Current Opinion in Chemical Biology最新文献

Carbohydrate-active enzymes (CAZymes) are responsible for the biosynthesis, modification and degradation of all glycans in Nature. Advances in genomic and metagenomic methodologies, in conjunction with lower cost gene synthesis, have provided access to a steady stream of new CAZymes with both well-established and novel mechanisms. At the same time, increasing access to cryo-EM has resulted in exciting new structures, particularly of transmembrane glycosyltransferases of various sorts. This improved understanding has resulted in widespread progress in applications of CAZymes across diverse fields, including therapeutics, organ transplantation, foods, and biofuels. Herein, we highlight a few of the many important advances that have recently been made in the understanding and applications of CAZymes.

Heparan sulfate (HS) is a linear, sulfated and highly negatively-charged polysaccharide that plays important roles in many biological events. As a member of the glycosaminoglycan (GAG) family, HS is commonly found on mammalian cell surfaces and within the extracellular matrix. The structural complexities of natural HS polysaccharides have hampered the comprehension of their biological functions and structure–activity relationships (SARs). Although the sulfation patterns and backbone structures of HS can be major determinants of their biological activities, obtaining significant amounts of pure HS from natural sources for comprehensive SAR studies is challenging. Chemical and enzyme-based synthesis can aid in the production of structurally well-defined HS oligosaccharides. In this review, we discuss recent innovations enabling the syntheses of large libraries of HS and how these libraries can provide insights into the structural preferences of various HS binding proteins.

Sialic-acid-binding immunoglobulin-type lectins (Siglecs) are a family of cell-surface immunomodulatory receptors that recognize sialic-acid-containing glycans. The majority of Siglecs have an inhibitory motif in their intercellular domain and can regulate the cellular activation of immune cells. Importantly, the immunomodulatory role of Siglecs is regulated by engagement with distinct sialoglycan ligands. However, there are still many unanswered questions about the precise ligand(s) recognized by individual Siglec family members. New tools and approaches to study Siglec-ligand interactions are rapidly filling this knowledge gap. This review provides an overview of recent advances in discovering Siglec ligands as well as the development of approaches to modulate the function of Siglecs. In both aspects, chemical biology approaches are emphasized with a discussion on how these are complementing biochemical and genetic strategies.

Bacterial cells are covered by a complex carbohydrate coat of armor that allows bacteria to thrive in a range of environments. As a testament to the importance of bacterial glycans, effective and heavily utilized antibiotics including penicillin and vancomycin target and disrupt the bacterial glycocalyx. Despite their importance, the study of bacterial glycans lags far behind their eukaryotic counterparts. Bacterial cells use a large palette of monosaccharides to craft glycans, leading to molecules that are significantly more complex than eukaryotic glycans and that are refractory to study. Fortunately, chemical tools designed to probe bacterial glycans have yielded insights into these molecules, their structures, their biosynthesis, and their functions.

The development of a robust chemical toolbox to interrogate the activity of heparanase-1 (HPSE-1), an endo-β-d-glucuronidase and the only known enzyme that cleaves heparan sulfate (HS), has become critically important. The primary function of HPSE-1, cleaving HS side chains from heparan sulfate proteoglycans (HSPGs), regulates the integrity of the extracellular matrix (ECM) and the bioavailability of active, heparan sulfate-binding partners such as enzymes, growth factors, chemokines, and cytokines. HPSE-1 enzymatic activity is strictly regulated and has been found to play fundamental roles in pathophysiological processes. HPSE-1 is significantly overexpressed under various conditions including cancer, metastasis, angiogenesis, and inflammation, making HPSE-1 a promising therapeutic and diagnostic target. Chemical tools that can detect and image HPSE-1 activity in vitro and/or in vivo can help drive the discovery of novel and efficacious anti-HPSE-1 drugs, investigate the basic biology of HPSE-1, and help serve as a diagnostic tool in clinical applications. Here, we will give an overview of the common chemical tools to detect HPSE-1 activity and highlight the novel heparanase probes recently developed in our lab.

Fluorescence imaging plays a pivotal role in the study of biological processes, and cell-permeable fluorogenic dyes are crucial to visualize intracellular structures with high specificity. Polymethine dyes are vitally important fluorophores in single-molecule localization microscopy and in vivo imaging, but their use in live cells has been limited by high background fluorescence and low membrane permeability. In this review, we summarize recent advances in the development of fluorogenic polymethine dyes via intramolecular cyclization. Finally, we offer an outlook on the prospects of fluorogenic polymethine dyes for bioimaging.

New applications for biomolecules demand novel approaches for their synthesis and modification. Traditional methods for modifying proteins and cells using non-specific labeling chemistry are insufficiently precise to rigorously interrogate the mechanistic biological and physiological questions at the forefront of biomedical science. Site-specific catalytic modification of proteins promises to meet these challenges. Here, we describe recent applications of the enzyme sortase A in facilitating precise biomolecule labeling. We focus on describing new chemistries to broaden the scope of sortase-mediated labeling (sortagging), the development of new probes for imaging via enzymatic labeling, and the modulation of biological systems using probes and reactions mediated by sortase.

Super-resolution microscopy (SRM) has transformed our understanding of proteins' subcellular organization and revealed cellular details down to nanometers, far beyond conventional microscopy. While localization precision is independent of the number of fluorophores attached to a biomolecule, labeling density is a decisive factor for resolving complex biological structures. The average distance between adjacent fluorophores should be less than half the desired spatial resolution for optimal clarity. While this was not a major limitation in recent decades, the success of modern microscopy approaching molecular resolution down to the single-digit nanometer range will depend heavily on advancements in fluorescence labeling. This review highlights recent advances and challenges in labeling strategies for SRM, focusing on site-specific labeling technologies. These advancements are crucial for improving SRM precision and expanding our understanding of molecular interactions.

Glycoproteome analysis at a site-specific level and proteome scale stands out as a highly promising approach for gaining insights into the intricate roles of glycans in biological systems. Recent years have witnessed an upsurge in the development of innovative methodologies tailored for precisely this purpose. Breakthroughs in mass spectrometry–based glycoproteomic techniques, enabling the identification, quantification, and systematic exploration of site-specific glycans, have significantly enhanced our capacity to comprehensively and thoroughly characterize glycoproteins. In this short review, we delve into novel tools in advancing site-specific glycoproteomic analysis and summarize pertinent studies published in the past two years. Lastly, we discuss the ongoing challenges and outline future prospects in the field, considering both the analytical strategies of mass spectrometry and the tools employed for data interpretation.

Among molecular imaging modalities that can monitor enzyme activity in vivo, optical imaging provides sensitive, molecular-level information at low-cost using safe and non-ionizing wavelengths of light. Yet, obtaining quantifiable optical signals in vivo poses significant challenges. Benchmarking using ratiometric signals can overcome dependence on dosing, illumination variability, and pharmacokinetics to provide quantitative in vivo optical data. This review highlights recent advances using fluorescent probes that are processed by enzymes to induce photophysical changes that can be monitored by ratiometric imaging. These diverse strategies include caged fluorophores that change photophysical properties upon enzymatic cleavage, as well as multi-fluorophore systems that are triggered by enzymatic cleavage to alter optical outputs in one or more fluorescent channels. The strategies discussed here have great potential for further development as well as potential broad applications for targeting diverse enzymes important for a wide range of human diseases.