Current Beckman Scholars
Timothy Fernandez (UAB Beckman Scholar)
Structural, Biophysical, Biochemical Characterization of the Interactions between Fas and Calmodulin
Apoptosis is a strictly regulated process by which abnormal cells are removed from the body without altering the immune system or generating an inflammatory response. Inappropriate apoptosis (enhanced or diminished) is linked to many human diseases including neurodegenerative and autoimmune disorders, AIDS, and many types of cancers. The apoptotic pathway is normally initiated by cell surface death receptors such as Fas. These receptors undergo a conformational change in response to their cognate ligands (FasL), allowing them to interact with adaptor proteins such as Fas-associated death domain (FADD). Fas-FasL interaction leads to activation of caspase 8 (by FADD) and formation of death-inducing signaling complex (DISC). DISC formation and subsequent protein recruitment is a critical initial step in regulating Fas-mediated apoptosis. There is compelling evidence that Fas interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Among these is calmodulin (CaM), which is recruited into DISC in cholangiocarcinoma cells. It has been hypothesized that Fas-CaM interaction may affect Fas-FADD interaction. Thus, this interaction regulates DISC assembly and inhibits apoptosis in cholangiocarcinoma and other cancer cells. Fas-CaM interaction appears to be an inhibitory component of DISC and may play a vital role in obstruction of caspases activation. I am interested in understanding the structural determinants of Fas-CaM interaction, which will be critical to understanding the precise molecular mechanism of Fas-mediated apoptosis and mechanism of inhibition. The interaction between Fas and CaM will be characterized using a combination of structural, biochemical and biophysical methods. These studies will likely lead to new strategies to develop inhibitors of these interactions and thus to cancer treatment. Isothermal titration calorimetry (ITC) will be used to characterize the binding of CaM to Fas. Nuclear Magnetic Resonance (NMR) methods, protease digestion, and mass spectrometry methods will be used to identify specific residues in the CaM-Fas interface. The proposed studies will establish a potential link between CaM/cell signaling pathway and aid in the development of osteoporosis and cancer treatments.
Michael Longmire (UAB Beckman Scholar)
The Elucidation of the Ethidium-DNA Complex Structure: The DNA Intercalator
My project is focused on answering a question that has eluded structural biologists for some time. Ethidium bromide is a chemical that is known to bind to DNA and has found a home in biotechnology and molecular biology research labs around the world as a fluorescent probe. Work done more than half a century ago characterizes the binding event. This event is believed to be the result of intercalation, where the drug inserts itself between the base pairs of duplex DNA. This was later confirmed in a crystal of ethidium complexed with an RNA dimer. However, this structure fails to demonstrate the structural effects of ethidium bound to DNA.
A challenge of the ethidium-DNA interaction comes in the promiscuity of the binding event; ethidium has minimal preferences for sequence and may bind and release from the same site frequently. My projects focus is to create a singular species of ethidium bound to DNA irreversibly. To accomplish this, I must develop a method of directing the binding event and securing it. This is to be accomplished with the use of distamycin A – a sequence-specific drug that binds to the minor groove of DNA – and ethidium monoazide – a derivative of ethidium that creates a covalent bond when exposed to visible light.
The first part of my project involves the novel synthesis of distamycin. With the help of Dr. Sadanandan Velu, a synthetic strategy has been developed that proposes to be more efficient than previously published works. The distamycin synthesized in this process will then be used to form a complex with a ten base pair DNA sequence, essentially creating a single open site for ethidium to bind.
The following step is to incorporate ethidium monoazide into this complex. Initially, this must be done in the absence of light to allow the drug to intercalate identically to the parent ethidium. This is followed by photolysis of the complex, creating an ethidium adduct situated between the base pairs. Removal of the non-covalently bound distamycin is then performed by dialysis and the free drugs are separated from the ethidium-DNA complex.
Following isolation of the complex, structures may be obtained by a combination of X-ray crystallography and NMR spectroscopy. This allows for a complete profile of the ethidium-DNA complex by utilizing the two most accepted methods of structure determination. All of this work thus serves to do the following: develop a potentially better synthesis for distamycin, create a method of characterizing promiscuous intercalating drugs, and provide some missing information for the effects of ethidium on DNA, allowing for potentially better approaches to drug discovery.
Dhruv Patel (UAB Beckman Scholar)
The Dynamic Interactions Between Biological, Organic, Inorganic, and Vascularized Components in Compsite Nanomatrix Gels in Vitro
In 2005, there were an estimated 1.6 million bone grafting procedures performed in the United States. Approximately 5 to 10 percent of the procedures were the result of non-union or delayed-union healing. Non-union fractures are characterized by improper healing that results from poor re-vascularization and limited blood supply. To date, bioengineered scaffolds for bone tissue regeneration have lacked the ability to provide an osteoconductive, osteoinductive, and angiogenic environment that works synergistically to encourage bone healing.
In Dr. Jun’s lab, we have already shown that the self-assembled peptide amphiphile (PA) nanomatrix can enhance the osteogenic differentiation of human mesenchymal stem cells, however we have yet to incorporate a vascularizing component. Peptide amphiphiles are self-assembling molecules that can be inscribed with cellular adhesive ligand sequences. These ligand sequences can be used to mimic the function of key extracellular matrix proteins and in turn guide cellular behavior. The overall goal of this study is to assess the effect that co-culturing human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) on a peptide amphiphile scaffold will have on the overall bone tissue regeneration response. The objective will be met by achieving the following aims:
Specific Aim 1: The assessment of the crosstalk between hMSCs and HUVECs in a PA-RGDS scaffold through the measure of soluble factor release. Hypothesis: There will be an increase in bone morphogenic protein and vascular endothelial growth factor secretion when hMSCs and HUVECs are co-cultured compared to when they are cultured separately. The secretion of bone morphogenic protein and vascular endothelial growth factor will be assessed for the co-culture of hMSCs and HUVECs. This value will be compared to single culture controls. Soluble factor release will be measured using an enzyme-linked immunosorbent assay. Gene expression will also be assessed through real-time polymerase chain reaction to determine which cell type is responsible for releasing each factor.
Specific Aim 2: The evaluation of the osteogenic differentiation of the hMSCs. Hypothesis: HMSCs that are cultured together with the HUVECs will show greater gene expression of key osteogenic markers compared to hMSCs that are cultured separately. The expression of key osteogenic genes will be assessed for hMSCs that were cultured individually and with HUVECs. Runt-related transcription factor 2 will be the first gene evaluated. The transcription is essentially an early marker for osteogenic differentiation as it activates other crucial genes needed for osteoblast formation. Alkaline phosphatase will be used as a mid stage marker for osteogenic differentiation, and osteocalcin will be used as a late stage marker for osteogenic differentiation. Expression for these three genes will be determined using real-time polymerase chain reaction.
Specific Aim 3: The morphological evaluation of co-cultured cells. Hypothesis: Over time, the HUVECs will congregate and form vessel like structures that will be surrounded by the mesenchymal stem cells. Immunohistochemical staining will be used to image the cells. The cytoskeleton, nucleus, and the endothelial cell surface marker, CD31, will be stained so that the cell morphologies can be visualized.
By accomplishing these specific aims, valuable information on the interactions between hMSCs and HUVECs in a PA scaffold can be obtained. This information can then be used to help find alternative strategies for bone tissue regeneration applications.