Pharmacology Of Neurotoxins

National Institute Of Mental Health

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Abstract

We are seeking to identify protein biomarkers of neuropsychiatric disorders, such as the obsessive compulsive syndrome that follows streptococcal infections in pediatric patients. A combination of immunoaffinity strategies will be used to separate proteins from patient sera. The mass spectra of peptides from these proteins will be compared to identify those characteristic of patient state and the disease trait (as well as other diseases). We have tested several strategies to isolate proteome fractions that are largely free from the most common circulating proteins (albumin, IgG, etc) and will now test the reproducibility of the proteome fraction profile using MALDI/TOF and ESI/TOF measurements of the intact proteins and their proteolytic digests. We have employed a tandem affinity purification (TAP) strategy to generate and isolate tagged yeast Tdp1( a tyrosine-DNA phosphodiesterase) protein. We confirmed the strategy using Rfc5 as a yeast target in parallel studies. LC/MS results demonstrated the expected isolation and characterization of multiple peptides from the known Rfc5 replication complex, confirming expected tightly associated protein interactions. However, Tdp1-TAP tagged did not bind consistently with other proteins. These observations are being pursued by determining whether complementary chemical methods could be used to covalently associate Tdp1 to its yeast partners early in the isolation process. Kynurenine 3-hydroxylase and indoleamine-2,3-dioxygenase are highly activated during CNS inflammation. These proteins have not been characterized in their native state. We are isolating both enzymes over-expressed in E. coli and comparing these primary structures with the same proteins affinity isolated from activated human cells in order to understand the protein regulation and post-translational modifications.