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Carolyn R Bertozzi

Publications and source records attributed to Carolyn R Bertozzi.

At least 19 recordsLinked to original sources
PubMed2022

Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

Animals↗
PubMed2006

A comparative study of bioorthogonal reactions with azides.

Detection of metabolites and post-translational modifications can be achieved using the azide as a bioorthogonal chemical reporter. Once introduced into target biomolecules, either metabolically or through chemical modification, the azide can be tagged with probes using one of three highly selective reactions: the Staudinger ligation, the Cu(I)-catalyzed azide-alkyne cycloaddition, or the strain-promoted [3 + 2] cycloaddition. Here, we compared these chemistries in the context of various biological applications, including labeling of biomolecules in complex lysates and on live cell surfaces. The Cu(I)-catalyzed reaction was found to be most efficient for detecting azides in protein samples but was not compatible with live cells due to the toxicity of the reagents. Both the Staudinger ligation and the strain-promoted [3 + 2] cycloaddition using optimized cyclooctynes were effective for tagging azides on live cells. The best reagent for this application was dependent upon the specific structure of the azide. These results provide a guide for biologists in choosing a suitable ligation chemistry.

Azides↗
PubMed2006

Synthetic glycobiology: Exploits in the Golgi compartment.

The challenge of engineering glycosylation has been confronted by synthetic chemists, biochemists and cell biologists, each with the primary goal of optimizing glycoconjugates for therapeutic applications. In nature, glycans are constructed by glycosyltransferases that are organized in an assembly line in the endoplasmic reticulum and Golgi compartment. Recent insights into the domain architecture, localization and regulation of glycosyltransferases have provided a platform for engineering their position within the secretory pathway and access to substrates. Using this knowledge, glycosyltransferase assembly lines have been redesigned for the production of specific glycan structures using protein engineering and chemical approaches. These efforts epitomize the emerging field of 'synthetic glycobiology'.

Glycoproteins↗
PubMed2006

Noncovalent complexes of APS reductase from M. tuberculosis: delineating a mechanistic model using ESI-FTICR MS.

ESI-FTICR MS was utilized to characterize a 4Fe-4S containing protein Mycobacterium tuberculosis APS reductase. This enzyme catalyzes the reduction of APS to sulfite and AMP with reducing equivalents from the protein cofactor, thioredoxin. Under nondenaturing conditions, a distribution of the apoprotein, a 2Fe-2S intermediate, and the 4Fe-4S holoprotein were observed. Accurate mass measurements indicated an oxidation state of +2 for the 4Fe-4S cluster, with no disulfide bond in the holoenzyme. Gas-phase stability of the 4Fe-4S cluster was investigated using both in-source and collision induced dissociation, which provided information regarding the relative gas-phase binding strength of iron towards protein ligands and inorganic sulfides. Noncovalent complexes of the holoprotein with several ligands, including APS, thioredoxin, and AMP, were also investigated. Calculated values of dissociation constants for the complexes indicate that AMP binds with a higher affinity to the enzyme intermediate than to the free enzyme. The implications of the binary and ternary complexes observed by gas-phase noncovalent interactions in the mechanism of APS reduction are discussed.

Adenosine Monophosphate↗
PubMed2006

A chemical reporter strategy to probe glycoprotein fucosylation.

Fucosylated glycoproteins are involved in many cell-cell recognition events and are markers of embryonic and malignant tissue. Here we report a method for rapid profiling of fucosylated glycoproteins from human cells using 6-azido fucose as a metabolic label.

Azides↗
PubMed2006

Using phage display to select antibodies recognizing post-translational modifications independently of sequence context.

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.

Amino Acid Sequence↗
PubMed2006

Chemical technologies for probing glycans.

Glycans are central to many biological processes, but efforts to define their functions at the molecular level have been frustrated by a lack of suitable technologies. Here we highlight chemical tools that are beginning to address this need.

Carbohydrate Conformation↗
PubMed2006

Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5'-phosphosulfate reductase.

APS reductase catalyzes the first committed step of reductive sulfate assimilation in pathogenic bacteria, including Mycobacterium tuberculosis, and is a promising target for drug development. We report the 2.7 A resolution crystal structure of Pseudomonas aeruginosa APS reductase in the thiosulfonate intermediate form of the catalytic cycle and with substrate bound. The structure, high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis, establish that the two chemically discrete steps of the overall reaction take place at distinct sites on the enzyme, mediated via conformational flexibility of the C-terminal 18 residues. The results address the mechanism by which sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P. aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for catalysis. The structure reveals an unusual mode of cluster coordination by tandem cysteine residues and suggests how this arrangement might facilitate conformational change and cluster interaction with the substrate. Assimilatory 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductases are evolutionarily related, homologous enzymes that catalyze the same overall reaction, but do so in the absence of an [Fe-S] cluster. The APS reductase structure reveals adaptive use of a phosphate-binding loop for recognition of the APS O3' hydroxyl group, or the PAPS 3'-phosphate group.

Amino Acid Sequence↗
PubMed2006

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids.

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 --> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

Amino Acids↗
PubMed2006

Interfacing carbon nanotubes with living cells.

We developed a polymer coating for carbon nanotubes (CNTs) that mimics the mucin glycoprotein coating of mammalian cells. CNTs coated with these mucin mimic polymers have two novel properties: they can bind to carbohydrate receptors, providing a means for biomimetic interactions with cell surfaces, and, importantly, they are rendered nontoxic to cells.

Animals↗
PubMed2006

Probing mucin-type O-linked glycosylation in living animals.

Changes in O-linked protein glycosylation are known to correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we report a technique for rapid profiling of O-linked glycoproteins in living animals by metabolic labeling with N-azidoacetylgalactosamine (GalNAz) followed by Staudinger ligation with phosphine probes. After injection of mice with a peracetylated form of GalNAz, azide-labeled glycoproteins were observed in a variety of tissues, including liver, kidney, and heart, in serum, and on isolated splenocytes. B cell glycoproteins were robustly labeled with GalNAz but T cell glycoproteins were not, suggesting fundamental differences in glycosylation machinery or metabolism. Furthermore, GalNAz-labeled B cells could be selectively targeted with a phosphine probe by Staudinger ligation within the living animal. Metabolic labeling with GalNAz followed by Staudinger ligation provides a means for proteomic analysis of this posttranslational modification and for identifying O-linked glycoprotein fingerprints associated with disease.

Acetylgalactosamine↗
PubMed2006

A sulfated metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis.

Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope of M. tuberculosis and negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate and a previously uncharacterized sulfotransferase, stf3. These findings extend the known functions of sulfated molecules as general modulators of cell-cell interactions to include those between a bacterium and a human host.

Animals↗
PubMed2006

Molecular basis for G protein control of the prokaryotic ATP sulfurylase.

Sulfate assimilation is a critical component of both primary and secondary metabolism. An essential step in this pathway is the activation of sulfate through adenylation by the enzyme ATP sulfurylase (ATPS), forming adenosine 5'-phosphosulfate (APS). Proteobacterial ATPS overcomes this energetically unfavorable reaction by associating with a regulatory G protein, coupling the energy of GTP hydrolysis to APS formation. To discover the molecular basis of this unusual role for a G protein, we biochemically characterized and solved the X-ray crystal structure of a complex between Pseudomonas syringae ATPS (CysD) and its associated regulatory G protein (CysN). The structure of CysN*D shows the two proteins in tight association; however, the nucleotides bound to each subunit are spatially segregated. We provide evidence that conserved switch motifs in the G domain of CysN allosterically mediate interactions between the nucleotide binding sites. This structure suggests a molecular mechanism by which conserved G domain architecture is used to energetically link GTP turnover to the production of an essential metabolite.

Amino Acid Sequence↗
PubMed2006

An alpha-formylglycine building block for fmoc-based solid-phase peptide synthesis.

[reaction: see text] Nearly all known sulfatases share a common active site modification that is required for their activity: conversion of cysteine to alpha-formylglycine. We report the synthesis of an alpha-formylglycine building block suitable for Fmoc-based solid-phase peptide synthesis. The building block was incorporated into a synthetic peptide derived from the active site of a Mycobacterium tuberculosis sulfatase.

Alanine↗
PubMed2006

Sulfate metabolism in mycobacteria.

Pathogenic bacteria have developed numerous mechanisms to survive inside a hostile host environment. The human pathogen Mycobacterium tuberculosis (M. tb) is thought to control the human immune response with diverse biomolecules, including a variety of exotic lipids. One prevalent M. tb-specific sulfated metabolite, termed sulfolipid-1 (SL-1), has been correlated with virulence though its specific biological function is not known. Recent advances in our understanding of SL-1 biosynthesis will help elucidate the role of this curious metabolite in M. tb infection. Furthermore, the study of SL-1 has led to questions regarding the significance of sulfation in mycobacteria. Examples of sulfated metabolites as mediators of interactions between bacteria and plants suggest that sulfation is a key modulator of extracellular signaling between prokaryotes and eukaryotes. The discovery of novel sulfated metabolites in M. tb and related mycobacteria strengthens this hypothesis. Finally, mechanistic and structural data from sulfate-assimilation enzymes have revealed how M. tb controls the flux of sulfate in the cell. Mutants with defects in sulfate assimilation indicate that the fate of sulfur in M. tb is a critical survival determinant for the bacteria during infection and suggest novel targets for tuberculosis drug therapy.

Carbohydrate Sequence↗
PubMed2006

A role for sulfation-desulfation in the uptake of bisphenol a into breast tumor cells.

Bisphenol A (BPA) is a widely used plasticizer whose estrogenic properties may impact hormone-responsive disorders and fetal development. In vivo, BPA appears to have greater activity than is suggested by its estrogen receptor (ER) binding affinity. This may be a result of BPA sulfation/desulfation providing a pathway for selective uptake into hormone-responsive cells. BPA is a substrate for estrogen sulfotransferase, and bisphenol A sulfate (BPAS) and disulfate are substrates for estrone sulfatase. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. Treatment of MCF-7 cells with BPAS leads to desulfation and uptake of BPA. No BPAS is found inside the cells. These findings suggest a mechanism for the selective uptake of BPA into cells expressing estrone sulfatase. Therefore, sulfation may increase the estrogenic potential of xenobiotics.

Benzhydryl Compounds↗
PubMed2006

Metabolic labeling of glycans with azido sugars for visualization and glycoproteomics.

The staggering complexity of glycans renders their analysis extraordinarily difficult, particularly in living systems. A recently developed technology, termed metabolic oligosaccharide engineering, enables glycan labeling with probes for visualization in cells and living animals, and enrichment of specific glycoconjugate types for proteomic analysis. This technology involves metabolic labeling of glycans with a specifically reactive, abiotic functional group, the azide. Azido sugars are fed to cells and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged, either ex vivo or in vivo, using one of two azide-specific chemistries: the Staudinger ligation, or the strain-promoted [3+2] cycloaddition. These reactions can be used to tag glycans with imaging probes or epitope tags, thus enabling the visualization or enrichment of glycoconjugates. Applications to noninvasive imaging and glycoproteomic analyses are discussed.

Animals↗