Progress 09/01/01 to 08/31/07
Outputs
OUTPUTS: We have analyzed experiments within two areas: Inflammation and anti-HIV strategies. Our group studies the structural biology of chemokines, which are important in both areas. We have presented our work in several seminars and posters, including: (1) Sealy Center for Structural Biology Symposium in May, 2007 (2 posters), (2) University of California Merced in February, 2007 (research seminar), (3) University of Colorado Health Sciences in October, 2007 (research seminar). Another output is in teaching. During 2007, I have taught both graudate students (BICH 624, entitled, "Proteins, Enzymes and Nucleic Acids; Spring 2007) and undergraduates (BICH 411, entitled, "Biochemistry II".
PARTICIPANTS: Several graduate students have contributed to the work: Li Zhang Hongjun Jin Bo Zhao Ioannis Kagiampakis Wein Tian
TARGET AUDIENCES: Most of our research and teaching output is geared toward scientists-in-training.
PROJECT MODIFICATIONS: We will probably not have major changes to our projects, except that our structural biology studies may extend more into X-ray crystallography rather than NMR if we can obtain crystals of our large protein complexes.
Impacts
We have had success in developing a protein that has potent anti-HIV activity, and have started work on an attempt to patent the protein.
Publications
- Sinae Kim, Jerry Tsai, Ioannis Kagiampakis, Patricia J. LiWang, and Marina Vanucci. 2007. Detecting protein dissimilarities in multiple alignments using Bayesian variable selection. Bioinformatics 23, 245-246.
- Hongjun Jin, Xiaohong Shen, Brandi Renee Baggett, Xiangming Kong and Patricia J. LiWang. 2007. The human CC chemokine MIP-1 dimer is not competent to bind to the CCR5 receptor. Biol. Chem. 282, 27976-27983.
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Progress 01/01/06 to 12/31/06
Outputs
We have two major accomplishments for 2006. First is the completion of the solution structure of the complex between the chemokine MIP-1b and vCCI, a powerful anti-inflammatory protein. This structure was determined using state of the art NMR techniques and is one of the few known complexes involving chemokines. Analysis of the complex structure will allow us to understand and possibly modulate the therapeutic properties of vCCI, which include the potential as an anti-allergy, anti-asthma medication. In addition to the structure we have also developed an ELISA binding assay and are working toward a quantitative fluorescent-based binding assay. Our second major accomplishment of 2006 is the development of in vitro techniques to measure the function of various chemokines, both in their ability to bind the receptor (with promising results for the chemokine homolog vMIP-II), and in their ability to inhibit HIV infection (with new strategies aimed at better HIV
inhibitors).
Impacts
Chemokines are small proteins that are involved in causing movement of immune system cells in the body. Our understanding of their function will help us to combat inflammation-related diseases such as rheumatoid arthritis and asthma. In particular, our recent study of the chemokine inhibitor vCCI gives us a structural picture of a protein that could be used as a drug to alleviate inflammation. In addition, we are attempting to harness the properties of chemokines to devise a strategy for inhibition of HIV infection.
Publications
- Li Zhang, Michele DeRider, Melissa A. McCornack, Chris Jao, Traci Ness, Richard Moyer, and Patricia J. LiWang Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1b Proc. Natl. Acad. Sci. USA 103, 13985-13990 (2006).
- Michele L. DeRider, Li Zhang, and Patricia J. LiWang Resonance assignments and secondary structure of vCCI, a 26 kDa soluble secreted chemokine inhibitor from rabbitpox virus J. Bio. NMR (March 2006).
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Progress 01/01/05 to 12/31/05
Outputs
Our major accomplishment this year has been to determine the structure of the complex between the chemokine MIP-1b and vCCI, a powerful anti-inflammatory protein. This structure was determined using state of the art NMR techniques and is one of the few known complexes involving chemokines. Analysis of the complex structure will allow us to understand and possibly modulate the therapeutic properties of vCCI, which include the potential as an anti-allergy, anti-asthma medication. Other progress in 2005 includes a detailed study of the quaternary state of the chemokines MIP-1b and IL-8. We have published one paper in this area in 2005, and continue to work toward another in 2006. Our experiments are geared toward changing the quaternary state of the chemokines, and obtaining a structural understanding of they types of amino acid changes that lead to monomeric and dimeric chemokines.
Impacts
Chemokines are small proteins that are involved in causing movement of immune system cells in the body. Our understanding of their function will help us to combat inflammation-related diseases such as rheumatoid arthritis, asthma, and multiple sclerosis. In particular, our recent study of the chemokine inhibitor vCCI gives us a structural picture of a protein that could be used as a drug to alleviate inflammation.
Publications
- Paola Casarosa, Maria Wahldhoer, Patricia J. LiWang, Henry Vischer, Thomas Kledal, Henk Timmerman, Thue W. Schwartz, Martine J. Smit, and Rob Leurs. (2005). CC and CX3C chemokines differentially interact with the N-terminus of the human cytomegalovirus-encoded US28 receptor J. Biol. Chem., 280: 3275 - 3285.
- Hongjun Jin, Garret L. Hayes, Nithyanada S. Darbha, Erik Meyer and Patricia J. LiWang. (2005). Investigation of CC and CXC chemokine quaternary state mutants (in press).
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Progress 01/01/04 to 12/31/04
Outputs
One of our accomplishments this year has been to quantitate the ability of sulfated sugars to manipulate the quaternary structure of the chemokine MIP-1?. This protein is an important component of the immune system, and our work supports our long-term goal of understanding the role quaternary structure plays in chemokine immune function. Another major accomplishment this year has been our progress toward determining the structure (using nuclear magnetic resonance, NMR) of a complex between the chemokine MIP-1? and a chemokine binding protein called vCCI. Currently there is only one reported structure of a chemokine in complex with any other complete protein. We hope to start publishing these results in 2005.
Impacts
MIP-1? is an immune system protein that is involved both in blocking HIV infection and in causing movement of immune system cells in the body. Our progress in the past year has been to allow a greater understanding of how MIP-1? binds its biological partners, including cell surface sugars and viral binding proteins. These results help clarify the mechanism behind general immune defenses as well as the mechanism of inflammation and inflammatory diseases.
Publications
- Melissa A. McCornack, Danielle M. Boren, and Patricia J. LiWang, Glycosaminoglycan Disaccharide Alters the Dimer Dissociation Constant of the Chemokine MIP-1?_ Biochemistry 43 10090-10101 (2004).
- Paola Casarosa, Maria Wahldhoer, Patricia J. LiWang, Henry Vischer, Thomas Kledal, Henk Timmerman, Thue W. Schwartz, Martine J. Smit, and Rob Leurs, CC and CX3C chemokines differentially interact with the N-terminus of the human cytomegalovirus-encoded US28 receptor. J. Biol. Chem., 280: 3275 - 3285 (2005).
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Progress 01/01/03 to 12/31/03
Outputs
In the past year, we have made progress toward characterizing the binding of the chemokine MIP-1b with cell surface sugars. This binding is critical for the activity of MIP-1b in the immune system, and probably plays a role in the ability of MIP-1b to block HIV infection. We have used nuclear magnetic resonance (NMR) to delineate the binding site of MIP-1b for cell surface sugars, and have shown that the interaction with these sugars involves both positively charged amino acids and non-charged amino acids. The results clearly show one binding cleft per MIP-1b monomer. More recently we have quantitated the ability of sulfated sugars to actually manipulate the quaternary structure of MIP-1b. The ability of MIP-1b to take on multiple quaternary forms (likely in response to biological cues such as the presence of cell surface sugars) appears to be an interesting facet of how this protein carries out its role in causing chemotaxis of immune system cells. A second
accomplishment this year has been our ability to obtain high quality NMR data of a complex between the chemokine MIP-1b and a chemokine binding protein called p35. Currently there is only one reported structure of a chemokine in complex with another complete protein. We are therefore using our NMR data to determine the structure of the complex, which will allow a greater understanding of how proteins and receptors bind chemokines.
Impacts
MIP-1b is an immune system protein that is involved both in blocking HIV infection and in causing movement of immune system cells in the body. Our progress in the past year has been to allow a greater understanding of how MIP-1b binds cell surface sugars. This binding interaction is critical for the ability of MIP-1b to function, and helps to clarifiy how immune system cells are able to reach sites of infection and inflammation.
Publications
- Melissa A. McCornack, Craig K. Cassidy, and Patricia J. LiWang "The binding surface and affinity of monomeric and dimeric chemokine MIP-1b for various glycosaminoglycan disaccharides" J. Biol. Chem. 278, 1946-1956 (2003).
- Amanda E. I. Proudfoot, Zoe Johnson, Tracy Handel, Elaine Lau, Patricia LiWang, Ian Clark-Lewis, Frederic Borlat, Timothy N.C. Wells and Marie Kosco-Vilbois "Glycosaminoglycan binding and oligomerization is essential for the in vivo activity of certain chemokines" Proc Natl. Acad. Sci. USA 100,1885-1890 (2003).
- Yufeng Zhang, Huangshang Lu, Patricia LiWang, Uluhan Sill, and Nancy S. Templeton "Optimization of Gene Expression in Nonactivated Circulating Lymphocytes" Molecular Therapy 8, 629-636 (2003).
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Progress 01/01/02 to 12/31/02
Outputs
MIP-1b is a member of the chemokine family of small immune system proteins that has also been shown to block infection by HIV. In the past year we have made progress toward determining how MIP-1b binds its receptor by mutating several residues in the region of amino acids 14-22, and measuring the affinity of the resulting variant for the CCR5 receptor as well as carrying out NMR structural characterizations. In the area of investigating chemokine quaternary structure, we have recently shown that the chemokine dimer, rather than having a role in binding the chemokine receptor, may instead be involved in the glycosaminoglycan (GAG) binding function of the protein. The binding of chemokines to GAGs has been shown to be essential to their in vivo activity, although few structural details are clear regarding the chemokine-GAG interaction. Specifically, we showed that the wild type chemokine MIP-1b binds heparin disaccharides with greater affinity than monomeric variants of
MIP-1b. In this work, we also showed GAG selectivity for this chemokine and used NMR data to delineate a heparin binding site on MIP-1b. We have consequently developed the hypothesis that GAG binding stabilizes the MIP-1b dimer and increases the MIP-1b dimer affinity.
Impacts
These basic-research projects will allow us to better understand the structural fundamentals of protein-protein and protein-GAG interactions. It is hoped that this knowledge will aid in the development of anti-HIV therapeutics.
Publications
- Bondue, Antoine, Jao, Shu-chuan, Blanpain, Cedric, Parmentier, Marc, and LiWang, Patricia. 2002. Characterization of the role of the N-Loop of MIP-1 beta in CCR5 binding. Biochemistry. 41: 13548-13555.
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Progress 01/01/01 to 12/31/01
Outputs
MIP-1b is a member of the chemotactic cytokine (chemokine) family of proteins whose members mediate inflammation and host defense. Several chemokines, including MIP-1b, have been shown to function as anti-HIV agents due to their association with chemokine receptors that are used by the virus to gain entry to the cell. Our long-term goal is to elucidate at the atomic level the interaction between the chemokine MIP-1b and its binding partners, particularly CCR5 (its natural receptor), cell surface glycosaminoglycans (GAGs), and p35 (a virally encoded chemokine binding protein) so as to reveal the structural determinants of the biological and anti-HIV properties of MIP-1b. In the past year we have demonstrated the importance of particular basic residues in binding glycosaminoglycans. In addition, we have obtained nuclear magnetic resonance (NMR) data for wild type MIP-1b and several variants in the presence of GAGs, and have defined a previously-unknown GAG binding
groove in MIP-1b. Other work includes the full structure determination of a monomeric variant of MIP-1b and the comparison of this variant to the wild type dimer.
Impacts
Our work is basic science toward understanding the molecular details of the interaction between the chemokine MIP-1b and its binding partners. This work has relevance to AIDS research and to the understanding of protein-protein interactions.
Publications
- "Structural comparison of monomeric variants of the chemokine MIP-1b having differing ability to bind the receptor CCR5" Seho Kim, Shu-chuan Jao, Jennifer S. Laurence, and Patricia J. LiWang, Biochemistry 40, 10782-10791 (2001).
- "The Importance of Basic Residues and Quaternary Structure in the Function of MIP-1b: CCR5 Binding and Cell Surface Sugar Interactions" Jennifer S. Laurence, Cedric Blanpain, Anne De Leener, Marc Parmentier, and Patricia J. LiWang, Biochemistry 40, 4990-4999 (2001).
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