Metalloprotein Research
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Lydia Finney Biosciences Office: X-ray Science Office:
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Biographical Sketch> Research Highlights
Metalloproteomics: X-ray Imaging of the Multi-Dimensional Proteome Coinvestigators and Collaborators: Stefan Vogt, Janet Wolford, Carol Giometti, and Tripti Khare.
An estimated one third of all proteins bind metal ions, and many of these proteins have regulatory or catalytic functions. Metals, including calcium, iron, copper and zinc, are imported first into the cell and then further into various cellular compartments, carefully directed into the correct metalloproteins. There, they serve numerous roles as enzymatic reaction centers, sites of structural scaffolding, as well as in signal transduction. Their activity is subject to regulation through coordinated changes in metal cofactor availability. Capturing these changes, by visualizing the extent of metal-binding to cellular proteins, is critical to understanding the physiology of metalloproteins.
Yet, imaging technology has not enabled us to visualize metals bound to proteins that have been separated by gel electrophoresis as readily as we can stain and image the proteins themselves. This project takes a simple, comprehensive approach for identifying and quantifying metalloproteins in complex samples using native-PAGE and synchrotron x-ray fluorescence imaging. By pairing the development of specialized, non-coordinating, non-denaturing 2D gel electrophoresis techniques with the development of dedicated, rapid wide-area XRM imaging capabilities, we aim to identify the entire complement of metalloproteins in a given biological sample. Through the application of these approaches to both known, characterized mixtures of proteins, as well as uncharacterized systems, we will expand the capabilities for high-throughput identification and characterization of life processes involving metal ion metabolism.
The Role of Zinc in Stem Cell Pluripotency: Elemental Imaging of Human Embryonic Stem Cells Coinvestigators and Collaborators: Qiaoling Jin, Yasmin Chishti, Janet Wolford, Stefan Vogt, and Liaohai Chen.
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Zinc plays numerous cellular roles, both as a critical structural element and as an enzymatic reaction center in many proteins. We are working to elucidate the role of zinc in the dynamic cellular process of human embryonic stem cell differentiation at a systems level. This study takes advantage of unrivaled high-resolution hard x-ray fluorescence microscopy (XFM) capabilities at the Advanced Photon Source. Our initial work has shown a relationship of zinc to the delicate balance between proliferation and differentiation in stem cell colonies. Using XFM and our new metalloproteomics techniques, we aim to further delineate the role of zinc in this balance. A better understanding of this relationship could lead us to an intervention point in the system, and bring us closer to unlocking the potential of stem cell biology. |
Zinc map of a stem cell colony section. |
Copper and angiogenesis: unraveling a relationship key to cancer progression Coinvestigators and Collaborators: David Glesne and Stefan Vogt
Cancerous tumors are hazardous because of their ability to grow, metastasize, and spread throughout the body. This process relies upon angiogenesis, or the growth of new blood vessels, to feed and support the tumor. While copper is critical to angiogenesis, the underlying molecular mechanism for this sensitivity has been more elusive. Direct imaging of metals using x-ray microprobes has given us a completely new view of cellular copper. We have applied XFM to analyze the critical sensitivity of angiogenesis to copper, examining cellular distributions of copper as a function of time during this process. Using XFM, we see that copper is spatially sensitive, as it is translocated from perinuclear areas of the cell towards the tips of extending filopodia and across the cell membrane into the extracellular space. Such findings may explain the heightened sensitivity of this cellular process to this transition metal and establish the kinds of regulatory roles that the spatial dynamics of cellular transition metals may play. In better understanding and characterizing the translocation of copper during angiogenesis, we seek to establish the precise mechanism of this transient secretion. We hope to identify an important and specific intervention point for the entire angiogenic process, enabling the development of better cancer-fighting drugs.
