Showing 1 - 10 of 59 Items

• Restriction End Date: 2025-06-01

Date: 2020-01-01

Creator: Katharine Toll

Access: Access restricted to the Bowdoin Community

Date: 2009-04-15

Creator: Patsy S. Dickinson, Teerawat Wiwatpanit, Emily R. Gabranski, Rachel J. Ackerman, Jake S., Stevens, Christopher R. Cashman, Elizabeth A. Stemmler, Andrew E. Christie

Access: Open access

The allatostatins comprise three structurally distinct peptide families that regulate juvenile hormone production by the insect corpora allata. A-type family members contain the C-terminal motif -YXFGLamide and have been found in species from numerous arthropod taxa. Members of the B-type family exhibit a -WX6Wamide C-terminus and, like the A-type peptides, appear to be broadly conserved within the Arthropoda. By contrast, members of the C-type family, typified by the unblocked C-terminus -PISCF, a pyroglutamine blocked N-terminus, and a disulfide bridge between two internal Cys residues, have only been found in holometabolous insects, i.e. lepidopterans and dipterans. Here, using transcriptomics, we have identified SYWKQCAFNAVSCFamide (disulfide bridging predicted between the two Cys residues), a known honeybee and water flea C-typelike peptide, from the American lobster Homarus americanus (infraorder Astacidea). Using matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), a mass corresponding to that of SYWKQCAFNAVSCFamide was detected in the H. americanus brain, supporting the existence of this peptide and its theorized structure. Furthermore, SYWKQCAFNAVSCFamide was detected by MALDI-FTMS in neural tissues from five additional astacideans as well as 19 members of four other decapod infraorders (i.e. Achelata, Anomura, Brachyura and Thalassinidea), suggesting that it is a broadly conserved decapod peptide. In H. americanus, SYWKQCAFNAVSCFamide is capable of modulating the output of both the pyloric circuit of the stomatogastric nervous system and the heart. This is the first demonstration of bioactivity for this peptide in any species.

Date: 2013-09-01

Creator: Kanokwan Champasa, Scott A. Longwell, Aimee M. Eldridge, Elizabeth A. Stemmler, Danielle H., Dube

Access: Open access

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Date: 2018-07-01

Creator: Danielle H. Dube

Access: Open access

“Drug Discovery” is a 13-week lecture and laboratory-based course that was developed to introduce non-science majors to foundational chemistry and biochemistry concepts as they relate to the unifying theme of drug discovery. The first part of this course strives to build students' understanding of molecules, their properties, the differences that enable them to be separated from one another, and their abilities to bind to biological receptors and elicit physiological effects. After building students' molecular worldview, the course then focuses on four classes of drugs: antimicrobials, drugs that affect the mind, steroid-based drugs, and anti-cancer drugs. During each of these modules, an emphasis is placed on how understanding the basis of disease and molecular-level interactions empowers us to identify novel medicinal compounds. Periodic in class discussions based on articles pertinent to class topics ranging from the spread of antibiotic resistance, to the molecular basis of addiction, to rational drug design, are held to enable students to relate course material to pressing problems of national and daily concern. In addition to class time, weekly inquiry-based laboratories allow students to critically analyze data related to course concepts, and later in the semester give students an opportunity to design and implement their own experiments to screen for antimicrobial activity. This course provides students with an understanding of the importance of chemistry and biochemistry to human health while emphasizing the process, strategies, and challenges related to drug discovery. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:327–335, 2018.

Date: 2020-12-11

Creator: Karen D. Moulton, Adedunmola P. Adewale, Hallie A. Carol, Sage A. Mikami, Danielle H., Dube

Access: Open access

Bacterial cell surface glycans are quintessential drug targets due to their critical role in colonization of the host, pathogen survival, and immune evasion. The dense cell envelope glycocalyx contains distinctive monosaccharides that are stitched together into higher order glycans to yield exclusively bacterial structures that are critical for strain fitness and pathogenesis. However, the systematic study and inhibition of bacterial glycosylation enzymes remains challenging. Bacteria produce glycans containing rare sugars refractory to traditional glycan analysis, complicating the study of bacterial glycans and the identification of their biosynthesis machinery. To ease the study of bacterial glycans in the absence of detailed structural information, we used metabolic glycan labeling to detect changes in glycan biosynthesis. Here, we screened wild-type versus mutant strains of the gastric pathogen Helicobacter pylori, ultimately permitting the identification of genes involved in glycoprotein and lipopolysaccharide biosynthesis. Our findings provide the first evidence that H. pylori protein glycosylation proceeds via a lipid carrier-mediated pathway that overlaps with lipopolysaccharide biosynthesis. Protein glycosylation mutants displayed fitness defects consistent with those induced by small molecule glycosylation inhibitors. Broadly, our results suggest a facile approach to screen for bacterial glycosylation genes and gain insight into their biosynthesis and functional importance, even in the absence of glycan structural information.

Date: 2020-01-01

Creator: Michael Harris

Access: Open access

A significant market share of modern plastics is held by long-chain hydrocarbon polymers, such as polyethylene and polypropylene, properties of which can be dramatically changed by addition of linear α-olefins. Production of linear α-olefins involves the creation of many unwanted byproducts, representing significant quantities of both economic and ecological waste. While catalysts have been designed to selectively produce industrially useful olefins, these catalysts often encounter challenges such as synthesis of other unwanted byproducts, slow reaction times, and difficulty of synthesis. Based on one such prior catalyst, we report here synthetic work towards a cobalt catalyst with a constrained N-heterocyclic carbene supporting ligand predicted to allow for more favorable product distributions. Synthesis of two precursors to a sterically unhindered N-heterocyclic carbene, as well as development of a synthetic protocol for the coordination of N,N’- dimethylimidazolium-2-carboxylate to Cp*Co(ethene)2 was completed. Activation of the precatalyst and preliminary catalytic experiments were performed, though abbreviated research periods made complete analysis impossible. Finally, we report evidence of the formation of a novel cobalt-NHC dimer as a temperature controlled byproduct of the desired catalyst synthesis.

Date: 2016-12-16

Creator: Emily L. Clark, Madhu Emmadi, Katharine L. Krupp, Ananda R. Podilapu, Jennifer D., Helble, Suvarn S. Kulkarni, Danielle H. Dube

Access: Open access

Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.

Date: 2010-12-01

Creator: Danielle H. Dube, Bin Li, Ethan J. Greenblatt, Sadeieh Nimer, Amanda K., Raymond, Jennifer J. Kohler

Access: Open access

Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H), a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA) binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway. © 2010 Dube et al.

• Restriction End Date: 2028-06-01

Date: 2023-01-01

Creator: Kevin Jairre Fleshman

Access: Access restricted to the Bowdoin Community

Date: 2013-09-01

Creator: Kanokwan Champasa, Scott A. Longwell, Aimee M. Eldridge, Elizabeth A. Stemmler, Danielle H., Dube

Access: Open access

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.