John David Dignam, Ph.D.
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John David Dignam, Ph.D. Professor Emeritus David.Dignam@utoledo.edu |
RESEARCH INTERESTS:
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 (Cell
and Cancer Biology Track).
EDUCATION:
Ph.D. 1977 University of Texas College of Graduate Studies of Biomedical Sciences
(Biochemistry)
B.S. 1972 University of Houston (Microbiology)
ACADEMIC APPOINTMENTS:
2004 - 2014 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
REPRESENTATIVE PUBLICATIONS:
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.