Although the three-dimensional structure of t-PA 
has not been elucidated, Dr. Gething and her col- 
leagues have been able to model all the domains, 
using the known structures of homologous domains 
in other proteins. Site-directed mutants designed us- 
ing these proposed structures have provided infor- 
mation about the role of individual amino acid se- 
quences of t-PA. Variant enzymes have been 
generated that are efficient, fibrin-stimulated plas- 
minogen 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 circu- 
lation, they may have significant potential 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 simian virus 40 
(SV40) T antigen from the tyrosinase promoter de- 
velop nonmetastatic melanoma with high fre- 
quency. Orthotopic translation of these tumors to 
nontransgenic animals results in progressive growth 
but not metastasis. Similar transplantation into nude 
mice, however, results in a frequent occurrence of 
metastasis to the liver, as is seen in human ocular 
melanoma. Current experiments involve investiga- 
tions of the mechanisms by which these tumors ac- 
quire metastatic potential by crossing these animals 
with other transgenic mice that express t-PA (wild- 
type or inhibitor-resistant forms) or PAI- 1 from the 
same tyrosinase promoter. 
Transgenic Models of Autoimmune Disease 
Dr. Gething and her colleagues are using trans- 
genic mice that express the HA glycoprotein of influ- 
enza virus to study the mechanisms underlying the 
development of autoimmunity. RIPHA mice, which 
express HA from the rat insulin II promoter/en- 
hancer only in the ^ cells of the pancreas, can de- 
velop an immune response to HA and other antigens 
of pancreatic (5 cells. However, the immune re- 
sponse is not as penetrant or as active as that ob- 
served in non-obese diabetic (NOD) mice, and 
current studies focus on a comparison of these two 
models. To compare the specific immune response 
to the well-characterized HA antigen in the two 
lines, the RIPHA gene has been introduced onto the 
NOD genetic background by crossing of RIPHA 
and NOD mice followed by extensive back- 
crossing to NOD. This study is being complemented 
by an analysis of the role of the inflammatory cyto- 
kines interleukin-1 (IL-1), tumor necrosis factor 
(TNF), and IL-6 during autoimmune diabetes. Re- 
cent studies by others indicate that IL- 1 and TNF can 
inhibit the onset of diabetes in NOD mice, probably 
via immunoregulation. The effects of immunostimu- 
lating RIPHA and RIPHA/NOD mice with influenza 
virus will be examined. 
Role of Stress-70 Proteins in Folding 
and Assembly of the Tumor-Suppressor 
Protein p53 
Until recently it was widely assumed that the fold- 
ing and oligomerization of newly synthesized poly- 
peptides and their subsequent molecular rearrange- 
ments are spontaneous processes that do not require 
the intervention of other cellular proteins. How- 
ever, it is now apparent that members of the stress- 
70 (hsp70; heat-shock protein 70) protein family 
are intimately involved in facilitating protein fold- 
ing and assembly within prokaryotic and eukaryotic 
cells. 
Dr. Gething's previous studies of the interaction 
of the endoplasmic reticulum (ER) stress-70 protein 
BiP with newly synthesized membrane and secre- 
tory proteins has led to an understanding of the role 
of stress-70 proteins in stabilizing unfolded or 
partly folded polypeptides in a form competent for 
further folding and oligomeric assembly. Current 
studies focus on the role of the cytosolic stress-70 
protein hsc70 in modulating the structure or activ- 
ity of mutant forms of the tumor-suppressor protein 
p53. Others have shown that p53 plays a role in 
regulation of normal cell growth and that it binds to 
a number of viral transforming proteins, including 
SV40 T antigen. Mutations in p53 convert the pro- 
tein to an oncogenic form capable of cooperating 
with activated ras to transform cells. These mutant 
forms of p53 appear to be altered conformationally, 
as indicated by loss of T antigen recognition, altered 
reactivity with monoclonal antibodies, and cotrans- 
lational binding to hsc70. 
Dr. Gething's group is using the fd bacteriophage 
expression system to analyze the interaction be- 
tween p53 and hsc70. Oligonucleotide sequences 
encoding peptides from wild-type and mutant forms 
of p53 have been synthesized as amino-terminal fu- 
sions to the phage adsorption protein pIII and ex- 
pressed on the surface of phage particles. By screen- 
ing p53-peptide-expressing phage for binding to 
hsc70, with subsequent enrichment and amplifica- 
tion in bacteria of those expressing hsc70-binding 
epitopes, the region (s) in p53 recognized by hsc70 
will be identified. 
Protein Folding in the ER: Role of BiP 
In the cell, as in vitro, the final conformation of a 
protein is determined by its amino acid sequence. 
60 
