Main Menu (Mobile)- Block

Main Menu - Block

janelia7_blocks-janelia7_secondary_menu | block
More in this page
janelia7_blocks-janelia7_fake_breadcrumb | block
Pedram Lab / Publications
custom | custom

Filter

facetapi-Q2b17qCsTdECvJIqZJgYMaGsr8vANl1n | block
facetapi-W9JlIB1X0bjs93n1Alu3wHJQTTgDCBGe | block

Associated Project Team

facetapi-PV5lg7xuz68EAY8eakJzrcmwtdGEnxR0 | block
facetapi-021SKYQnqXW6ODq5W5dPAFEDBaEJubhN | block
general_search_page-panel_pane_1 | views_panes

3 Publications

Showing 1-3 of 3 results
Your Criteria:
    08/17/20 | An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins
    Shon DJ, Malaker SA, Pedram K, Yang E, Krishnan V, Dorigo O, Bertozzi CR
    Proceedings of the National Academy of Sciences. Jan-09-2020;117(35):21299 - 21307. doi: 10.1073/pnas.2012196117

    Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.

     

    View Publication Page
    07/29/20 | Lysosome-targeting chimaeras for degradation of extracellular proteins
    Banik SM, Pedram K, Wisnovsky S, Ahn G, Riley NM, Bertozzi CR
    Nature. Jan-08-2021;584(7820):291 - 297. doi: 10.1038/s41586-020-2545-9

    The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein—for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins—the products of 40% of all protein-encoding genes7—are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified—but essential—component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.

     
     

    View Publication Page
    01/06/20 | Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells
    Schumann B, Malaker SA, Wisnovsky SP, Debets MF, Agbay AJ, Fernandez D, Wagner LJ, Lin L, Li Z, Choi J, Fox DM, Peh J, Gray MA, Pedram K, Kohler JJ, Mrksich M, Bertozzi CR
    Molecular Cell. Jan-06-2020;78(5):824 - 834.e15. doi: 10.1016/j.molcel.2020.03.030

    Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

     
     

    View Publication Page