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Characterization and Hybridization of
Disease-Associated Human Adenylosuccinate Lyase Mutants

Peychii Lee, Jenna M. Currier, and Roberta F. Colman.
Department of Chemistry & Biochemistry

Hydrogels are heavily hydrated materials that show considerable promise as artificial extracellular matrices for use in tissue regenerative therapies. The development antibacterial hydrogels has been of great interest to the hydrogel research community as a means to combat the threat of infection during material implantation.  We have developed MAX1, a self-assembling b-hairpin peptide hydrogel whose surface exhibits inherent antibacterial activity against several pathogens prevalent in hospital settings.  Under physiological conditions, MAX1 self-assembles into a highly crosslinked, mechanically rigid hydrogel whose solvent-exposed fibrils display positive charge, which is thought to be important for antibacterial activity.  This study aims to investigate the contributions of a cation-p interaction to the antibacterial activity of a newly designed peptide hydrogel.  Cation-p interactions are a common feature of many antibacterial peptides, where they assist in the disruption of bacterial membranes.  Thus, a new b-hairpin peptide (RWMAX1) was designed, incorporating a cross-strand Tryptophan/Arginine pair, in the hope of creating a more potent antibacterial hydrogel.  The folding and self-assembly properties were assessed using circular dichroism and rheology and the antibacterial activity was investigated against E. coli and S. aureus.  Supported by the Beckman Foundation.

Structure of Voltage Sensitive Calcium Channels
in Mechanosensitive Osteocytes

Amber Sobia Majid¹, William R. Thompson², Kirk J. Czymmek¹, Randall L. Duncan^1,2, Mary C. Farach-Carson^1,2.
¹Department of Biological Sciences, ²Biomechanics and Movement Science Program

Regulation of skeletal remodeling is a major focus of attention in translational osteoporosis research. A recent association study including 200 single-nucleotide polymorphisms on human chromosome 3p21 revealed CACNA2D2, (one of four α2δ subunits of the voltage sensitive calcium channel (VSCC) complex), as a novel susceptibility marker for bone mineral density (BMD) variation. VSCCs are a complex of polypeptides that influence osteoblast mechanosensitivity and consist of a pore forming α1 subunit, an intracellular β subunit, a dimer of α2 and δ subunits, and a γ subunit in some tissues. This study characterized the structure of the VSCC complex in an osteocyte-like cell line, MLO-Y4, using RT-PCR, Western blot, and immunostaining. We demonstrated that the T-type Cav3.2 (α1H) subunit is the predominant α1 subunit expressed in osteocytic cells in vitro and in cortical bone.  Additionally, MLO-Y4 cells express α2δ1, γ7, and β14 subunits. These findings represent a shift in the expression of VSCC from long lasting (L-type) in osteoblasts to transient (Ttype) channels in osteocytes. An association of the α2δ subunit with T-type channels in osteocytes could stabilize the functional channel and provide a mechanism for interaction with the extracellular environment. This key protein complex may provide a crucial function for mechanosensitive osteocytes in bone, having implications in skeletal remodeling and BMD.  (Supported in part  by HHMI Undergraduate Science Education Grant)

Recipient of First Place Award in the ASBMB Undergraduate Poster Competition

Recipient of a FASEB MARC Program Poster/Oral Presentation Travel Award.

Design of a Cell-responsive de-PEGylation DNA Delivery System

Stephanie L. Myrick, Peter G. Millili, and Millicent O. Sullivan
Department of Chemical Engineering

We are interested in formulating efficient non-viral DNA delivery vehicles by incorporating multiple functional layers that can be sequentially cleaved off during the delivery process to expose new features. We have established a simplified model vehicle consisting of a polycation-condensed DNA core attached via linker peptides to a layer of poly(ethylene) glycol (PEG). PEG has been shown to protect biomaterials from salt-induced aggregation and immune recognition, but PEG coatings can inhibit DNA delivery at the target cell. This project is focused on validating cell-responsive de-PEGylation of the vehicle prior to delivery via the cleavage of linker peptides sensitive to matrix metalloproteinase-1 (MMP-1), a collagen-degrading enzyme upregulated by fibroblasts as they migrate through the extracellular matrix.  This migration is observed in tumor stroma and at sites of injury, making MMP-1 secretion a useful targeting signal. Having established the conditions that stimulate MMP-1 expression by human dermal fibroblasts via Western blotting and immunostaining, we are now investigating the efficiency of vehicle cleavage using light scattering in combination with salt aggregation assays (Figure 1) to detect PEG layer release. Further experiments in this area and cell studies are ongoing. Supported by HHMI, NSF, and UDRF.

Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition

Matthew Richards, Benjamin Rohe, and Mary C. Farach-Carson 
Department of Biological Sciences

Perlecan, also called HSPG2, is a heparan sulfate proteoglycan predominantly located in basement membranes and the matrix surrounding endothelial, mesenchymal and stromal cells.  The reactive stroma surrounding prostate cancer cell lines produces high levels of the protein which may play a role in delivery of growth and angiogenic factors, aiding survival and growth of metastatic tumors.  The overall goal of this project was to study the promoter in order to understand the up-regulation of perlecan in the tumor reactive stroma which occurs via transcriptional increases in perlecan biosynthesis.  The sequence for the human HSPG2 promoter was found using public databases and compared to a published human sequence (Iozzo et al., 1997) and a mouse promoter sequence found in online databases.  Several transcription factor binding sites of interest were identified for further study including NFkB, CREB, Smad3, Elk-1, c-Jun and TCF/LEF-1.  Currently we are working to build a promoter-reporter construct by isolating the promoter region using polymerase chain reaction (PCR) amplification.  Our current strategy seeks to optimize the PCR conditions with different sets of primers.  The next step will be creating a fluorescence protein reporter construct of the full promoter sequence in order to test the effects of the identified pathways.  This research was funded by the HHMI program and NIH/ NCI P01 CA098912.

Recipient of an ASBMB Undergraduate Travel Award.

The Screening of PDZ Domain Array
to Identify JAM-B Interacting Proteins

Tight junctions (TJs) host complexes that include PDZ domain-containing proteins. PDZ proteins participate in transmembrane molecule organization and TJ formation. Transmembrane protein Junctional Adhesion Molecule-A (JAM-A) is found in TJs and interacts with PDZ proteins AF-6 and ZO-1 via its PDZ binding motif. Another JAM family member, JAM-B displays such a motif but PDZ binding partners are unknown. Because of the similar roles and cytoplasmic regions of JAM-A and JAM-B, we hypothesize that JAM-B also binds to PDZ proteins. To identify these interactions, HA-tagged JAM-B will be used to probe a protein microarray spotted with PDZ domains. A cDNA construct encoding a HA-tagged GST JAM-B protein was created. E. coli BL21 cells transformed with this construct were induced by IPTG, and recombinant protein expression and solubility was analyzed by SDS-PAGE. JAM-B – PDZ domain interactions on the array will be detected by chemiluminescence. This analysis of JAM-B and PDZ protein interactions will further our understanding of signaling pathways particularly pertaining to TJ formation and regulation. Supported by an HHMI Undergraduate Science Education Grant, Beckman Foundation and the NIH.

Deletion of a single ribosomal protein, RPL29, increases bone fragility due to diminished protein synthesis and poor tissue quality, suggesting an important link between skeletal tissue growth and efficient protein production.  The goal of this study is to determine if reduced capacity to synthesize large volumes of proteins modifies bone anabolic response to mechanical load.  To test this idea, tibiae of RPL29-null mice and age-matched wild type (WT) controls were loaded at 20% maximal load, 0.5 Hz for 50 cycles/day, with a five-second rest period inserted between two cycles for two weeks. The cortical bone microstructure and mechanical properties of the non-loaded and loaded tibiae of both RPL29-null and WT mice were analyzed using micro-computed tomography (microCT) and three-point-bending tests, respectively.  Results indicate that following loading minor changes occur at the structural and material property levels in both control and null tibiae.  Ongoing studies are using dynamic histomorphometry to quantify differences in bone formation capacity between loaded and non-loaded conditions.  These studies will establish the importance of high volume protein synthesis for the regulation of bone formation. This work is supported by NIH P20 RR016458-06 and an HHMI Undergraduate Science Education Grant.

Our lab has shown that MDA-MB-231 human breast cancer cells can be isolated from the chick embryo brain after injection into the extra-embryonic vasculature.  Others have demonstrated with nude mice that re-injection of these cells results in sublines with enhanced capability to metastasize to the brain.  It remained unknown whether this was the result of cells specifically targeting the brain, or increased survival in the brain compared to other organs.  We transfected MDA-MB-231 cells with neomycin resistance and lacZ genes to enable drug-selection and visualization.  This allowed us to quantitate cells which extravasated into the brain and grew as colonies after dissection, dissociation, and drug selection following injection.  To determine the sensitivity of the chick embryo system for detecting brain metastases, cells were injected at high and low concentrations.  Colonies were detected following injection of as few as 5,000 cells (average of 32.2 colonies/brain).  Sublines of cells which had been through the brain up to five times were created.  The number of colonies isolated from the brain and liver after re-injection was compared to that of previously uninjected cells.  Re-injected cells produced a greater number of colonies, suggesting that these cells specifically target the brain.  This project was supported by NIH grant 2 P20 RR016472-07 under the INBRE Program of the National Center for Research Resources.