ABSTRACT
All human cells use O-GlcNAc protein modifications (O-linked N-acetylglucosamine) to rapidly adapt to changing nutrient and stress conditions through signaling, epigenetic, and proteostasis mechanisms. A key challenge for biologists in defining precise roles for specific O-GlcNAc sites is synthetic access to homogenous isoforms of O-GlcNAc proteins, a result of the non-genetically templated, transient, and heterogeneous nature of O-GlcNAc modifications. Toward a solution, this review details the state of the art of two strategies for O-GlcNAc protein modification: advances in "bottom-up" O-GlcNAc peptide synthesis and direct "top-down" installation of O-GlcNAc on full proteins. We also describe key applications of synthetic O-GlcNAc peptide and protein tools as therapeutics, biophysical structure-function studies, biomarkers, and as disease mechanistic probes to advance translational O-GlcNAc biology.
Subject(s)
Acetylglucosamine/metabolism , Peptides/metabolism , Proteins/metabolism , Acetylglucosamine/chemistry , Carbohydrate Conformation , Humans , Models, Molecular , Peptides/chemistry , Protein Processing, Post-Translational , Proteins/chemistryABSTRACT
Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.
Subject(s)
Click Chemistry/methods , Glycolipids/chemistry , Mycobacterium/pathogenicity , Proteins/metabolism , HumansABSTRACT
Trehalosamine (2-amino-2-deoxy-α,α-d-trehalose) is an aminoglycoside with antimicrobial activity against Mycobacterium tuberculosis, and it is also a versatile synthetic intermediate used to access imaging probes for mycobacteria. To overcome inefficient chemical synthesis approaches, we report a two-step chemoenzymatic synthesis of trehalosamine that features trehalose synthase (TreT)-catalyzed glycosylation as the key transformation. Soluble and recyclable immobilized forms of TreT were successfully employed. We demonstrate that chemoenzymatically synthesized trehalosamine can be elaborated to two complementary imaging probes, which label mycobacteria via distinct pathways.
