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¡¡ James F. Riordan, Deputy Director Research
Activities Metallobiochemistry Dr.
Riordan has been studying the structure, function and mechanism of action of angiotensin
converting enzyme, ACE, for the past 20 years. ACE
is a zinc dipeptidylcarboxypeptidase that is central to the control of blood pressure by
virtue of its activity toward angiotensin I and bradykinin.
Inhibitors directed against the active site zinc atom of ACE are widely used
in anti-hypertensive therapy. Somatic
ACE is synthesized in endothelial, epithelial and neuroepithelial cells as a precursor
protein that is transported to the plasma membrane where it resides as a class I
ectoenzyme. The extracellular portion of the
molecule consists of two N-terminal tandem catalytic domains of about 600 residues each,
followed by a 22-residue transmembrane domain and a C-terminal 28-residue cytosolic
domain. A second membrane-bound form of ACE
is generated by alternative transcription initiation and is found only in post-meiotic and
generating sperm cells. The biological
function of this so-called testicular ACE, which has only a single catalytic domain, is
unknown. ACE was
chosen initially as a target for investigation by Dr. Riordan because it was a zinc enzyme
of major physiological significance whose mechanism of action was unclear and for which
potent inhibitors had not yet been identified. In
the course of these studies ACE has emerged as a model system for a number of important
biological problems. It was among the first
membrane proteins recognized to undergo solubilization from the cell surface by means of
proteolytic cleavage, a process now referred to as shedding. The enzymology of shedding is a rapidly growing
area of research with implications for inflammation, cyokine regulation and Alzheimer's
disease, among others. The two active sites
of ACE exemplify the divergence of biological functions by gene duplication and
independent evolution. The specific functions
of the two sites are still under investigation. Similarly,
the functional relationship between the somatic and testicular forms of ACE provides an
alternative means to study this problem. ACE
is also a useful system to study the biosynthesis of zinc enzymes, in particular the steps
leading to the insertion of zinc into the protein. Gene
knock-out experiments have shown that the testicular ACE is essential for fertility but
the peptide substrates of the enzyme have not been identified. During the
past year Dr. Riordan and his colleagues completed a study of the structural organization
of somatic ACE by the use of proteolytic enzymes. Proteolytic
cleavage by endoproteinase Asp-N breaks the T615 - D616 peptide bond and separates the two
catalytic domains. This enzyme also breaks
the L1219 - D1220 peptide bond to remove the transmembrane and cytosolic domains. The two catalytic domains were isolated by
immunoaffinity chromatography and their enzymatic characteristics toward a variety of
substrates was established. Further
studies on the shedding of ACE from genetically transformed CHO cells were pursued by Dr.
Riordan's colleagues, Drs. Mario Ehlers and Edward Sturrock at the University of Cape
Town. In order to determine the structural
features that permit some cell surface proteins to be shed while others remain membrane
bound, these investigators introduced sequence variants into the juxtamembrane region of
ACE, including sequences from the EGF-receptor which normally does not undergo cleavage. Remarkably, the variant protein was also cleaved,
suggesting that structural features outside the juxtamembrane region are important for
mediating cell surface peptidase activity. The
protease responsible for the shedding of ACE, and other membrane proteins as well, has
been identified as a member of the adamalysin family known as TACE (TGF-alpha converting
enzyme). Dr. Riordan served as a consultant
in these studies. Work also
continued on the x-ray crystallography of testicular ACE.
Support for this was was provided by a grant from the Wellcome Research
Trust to Drs. Ehlers and Sturrock in South Africa, and Dr. Ravi Acharya in Bath, England. Angiogenin When
angiogenin is added to the culture medium of sparsely growing human endothelial cells, it
binds to the cell surface (presumably to specific angiogenin receptors), is rapidly
internalized and is translocated to the cell nucleus where it accumulates in the
nucleolus. In order to determine the cellular
components involved in the process of nuclear translocation the effect of various
inhibitors was examined. Treatment of cells
with colchicine, nocodazole and taxol, which disrupt the microtubule system, does not
affect the nuclear translocation process, suggesting that cells transport internalized
angiogenin in a microtubule independent fashion. Lysosomal
inhibitors, chloroquine and leupeptin, neither inhibit nor enhance the nuclear
translocation of angiogenin, indicating that lysosomal targeting and processing are not
required for, and do not compete with, nuclear translocation. Moreover, treatment of cells with a tyrosine
kinase antagonist, genestein, does not change the ability of the cells to translocate
angiogenin into the nucleus. These results
suggest that exogenous angiogenin is translocated by a mechanism that does not require
activation of tyrosine kinase, but includes receptor-mediated endocytosis, microtubule and
lysosomal independent transport across the cytoplasm and nuclear localization
sequence-assisted nuclear import. In order
to understand the mechanism of action of angiogenin and to search for angiogenin
inhibitors with therapeutic potential, Dr. Riordan and his colleagues employed the
systematic evolution of ligands by exponential enhancement (SELEX) procedure of Gold and
coworkers to isolate oligodeoxynucleotide aptamers for angiogenin. They isolated an oligonucleotide that inhibits the
ribonucleolytic, mitogenic and angiogenic activity but does not affect its nuclear
localization in endothelial cells. Remarkably,
the oligonucleotide ligand undergoes nuclear cotranslocation with angiogenin and
accumulates in the nucleus in a stoichiometric ratio, ¡¡
suggesting
that angiogenin exerts its ribonucleolytic activity in the cell nucleus and that
inhibition of this activity within the nucleus is an effective means to abolish its
angiogenic activity. Dr.
Riordan, together with Dr. Hu and co-workers, has been attempting to identify the 170 kDa
cell surface protein that is thought to be the angiogenin receptor. Nanogram amounts of this protein obtained from
sparse cell cultures were proteolytically digested and the resultant peptides were
separated by HPLC. Several of the peptides
were subjected to microsequencing and oligonucleotide probes were synthesized based on
these sequences. A cDNA library made from
mRNA isolated from sparse HUVE cells was cloned and screened with labelled angiogenin but
without success. Further attempts to identify
the angiogenin receptor using this and alternative strategies are now in progress. Support Dr.
Riordan's ACE research was supported by the Endowment for Research in Human Biology, Inc. Publications Metallobiochemistry Sturrock,
E.D., Danilov, S.M., Riordan, J.F. (1997) Limited
Proteolysis of Human Kidney Angiotensin Converting Enzyme and Generation of Catalytically
Active N- and C-terminal Domains. Biochem.
Biophys. Res. Commun. 236, 16-19. Angiogenin Li, R.,
Riordan, J.F., Hu, G.-F. (1997). Nuclear Translocation of Human Angiogenin in Cultured
Human Umbilical Artery Endothelial Cells is Microtubule and Lysosome Independent. Biochem.
Biophys. Res. Commun. 238, 305-312. Nobile,
V., Russo, N., Hu, G.-F., Riordan, J.F. (1998). Inhibitors of Human Angiogenin by DNA
Aptamers: Nuclear Colocalization of an
Angiogenin-Inhibitor Complex. Biochemistry 37, 6857-6863. Hu, G.-F.,
Riordan, J.F., Vallee, B.L. (1998). Angiogenin. In: Human Cytokines: Handbook for
Basic and Clinical Research (ed: B.B. Aggarwal) Blackwell
Science, Vol. III, 5, 67-91. Vallee,
B.L., Riordan, J.F. (1997) Organogenesis and Angiogenin. Cell. Mol. Life Sci. 53,
803-815. D¡¯Alessio,
G., and Riordan, J.F. (1997) Ribonuclease Structures and Functions. Academic Press, New York. ¡¡
Other
Professional Activities Dr.
Riordan was again a tutor in the Integrated Human Physiology course for first year medical
students, his eleventh consecutive year as a Holmes Society tutor. Dr.
Riordan attended a National Academy of Sciences Colloquium entitled Proteolytic Processing
and Physiological Regulation held at the NAS Beckman Center in Irvine, CA in February
1999. He also chaired the session on
Angiogenin at the Fifth International Meeting on Ribonucleases held at the Airlie Center
in Warrenton, VA, in May 1999. Dr.
Riordan continued as an Associate Editor of Biochemistry, a position he has held since
1994. Owing to the heavy workload of this
position, he resigned as Editor-in-Chief of the Journal of Inorganic Biochemistry and as
an Executive Editor of Analytical Biochemistry, positions he held since 1979. He remained a member of the Editorial Boards of
the Journal of Protein Chemistry and the European Journal of Biochemistry, and he
continues to serve as a reviewer for numerous other journals. Dr.
Riordan served as Chairman of SSS-6, an NIH Special Emphasis Panel on Chemistry and
Related Sciences that reviews Small Business Innovative Research grant applications. He also chaired a Shared Instrumentation (Mass
Spectroscopy) Special Emphasis panel that reviews applications to the Center for
Scientific Review of the NIH. Collaborations Dr. K.
Ravi Acharya, Univ. Bath, U.K. - Crystallization and
structure characterization of angiotensin converting enzyme. Dr. Peter
Bünning, Hoechst Marion Roussel, Frankfurt, Germany - Cell biology of angiogenin. Dr. Sergei
Danilov, Univ. Illinois Chicago - Structure and function of angiotensin converting enzyme. Dr. Mario
Ehlers, Univ. Cape Town, South Africa - Crystallization, structure characterization and
solubilization of angiotensis converting enzyme. Dr.
Stanislaw Mikulski, Alfacell Corp. Bloomfield, NJ - Studies on the nuclear translocation
of onconase, a member of the ribonuclease family. Dr. Edward
Sturrock, Univ. Cape Town, South Africa - Crystallization, structure characterization and
solubilization of angiotensin converting enzyme. ¡¡
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