nonenzymatic glycation on histones plays pathophysiologically important roles in regulating the three-dimensional architecture of cellular chromatin and cancer development. In our previous studies, we have uncovered that enzymes DJ-1 and PAD4 exhibit “glyoxalase” and “deglycase” activities to antagonize the MGO/GO-modifications of histones. We also found that the general inhibition of histone deacetylases using suberoylanilide hydroxamic acid (SAHA) antagonized histone MGO-glycation due to the direct competition of reactive sites (i.e., lysine residues). Here, we report that a histone deacetylase, sirtuin 2 (SIRT2), functions as a “semi-deglycase” that removes lactic and glycolic acids from ε-N-l-lactyllysine and hydroxyacetyllysine residues, which are derived from MGO/GO-lysine adducts through the isomerization catalyzed by DJ-1. Overall, SIRT2 is a newly identified regulator for histone glycation, which can prevent the cytotoxicity of MGO and GO by eventually converting them into lactate and glycolate with the assistance of an enzymatically inactive DJ-1 mutant (i.e., DJ-1-C106A).