John David Dignam, Ph.D.
|John David Dignam, Ph.D.
The research of my laboratory is focused on nucleic acid enzymology, nucleic acid protein interaction and more recently on the development of modified human albumin for the treatment of shock.
Aminoacyl tRNA synthetases are a class of enzymes that ensure the fidelity of protein synthesis by attaching amino acids to their cognate tRNAs. Studies of the thermodynamics of binding of ligands to glycyl tRNAs suggest that the formation of glycyl adenylate, an obligate intermediate on the reaction pathway, is accompanied by a significant conformation change in the protein that alters the affinity of the enzyme for tRNA. We are extending these studies to alanyl-tRNA synthetase, an enzyme with editing activities for both noncognate adenylates (glycyl- and seryl-adenylates) and for misacylated tRNAala (acylated with serine or glycine). Our thinking is that enzyme ligated with the noncognate adenylate will have reduced affinity for tRNAala, resulting in a reduction in misacylation with serine or glycine and increased specificity.
Adenoassociated virus 2, a nonpathogenic parvovirus, has attracted interest as a potential gene therapy vector. It has a small (4500 nucleotides) genome that encoding four DNA helicases that are required for viral DNA replication and efficient packaging of single stranded DNA into virions. Rep78, Rep68, Rep52 and Rep40 share common structural elements in their helicase domains, but differ at their N-termini and C-termini as a result of differential splicing and different mRNAs arising from the use of different transcription start sites. The larger Rep proteins, Rep78 and Rep68, assemble into stable, hexameric oligomers on specific secondary structures on the 3’ and 5’ termini of the single stranded viral DNA. Rep78 and Rep68 also have a site specific nuclease activity, residing in an N-terminal structure, that creates a priming site in the template for DNA replication. The smaller replication proteins, Rep40 and Rep52, require ATP to bind DNA, show no sequence specificity in DNA binding and lack the N-terminal nuclease domains. Rep52 and Rep40 are implicated in packaging plus and minus single stranded DNA into virions. Our studies are aimed at understanding how these proteins assemble on DNA structures and the specificity of their interaction with DNA.
A third project is the development of a modified form of human albumin as a treatment for hypovolemia resulting from increased permeability of capillaries that occurs in shock. Increased permeability of capillaries (also called capillary leak) to macromolecules, such as albumin, occurs in number of clinical conditions including sepsis and trauma. Albumin extravasates into the extracellar space with a resulting loss of the oncotic gradient that draws water back from tissues into blood vessels. We have demonstrated that polyethylene glycol-modified albumin is effective in animal models of sepsis and hemorrhagic shock in improving organ perfusion and maintaining blood pressure. The rationale behind using polyethylene glycol-modified albumin is that this modified protein has a sufficiently large hydrodynamic radius that precludes its passing through defects in capillaries that occur in shock and is thus retained within blood vessels to maintain the oncotic gradient.
Member of the mentoring faculty for the Biomedical Sciences Graduate Program (Cancer
Ph.D. 1977 University of Texas College of Graduate Studies of Biomedical Sciences (Biochemistry)
B.S. 1972 University of Houston (Microbiology)
2004 - pres. Professor, Biochemistry and Cancer Biology, University of Toledo College of Medicine
1988 - 2004 Associate Professor, Biochemistry and Molecular Biology, Medical University of Ohio
1982 - 1988 Assistant Professor, Biochemistry, The University of Mississippi
1980 - 1982 Postdoctoral Fellow, Biological Chemistry, Washington University
1977 - 1980 Postdoctoral Fellow, Biochemistry, University of Connecticut
Assaly, R.A., Habib, R.H., Azizi, M., Shapiro, J.I. and Dignam, J.D. (2008) Use of multiple fluorophores for evaluating microvascular permeability in control and septic rats. Clin. Sci. (Lond.) 114:123-130.
Dignam, S., Collaco, R.F., Bieszczad, J., Needham, P., Trempe, J.P. and Dignam, J.D. (2007) Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase. Biochemistry 46:568-576.
Needham, P.G., Casper, J., Dignam, J.D. and Trempe, J.P. (2006) Characterization of adeno-associated virus Rep protein-mediated inhibition of transcription of the adenovirus major late promoter in vitro. J. Virology 80:6207-6217.
Timpe, J., Bevington, J., Casper, J., Dignam, J.D. and Trempe, J.P. (2005) Mechanisms of adeno-associated virus genome encapsidation. Current Gene Therapy 5:273-285.
Casper, J., Timpe, J., Dignam, J.D. and Trempe, J.P. (2005) Identification of an adeno-associated virus Rep protein binding site in the adenovirus E2a promoter. J. Virology 79:28-38.
Sampath, P., Mazumder, B., Seshadri, V., Gerber, C.A., Chavatte, L., Kinter, M., Ting, S.M., Dignam, J.D., Kim, S., Driscoll, D.M. and Fox, P.L. (2004) Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation. Cell 119:195-208.
Assaly, R.A., Azizi, M., Kennedy, D.J., Amauro, C., Zaher, A., Houts, F.W., Habib, R.H., Shapiro, J.I. and Dignam, J.D. (2004) Plasma expansion by polyethylene-glycol-modified albumin. Clin. Sci. (Lond.) 107:263-272.
Collaco, R.F., Kalman-Maltese, V., Smith, A.D., Dignam, J.D. and Trempe, J.P. (2003) A Biochemical Characterization of the Adeno-Associated Virus Rep40 Helicase. J. Biol. Chem. 278(36):34011-34017.
Dignam, J.D., Nada, S. and Chaires, J.B. (2003) Thermodynamic Characterization of
the Binding of Nucleotides to Glycyl-tRNA Synthetase. Biochemistry 42(18):5333-5340.
Dignam, J.D., Qu, X. and Chaires, J.B. (2001) Equilibrium Unfolding of Bombyx mori Glycyl-tRNA Synthetase. Journal Biol. Chem. 276(6):4028-4037.
Trempe Norcum, M. and Dignam, J.D. (1999) Immunoelectron Microscopic Localization of Glutamyl-/ Prolyl-tRNA Synthetase within the Eukaryotic Multisynthetase Complex. Journal Biol. Chem. 274(18):12205-12208.
Ren, J., Qu, X., Chaires, J.B., Trempe, J.P., Dignam, S.S. and Dignam, J.D. (1999) Spectral and Physical Characterization of the Inverted Terminal Repeat Structure of Adenoassociated Virus 2. Nucl. Acids Res. 27 (9):1985-1990.