Category D. Protein Structure & Function
D1. Identification of BAG3 as a Novel SUMOylation Target in Liver
Wenqi Cui1, Nadezhda Galeva2, Todd Williams2, and Jeff L. Staudinger1
1Department of Pharmacology and Toxicology, School of Pharmacy, 2Mass spectrometry & Analytical proteomics Lab, University of Kansas, Lawrence, Kansas, USA
Covalent modification by SUMO is an important regulator of the functional properties of many proteins implicated in human diseases. While there are many examples of individual specific proteins regulated by SUMOylation, there has been no comprehensive survey of the targets of SUMOylation in a human disease. The major reason for this is the lack of a facile and general method for comprehensive identification and quantitating SUMOylated proteins in cells or live animal models. We have generated a novel mass spectrometry-based proteomic strategy to identify SUMO substrate proteins in the liver. Moreover, we used two adenoviral expression vectors to co-express the E3 ligase PIAS1 protein and SUMO3 to drive SUMOylation in primary cultures of hepatocytes. Current preliminary data showed that our innovative tools are valid, and we have identified numbers of unknown SUMO substrate proteins in the liver including a HSP70 cochaperone protein named Bcl2-associated athanogene 3 (BAG3). BAG3 is an anti-apoptotic protein, and is overexpressed in human hepatocellular carcinoma. SUMOylation of BAG3 is highly likely involves in regulating cancer cell survival. Our exploratory research will likely provide a new therapeutic target and innovative strategy to develop drug that aiming HCC.
D2. Protein Production Group: Phase III of the COBRE Protein Structure and Function
Anne Cooper and Philip Gao
Protein Structure Group, Center of Biomedical Research Excellence in Protein Structure and Function, University of Kansas, Lawrence, KS, USA
The Protein Production Group is one of the three core laboratories within the KU COBRE in Protein Structure and Function. It provides services at all stages of protein production and purification including: construction of the plasmid expression vector, protein expression, purification, and modification processes, mammalian stable cell line construction, and binding analysis via Surface Plasmon Resonance. Both prokaryotic and eukaryotic proteins can be produced in E. coli, insect cell, mammalian and cell-free expression systems. The facility also has the capacity for expanding on existing purifications schemes for large scale preparations. Most of the preparations and purifications are conducted in conjunction with automated equipment to ensure precision and efficiency. The Protein Production Group’s objective is to over-express and purify properly folded functional proteins in quantities sufficient for functional studies (catalytic or biological), binding assays (small ligand or macromolecular), structural analysis (X-ray, NMR), and high throughput (HTP) screening. In addition, the facility also provides advice on the generation and analysis of proteins depending on their physicochemical properties and the needs of the investigator.
D3. Effects of N-Glycosylation on Protein Conformation and Dynamics: Protein Data Bank Analysis and Molecular Dynamics Simulation Study
Hui Sun Lee1 and Wonpil Im2
1Higuchi Biosciences Center, 2Department of Molecular Biosciences and Center for Computational Biology, The University of Kansas, Lawrence, KS, USA
N-linked glycosylation is one of the most important, chemically complex, and ubiquitous post-translational modifications in all eukaryotes. The N-glycans that are covalently linked to proteins are involved in numerous biological processes. There is considerable interest in developments of general approaches to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in protein design with advantageous properties. In this study, the impacts of N-glycans on protein structure and dynamics are systematically investigated using an integrated computational approach of the Protein Data Bank structure analysis and atomistic molecular dynamics simulations of glycosylated and deglycosylated proteins. Our study reveals that N-glycosylation does not induce significant changes in protein structure, but decreases protein dynamics, likely leading to an increase in protein stability. Overall, these results suggest not only a common role of glycosylation in proteins, but also a need for certain proteins to be properly glycosylated to gain their intrinsic dynamic properties.
D4. The University of Kansas Protein Structure Laboratory
Nurjahan Mehzabeen and Scott Lovell
Center of Biomedical Research Excellence in Protein Structure and Function, The University of Kansas, Lawrence, KS, USA
The Center of Biomedical Research Excellence in Protein Structure and Function (COBRE-PSF) at The University of Kansas University supports health-related basic research efforts to obtain structural/functional information for proteins. One of the three core laboratories at the COBRE-PSF, the Protein Structure Laboratory (PSL), collaborates with investigators from various institutions in an effort to obtain the 3-dimensional structures of proteins using X-ray crystallography. The capabilities and infrastructure of the PSL are presented here along with examples of collaborative projects that have been completed. The results from these projects highlight the importance of obtaining structural information to provide mechanistic/functional insight for particular proteins and demonstrate the significance of structural biology to facilitate and support drug discovery efforts.
D5. Investigating “Stuffed” Domains of NRPS Assembly Line
Trey Ronnebaum1, Thomas E. Prizinzano3,1, Audrey L. Lamb2
1Deparment of Chemistry, 2Department of Molecular Biosciences, 3Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA
Microbes generate bioactive peptides using nonribosomal peptide synthetases (NRPS). These bioactive peptides are not only used as secondary metabolites (toxins, pigments, siderophores – iron scavenging molecules), but have found their way into the clinic as antibiotics, anticancer drugs, and immunosuppressants. To produce their unique bioactivity, these peptides must be tailored. Natural product chemists, metabolic engineers, and researchers in biochemistry and biotechnology work to exploit the biosynthesis of these secondary metabolites in order to generate new compounds for clinical use. The long term goal of this project is to understand the structure-function relationships of epimerases and methyltransferases that are incorporated into these assembly lines. Structural biology and mechanistic enzymology can provide novel insight and assist natural product investigations, protein engineering projects, antimicrobial development, and other therapeutic design.