1997 — 2002 |
Bertozzi, Carolyn R |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Synthesis of Glycoconjugates For Study of Carbohydrate Processing Enzymes @ University of California San Francisco
Cell surface oligosaccharides play essential roles in the cell-cell recognition events associated with bacterial and viral infection, tumor cell metastasis and leukocyte adhesion at sites of inflammation. These important biological functions obviate importance of studying the enzymes that modulate oligosaccharide structures. We have developed a multidisciplinary program aimed at the study of two oligosaccharide-processing enzymes: carbohydrate sulfotransferases and proximal glycanases. The sulfotransferases have been implicated in the regulation of leukocyte adhesion to endothelium at sites of inflammation, but have not yet been identified at the molecular level. The proximal glycanases liberate N-linked oligosaccharides fromglycoproteins, yet their biological functions remain undefined. Our goals are to identify the mechanisms and biological functions of these enzymes using organic chemistry as a tool. We are synthesizing inhibitors of both classes of enzymes, which will be used to observe the effects of enzyme inhibition on the cellular expression of glycoconjugates. In the case of the sulfotransferases, the inhibitors we synthesize may have anti-inflammatory activity and serve as leads for a new generation of anti-inflammatory drugs. Our synthetic targets comprise complex glycoconjugates related to cell-associated glycoproteins. These molecules are among the most difficult to synthesize and characterize. Mass spectrometry willbe an essential tool for the characterization of our synthetic compounds due to their high molecular weight, their structural complexity and their chemical liability. The UCSF Mass Spectrometry Facility is the only facility in the area with state-of-the-art instrumentation and a staff with expertise in glycoconjugate characterization, both of which will be necessary to these projects.
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0.976 |
1998 — 2002 |
Bertozzi, Carolyn |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Engineering Glycoforms On Proteins and Cells by Chemoselective Ligation @ University of California-Berkeley
The focus of this research will be the development of strategies for engineering homogeneous glycoforms on proteins and cells using the principle of chemoselective ligation. For gycoprotein engineering, a three-step process is envisioned. First, recombinant expression of O-linked glycoproteins in the presence of an oligosaccharide elongation inhibitor yields truncated glycoproteins with single GalNAc residues at the O-linked sites. Second, an aldehyde group is introduced onto truncated glycoproteins using galactose oxidase. Third, synthetic oligosaccharides functionalized with complementary reactive groups are ligated onto the truncated scaffold, affording glycoproteins with rationally designed, homogeneous glycoforms. The focus of the educational activities involves course and web site development. With this Faculty Early Development (CAREER) award, the Organic and Macromolecular Chemistry Program is supporting the research and educational activities of Dr. Carolyn Bertozzi of the Department of Chemistry at the University of California, Berkeley. Professor Bertozzi will focus her research efforts on developing strategies for engineering homogeneous glycoforms on proteins and cells using the techniques of synthetic chemistry, recombinant expression and metabolic engineering. This approach will be used to investigate glycoproteins involved in leukocyte activation and cell-cell interactions essential to the inflammatory response. Course and curriculum development will be stressed in the educational efforts supported by the award.
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0.915 |
1999 — 2002 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Carbohydrate Sulfotransferases @ University of California Berkeley
L-Selectin mediates the initial attachment of blood-borne lymphocytes to endothelial cells during lymphocyte homing to secondary lymphoid organs. In addition, L-selectin participates in the similar process of leukocyte adhesion and extravasation at sites of chronic inflammation. The L-selectin ligands are mucin-like glycoproteins adorned with the unusual sulfated carbohydrate epitopes, 6-sulfo sialyl Lewis x and 6'-sulfo sialyl Lewis x. Sulfation of these epitopes on the N-acetylglucosamine (GlcNAc) and galactose (Gal) residues, respectively, converts inactive glycoforms to high-avidity L-selectin ligands. Furthermore, sulfation of these ligands is restricted in the vasculature to sites of sustained lymphocyte recruitment such as peripheral lymph nodes and chronically inflamed tissues. Therefore, the GlcNAc-6- and Gal-6-sulfotransferases that install the sulfate esters may be key modulators of lymphocyte recruitment to lymph nodes and chronically inflamed tissues, and potential targets for anti-inflammatory therapy. Through a collaborative effort with two other laboratories, three human carbohydrate sulfotransferase clones that may be involved in L- selectin ligand biosynthesis have been identified. The broad objectives of this proposal are the biochemical characterization of these enzymes and the design and synthesis of selective inhibitors. The first Aim of the proposed research is to develop a modular approach to inhibitor design based on the conjugation of two independently-derived compounds, one optimized to bind the carbohydrate binding site and the other optimized to bind the 3'-phosphoadenosine-S'-phosphosulfate (PAPS) binding site. In order to establish a framework for the design of carbohydrate binding site inhibitors, the substrate specificity of each enzyme and the structural features required for recognition will be defined. Preferred carbohydrate substrates will then serve as leads for the design of glycomimetic inhibitors. In parallel, PAPS binding site inhibitors will be identified through the synthesis and screening of aromatic heterocycle libraries. The pharmacophores derived from these parallel efforts will be tethered to produce potent and selective sulfotransferase inhibitors. The second Aim of the proposal is to define the features of carbohydrate sulfotransferase sequences that relate to function by site-directed and domain swapping mutagenesis. The results will contribute to a model for predicting sulfotransferase specificity based on genomic sequence analysis.
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1 |
1999 — 2005 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Metabolic Engineering of Reactive Glycoconjugates @ University of California Berkeley
Aberrant glycosylation patterns are a hallmark of the tumor phenotype. While highly variable in structure, many tumor-associated oligosaccharides share one important feature: they contain sialic acid residues. Indeed, the overexpression of sialic acid is highly correlated with the malignant phenotype in gastric, colon, pancreatic, liver, lung, prostate and breast cancers, as well as several types of leukemia. Consequently, new strategies for targeting cells on the basis of differential sialic acid expression levels may have widespread utility in the treatment and diagnosis of cancer. This proposal describes a chemicAL approach to the selective targeting of highly sialylated cells with therapeutic and diagnostic agents. The strategy is predicated on the remarkable tolerance of the sialic acid biosynthetic machinery for modified substrates. We have shown that a uniquely reactive functional group, the ketone, can be delivered to cell surface sialic acids by feeding the cells the unnatural metabolic precursor N-levulinoyl mannosamine (ManLev). The ketone provides the ideal mechanism for targeting cells in their native environment because it is chemically orthogonal to all other cell surface components, yet will react selectively with hydroxylamines and hydrazides under physiological conditions. Thus, in the context of the biological milieu, the ketone introduces a unique functional group which permits covalent targeting with molecules bearing complementary functionality. The objective of the proposed research is to explore the potential application of unnatural sialic acid biosynthesis to the selective delivery of therapeutic and diagnostic agents to human tumor cells. A positive correlation between sialic acid expression level and ManLev metabolism is critical for the proposed application, and will be established using tumor cell lines selected for defined sialic acid levels. Next, hydroxylamine-conjugated toxins, imaging reagents and small molecular antigens will be synthesized, and their selectivity for cells rich in sialic acid will be evaluated. As a prelude to future in vivo targeting studies, unnatural sialic acid biosynthesis in laboratory animals will be investigated. Finally, the biosynthetic pathway for cell surface fucosides will be explored as an alternative vehicle for the cell surface delivery of unique chemical targets. This project is the first phase of a long-term program focusing on applications of unnatural oligosaccharide biosynthesis.
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1 |
2000 — 2002 |
Majumdar, Arunava (co-PI) [⬀] Keaveny, Tony (co-PI) [⬀] Bertozzi, Carolyn Pruitt, Lisa [⬀] Lee, Luke (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mri: Instrumentation Acquisition For the Development of a Wet Facility For Nano-Bioengineering @ University of California-Berkeley
0079243 Pruitt The objective of this NSF MRI Award is to develop a Wet Facility for Nano-Bioengineering that employs nanoengineering for the characterization of cellular to molecular-level biological systems. The facility will be unique in that it will provide laboratory equipment that enables characterization of living biological systems in their physiological environments. The research focus of the facility is based on the merger of nanotechnology and bioengineering which are both emerging fields with promise of great scientific breakthroughs in the area of tissue engineering, cell mechanics, biological processes, and imaging. The facilities will provide the resources for creating a biomimetic framework necessary for novel synthesis of engineered materials or devices. The impact of this facility will span the disciplines of mechanical engineering, electrical engineering, bioengineering, and materials science, as well as the general areas of chemistry, biology and medicine. The acquired equipment for this facility will provide the necessary tools for nanoscale characterization of cells, proteins, interfaces, and BioMEMs structures. The approach to developing this facility is to acquire several large pieces of complementary equipment that would be difficult for any single faculty member to acquire alone because of the large initial cost and associated long-term maintenance. Thus, they propose to purchase the following systems using funds from this NSF initiative: an atomic force microscope and nanoindenter with fluid chambers for cell and tissue manipulation studies, a confocal microscope for cell imaging and manipulation, an infrared spectrometer and high pressure liquid chromatography system for characterization of protein binding, and an environmental scanning electron microcope to study substructure morphology of biomaterials.
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0.915 |
2002 — 2010 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mycobacterial Sulfation Pathways @ University of California Berkeley
DESCRIPTION (provided by applicant): Sulfated are central mediators of extracellular traffic and cell-cell communication in humans. The enzymes that install and remove sulfate esters, sulfotransferases and sulfatases, respectively, are now appreciated as major contributors to human health and disease. By contrast, the roles of sulfated sugars and the associated enzymes in bacteria remain relatively unexplored. Mycobacterial pathogens have been declared a global emergency by the World Health Organization, particularly in regard to the deadly synergy of Mycobacterium tuberculosis with AIDS, but also due to the emergence of drug-resistant strains. In Mycobacteria, several sulfated molecules have been identified. These include a sulfated glycolipid, SL- 1 that has been implicated as a virulence factor for M. tuberculosis. Another sulfated carbohydrate, part of a glycopeptidolipid, has been detected in a drug resistant strain of M. aviurn isolated from an AIDS patient. Recently, the complete genome sequences of M. tuberculosis, M. avium, and M. smegmatis have become available, enabling the search for genes that participate in sulfation pathways. We have identified an extensive family of sulfotransferases and sulfatases from the completed genomes of these three Mycobacteria. The enzymes may be critical determinants of Mycobacterial virulence and potential targets for anti-Mycobacterial therapy. Through a collaborative effort, our laboratories (Prof. Carolyn Bertozzi and Prof. Lee Riley, UC Berkeley) have initiated a program aimed at the genetic and biochemical characterization, and small molecule inhibition of the sulfotransferases from M. tuberculosis and M. smegmatis. In addition, we have identified several sulfatases that have considerable similarity to mammalian carbohydrate sulfatases, suggesting a role for these enzymes in host/pathogen interactions. Finally, in order to define the sulfur incorporation pathways of Mycobacteria, we have begun the characterization of enzymes involved in the early stages of cysteine biosynthesis. The aims of this proposal are threefold: (1) to determine the functions of the carbohydrate sulfotransferases in M. tuberculosis and M. smegmatis using genetic, biochemical and chemical approaches; (2) to investigate the involvement of bacterial sulfatases in host/pathogen interactions; and (3) to define the sulfur assimilation pathway of M. tuberculosis.
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1 |
2002 — 2010 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Studies of O-Linked Glycosylation @ University of California Berkeley
DESCRIPTION (provided by applicant): The objectives of this project are: (1) the development of chemical tools for fundamental studies of O-linked glycosylation, and (2) the identification of glycoprotein cancer biomarkers. Mucin-type O-linked glycosylation is a posttranslational modification of membrane and secreted proteins in higher eukaryotes. Its functions are poorly understood, but altered mucin-type O-linked glycosylation has been correlated with cancer growth and metastasis. Chemical tools are needed both for perturbing and detecting O-linked glycosylation on proteins and cells. The first major goal of this renewal application is to develop small molecule inhibitors of mucin-type O-linked glycosylation by targeting the polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) and UDP-GlcNAc/GalNAc C4-epimerase (GALE), enzymes that are required for O-glycan biosynthesis. The inhibitors will be used to probe the importance of O-linked glycans in tumor growth and metastasis. The second goal is to develop a chemical approach for rapid profiling of changes in mucin-type O-linked glycosylation associated with cancer. The approach involves metabolic labeling of O-linked glycoproteins within living animals using an azido analog of N-acetylgalactosamine (termed GalNAz). The labeled glycoproteins from serum and tissue samples will be chemically tagged with phosphine probes via Staudinger ligation, permitting their detection and identification using proteomic methods. Comparison of labeled species from normal and tumor-bearing mice may reveal new serum biomarkers of disease. The final goal is to develop an analogous method for detection of protein O-fucosylation, a recently discovered form of O-linked glycosylation with mysterious functions. Cell-surface sugars are known to participate in many normal and disease processes, but have not yet been exploited as targets for drugs or clinical diagnostics. This research will advance our understanding of the roles cell-surface sugars play in tumor growth and metastasis. The chemical tools developed in this project may produce a new generation of anti-cancer drugs and new clinical tests for early diagnosis.
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1 |
2004 — 2008 |
Bertozzi, Carolyn R |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Ucb Chemistry-Biology Interface Training Program @ University of California Berkeley
The aim of this Chemistry-Biology Interface predoctoral training program at UC Berkeley is to provide graduate Ph.D. students with a unique depth of training in the application of chemical principles and techniques to the investigation and modulation of biological systems. This program [Chemical Biology Graduate Program (CBGP)], has now been operating for three years with a present total of 14 students and 24 anticipated by August 2003. This program was created in response to a recognized need expressed by the graduate students and faculties of the Chemistry, Molecular and Cell Biology, Bioengineering, and Chemical Engineering Departments at UC Berkeley. The importance of chemical approaches in biological research is becoming ever more appreciated, and the interface between the disciplines holds enormous opportunities for advancing biomedical science. However, the physical and cultural boundaries that separate the disciplines in most universities have created a divide between chemical and biological research, and between the corresponding graduate training programs. The goal of the Chemical Biology Graduate Program (CBGP) to provide the structure and resources for a rigorous training experience in the principles and techniques of both chemistry and biology. This will encourage further integration of the fields and prepare students for a future in research at the interface. The program offers a curriculum of graduate courses in both disciplines, and opportunities for laboratory training in numerous techniques. The goals of the program will be accomplished through: 1) lab rotations for first-year graduate students, chosen from at least 34 participating laboratories, 2) a core of didactic courses, with specific additional courses to be selected based on the student's individual interests, 3) numerous seminar programs already in place in the participating departments with speakers whose research spans chemistry and biology, 4) an annual retreat to foster interactions among its students and faculty, 5) poster sessions at the end of each rotation period during which first-year graduate students present their research results, and 6) a Ph.D. dissertation at the chemistry-biology interface. We request support for 10 students each year for their first year rotation period. Students will be recruited with backgrounds in chemistry, biology, biochemistry and chemical engineering. Minority recruitment will be aggressively accomplished through the Berkeley Edge Program and other related campus programs.
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1 |
2004 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
"Investigations of Golgi Enzymes" @ University of California Berkeley
[unreadable] DESCRIPTION (provided by applicant): Cell surface glycans are major determinants of cell-cell and cell-matrix interactions. Their structures reflect the expression of glycosyltransferases and sulfotransferases that act in an assembly line within the Golgi compartment. The broad objective of this project is to develop chemical tools for studying Golgi enzymes and the functions of the glycans they produce on cells. The last granting period focused on a newly discovered family of GlcNAc-6-sulfotransferases. The goals were to (1) develop small molecule inhibitors of the sulfotransferases as tools for biological studies and leads for drug discovery, (2) identify residues involved in substrate binding and catalysis, and (3) determine the preferred cellular substrates as a step toward elucidating biological function. An exciting discovery from this work was that the substrate preference of each enzyme in vivo is governed largely by its distribution among the Golgi cisternae. Thus, Golgi localization was identified as a major determinant of biological function. The Specific Aims of the next granting period build from this discovery. The major objective of the next granting period is to develop an approach for modulating Golgi enzyme activity with small molecules that target their common requirement of Golgi localization. The proposed strategy is based on the chemical dimerizer-induced assembly of the enzymes' modular catalytic and localization domains. The approach was validated with fucosyltransferase 7 (FucT7) and the GlcNAc-6- sulfotransferases GST-2 and GST-3, using the rapamycin/FRB/FKBP system for inducible domain assembly. The Specific Aims of the next granting period expand upon this discovery in three directions. The first Aim is to investigate the FucT7 system in more detail in order to define those parameters that affect the cellular activity of the reconstituted domains. The goal is to optimize the current FucT7 system for application to studies of tumor cell metastasis and for use in transgenic mice. The second Aim is to apply the approach to other Golgi enzymes, chosen for their diversity of substrates and functions. The third and final Aim is to determine whether chemical dimerizers can be used to modulate associations between two glycosyltransferases. Such associations are thought to be important for the efficiency of glycolipid biosynthetic pathways. The ability to modulate glycosyltransferase associations will provide means to control glycolipid expression on cells. [unreadable] [unreadable]
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1 |
2005 — 2008 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Metabolic Oligosaccharide Engineering @ University of California Berkeley
DESCRIPTION (provided by applicant): This is a competing renewal application of RO1 GM58867 ("Metabolic Oligosaccharide Engineering"). The long-term objective of this project is to develop metabolic oligosaccharide engineering as a tool for fundamental studies of glycan function, particularly with respect to human disease. In the last granting period we discovered that unnatural sugars bearing bioorthogonal functional groups, such as ketones and azides, are metabolized by cells and incorporated into cellular glycans. When displayed on the cell surface, the functionalized sugars can be modified by covalent reactions with exogenous reagents. We employed this new technique in numerous studies of glycobiology, including cell surface targeting as a function of glycan expression pattern. An integral component of the project was the development of bioorthogonal reactions that can be performed on modified glycans expressed on living cells. We developed a reaction of azides and phosphines, termed the Staudinger ligation, with such high selectivity that it can be executed on cultured cells without detriment to their physiology. We further demonstrated that the Staudinger ligation proceeds in living animals, permitting the covalent targeting of cell-surface azidosugars with phosphine probes in vivo. The first major objective of the next granting period is to explore applications of metabolic oligosaccharide engineering to tumor imaging and immunotherapy. This goal is reflected in Aims 1-4. Aim 1 will define the scope and consequences of azidosugar metabolism in laboratory mice. This includes identifying the specific glycoconjugates that are labeled with azidosugars in vivo and comparative studies of azidosugar metabolism in normal and tumor tissues. Aim 2 will apply azidosugar metabolism and the Staudinger ligation to noninvasive imaging of tumor glycosylation. Aim 3 focuses on the development of caging strategies to enhance the selectivity of tumor labeling with azidosugars. Aim 4 outlines a new approach to tumor vaccine therapy that, exploits unnatural sugars as neoantigens capable of breaking immune self-tolerance. The second major objective is to expand metabolic oligosaccharide engineering to encompass new carbohydrate pathways and new bioorthogonal chemistries. This goal is reflected in Aims 5 and 6. Aim 5 will probe the metabolism of azido fucose analogs. Aim 6 describes a modification to the Huisgen azide-alkyne cycloaddition that may render the reaction suitable for in vivo applications.
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1 |
2005 — 2007 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Investigations of Golgi Enzymes @ University of California Berkeley
[unreadable] DESCRIPTION (provided by applicant): Cell surface glycans are major determinants of cell-cell and cell-matrix interactions. Their structures reflect the expression of glycosyltransferases and sulfotransferases that act in an assembly line within the Golgi compartment. The broad objective of this project is to develop chemical tools for studying Golgi enzymes and the functions of the glycans they produce on cells. The last granting period focused on a newly discovered family of GlcNAc-6-sulfotransferases. The goals were to (1) develop small molecule inhibitors of the sulfotransferases as tools for biological studies and leads for drug discovery, (2) identify residues involved in substrate binding and catalysis, and (3) determine the preferred cellular substrates as a step toward elucidating biological function. An exciting discovery from this work was that the substrate preference of each enzyme in vivo is governed largely by its distribution among the Golgi cisternae. Thus, Golgi localization was identified as a major determinant of biological function. The Specific Aims of the next granting period build from this discovery. The major objective of the next granting period is to develop an approach for modulating Golgi enzyme activity with small molecules that target their common requirement of Golgi localization. The proposed strategy is based on the chemical dimerizer-induced assembly of the enzymes' modular catalytic and localization domains. The approach was validated with fucosyltransferase 7 (FucT7) and the GlcNAc-6- sulfotransferases GST-2 and GST-3, using the rapamycin/FRB/FKBP system for inducible domain assembly. The Specific Aims of the next granting period expand upon this discovery in three directions. The first Aim is to investigate the FucT7 system in more detail in order to define those parameters that affect the cellular activity of the reconstituted domains. The goal is to optimize the current FucT7 system for application to studies of tumor cell metastasis and for use in transgenic mice. The second Aim is to apply the approach to other Golgi enzymes, chosen for their diversity of substrates and functions. The third and final Aim is to determine whether chemical dimerizers can be used to modulate associations between two glycosyltransferases. Such associations are thought to be important for the efficiency of glycolipid biosynthetic pathways. The ability to modulate glycosyltransferase associations will provide means to control glycolipid expression on cells. [unreadable] [unreadable]
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1 |
2009 — 2013 |
Bertozzi, Carolyn R |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Univ of Calif Berkeley Chemistry-Biology Interface Program @ University of California Berkeley
This is the first renewal application for the Chemistry-Biology Interface predoctoral training grant at UC Berkeley. The program provides graduate PhD students with a unique depth of training in the application of chemical principles and techniques to the investigation and modulation of biological systems. The program's goal is to encourage further integration of the fields of chemistry and biology and prepare students for a future of research at this interface. The graduate program provides: 1.) three 10-week rotations for first year students 2.) A core of didactic courses with additional courses selected based on individual student interest, 3.) seminars, 4.) annual retreat, 5.) Poster sessions at the end of each rotation period, 6.) career day, 7.) supergroup meetings, 8.) a PhD dissertation at the chemical biology interface, and 9.) aggressive diversity recruitment efforts. The program selects students from the matriculating pools of first year students in the 4 departments of Chemistry, Molecular and Cell Biology, Chemical engineering, and Bioengineering. Students are selected on the basis of their stated interest in chemical biology research, undergraduate scholastic performance, scores on GRE tests, recommendations from faculty at their undergraduate institutions, and previous research experience. The program has thrived during the first five years with 53 current students, 36 participating faculty, and 12 PhD alumni. Each year the pool of qualified applicants grows larger. The program is positioned to expand to keep up with demand. Annual support for 12 first-year students during their rotation period is requested. RELEVANCE (See instructions): This proposal trains students to develop competence in the fields of chemistry and biology. Students learn the language and techniques to manipulate biological systems at the molecular level, which is key for the design of rational means of diagnosis and treatment of pathological processes. Reciprocally, study of biological problems leads to the development of new synthetic and mechanistic tools in the chemical
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1 |
2009 — 2018 |
Bertozzi, Carolyn R |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Metabolic Engineering With Bioorthogonal Chemical Reporters @ University of California Berkeley
DESCRIPTION (provided by applicant): The broad objective of this project is to apply a technique developed in our lab termed metabolic oligosaccharide engineering to in vivo imaging of global changes in the glycome associated with embryonic development and cancer. The "glycome" is the totality of glycans that cells produce under specified conditions of time, space and environment. Changes in the glycome's composition and distribution are associated with embryogenesis and cancer progression. We seek to develop chemical tools for imaging the dynamic cell-surface glycome in living organisms. In the last granting period, we demonstrated that three important sectors of the glycome - sialylated glycans, mucin-type O-glycans and fucosylated glycans, can be metabolically labeled with azido analogs of their biosynthetic precursors. The azide served as a chemical reporter that was visualized by Staudinger ligation with phosphine probes. We performed non-invasive imaging of sialic acids in healthy mice by metabolic labeling with N-azidoacetylmannosamine (ManNAz) followed by sequential injection of biotinylated phosphine and fluorescent streptavidin conjugates. For direct labeling of azidosugars, we designed fluorescent phosphine probes with a variety of spectral properties. In order to improve the sensitivity and time resolution of glycan imaging, we developed a new bioorthogonal reaction with faster kinetics than the Staudinger ligation: the strain-promoted cycloaddition of azides and cyclooctynes ("Cu-free click chemistry"). We employed a difluorinated cyclooctyne (DIFO) to image spatiotemporal changes in the glycomes of live cells and developing zebrafish. In the next granting period we plan to build upon these discoveries with four specific aims. First, we will expand our analysis of glycomic transformations during zebrafish development (Aim 1). We will image new sectors of the glycome (e.g., sialylated glycans, fucosylated glycans, glycosaminoglycans and N-glycans) at various stages of development. In addition, we will perturb the expression of certain glycosyltransferases and monitor concomitant changes in the glycome by in vivo imaging. We will develop new cyclooctyne imaging reagents with improved pharmacokinetic and fluorogenic properties (Aim 2). With the use of new phosphine and cyclooctyne probes, we will image glycans in mouse tumor models (Aim 3). Finally, we will develop new bioorthogonal reactions to expand the scope of the chemical reporter method (Aim 4). PUBLIC HEALTH RELEVANCE: All human cells are coated with complex sugar molecules termed "glycans". Each type of cell in the human body has its own collection of these glycans coating the cell surface. When cells transform from an embryonic state to a mature state, or from a healthy state to a cancerous state, the collection of glycans changes its makeup. The goal of this project is to develop tools from the field of chemistry that can help researchers and physicians monitor the changes in cell- surface glycans inside the body using imaging techniques. These chemical tools could be useful for cancer detection and diagnosis.
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1 |
2009 — 2016 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chemical Cell Surface Engineering
DESCRIPTION (provided by applicant): Glycans attached to cell-surface proteins and lipids mediate interactions with receptors on other cells, the extracellular matrix (ECM), or molecules on the same cell membrane. As well, cell-surface glycoconjugates collectively form the glycocalyx, a structure with bulk physical properties that can influence extracellular interactions Cell-surface glycosylation patterns often shift in response to cellular changes, most notably during malignant transformation. Two frequently observed cancer-associated phenotypes are cell-surface mucin overexpression and hypersialylation, but the functional significance of these altered glycoprofiles is not well understood. More broadly, while much work has been devoted to characterizing cancer glycomes, there are very few examples in which tumor-associated glycoconjugates have been ascribed specific cancer-related functions. The broad objectives of this project are to (1) develop chemical approaches for engineering structurally defined glycoconjugates on cells, and (2) shed light on the functional significance of cancer-associated glycosylation motifs using these methods. We aim to generate synthetic glycopolymers and protein/glycopolymer chimeras that emulate the structures and biological properties of cell surface mucin glycoproteins (Aim 1). We will use these materials and methods to engineer the display of chemically defined mucin mimetics on live cells, where we can probe their contribution to cancer-related processes. Specifically, we will test the emerging hypothesis that mucin overexpression alters the physical properties of the cell surface glycocalyx so as to promote integrin clustering and cell survival in non-adherent settings (Aim 2). This component of the project is a collaborative effort with Prof. Valerie Weaver's laboratory at UCSF. Finally, we will determine whether hypersialylation engineered via cell surface glycopolymer display protects cancer cells from innate immune destruction by NK cells (Aim 3). Such protection, and the selective advantage it confers, could explain the widespread occurrence of hypersialylation among disparate cancer types.
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1 |
2010 — 2011 |
Bertozzi, Carolyn R |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Chemical Biology Approaches to Study O-Glcnac in Mammalian Cells @ University of California, San Francisco
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The dynamic modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is a critical and ubiquitous cell signaling paradigm, regulating substrates'function, localization and stability. Protein OGlcNAcylation controls numerous processes in a wide range of mammalian tissues, and its dysregulation is implicated in human diseases, such as type II diabetes and neurodegeneration. Despite its broad functional significance, major aspects of O-GlcNAc signaling are poorly understood. Specifically, three key questionsremain unanswered: 1. What are the functionally important substrates in pathways that O-GlcNAc regulates? 2.What biochemical effect does O-GlcNAc have on these substrates? 3. How are proteins targeted for signal dependent O-GlcNAcylation? Because O-GlcNAc is a transient post-translational modification not under direct genetic control, these questions are challenging to answer using traditional molecular biology techniques alone. Therefore, we have pioneered chemical biology approaches to studying O-GlcNAc that make use of unnatural sugar reagents and cognate detection probes. We will apply these tools to study the role of O-GlcNAc in regulating two model cell biological processes: the DNA damage response and mitochondrial metabolism. Importantly, the identification of glycosylation sites on O-GlcNAc targets is critical for understanding the role of the modification in both processes. The UCSF Mass Spectrometry Facilty will provide vital expertise and collaborative resources for identifying O-GlcNAcylation sites on proteins of interest. Together, the project will exploit both novel chemical methods and mass spectrometry to dissect the functional role of O-GlcNAc in mammalian cell physiology.
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0.976 |
2011 — 2014 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Studies of Protein Glycosylation @ University of California Berkeley
DESCRIPTION (provided by applicant): This is a competing renewal application of RO1 GM66047 (Studies of Protein Glycosylation). The broad objective of this project is to develop chemical tools for studies of protein glycosylation, the most prevalent and structurally diverse form of posttranslational modification. The next granting period is focused on two forms of Ser/Thr O-glycosylation for which chemical tools are needed: mucin-type O-glycosylation characterized by a conserved core N-acetylgalactosamine (GalNAc) residue, and O-GlcNAcylation in which Ser/Thr residues are modified with a single 2-N-acetylglucosamine (GlcNAc) residue. Changes in mucin-type O-glycoprotein expression have been associated with cancer. We hypothesize that prostate cancer biomarkers exist among this class of glycoproteins. Protein O-GlcNAcylation is a reversible signaling modification that is known to be critical for viability of embryos. We hypothesize that O-GlcNAcylation regulates proteins involved in human embryonic stem cell (hESC) differentiation and that differences in O-GlcNAcylation states contribute to the varied behavior of hESCs and induced pluripotent stem cells (iPSCs). We propose to develop new technologies for chemically-directed glycoproteomics (Aim 1) and to apply these tools to profile changes in mucin-type O-glycosylation associated with prostate cancer (Aim 2) and to profile O-GlcNAcylated proteins in differentiating hESC and iPSCs (Aim 3). The proposed glycoproteomics platform in Aim 1 combines two technologies developed in the last granting period: metabolic labeling of mucin-type O-glycans and O-GlcNAcylated proteins with azidosugars, and isotopic signature transfer and mass pattern prediction (IsoStamp) for chemically-directed mass spectrometry. IsoStamp exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be extracted from full-scan MS data sets using a computational algorithm we developed in-house. The method enables high- sensitivity detection of chemically tagged peptides from complex mixtures such as cell and tissue lysates. In addition to the use of metabolic labeling and IsoStamp, our glycoproteomics workflow includes glycosylation site-identification via ETD fragmentation, and quantitative comparative MS analysis with light and heavy IsoStamp tags. In Aim 2, these glycoproteomics tools will be used to identify mucin-type O-glycoproteins from human prostate cancer biopsy tissue as a function of Gleason grade, and for comparative studies with normal human prostate tissue. In Aim 3, we will probe changes in the O-GlcNAcylation states of cytosolic and nuclear proteins in human embryonic stem cells (hESCs) during neuronal differentiation. We will also perform a comparative analysis of O-GlcNAcylated proteins in hESCs and iPSCs.
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1 |
2011 — 2014 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chemical Mycobacteriology @ University of California Berkeley
DESCRIPTION (provided by applicant): This is a competing renewal application of R01 AI51622 entitled Chemical Mycobacteriology. Mycobacterium tuberculosis (Mtb) infections are difficult to treat owing to the requirement of multiple drugs administered over many months, the emergence of drug-resistant strains, and a complex lifecycle that can include a drug-refractory latent stage. Mtb adapts to diverse environments during disease progression by influencing host cells and altering its own metabolic state. Glycolipids of the outermost capsular layer are thought to contribute to host-pathogen interactions; their underlying biosynthetic machineries might offer new targets for Mtb therapy. Metabolic pathways essential for survival during latency are also attractive drug targets. The broad objectives of this program are (1) to investigate the functions of mycobacterial cell wall glycolipids, and (2) to explore sulfur metabolism as a new niche for drug discovery. During the last granting period we focused our studies on the Mtb-specific trehalose glycolipid sulfolipid-1 (SL-1), a putative Mtb virulence factor. We elucidated the complete genetic and biosynthetic machinery underlying SL-1 and probed its functions in host cells and animals. In the course of these studies we discovered a novel sulfated menaquinone metabolite in Mtb, termed S881, disruption of which produces a hypervirulent phenotype in the mouse infection model. We also validated ATP sulfurylase, the enzyme catalyzing the first committed step in sulfate assimilation, as an attractive drug target. The next granting period will focus on four specific aims, the first two of which builds directly from previous work. In Aim 1 we will develop small molecule inhibitors of ATP sulfurylase as anti-tuberculosis drugs with possible application toward latent Mtb. In Aim 2 we will investigate the biosynthesis and function of S881, which we propose to play a role in adaptive respiration. We will also initiate two new research directions. In Aim 3 we will study trehalose glycolipids in the Mtb relative Mycobacterium marinum, exploiting the zebrafish infection model to profile their expression during infection. Two emerging technologies, MALDI mass spectrometry imaging and metabolic/bioorthogonal labeling for fluorescence imaging, will be employed to probe the dynamics of trehalose glycolipids during the course of disease. Finally, in Aim 4 we will investigate pathways that modulate cell wall structure in response to osmotic stress. A chemical approach to imaging peptidoglycan in vivo will be developed to facilitate these studies.
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1 |
2015 |
Bertozzi, Carolyn R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dynamics of Tb and Mdr Tb Transmission in Areas With High Hiv Prevalence
DESCRIPTION (provided by applicant): This is a competing renewal application of R01 AI51622 entitled Chemical Mycobacteriology. Mycobacterium tuberculosis (Mtb) infections are difficult to treat owing to the requirement of multiple drugs administered over many months, the emergence of drug-resistant strains, and a complex lifecycle that can include a drug-refractory latent stage. Mtb adapts to diverse environments during disease progression by influencing host cells and altering its own metabolic state. Glycolipids of the outermost capsular layer are thought to contribute to host-pathogen interactions; their underlying biosynthetic machineries might offer new targets for Mtb therapy. Metabolic pathways essential for survival during latency are also attractive drug targets. The broad objectives of this program are (1) to investigate the functions of mycobacterial cell wall glycolipids, and (2) to explore sulfur metabolism as a new niche for drug discovery. During the last granting period we focused our studies on the Mtb-specific trehalose glycolipid sulfolipid-1 (SL-1), a putative Mtb virulence factor. We elucidated the complete genetic and biosynthetic machinery underlying SL-1 and probed its functions in host cells and animals. In the course of these studies we discovered a novel sulfated menaquinone metabolite in Mtb, termed S881, disruption of which produces a hypervirulent phenotype in the mouse infection model. We also validated ATP sulfurylase, the enzyme catalyzing the first committed step in sulfate assimilation, as an attractive drug target. The next granting period will focus on four specific aims, the first two of which builds directly from previous work. In Aim 1 we will develop small molecule inhibitors of ATP sulfurylase as anti-tuberculosis drugs with possible application toward latent Mtb. In Aim 2 we will investigate the biosynthesis and function of S881, which we propose to play a role in adaptive respiration. We will also initiate two new research directions. In Aim 3 we will study trehalose glycolipids in the Mtb relative Mycobacterium marinum, exploiting the zebrafish infection model to profile their expression during infection. Two emerging technologies, MALDI mass spectrometry imaging and metabolic/bioorthogonal labeling for fluorescence imaging, will be employed to probe the dynamics of trehalose glycolipids during the course of disease. Finally, in Aim 4 we will investigate pathways that modulate cell wall structure in response to osmotic stress. A chemical approach to imaging peptidoglycan in vivo will be developed to facilitate these studies.
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1 |
2015 — 2021 |
Bertozzi, Carolyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chemical Glycoproteomics
PROJECT SUMMARY Mucins are densely O-glycosylated proteins with extended regions of clustered Ser/Thr-linked O-glycans, a structural feature that imparts a rigid and extended conformation. Their range of biological functions include physical stiffening of the glycocalyx to modulate cell survival in low adhesion settings, and biochemical interactions with glycan-binding receptors on other cells. Altered mucin expression and glycosylation patterns have been strongly linked to cancer progression. Crude measurements of these changes are currently used for cancer diagnosis but are imperfect due to their lack of molecular-level detail. A detailed map of mucin O-glycan structures and sites has been impossible to obtain, as mucins are recalcitrant to conventional mass spectrometry-based glycoproteomics methods. As a consequence, the cellular pathways underlying aberrant mucin structures are not well defined. We are pursuing these questions with the long-term goal of identifying more accurate cancer biomarkers and new therapeutic targets. During the previous funding period, we developed new mass spectrometry-based glycoproteomics methods and used them in fundamental studies of the enzymes that initiate mucin-type O-glycosylation, the polypeptide GalNAc transferases. Examples of our accomplishments include (i) development of the IsoTaG method for intact glycoproteomics via isotopic recoding and mass-independent glycopeptide discovery; (ii) identification of an optimal tandem mass spectrometry method for O-glycosite discovery; and (iii) development of a bump/hole strategy to identify biological substrates of polypeptide GalNAc transferases that initiate mucin-type O- glycosylation. In preliminary work for this application, we repurposed mucin-specific proteases (?mucinases?) from gut-resident microbes as tools for mapping O-glycosites on mucin domains. In the next funding period, we plan to develop a comprehensive ?mucinomics? platform. We will use engineered mucinases as glycoform-sensitive probes of mucin expression on cells and tissues. We will also develop a mucinase-based enrichment strategy for mass spectrometry-based discovery of new mucin domain molecules as well as O-glycosite mapping. Integrated into this workflow will be newly developed ionization methods and search algorithms for O-glycosite identification. Finally, we will use the mucinomics platform to define pathways by which prevalent oncogenes drive altered mucin expression and glycosylation in cancer.
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1 |
2015 — 2016 |
Bertozzi, Carolyn R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Revolutionizing the Detection of O-Glcnac
? DESCRIPTION (provided by applicant): The N-acetylglucosamine (O-GlcNAc) modification is a reversible attachment of O-GlcNAc onto serine or threonine residues of intracellular proteins. This modification mediates cellular activities by regulating protein trafficking, conformational change, and by antagonizing phosphorylation. Many human pathologies exhibit aberrant O-GlcNAcylation of specific proteins. Current strategies for detecting O-GlcNAc, however, are insufficient. Immunoblotting and mass spectrometry are the state-of-the-art methods to study protein O-GlcNAcylation. To detect the modification of a specific protein, Western blots are typically paired with a prior precipitation step with either target specific antibodies or anti-O-GlcNAc affinity reagents. This enrichment process can be labor intensive and lead to major sample loss due to the relatively low affinity of lectins and anti-O-GlcNAc antibodies. Mass spectrometry is the most widely used tool for profiling protein-specific O-GlcNAc modification on a global level. The generation of high-quality glycoproteomics data, however, requires large quantities of sample followed by rigorous and labor intensive enrichment to ensure adequate representation of low-abundance glycoproteins. Also, the instrumentation to perform these analyses is possessed by only a few labs due to it being very expensive and requiring a high level of specialized expertise to operate. We herein propose to develop a low-tech, broadly accessible method for analyzing the O-GlcNAcylation state of specific proteins. Changes in O-GlcNAcylation are mechanistically implicated in many human diseases and thus represent a fertile ground for scientific investigation. The broad goal of this proposal is to develop and disseminate an accessible PCR-based glycoproteomics platform for monitoring changes in O-GlcNAcylation. We describe the development of a technique that will allow the detection of protein-specific glycosylation directly from lysate using chemical probes of O-GlcNAc in tandem with multiplexed proximity ligation assays and fluorescence-based quantitative PCR.
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1 |
2016 — 2019 |
Bertozzi, Carolyn R Pitteri, Sharon |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Making Glycoproteomics Via Mass Spectrometry More Accessible to the Greater Scientific Community
7. PROJECT SUMMARY/ABSTRACT We very recently published a method that, for the first time, allows glycan structures and sites of attachment to be discerned from complex biological samples for both N- and O-glycans alike. A major breakthrough is that this method does not require truncation of the glycans, allowing the rich glycomic information to remain intact during analysis. In addition, this method, termed Isotope-Targeted Glycoproteomics (IsoTaG) enriches glycopeptides and allows unprecedented detection of low abundance glycoproteins. IsoTaG also mitigates the need for extensive fractionation, mass spectrometer analysis time, and computation time. Also, we have since been able to transfer the IsoTag method to interested labs that have no prior glycobiology expertise, who were able to successfully perform glycoproteomic analysis on their samples of interest. With these barriers to large scale implementation of glycoproteomics substantially reduced, we propose herein to make IsoTaG accessible to the broader scientific community as a standard service offered by mass spectrometry (MS) core facilities. To accomplish this, we will improve the IsoTaG reagent and develop a large scale synthesis to enable distribution to the community. We will also standardize various aspects of the chromatography used to separate glycopeptides, including making standards that can be used to calibrate retention times. Also, we will develop protocols for use of these standards to tune instrument parameters for optimal fragmentation of the glycopeptides. Finally, we will provide the reagents and protocols to several mass spectrometry core facilities to trial use of the IsoTag system on their instruments. Ensuing dialog with the facilities will help us improve the method with the goal of it being adoptable by any facility that has high resolution MS instrumentation.
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1 |
2016 — 2021 |
Bertozzi, Carolyn |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Stanford Chem-H Chemistry/Biology Interface Predoctoral Training Program
Project Summary/Abstract This application seeks funding for a new Chemistry-Biology Interface predoctoral training program at Stanford University, aligned with the mission of the new Stanford ChEM-H institute. Stanford ChEM-H was formed with the mission of bringing chemists, biologists, engineers and clinicians together to pursue a molecular level understanding of the principles underlying human health and to devise innovative disease interventions. A major component of this initiative is cross-disciplinary student training, the flagship being this new predoctoral training program at the chemistry/biology interface. The program will provide PhD students with a diverse community of peers and mentors from the Schools of Humanities and Sciences, Engineering and Medicine. Graduate students in the program will be recruited from six home departments and PhD granting programs: Chemistry, Chemical Engineering, Chemical & Systems Biology, Biochemistry, Biology and Bioengineering. Mentors are affiliated with diverse departments and programs, including physician scientists who are practicing clinicians. Key components of the program include first-year laboratory rotations, core coursework in chemical biology, student and faculty seminars, career development activities and an annual retreat. Students will also be educated in the unmet needs of key therapeutic areas through bootcamps led by clinicians at the Stanford Medical School. As well, students will receive training in communication across scientific disciplines and with the community at large, in part through a newly created student/screenwriters' exchange event in partnership with the National Academies of Science. Students trained in this program will be exposed to a wide range of scientific concepts and techniques, meet diverse experts across the physical, life and medical sciences, and be uniquely situated to tackle challenges in human health from a molecular level perspective.
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1 |
2016 — 2021 |
Bertozzi, Carolyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Chemical Mycobateriology
7. PROJECT SUMMARY/ABSTRACT This is a competing renewal application of R01 AI51622 entitled ?Chemical Mycobacteriology?. The broad objectives of this project are (1) to study the effects of tuberculosis drugs on mycobacterial cell wall dynamics by in vivo imaging in the Mycobacterium marinum/zebrafish infection model; and (2) to develop a new point-of- care method for clinical detection of live Mtb in patient sputum samples. Tuberculosis (TB) is a chronic pulmonary disease caused by infection with Mycobacterium tuberculosis (Mtb). A variety of drugs have been identified that rapidly kill Mtb and its relatives in vitro, yet clinical treatment requires at least 6 months of combination therapy and resistance is rampant. The reasons that antibiotics are less effective in vivo remain unclear, and this knowledge gap is exacerbated by our inability to directly study the molecular effects of TB drugs on bacteria during infection. To do so would require an infection model amenable to noninvasive monitoring, and probes that report on bacterial systems affected by drug action. In the previous granting period, we developed chemical methods for imaging components of the mycobacterial cell wall, a target of several frontline TB drugs. We used metabolic and bioorthogonal labeling methods to image trehalose glycolipids of the mycomembrane, an essential cell wall layer disrupted by the TB drugs isoniazid and ethambutol. In parallel, we developed reagents for imaging newly synthesized peptidoglycan (PG) in bacteria residing within human host cells. These methods offer a newfound capability of monitoring how the cell wall responds to drug treatment during the course of infection. Our proposal for the next granting period comprises three specific aims. Aim 1: We will investigate the effects of TB drugs on cell wall dynamics in vitro and in vivo, with an eye for identifying stages of infection that perturb drug responses. We will image changes in trehalose mycolate production, subcellular localization and mobility as a function of infection stage and drug treatment using the M. marinum/zebrafish infection model, a natural and experimentally tractable host-pathogen system. Aim 2: We will develop a new method for point-of- care detection of Mtb in patient sputum samples using solvatochromic mycomembrane imaging agents. In collaboration with Prof. Bavesh Kana at Univ. Witswatersrand, South Africa, we will field test the method by analysis of sputum samples collected from HIV-1 coinfected and uninfected TB patients as well as naïve and drug-treated TB patients. Aim 3: Finally, we will explore how mycobacteria's unusual property of asymmetric growth contributes to virulence and drug sensitivity in vivo by imaging PG in the M. marinum/zebrafish infection model. Achievement of these aims will provide new insights into TB drug action and resistance, and deliver a new clinical tool for accurate diagnosis and drug efficacy monitoring.
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1 |
2019 — 2021 |
Bertozzi, Carolyn Brooks, James D. (co-PI) [⬀] Pitteri, Sharon |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Glycosylation and Immune Evasion in Urologic Tumors
PROJECT SUMMARY/ABSTRACT The ability of tumor cells to evade the immune system is a well-known, yet poorly understood phenomenon in early cancer development. Despite promising immunotherapy strategies that have emerged from targeting these interactions, there is relatively little known about the complete repertoire of receptor-ligand interactions that contribute to immune evasion. We seek to understand how glycosylation, a well-established aberrant modification in cancer, aids cancer cells in evading the immune system. Identification of glycoproteins that modulate immune function could lead to new types of therpaies and could also serve as companion diagnostic biomarkers to guide patient selection of immunotherapies at an early time point in prostate cancer and clear cell renal cell carcinoma. First, because sialic acid is known to be overexpressed on the surface of cancer cells, we will use intact glycoproteomics methods developed in-house to enrich and identify sialoglycoproteins from cancerous and matched healthy tissues from patients. Quantitative comparative analyses will reveal changes in sialoglycoprotein expression and illuminate candidate ligands for sialic acid-binding proteins in the tumor microenvironment that potentially contribute to immune inactivation. Correlation of these glycoproteomic datasets with RNA-seq data focused on glycogene expression will bolster the assignment of specific glycoforms as cancer biomarkers. Second, using immunohistochemistry and CODEX methods, we will analyze expression levels of sialic acid-binding immunoglobulin-type lectin (Siglec) receptor proteins on tumor- resident immune cells and cross-correlate the findings with RNA-seq data as well as immune cell markers. We will also probe for the presence of ligands for various Siglec isoforms on tumor cell surfaces and obtain spatial information about their distribution on immune cells in intact tumor tissue. For any Siglecs identified as prominently displayed on immune cells in the tumor environment, we will develop cell-based assays to probe their contribution to tumor cell immunoreactivity. Third, we will perform a genome-wide screening using CRISPRi to identify genes that facilitate the binding of Siglecs to cancer cells. Finally, we will correlate the datasets from Aims 1, 2, and 3 with patient outcomes in a larger set of tissue samples contained on a tissue microarray, and evaluate their utility as prognostic indicators.
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0.915 |
2019 — 2021 |
Bertozzi, Carolyn Weaver, Valerie Marie |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Targeting the Cancer Glycocalyx
PROJECT SUMMARY Cell surface glycans mediate interactions with receptors on other cells, in the extracellular matrix, or on the same cell membrane. Altered glycosylation has long been known as a hallmark of cancer. Two frequently observed cancer-associated phenotypes are hypersialylation and mucin overexpression. These cancer glycosignatures strongly correlate with disease aggressiveness and poor patient outcomes, but their functional contribution to cancer progression has been unclear. The broad objective of this program is to bring chemical tools to bear on this important problem in oncology, with an eye for developing new modes of intervention. An enabling tool for these studies are synthetic glycopolymers that we used to engineer discrete glycosylation patterns on live cells, or to engage specific glycan-binding proteins in a multivalent manner. In the previous granting period we made three major discoveries regarding the roles of cancer glycosignatures in disease: (1) Hypersialylation is a mechanism of immune evasion mediated through the Siglec family of sialic acid-binding immune cell receptors. Accordingly, immune cell killing of cancer cells can be potentiated by targeted cleavage of their cell-surface sialosides using antibody-sialidase conjugates. (2) Mucin overexpression enhances the thickness and stiffness of the glycocalyx, which promotes integrin clustering and focal adhesion signaling. This, in turn, enhances cell survival in vitro and promotes metastasis in mouse tumor models. And finally, (3) a glycan switching mechanism modulates partitioning of galectin-1, a prominent breast cancer marker, between a cell's glycocalyx and nucleus. Nuclear localization of galectin-1 drives breast cancer invasion, and this is inhibited by glycopolymers that sequester galectin-1 extracellularly. These discoveries form the foundation of the aims proposed in this renewal application. Aim 1 is a corollary to our discovery that cancer mucins drive oncogenesis. We will develop antibody-enzyme conjugates comprising mucin-specific proteases (aka ?mucinases?) to deforest cancer cells. We will generate tool molecules using known bacterial mucinases, and also identify human mucinases for incorporation into therapeutic candidates. In Aim 2, we will construct next-generation glycopolymers with native polypeptide backbones. These will be employed for fundamental studies of cancer glycobiology and for translational applications in Aim 3. Finally, in Aim 3 we introduce a new strategy for targeting extracellular proteins for degradation using glycopolymers that hijack the mannose-6-phosphate receptor (M6PR) lysosomal trafficking pathway. We will construct antibody-M6P glycopolymer conjugates that bind oncogenic cell-surface molecules such as growth factor receptors and the cancer-associated mucin MUC1 and target them for lysosomal degradation via engagement of M6PR. This new therapeutic modality complements the popular PROTAC approach for targeting intracellular proteins for proteasomal degradation.
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0.915 |
2020 — 2025 |
Bao, Zhenan [⬀] Deisseroth, Karl (co-PI) [⬀] Bertozzi, Carolyn |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Fmrg: Genetically-Targeted Chemical Assembly (Gtca) of Functional Structures in Living Cells, Tissues, and Animals
The broad vision for this project is to develop new tools for future biomanufacturing through cross-cutting collaborations of scientists?chemists, biologists, physicians, and engineers?united by the immense opportunity of building functional materials with, and within, biological life. The proposed methodologies establish the biomanufacturing toolbox for genetically-targeted chemical synthesis of a variety of functional materials within living cells, tissues and animals. Diverse cell-specific chemical syntheses enable a broad array of functional characteristics and assembled structures. In the long term, such techniques enable building electronics directly within biological systems by harnessing the complex assembly structures within cells. The application of these techniques to develop the capability to create new conductive pathways within the brain may lead to rewiring of neural circuits. Moreover, genetically-targeting the peripheral nerve may allow cell-specific nerve stimulation and recording for neuroprosthesis. Further investigation of the deposited material on neural activity may lead to treatments for diseases such as Alzheimer?s disease (AD) and amyotrophic lateral sclerosis (ALS), or selectively repair demyelinated areas for treatment of multiple sclerosis (MS). Even though current work only focuses on basic tool development and initial understanding of the impact of the modifications on neural activities, the tools can be potentially expanded to diverse cell types for therapeutics and creation of new materials and assemblies. This project offers direct training opportunities for the students and postdocs involved in terms of research as well as important skills for interdisciplinary collaboration. These trainees subsequently further the development of biomanufacturing and their method of collaboration by running their own independent research groups in academia or by incorporating their knowledge into future industrial developments. A Training Core program in this project provides hands-on training on basic biomanufacturing techniques for hundreds of students, instructors and researchers.
Despite existing ability to engineer materials with diverse form and functionality, a high-level of structural and functional complexity found in multicellular living systems are still challenging to realize. The capability of genetically targeting enzymes and other proteins to specific cell types has yet to be harnessed to direct complex assembly of functional structures instructed by biological systems. This project integrates the fields of molecular genetics, tissue biology, chemistry, and materials science in unprecedented ways to transform the biomanufacturing of complex structures. The project focuses on building novel structures in vivo, creating natural and unnatural polymers within targeted cell-types of living organisms. This approach is extended to the development of a universal shared methodology for targeted chemistry within living beings. The work proposed focuses on developing and applying novel toolboxes for diverse genetically-targeted synthetic processes while engineering for biocompatibility, characterizing the synthesized molecules/materials, and understanding the mechanisms and implications of forming synthetic materials for eliciting natural and novel biological functions.
This award is co-funded by the Division of Molecular and Cellular Biosciences, the Division of Chemical, Bioengineering, Environmental and Transport Systems and the Division of Chemistry, and also by the Division of Industrial Innovation and Partnerships, the Division of Engineering Education and Centers, and the Division of Materials Research.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
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0.915 |
2020 — 2021 |
Bertozzi, Carolyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chemical Glycobiology Tool Development: Lytacs
PROJECT SUMMARY This is a renewal application of R37 GM058867 which has supported our foundational efforts in chemical glycobiology tool development since 1999. In the next granting period we will focus our efforts on a new chemical biology platform for targeted degradation of extracellular proteins. Targeted protein degradation platforms such as proteolysis targeting chimeras (PROTACs) are now well-established as powerful strategies to address canonically ?undruggable? proteins. However, canonical PROTAC approaches involve manipulation of a cytosolic protein degradation machinery and therefore are fundamentally limited to targets with ligandable cytosolic domains. This requirement excludes most secreted and cell-surface membrane-associated proteins, which are estimated to comprise 40% of protein-encoding genes and are key agents in cancer, aging-related diseases, and autoimmune disorders. Thus, there has been a recent surge of interest in new approaches for targeted degradation of extracellular proteins, with a particular focus on harnessing the endosome-lysosome pathway. The work proposed herein focuses on what we believe to be a leading technology in this space. We developed ?lysosome targeting chimeras? (LYTACs) that direct proteins of interest to lysosomes via engagement of the cation-independent mannose-6-phosphate receptor (CI-M6PR). LYTACs comprise a binding element (e.g., an antibody or small molecule ligand) specific to the extracellular target protein, conjugated to mannose-6-phosphate (M6P) analogs that engage CI-M6PR. The receptor endogenously transports lysosomal enzymes marked with M6P caps on N-glycans residues to their destination organelle by cycling continuously between endosomes, the cell surface, and the Golgi complex. CI-M6PR has been exploited to deliver therapeutic enzymes for treatment of lysosomal storage disorders. However, prior to our work, this lysosome delivery system had not been contemplated as a vehicle for targeted degradation. In preliminary work we used bioorthogonal chemistries to conjugate ligands or antibodies that bind a protein of interest to synthetic CI-M6PR engagers. We demonstrated that both soluble extracellular proteins and membrane-bound cell-surface proteins can be targeted for degradation by LYTACs. These preliminary studies set the stage for expansion of the program to include fundamental studies of LYTAC scope and mechanism as well as translational therapeutic applications. The Specific Aims of this project are to (1) synthesize homogeneous LYTACs and optimize structures for in vitro and in vivo applications, (2) characterize the LYTACable proteome, and (3) apply LYTACs in therapeutic models that involve soluble and cell-surface membrane-bound targets.
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0.915 |
2021 |
Bertozzi, Carolyn |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Aptamer-Based Lytacs to Screen For Critical Determits of Lysosomal Degradation
PROJECT ABSTRACT Extracellular and transmembrane proteins are central pathological components in cancer, aging-related disorders, and autoimmune disease. These proteins can be degraded using lysosome targeting chimeras (LYTACs), antibodies conjugated to polymeric chemical moieties engaging the cation-independent mannose-6- phosphate receptor (CI-M6PR). As these molecules require careful chemical synthesis, we will produce alternate LYTACs in high throughput using aptamers targeting CI-M6PR linked to nanobodies via enzymatic autoconjugation. This method will help elucidate optimal LYTAC binding affinities to CI-M6PR. We will further use enzymatic autoconjugation to produce aptamer-LYTACs within bacterial cells in a genetically-encoded fashion, enabling future work to define the LYTACable proteome.
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0.915 |