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Metabolically labeling proteins with glycans that enable attachment of an isotopically encoded tag allows for the identification of N- and O- glycopeptides and their glycan structures. Protein glycosylation is a heterogeneous post-translational modification (PTM) that plays an essential role in biological regulation. However, the diversity found in glycoproteins has undermined efforts to describe the intact glycoproteome via mass spectrometry (MS). We present IsoTaG, a mass-independent chemical glycoproteomics platform for characterization of intact, metabolically labeled glycopeptides at the whole-proteome scale. In IsoTaG, metabolic labeling of the glycoproteome is combined with (i) chemical enrichment and isotopic recoding of glycopeptides to select peptides for targeted glycoproteomics using directed MS and (ii) mass-independent assignment of intact glycopeptides. We structurally assigned 32 N-glycopeptides and over 500 intact and fully elaborated O-glycopeptides from 250 proteins across three human cancer cell lines and also discovered unexpected peptide sequence polymorphisms (pSPs). The IsoTaG platform is broadly applicable to the discovery of PTM sites that are amenable to chemical labeling, as well as previously unknown protein isoforms including pSPs.

Biological receptors distinguish between S - and R -enantiomers and these subtle differences in chirality can lead to vastly different protein affinities. Now, a proteomics approach has been developed that capitalizes on chirality to map enantiomeric probe pairs and provides a rapid and global view of protein ligandability within the cell.

The metabolite (−)-lomaiviticin A, which contains two diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups, inhibits the growth of cultured human cancer cells at nanomolar–picomolar concentrations; however, the mechanism responsible for the potent cytotoxicity of this natural product is not known. Here we report that (−)-lomaiviticin A nicks and cleaves plasmid DNA by a pathway that is independent of reactive oxygen species and iron, and that the potent cytotoxicity of (−)-lomaiviticin A arises from the induction of DNA double-strand breaks (dsbs). In a plasmid cleavage assay, the ratio of single-strand breaks (ssbs) to dsbs is 5.3 ± 0.6:1. Labelling studies suggest that this cleavage occurs via a radical pathway. The structurally related isolates (−)-lomaiviticin C and (−)-kinamycin C, which contain one diazofluorene, are demonstrated to be much less effective DNA cleavage agents, thereby providing an explanation for the enhanced cytotoxicity of (−)-lomaiviticin A compared to that of other members of this family. (−)-Lomaiviticin A inhibits the growth of cancer cells at nanomolar to picomolar concentrations; however, the basis for this potent cytotoxicity is not known. This natural product has now been shown to induce production of DNA double-strand breaks at nanomolar concentrations. Evidence demonstrates that strand cleavage proceeds via reactive carbon-centred free radical intermediates.

(–)-Lomaiviticin A (1) is a complex antiproliferative metabolite that inhibits the growth of many cultured cancer cell lines at low nanomolar–picomolar concentrations. (–)-Lomaiviticin A (1) possesses a C₂-symmetric structure that contains two unusual diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups. Nucleophilic activation of each diazofluorene within 1 produces vinyl radical intermediates that affect hydrogen atom abstraction from DNA, leading to the formation of DNA double-strand breaks (DSBs). Certain DNA DSB repair-deficient cell lines are sensitized toward 1, and 1 is under evaluation in preclinical models of these tumor types. However, the mode of binding of 1 to DNA had not been determined. Here we elucidate the structure of a 1:1 complex between 1 and the duplex d(GCTATAGC)₂ by NMR spectroscopy and computational modeling. Unexpectedly, we show that both diazofluorene residues of 1 penetrate the duplex. This binding disrupts base pairing leading to ejection of the central AT bases, while placing the proreactive centers of 1 in close proximity to each strand. DNA binding may also enhance the reactivity of 1 toward nucleophilic activation through steric compression and conformational restriction (an example of shape-dependent catalysis). This study provides a structural basis for the DNA cleavage activity of 1, will guide the design of synthetic DNA-activated DNA cleavage agents, and underscores the utility of natural products to reveal novel modes of small molecule–DNA association.

Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N - and 576 O -glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

To use this as a TRAM, the authors converted ecDHFR into a shuttle protein by attaching a nuclear export sequence (NFS) - a short amino-acid sequence that causes proteins to be exported from the nucleus to the cytoplasm - and added a fluorescent tag for detection. Because ecDHFR is a bacterial protein, Ng etai, next examined whether mammalian shuttles can be used to relocalize target proteins. The authors then used gene-editing techniques to construct cells that expressed one of three protein targets fused to FKBP12F36V, and showed that the TRAMs engage with the shuttle proteins to alter the localization of the tagged target proteins. [...]Ng et al. demonstrated that TRAMs can be used ex vivo to export an engineered neuroprotective protein from the nucleus of mouse neurons to the neurons' axon projections.

Many diseases arise from the misplacement of proteins in cells. A potential solution to this problem has been developed: small molecules that help displaced proteins catch a ride with other proteins to return to their proper location. Proteins induced to shuttle between the nucleus and cytoplasm.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide 1 , therapeutic agents used in the treatment of haematopoietic malignancies 2 – 4 and as ligands for targeted protein degradation 5 – 7 . 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. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN. C-terminal cyclic imides are physiological degrons that enable the ubiquitin E3 ligase adapter protein cereblon to target substrates for degradation.

O-linked N -acetylglucosamine ( O -GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O -GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O -GlcNAcase (OGA) as an O -GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O -GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O -GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O -GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O -GlcNAc via the selective removal of O -GlcNAc from individual proteins directly in cells. Fusion of a split form of the protein O -GlcNAcase with nanobodies enables the targeted removal of O -GlcNAc protein modifications, providing a tool for probing the functional roles of specific O -GlcNAc modifications in a cellular context.

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-β1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-β1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.