Photocrosslinking and capture for the analysis of carbohydrate-dependent interactions.

Carbohydrates play crucial roles in biological systems, including by mediating cell and protein interactions. The complexity and transient nature of carbohydrate-dependent interactions pose significant challenges for their characterization, as traditional techniques often fail to capture these low-affinity binding events. This review highlights the increasing utility of photocrosslinkers in studying carbohydrate-mediated interactions.

The post-translational modification O-GlcNAc is a sensor and regulator of metabolism.

Cells must rapidly adapt to changes in nutrient conditions through responsive signalling cascades to maintain homeostasis. One of these adaptive pathways results in the post-translational modification of proteins by O-GlcNAc. O-GlcNAc modifies thousands of nuclear and cytoplasmic proteins in response to nutrient availability through the hexosamine biosynthetic pathway.

O-GlcNAc modification of HSP27 alters its protein interactions and promotes refolding of proteins through the BAG3/HSP70 co-chaperone.

Almost all types of cellular stress induce post-translational O-GlcNAc modifications of proteins, and this increase promotes cell survival. We previously demonstrated that O-GlcNAc on certain small heat shock proteins (sHSPs), including HSP27, directly increases their chaperone activity as one potential protective mechanism. Here, we furthered our use of synthetic proteins to prepare biotinylated sHSPs and show that O-GlcNAc modification of HSP27 also changes how it interacts within the sHSP system and the broader HSP network.

Potential for targeting small heat shock protein modifications.

Small heat shock proteins (sHSPs) play key roles in cellular stress and several human diseases. The direct effects of some post-translational modifications (PTMs) on certain sHSPs have been characterized, raising the possibility that small molecules could be used to modulate these modifications and indirectly up- or downregulate sHSP activity.

Backbone Modification Provides a Long-Acting Inverse Agonist of Pathogenic, Constitutively Active PTH1R Variants.

Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R-H223R mutant both in vitro and in vivo.

O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology.

Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer's and Parkinson's disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease.

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked -acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies.

Understanding and exploiting the roles of O-GlcNAc in neurodegenerative diseases.

O-GlcNAc is a common modification found on nuclear and cytoplasmic proteins. Determining the catalytic mechanism of the enzyme O-GlcNAcase (OGA), which removes O-GlcNAc from proteins, enabled the creation of potent and selective inhibitors of this regulatory enzyme. Such inhibitors have served as important tools in helping to uncover the cellular and organismal physiological roles of this modification.

Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of α-synuclein post-translational modifications.

The Parkinson's disease associated protein α-synuclein (αS) has been found to contain numerous post-translational modifications (PTMs), in both physiological and pathological states. One PTM site of particular interest is serine 87, which is subject to both O-linked β-N-acetylglucosamine (gS) modification and phosphorylation (pS), with αS-pS enriched in Parkinson's disease. An often-overlooked aspect of these PTMs is their effect on the membrane-binding properties of αS, which are important to its role in regulating neurotransmitter release.

O-GlcNAc Modification Alters the Chaperone Activity of HSP27 Charcot-Marie-Tooth Type 2 (CMT2) Variants in a Mutation-Selective Fashion.

Increased O-GlcNAc is a common feature of cellular stress, and the upregulation of this dynamic modification is associated with improved survival under these conditions. Likewise, the heat shock proteins are also increased under stress and prevent protein misfolding and aggregation. We previously linked these two phenomena by demonstrating that O-GlcNAc directly increases the chaperone of certain small heat shock proteins, including HSP27.

A photoaffinity glycan-labeling approach to investigate immunoglobulin glycan-binding partners.

Glycans play a pivotal role in biology. However, because of the low-affinity of glycan-protein interactions, many interaction pairs remain unknown. Two important glycoproteins involved in B-cell biology are the B-cell receptor and its secreted counterpart, antibodies.

HSP27 Inhibitory Activity against Caspase-3 Cleavage and Activation by Caspase-9 Is Enhanced by Chaperone O-GlcNAc Modification .

One of the O-GlcNAc modifications is the protection of cells against a variety of stressors that result in cell death. Previous experiments have focused on the overall ability of O-GlcNAc to prevent protein aggregation under stress as well as its ability to affect stress-response signaling pathways. Less attention has been paid to the potential role for O-GlcNAc in the direct inhibition of a major cell-death pathway, apoptosis.

O-GlcNAc modification forces the formation of an α-Synuclein amyloid-strain with notably diminished seeding activity and pathology.

The process of amyloid fibril formation remains one of the primary targets for developing diagnostics and treatments for several neurodegenerative diseases (NDDs). Amyloid-forming proteins such α-Synuclein and Tau, which are implicated in the pathogenesis of Alzheimer's and Parkinson's disease, can form different types of fibril structure, or strains, that exhibit distinct structures, toxic properties, seeding activities, and pathology spreading patterns in the brain. Therefore, understanding the molecular and structural determinants contributing to the formation of different amyloid strains or their distinct features could open new avenues for developing disease-specific diagnostics and therapies.

The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide, therapeutic agents used in the treatment of haematopoietic malignancies and as ligands for targeted protein degradation. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN.

Synthesis of O-GlcNAcylated small heat shock proteins.

A protein's structure and function often depend not only on its primary sequence, but also the presence or absence of any number of non-coded posttranslational modifications. Complicating their study is the fact that the physiological consequences of these modifications are context-, protein-, and site-dependent, and there exist no purely biological techniques to unambiguously study their effects. To this end, protein semisynthesis has become an invaluable chemical biology tool to specifically install non-coded or non-native moieties onto proteins in vitro using synthetic and/or recombinant polypeptides.

Exo-Enzymatic Addition of Diazirine-Modified Sialic Acid to Cell Surfaces Enables Photocrosslinking of Glycoproteins.

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions ; however, use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types.

Generation of Potent and Stable GLP-1 Analogues Via "Serine Ligation".

Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood.

A 2D and 3D cell culture protocol to study O-GlcNAc in sphingosine-1-phosphate mediated fibroblast contraction.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification involved in a wide range of signaling pathways, but its specific role in regulating biological processes remains unclear. This protocol describes approaches to understand O-GlcNAc's role in fibroblast contraction. Specifically, cellular O-GlcNAc levels are controlled through treatment of fibroblasts with inhibitors in both 2D and 3D cultures.

Methods for Studying Site-Specific O-GlcNAc Modifications: Successes, Limitations, and Important Future Goals.

O-GlcNAcylation is a dynamic post-translational modification which affects myriad proteins, cellular functions, and disease states. Its presence or absence modulates protein function via differential protein- and site-specific mechanisms, necessitating innovative techniques to probe the modification in highly selective manners. To this end, a variety of biological and chemical methods have been developed to study specific O-GlcNAc modification events both and , each with their own respective strengths and shortcomings.

Hyaluronic acid fuels pancreatic cancer cell growth.

Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in human PDA cell lines, which completely blocked proliferation in vitro and led to cell death.

Multifaceted Regulation of Akt by Diverse C-Terminal Post-translational Modifications.

Akt is a Ser/Thr protein kinase that regulates cell growth and metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2.