John E. Wiktorowicz, Ph.D., Professor
The comparatively new field of differential proteomics promises the design and implementation of pharmacological, immunological, or other interventional strategies affecting the expression and function of proteins. The operative assumption is that the pool of proteins differentially expressed as a result of cellular perturbation is the ultimate cause of the cellular condition. The challenge implicit in this task involves the detection and quantification of the uniquely expressed proteins that represent less than 1% of the
total numbers of protein expressed. The effective solution therefore requires the separation and quantification of all of the proteins expressed in normal and affected cells, and the isolation and identification of those that are uniquely expressed and therefore causative of the cell’s altered state. Having recently joined the Department from Lynx Therapeutics, where I was Director of Proteomics, my lab is engaged in completing the development of a technology that automatically accomplishes the separation, accurate quantification, and isolation of the uniquely expressed low-abundance proteins, such as those that appear in nuclear domain-10 (ND-10) structures. ND-10 structures, found associated with the nuclear matrix, are constructs of electro-diffuse and electro-dense material observed near heterochromatin. Biochemically, they have been characterized as repositories of transcription factors organized by the promyelocytic leukemia protein (PML) in its sumoylated form (small ubiquitin-like modifier) and thus can serve as structures naturally enriched in low-abundance proteins. The composition of ND-10 structures is incomplete but is known to consist of at least 40 proteins under varying conditions. In its role as the putative depot of transcription factors and ancillary proteins, the complexes serve as sites for replication of various viruses. The understanding of the dynamic processes that lead to ND-10 protein accumulation and dispersal requires the detection, quantification, and temporal cellular distribution of the ND-10 proteins. This accomplishment will yield important insights into viral control of host metabolic processes, as well as cellular response to environmental stress. My research interests and goals exhibit a two-tiered purpose: to develop new proteomics technology to address the low abundance protein issue, and to apply the new technology to the characterization of ND-10 complexes. My first objective requires the completion of a device that is a liquid-based two-dimensional automated electrophoretic protein separation system (the Protein ProFiler™) that will enable sensitive (fmol), reproducible, and automated high-throughput protein separations (30 min). It will replace or considerably extend current electrophoretic and chromatographic technologies generally acknowledged to be technically difficult, semi-quantitative at best, and time-consuming. My subsequent goal is the enumeration and quantification of the low-abundance proteins that constitute the nuclear ND-10 structures with the goal of understanding the dynamic processes that lead to their accumulation and dispersion as the result of viral stimulation. The hypothesis that I wish to address in the long-term states that these structures are a critical component in the propogation of viral infection, and interference in the accumulation and/or dispersion of the constituent proteins will affect the course and prognosis of viral disease.