Molecular Analysis of Proteins Involved 
in Human Disease 
Mary-Jane H. Gething, Ph.D. — Investigator 
Dr. Gething is also Professor of Biochemistry at the University of Texas Southwestern Medical Center at 
Dallas. She received her bachelor 's and Ph.D. degrees from the University of Melbourne. After holding 
research fellowships in Cambridge, England, with Brian Hartley and in London with Michael Waterfield, 
she joined the scientific staff of the Imperial Cancer Research Fund, London. After that she was a senior 
staff investigator at the Cold Spring Harbor Laboratory, New York, before going to the Southwestern 
Medical Center. 
OUR studies on experimental models of hu- 
man disease involve three systems: 1) hu- 
man tissue-type plasminogen activator (t-PA), a 
serine protease involved in fibrinolysis, tissue re- 
modeling, and metastasis; 2) the hemagglutinin 
of influenza virus, which is being used to develop 
models of autoimmune disease in transgenic 
mice; and 3) the tumor-suppressor protein p53 
and its interaction with cytosolic stress-70 
proteins. 
Role of t-PA in Thrombolysis and Metastasis 
Many normal and abnormal biological pro- 
cesses that require extracellular proteolysis, in- 
cluding thrombolysis, tissue remodeling, and 
metastasis, are mediated by plasminogen activa- 
tors that cleave plasminogen to the active pro- 
tease plasmin. One such activator, t-PA, is the 
principal thrombolytic agent in the circulation, 
and its elevated expression is thought to be 
linked to increases in the metastatic potential of 
some types of tumor cells, including malignant 
melanomas. 
The t-PA protein is composed of a number of 
independent structural domains. The finger do- 
main and an epidermal growth factor (EGF)-like 
domain are involved in the initial, high-affinity 
binding of t-PA to fibrin, while stimulation of t-PA 
activity requires secondary, lower-affinity inter- 
actions of fibrin with either of two kringle do- 
mains of the molecule. The binding of t-PA to 
specific clearance receptors on hepatic cells also 
involves sequences within the finger and/or EGF- 
like domains. Finally, a specific inhibitor, plas- 
minogen activator inhibitor ! (PAI-1), interacts 
with the active site in the carboxyl-terminal cata- 
lytic domain. 
Although the three-dimensional structure of 
t-PA has not been solved, we have been able to 
model all the domains through use of known 
structures of homologous domains in other pro- 
teins. Site-directed mutants using these proposed 
structures have provided information about the 
role of individual amino acid sequences of the 
protein, and variant enzymes have been gener- 
ated that are efficient, fibrin-stimulated plasmin- 
ogen activators but are resistant to inhibition by a 
variety of serpins, including PAI- 1 , or do not bind 
to the t-PA receptor(s) involved in clearance of 
the enzyme in the liver. Because these mutant 
enzymes should have an extended effective life in 
the circulation, they may have significant poten- 
tial for use in thrombolytic therapy of patients 
with myocardial infarction. 
The variant enzymes are also being utilized to 
test the role of t-PA in metastasis of malignant 
melanoma cells. Transgenic mice expressing the 
T antigen oncogene from simian virus 40 (SV40) 
under the control of the mouse tyrosinase pro- 
moter develop ocular melanoma with high fre- 
quency. These tumors are transplantable to non- 
transgenic animals but are not metastatic. We are 
currently developing additional lines of trans- 
genic mice that express wild-type or inhibitor- 
resistant forms of t-PA from the same tyrosinase 
promoter. Crossing of the Ty-Tag and Ty-tPA 
transgenic mice will reveal whether an increased 
level of t-PA production in melanoma cells would 
result in increased metastatic potential. 
Transgenic Models of Immune Tolerance 
and Autoimmune Disease 
We are using transgenic mice to study the de- 
velopment of immunological responses to the 
hemagglutinin (HA) of influenza virus. RIPHA 
mice, which express HA from the rat insulin II 
promoter/enhancer only in the /3-cells of the pan- 
creas, promise to provide a valuable model for 
the study of immune tolerance and autoimmune 
diabetes. These mice display no physiological 
problems until 4-5 months of age. After that, in- 
creases in the blood glucose levels begin to ap- 
pear, and shortly thereafter these mice develop a 
severe hyperglycemia that is responsive to 
insulin. 
In the RIPHA-33 line, both male and female 
animals can develop an immune response to HA 
and other antigens of pancreatic |8-cells. How- 
ever, male mice become hyperglycemic with 
much higher frequency (45 percent ) than female 
mice ( 5 percent ) . Recent studies have shown that 
the amount of fat in the diet is an important regu- 
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