Note: Descriptions are shown in the official language in which they were submitted.
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1 METHOD FOR PR-39 PEPTIDE REGULATED STIMULATION OF
ANGIOGENESIS
6
RESEARCH SUPPORT
The research effort for the invention was supported in part by grants from
the National Institutes of Health, grants ROl HL 53793 and P50 HL 56993 (MS),
11 F32 HL 10013 (RV); and by a grant from Chiron Corporation. The government
has
certain rights in the invention.
CROSS REFERENCE
The present application is a Continuation-In-Part of prior pending United
16 States Patent Application Serial No. 09/276,868 filed March 26, 1999.
FIELD OF THE INVENTION
The present invention is concerned generally with the induction of
21 angiogenesis within viable cells comprising living tissues and organs; and
is
particularly directed to mechanisms regulated by PR-39 peptides which result
in a
stimulation of angiogenesis on-demand and may be used as a controlled
therapeutic
treatment.
26 BACKGROUND OF THE INVENTION
Angiogenesis, by definition, is the formation of new capillaries and blood
vessels within living tissues; and is a complex process first recognized in
studies of
wound healing and then within investigations of experimental tumors.
Angiogenesis
31 is thus a dynamic process which involves extracellular matrix remodeling,
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1 endothelial cell migration and proliferation, and functional maturation of
endothelial
cells into mature blood vessels [Brier, G. and K. Alitalo, Trends ~l Biology
6:
454-456 (1996)]. Clearly, in normal living subjects, the process of
angiogenesis is a
normal host response to injury; and as such, is an integral part of the host
body's
homeostatic mechanisms.
6 It will be noted and appreciated, however, that whereas angiogenesis
represents an important component part of tissue response to ischemia, or
tissue
wounding, or tumor-initiated neovascularization, relatively little new blood
vessel
formation or growth takes place in most living tissues and organs of mature
adults
(such as the myocardium of the living heart) [Folkman, J.and Y. Shing, ~, B_
iol.
11 hem. 2 7: 10931-10934 (1992); Folkman, J., Nat. Med. _l: 27-31 (1995);
Ware,
J.A. and M. Simons, Na re Med. ~: 158-164 (1997)]. Moreover, although
regulation of an angiogenic response in-vivo is a critical part of normal and
pathological homeostasis, relatively little is presently known about the
control
mechanisms for this process.
16 Overall, a number of different proteins, growth factors and growth factor
receptors have been found to be involved in the process of stimulation and
maintenance of angiogenic responses. For example, a number of cell membrane-
associated proteins are thought to be involved in the processes of
angiogenesis.
Such proteins include SPARC [Sage etet al., ~ ~l_l Biol. 19~: 341-356 (1989);
21 Motamed K. and E.H. Sage, Ki n I~ .~: 1383-1387 (1997)]; thrombospondin 1
and 2 respectively [Folkman, J., ~ Med. _l: 27-31 (1995); Kyriakides et al.,
Cell Biol. 140: 419-430 (1998)]; and integrins av~35 and av~i3 [Brooks etet
al.,
Science 2 4: 569-571 (1994); Friedlander gt al., i n 2~7 : 1500-1502 (1995)].
In
addition, a major role is played by heparin-binding growth factors such as
basic
26 fibroblast growth factor (bFGF) and vascular endothelial growth factor
(VEGF); and
thus the regulation of angiogenesis is believed today to involve matrix
components
such as extracellular heparin sulfate and core proteins such as syndecans
which are
found at the surface of endothelial cells.
However, while a number of heparin binding growth factors (including
31 VEGF, FGF1 and FGF2) have been shown to promote angiogenesis in-vitro and
in-
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1 vivo, their process involvement appears limited to tissues demonstrating
some form
of inflammatory response to trauma (as defined by the presence of blood-
derived
macrophages), be it a direct tissue injury (such as wounding) or ischemia.
Moreover, the presence of blood-derived macrophages is also routinely
associated
with localized secretion of a number of proteins including cytokines such as
IL-2
6 and TNF-a, growth factors such as VEGF and FGF-2, matrix metalloproteinases
as
well as many other biologically active molecules. Accordingly, although there
have
been many investigations, publications, and developments of these entities,
there
remains a general ignorance and failure of understanding by research
investigators
and clinicians alike regarding useful and effective specific means and methods
for
11 inducing angiogenesis on-demand within living cells, tissues, and organs.
Thus,
while the value and desirability of initiating new vascularization -
especially using
cells in localized areas on an as needed basis as well as a therapeutic
treatment for
individual patients - is well recognized, these aims remain a long sought goal
yet to
be achieved in a practical manner.
16
SUMMARY OF THE INVENTION
The present invention has multiple aspects and uses. A first aspect provides
a method for stimulating angiogenesis within a targeted collection of viable
cells in-
21 situ, said method comprising the steps of:
identifying a collection of cells comprising viable cells in-situ as a target
for
stimulation of angiogenesis;
providing means for effecting an introduction of at least one member selected
from the group consisting of the PR-39 oligopeptide collective to the
cytoplasm of
26 said targeted collection of cells;
introducing at least one member of the PR-39 oligopeptide collective to the
cytoplasm of said targeted collection of cells using said effecting means;
allowing said introduced PR-39 oligopeptide collective member to interact
with such proteasomes as are present within the cytoplasm of said targeted
collection
31 of cells whereby
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1 (a) at least some of the proteasomes interact with said PR-39
oligopeptide collective member, and
(b) at least a part of the proteolytic activity mediated by said
interacting proteasomes becomes selectively altered, and
(c) the selectively altered proteolytic activity of said interacting
6 proteasomes results in a stimulation of angiogenesis in-situ within the
targeted
collection of viable cells.
A second aspect of the invention provides a method for selective inhibition of
proteasome-mediated degradation of peptides in-situ within a collection of
viable
cells, said method comprising the steps of:
11 identifying a collection of cells comprising viable cells in-situ as a
target;
providing means for effecting an introduction of at least one member selected
from the group consisting of the PR-39 oligopeptide collective to the
cytoplasm of
said targeted collection of cells;
introducing at least one member of the PR-39 oligopeptide collective to the
16 cytoplasm of said targeted collection of cells using said effecting means;
allowing said introduced PR-39 oligopeptide collective member to interact
with such proteasomes as are present within the cytoplasm of said targeted
collection
of cells whereby
(a) at least some of the proteasome interacts with the PR-39
21 oligopeptide collective member, and
(b) at least a part of the proteolytic activity mediated by said
interacting proteasomes becomes markedly altered, and
(c) the markedly altered proteolytic activity of said interacting
proteasomes results in a selective inhibition of proteasome-mediated
degradation of
26 peptides in-situ within the targeted collection of cells.
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1 BRIEF DESCRIPTION OF THE FIGURES
The present invention may be more fully understood and better appreciated
when taken in conjunction with the accompanying drawing, in which
Figs. lA-1D are presentations of empirical data showing the direct
interaction between PR-39 peptide and the a7 subunit of proteasomes
intracellularly;
6 Figs. 2A-2D are presentations of empirical data showing the effect of PR-39
peptide upon proteasome activity in-vivo;
Figs. 3A-3D are graphs demonstrating the results of in-vitro proteasome
activity assays;
Figs. 4A-4C are presentations of empirical data showing the in-vivo effects
11 of PR-39 peptide expression;
Figs. SA-SC are photographs of representative sections showing differences
in vascularity among control, PR-39 peptide and FGF2 impregnated Matrigel
pellets;
Fig. 6 is a graph providing a quantitative analysis of vascularity for the
16 representative sections of Fig. 5; and
Fig. 7 is a graph showing the induction of angiogenesis in-vivo using PR-39
peptide and short-length PRll peptide impregnated Matrigel pellets.
21 DETAILED DESCRIPTION OF THE INVENTION
The present invention is a method for stimulating angiogenesis via the
purposeful introduction of native PR-39 peptide or a member of the PR-39
derived
oligopeptide family to the cytoplasm of viable cells in-situ. The PR-39
peptide or
the derived member of the family will interact with such proteasomes as are
present
26 intracellularly; and the consequence of PR-39 peptide/proteasome
interaction is the
selective inactivation of proteasomes such that intracellular degradation of
proteins
such as HIF-la and IxBa is diminished and a marked stimulation of angiogenesis
in-
situ consequently results.
A number of major benefits and advantages are therefore provided by the
31 means and methods comprising the present invention. These include the
following:
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1 1. The present invention provides an in-situ stimulation of angiogenesis. By
definition, therefore, both in-vivo and in-vitro circumstances of use and
application
are envisioned and expected. Moreover, the viable cells which are the location
of
PR-39 peptide and proteasome interaction, alternatively may be isolated cells;
be
part of living tissues comprising a variety of different cells such as
endothelial cells,
6 fibrocytes and muscle cells; and may also comprise part of specific organs
in the
body of a living human or animal subject. While the user shall choose the
specific
conditions and circumstances for practicing the present invention, the
intended scope
of application and the envisioned utility of the means and methods described
herein
apply broadly to living cells, living tissues, functional organs and systems,
as well
11 as the complete living body unit as a viable whole.
2. The present invention has a variety of different applications and uses. Of
clinical and medical interest and value, the present invention provides the
opportunity to stimulate angiogenesis in tissues and organs in a living
subject which
16 has suffered defects or has undergone anoxia or infarction. A common
clinical
instance is the myocardial infarction or chronic myocardial ischemia of heart
tissue
in various zones or areas of a living human subject. The present invention
thus
provides opportunity and means for specific site stimulation and inducement of
angiogenesis under controlled conditions. The present invention also has major
21 research value for research investigators in furthering the quality and
quantity of
knowledge regarding the mechanisms controlling angiogenesis under a variety of
different conditions and circumstances.
3. The present invention envisions and permits a diverse range of means for
26 introducing native PR-39 peptide or a shorter-length peptide of the
oligopeptide
family to a specific location, site, tissue, organ, or system in the living
body. A
variety of different routes of administration are available to the
practitioner; and a
wide and useful choice of delivery systems are conventionally available, and
in
accordance with good medical practice are adaptable directly for use. In this
31 manner, not only are the means for PR-39 peptide introduction under the
control of
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7
1 the user, but also the manner of localized application and the mode of
limiting the
area of peptide introduction can be chosen and controlled.
I. Underlying Direct And Indirect Mechanisms For Initiating A
6 Stimulation Of Angiogenesis
The present invention utilizes and relies upon novel and previously unknown
direct and indirect mechanisms of interaction between PR-39 peptide (or its
shorter-
length homologs) and proteasomes in-situ as the basis for stimulation of
11 angiogenesis in cells, living tissues, and organs. Evidence of such
multiple
intracellular interactions is provided by the experiments and empirical data
described hereinafter. Such interactions between proteasomes (and its a7
subunit in
particular) and the PR-39 peptides collectively (of any size) are previously
unknown; in fact, no meaningful relationship or interaction between any
peptide
16 whatsoever and intracellular proteasome function has ever been proposed or
envisioned before the present invention was conceived or demonstrated
empirically.
As shown experimentally hereinafter, the PR-39 peptide (and the shorter-
length PR-39 derived oligopeptide family members) when introduced into the
cytoplasm of viable cells will interact, directly and indirectly, with
proteasomes. In
21 some instances, the interaction between the collective of PR-39
oligopeptides and
the proteasome is direct and a direct binding with the a7 subunit of the
proteasome
occurs. In these instances, no intermediaries or cofactors are involved in the
binding reaction; and such direct a7 subunit binding interactions result in a
selective
inactivation and inhibition of proteasome function intracellularly such that
26 expression of certain proteins such as HIF-la is increased and stimulation
of
angiogenesis subsequently occurs.
In addition, however, alternative mechanisms of interaction in addition to
and other than direct inactivation of a proteasome subunit are deemed to exist
and be
in effect in-situ for members of the PR-39 peptide collective (including
native PR-39
31 and its substituted or homologous forms). As merely an exemplary and
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8
1 representative instance illustrating an alternative mechanism of action, the
experiments and empirical data presented hereinafter demonstrate that: the PR-
39
peptide acts as a selective inhibitor of tumor necrosis factor-a induced IxBa
degradation by proteasomes; and that PR-39 peptide inhibition of IxBa
degradation
is rapidly reversible (unlike the action of known inhibitory compounds such as
6 lactacysin); and that even in the presence of PR-39 peptide, the
intracellular
expression of other proteasome-regulated proteins such as p105 and p50 NFKB
was
unchanged. These empirical findings are meaningful evidence of, harmonious
with,
and more consistent as demonstrating indirect mechanisms of interaction
(rather than
any direct action effect). Accordingly, the selective and reversible nature of
PR-39
11 peptide activity with regard to inhibiting IKBa degradation is perceived to
be due to
a prevention of recognition of ubiquitinated IKBa by the proteasome and/or by
a
physical blockage of its entry into the interior of the proteasome in-situ.
These
indirect mechanisms of action are functional alternatives available on-demand
and
effective concurrently in addition to any direct action inactivation
mechanisms.
16 To obtain a demonstrable proteasome interaction and effect in-situ, the
introduction of native PR-39 peptide (or one of its substituted forms or its
shorter-
length homologs) is a necessary prerequisite; and the presence of sufficient
PR-39
peptide (or its substituted or homologic equivalent) quantitatively to
interact
selectively with proteasomes intracellularly within viable cells can be
achieved
21 under both in-vivo conditions and in-vitro experimental circumstances.
The methodology and means provided by the present invention for
selectively inhibiting proteolysis and stimulating angiogenesis within viable
cells is
therefore directed at and focused upon the intracellular degradation
capability and
functional activity of proteasomes. Such selective inhibition and/or
disruption of
26 proteasome-mediated degradation is achieved via the introduction of native
PR-39
peptide or a member of the shorter-length PR-39 derived oligopeptide family in
a
therapeutic regimen of treatment.
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1 II. Proteasomes
The proteasome is a component of the ubiquitin-proteasome-dependent
proteolysis system. This system plays a major role in the turnover of
intracellular
proteins, of misfolded proteins, and in the selective degradation of key
proteins.
Controlled protein degradation is an important and efficient way to remove
6 nonfunctional proteins and/or to regulate the activity of key proteins.
Target
proteins are selectively recognized by the ubiquitin system and subsequently
marked
by covalent linkage of multiple molecules of ubiquitin, a small conserved
protein.
The polyubiquitinated proteins are degraded by 26S proteasome. This complex,
however, is composed of two large subcomplexes: the 20S proteasome
constituting
11 the proteolytic core and the 19S regulatory complex which confers
polyubiquitin
binding and energy dependence. A simplified scheme of the ubiquitin pathway is
depicted by Flow Scheme A below.
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- 10-
Flow Scheme A
Ubiquitin
E1
E1-S-Ubiquitin Protein
E, E2 modification
E2-S-Ubiquitin anallary proteins
Ez E3 Substrate
E3-S-Ubiquitin
E2 ~I I_'~ E7
Substrate-(Ubiquitin)"
~ I 19S complex
26S proteasome ~ 20S proteasome
19S complex
Ubiquitin ~ + ~ Peptides
Schematic representation of the pro tea some-ubiquitin
p;uhwuv. Ubiquitin is first activated by a ubiquitin-activating
enzyme~(UBA or EI) and passed on to a ubiquitin-conjugating
protein f UBC ur E3). Ubiquitin is then linked directly, or with the
help of ubiyuitin ligases (E)s. via an isopeptide bond to a lysine
residue of the substrate protein. Polyubiquitinated proteins are
recuenized and selectively degraded by the ?6S proteusome. yield-
in~_ reusable ubiquitin molecules and peptides of > to I > amino
acids. Conversion of a protein into a substrate for ubiquitination
can in cenain races occur ;titer pos«ranslational modification or
association with ancillarv factors. Proteins can also be recoenized
by an E3 ubiquitin liease without prior modification or associa-
tion
* Reproduced from Gerards et al., CMLS 54: 253-262 (1998)
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11
1 A substantial quantum of research has been conducted to understand the
architecture, assembly, and molecular biology of the proteasome. Merely
representative of scientific publications in this field are the following, the
individual
texts of which are expressly incorporated by reference herein: Goldberg et al.
,
Biol. hem. 37$: 131-140 (1997); Tanaka, K., Biochem. Bioph,~ Res, mmun.
6 247: 537-541 (1998); Baumeister et al., ell 92: 367-380 (1998); Gerards et
al.,
CMLS 54: 253-262 (1998); Maurizi, M.R., Curr. Biol. $: 8453-8456 (1998);
Rechsteiner etet al., J. Biol. h m. ~: 6065-6068 (1993); Gerards et al.,~ Mol.
Biol. 275: 113-121 (1998); Fenteany, G. and S. Schreiber, ~ Biol. h m. ~:
8545-8548 (1998); and Oikawa a al., Biochem. Bio~phvs. Res. Commun. 24~: 243-
11 248 (1998).
The 20S proteasome
The degrading component in ubiquitin-dependent protealysis is the 26S
proteasome. The catalytic core of this complex is the 20S proteasome, which is
16 highly conserved and can be found in eukaryotes, archaebacteria, and some
eubacteria. In eukaryotes, the amount of proteasomes can constitute up to 1 %
of the
cell content, depending on the average protein breakdown rates of the organ.
Proteasomes are localized in the nucleus and the cytosol, sometimes
colocalizing or
associating with the cytosketon. [See for example: Hilt, W. and D.H. Wolf,
21 Trends Biochem. ci. 21: 96-102 (1996); Ciechanover, A. , Cell ~: 13-21
(1994);
Jentseh, S. and S. Schlenker, ~l_l $2: 881-884 (1995); Coux ~t , Annu. Rev.
Biochem. ~5: 807-847 (1996); Dahlmann etet al., FEBS Lett. ~: 125-131 (1989);
Tamura et al., urr Biol. ~: 766-774 (1995); Machiels et al., Eur. ~ X11 Biol.
66:
282-292 (1995); Schemer, K. and F. Bey, Proe. Nucleic Acid Res. Mol. Biol. 49:
26 1-64 (1994); and Gerards et al., CMLS 54: 253-262 (1998)].
The first description of a "cylinder-shaped" complex with proteasome-like
features dates back to the late 1960s. The plethora of names given to it
subsequently is a reflection of the problems that were encountered over a
period of
two decades in trying to define its biochemical properties and cellular
functions.
31 Enzymological studies revealed an array of distinct proteolytic activities
and led to a
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12
1 consensus name, 'multicatalytic proteinase' . This name, however, was soon
replaced by a new one, the 'proteasome' emphasizing its character as a
molecular
machine.
At about the same time, it was found that the occurrence of proteasomes was
not restricted to eukaryotic cells. A compositionally simpler, but
structurally
6 strikingly similar proteolytic complex was found in the archaeon
Thermoplasma
acidophilum, which later took a pivotal role in elucidating the structure and
enzymatic mechanism of the proteasome.
Nomenclature
11 The 20S proteasome was independently discovered by groups working in
different fields, and hence was given a variety of different names. In 1970,
Scherrer and colleagues observed ring-shaped particles in ribosome-free
messenger
RNA (mRNA) preparations [Sporh ~t al. , Eur. J_. Biochem. 17: 296-318 (1970)]
.
Subsequently, in 1979, DeMartino and Goldberg isolated a 700-kDa 'neutral
16 protease' from rat liver [DeMartino, G.N. and A.L. Goldberg, J. Biol. hem
2~4:
3712-3715 (1997)). Then, in 1980 Wilk and Orlowski isolated a large protease
complex from the pituitary that possessed three different catalytic
activities. They
called it multicatalytic protease [Wilk, S. and M. Orlowski, ~ Neurochem. ~5:
1172-1182 (1980); Wilk, S. and M. Orlowski, ~ Neurochem. 40: 842-849 (1983)].
21 Later, Monaco and McDevitt immunoprecipitated complexes consisting of low
molecular weight proteins (LMPs) with a possible role in antigen presentation
[Monaco, J.J. and H.O. McDevitt, Nature ~0 : 797-799 (1984)]. Also, in 1984
this
particle was called prosome, referring to its presumed role in programming
mRNA
translation [Schmid et al., EMBO ~: 29-34 (1984)]. Altogether, this complex
has
26 been given 21 different names in the literature. Since all particles were
shown to be
identical the name 'proteasome' (which is now generally accepted) was proposed
first, referring to its proteolytic and particulate nature [Amigo et al . , Na
re
192-194 (1988); Faulkenburg et al., Nature ~: 190-192 (1988); Brown et al.,
Nature ~: 355-357 (1991)].
31
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1 Overall characteristics and properties
The 20S proteasome is the major cytosolic protease in eukaryotic cells and is
the proteolytic component of the ubiquitin-dependent degradative pathway.
Proteasomes are also found in some, but not all, archaebacteria and
eubacteria, and
in eukaryotes. True proteasomes are composed of 28 subunits, 14 each of two
6 different classes - non-catalytic alpha (a) and catalytically-active beta
(~i) subunits.
The subunits are arranged in rings of seven subunits, all of a single type.
The 20S
proteasome is a stack of four rings, two inner beta rings flanked by the alpha
rings.
The junction between the beta rings produces a remarkable structural feature
of
proteasomes - an interior aqueous cavity large enough to accommodate about 70
11 kDa of protein and accessible only through narrow axial channels in the
rings. The
catalytic sites are located on the beta subunits within the aqueous cavity.
Isolation
of the catalytic sites in this way, and the limited access via narrow
channels, serves
to compartmentalize proteolysis, allowing degradation of only those proteins
that
can be actively translocated into the interior of the proteasome.
16
Structure and subunit components
The 20S proteasome has a cylindrical or barrel-like structure, typically 14.8
nm in length and 11.3 nm in diameter. It is composed of 28 subunits and
arranged
in four stacked rings, resulting in a molecular mass of about 700 kDa. This
overall
21 structural architecture is conserved from bacteria to man.
In eukaryotes, including humans, 14 different subunits, ranging from 21 kDa
to 32 kDa, are present in the complex. Based on the sequence homology with the
T.
acidophilum a- or ~i-subunit, the eukaryotic subunits are divided into a-type
and ~-
type, respectively [Zwicki a al., Biochemistry ~_l: 964-972 (1992); Heinemeyer
et
26 al.; Biochemistry ~: 12229-12237 (1994); Coux etet al., MQl_. .~ 245: 769-
780 (1994)]. Table 2 shows some characteristics and alternative names of the
subunits of the human and yeast 20S proteasome using the older and the new
nomenclature proposed by Groll and coworkers [Groll e, t al., Na r ,~$~: 463-
471
(1997)]. Immuno-electron microscopy (EM) studies also revealed that the
31 eukaryotic a-type subunits reside in the outer rings and the ~3-type
subunits in the
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14
1 inner rings. Furthermore, these studies indicated that in the eukaryotic 20S
proteasome seven different subunit constitute a ring, each subunit located at
a
defined position [Kopp et al., J. Mol. Biol. 229: 14-19 (1993); Kopp et al.,
J. Mol.
Biol. 24 : 264-272 (1995); Schauer et al., ~ Struct. Biol. X11: 135-147
(1993);
Kopp et al., Proc Natl Acad Ski USA,~4: 2939-2944 (1997)]. Therefore, the
6 eukaryotic proteasome assembles as an al_~(31-~~31-gal-~ particle. The
typical human
structure and assembly is illustrated by Table 3
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Table 2: Nomenclature and molecular masses of proteasomal subunits
Systematic Molecular mass
of
name Human ,gene Yeast gene human subunit
(kDa)
a 1 HsPROS27 HsIota C7 PRS2 27.4
a2 HsC3 Y7 25.9
a3 HsC9 Y13 29.5
a4 XAPC7 HsC6 PRE6 27.9
a5 HsZeta PUP2 26.4
a6 HsPROS30 HsC2 PRES 30.2
a7 HsC8 C 1 PRS 1 28.4
X31 HsDelta Y PRE3 25.3 (21.9)
~i 1 i LMP2 23 .2 (20. 9)
X32 Z PUP1 30.0 (24.5)
~32i MECL1 28.9 (23.8)
X33 HsC 10-11 PUPS 22. 9
X34 HsC7-1 PRE1 C11 22.8
X35 MB1 X PRE2 nd (22.4)
(35i LMP7 30.4 (21.2)
~6 HsCS CS PRS3 26.5 (23.3)
X37 HsBPROS26 HsN3 Pre4 29.2 (24.4)
* Reproduced from Gerards etet al., ML 54: 253-262 (1998)
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Table 3: Schematic representation of the human 20S proteasome*
a cr
,i
~a
cz
* Reproduced from Gerards et al., CMLS 54: 253-262 (1998)
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1 Proteolytic activitX
The first report on the multicatalytic properties of the proteasome stems from
1983, when three different proteolytic activities were distinguished: 'trypsin-
like',
'chymotrypsin-like' and 'peptidylglutamyl-peptide hydrolase' activity [Wilk,
S. and
M. Orlowski, J. Neurochem. 40: 842-849 (1983)]. These three proteasomal
6 activities refer to peptide bond cleavage at the carboxyl side of basic,
hydrophobic
and acidic amino acid residues, respectively. They were identified using short
synthetic peptide substrates and are believed to be catalyzed at independent
sites - in
part because the different proteolytic activities respond differentially to
various
activators and inhibitors. With similar approaches, at least two additional
11 proteolytic activities have been recently described [Orlowski et al. ,
Biochemistry
32: 1563-1572 (1993); Orlowski, M., BiochemistrX 2~: 10289-10297 (1990);
Riven, A.J., Biochem. ~ 291: 1-10 (1993)].
The Progressive Degradation Of Protein Substrates
16 Recent studies have also revealed a fundamental new property of the
proteasome that clearly distinguishes it from conventional proteases: i.e.,
this
particle degrades a protein substrate all the way to small peptides, before
attacking
another protein substrate [Akopian et al., ~ Biol. Chem. 272: 1791-1798
(1997)].
Because the proteasome's multiple active sites are located in its central
chamber and
21 because diffusion of a peptide substrate into this compartment must be a
slow
process, these particles function in a highly processive fashion; i.e., they
have
mechanisms of action to bind tightly protein substrates and to make multiple
cleavages in the polypeptide before releasing the peptide products. Moreover,
the
ratio of new peptides generated to the number of substrate molecules consumed
is
26 constant during the reaction. In other words, as peptides accumulated, they
were
not hydrolyzed further, even during prolonged incubations, where up to half of
the
substrate molecules were consumed. Equally important, the disappearance of
these
substrate molecules coincided exactly with the appearance of small peptide
products
[Goldberg et al., Biol. h m ,~7$: 131-140 (1997)]. These observations,
together
31 with the fording that the pattern of the products is independent of time,
established
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18
1 that processive degradation is a general feature of the 20S proteasome
[Gerard et al. ,
CMLS 54: 253-262 (1998)].
The contribution of each individual active center and proteolytic activity to
the degradation of longer peptides and complete proteins is presently unknown.
Nevertheless, proteasomes are able to cleave behind most amino acids in a
protein.
6 Thus, the 20S proteasome is in fact a nonspecific endopeptidase. In
addition,
however, the generated (degraded) peptides fall into a rather narrow size
range of 6
to 10 amino acids in length, demonstrating the existence of a kind of
'molecular
ruler' . The average length of the degradation products is typically 7 to 8
amino
acids; this finding is in agreement with the distance between the active sites
in the
11 proteasome. Similar nonspecific endopeptidase activity and size
distribution of
degration products from whole proteins was observed for proteasomes generally
and
by proteasomes of human origin in particular.
Other features of the 20S proteasome degradation are also unique. While
unfolded peptides are usually digested, most native proteins are resistant to
16 proteolytic degradation by the 20S proteasome in vitro. However,
denaturation of
the substrate protein by oxidation or reduction of disulphide bridges can
render it
accessible to degradation by proteasomes. Also, small gold particles with a
diameter of 2 nm containing unfolded substrate cannot enter the proteasome.
These
characteristics show that a relatively narrow opening controls access to the
inner
21 proteolytic compartment of the proteasome.
III. The PR-39 Oligopeptide Collective
Native PR-39 peptide is a substance belonging to the cathelin family of
26 proteins; the mature peptide is 39 amino acids in length in the naturally
occurring
state; and the peptide is able to exert a variety of activities and cause
different
cellular outcomes. Although first identified as a membrane permeating
antibacterial
peptide found in the intestine of pigs [Agerberth et al., Eur ~, Biochem. ~ :
849-
854 (1991)], this peptide was subsequently isolated from wounds where it could
31 simultaneously reduce infection and influence the action of growth factors,
matrix
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19
1 components, and other cellular effectors involved in wound repair [Gallo et
al. ,
Proc. Natl. Acad. Sci. ~J~A ~1: 11035-11039 (1994); Gallo et al., ~, Inves .
Dermatel. 104: 555 (1995)]. The structure and membrane interactions of native
PR-
39 peptide have also been elucidated [Cariaux et al., Eur. ~ Biochem. 224:
1019-
1027 (1994)] and the complete amino acid sequences of native PR-39 peptide and
its
6 various substituted forms have been reported [PCT Publication No. WO
92/22578
published 23 December 1992].
More recently, the native PR-39 peptide was shown to possess a syndecan-
inducing activity in furtherance of its wound healing capabilities; and while
renamed
a "synducin" , was shown to induce cellular production of two specific
11 proteoglycans, syndecan-1 and syndecan-4, within living mesenchymal cells
[U.S.
Patent No. 5,654,273]. Overall, native PR-39 peptide has been shown to play a
role
in several inflammatory events including wound healing and myocardial
infarction
[Gallo et al., Proc. Natl. Acad. ri ~A. 2: 11035-11039 (1994); Li etet al., it
.
Res. ~: 785-796 (1997)]; and the native peptide has been shown to be taken up
16 rapidly by a number of different cell types including meschymal cells and
endothelial cells [Chap; Y.R. and R.L. Gallo, J. Biol. hem. 273: 28978-28985
(1998)].
The PR-39 peptide gr~o ying
21 Native PR-39 peptide is composed of the 39 amino acid sequence shown
below (and also by Table 4).
PR-39: Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-
Pro-Phe-Phe-Pro-Pro-Arg-Leu-Pro-Pro-Arg-Ile-Pro-Pro-Gly-Phe-Pro-
26 Pro-Arg-Phe-Pro-Pro-Arg-Phe-Pro
As conventionally known and reported [see for example, U.S. Patent No.
5,654,273], the specific peptide can be substituted using conservative
substitutions
of amino acids having the same or functionally equivalent charge and
structure,
31 except for the required amino acid sequence "Arg-Arg-Arg" at the N-terminus
and
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1 the intermediate amino acid sequences "Pro-Pro-X-X-Pro-Pro-X-X-Pro" and "Pro-
Pro-X-X-X-Pro-Pro-X-X-Pro" where X can be substituted freely using any amino
acid. Thus, all of the preferred substituted amino acid sequences are of about
the
same size and each differ from the native PR-39 peptide sequence only by
substitutions in the intermediate portions of the structure.
6
The PR-39 derived oligopeptide familx
In addition to the conventionally known native PR-39 peptide amino acid
residue sequence and its readily recognizable substituted forms as described
above,
an entirely novel and unforeseen family of PR-39 derived oligopeptide
structures is
11 provided by the present invention for use. This previously unknown family
of PR-
39 derived oligopeptides is constituted of members which individually will
cause a
selective inhibition of proteasome-mediated degradation of peptides in-situ
after
introduction intracellularly to a viable cell.
Each member of this PR-39 derived oligopeptide family presents
16 characteristics and properties which are commonly shared among the entire
membership. These include the following:
(i) each peptide sequence is less than 39 amino acid residues in length in
every embodiment, and preferably is less than 20 residues in size in the best
mode;
(ii) each short-length peptide sequence is at least partially homologous (or
21 analogous) with the N-terminal amino acid residues of the native PR-39
peptide, and
preferably is completely identical or markedly similar to the N-terminal end
residues
of the native PR-39 peptide;
(iii) each short-length peptide is able to interact in-situ with at least the
a7
subunit of such proteasomes as are present within the cytoplasm of the cell;
and
26 (iv) each short-length peptide sequence is able to alter markedly the
proteolytic activity of proteasomes with an interacting a7 subunit such that a
selective increased expression of specific proteins (such as IxBa and HIF-la)
occurs
m-situ.
Merely as illustrative examples and preferred embodiments of the broad
31 membership constituting this PR-39 derived oligopeptide family, the members
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21
1 comprising 15, 11 and 8 amino acid residues respectively in length are
presented
below as the PR15, PR11, and PR8 entities respectively. For comparison
purposes
only, the complete amino acid sequence of the native PR-39 peptide is
presented as
well.
6 PR-39: 1 2 3 4 5 6 7 8 9 10 11 12 13
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-
14 15 16 17 18 19 20 21 22 23 24 25 26
Pro-Pro-Pro-Phe-Phe-Pro-Pro-Arg-Leu-Pro-Pro-Arg-Ile-
11
27 28 29 30 31 32 33 34 35 36 37 38 39
Pro-Pro-Gly-Phe-Pro-Pro-Arg-Phe-Pro-Pro-Arg-Phe-Pro
16 PR-15: 1 2 3 4 5 6 7 8 9 10 11 12 13
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-
14 15
Pro-Pro
21
26
PR-11 : 1 2 3 4 5 6 7 8 9 10 11
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg
PR-8: 1 2 3 4 5 6 7 8
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr
31 The PR-39 Oligonentide Collective
Terminology and nomenclature often pose problems for the reader as to what
precisely is meant. Accordingly, for definitional purposes, avoidance of
ambiguities, and clarity of understanding, the following terms and titles will
be
employed herein. The term "PR-39 peptides grouping" includes by definition the
36 native PR-39 structure and all substituted forms conventionally known of
the
naturally occurring 39 length amino acid sequence. In distinction, the term
"PR-39
derived oligopeptide family" and its members includes by definition all the
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22
previously unknown shorter-length homologs and analogs of the native PR-39
structure as described above. Finally, the umbrella term and category title
"PR-39
oligopeptide collective" includes by definition both the 'PR-39 peptide
grouping' as
well as the 'PR-39 derived oligopeptide family' members, and identifies any
and all
individual structures falling into either of the two subset categories.
6
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23
Table 4:
(1) GBZ1SRAL INFORMATION:
(1) APPLIC3l2iT~ Children's Medical Center Corporaton
(ii) TITLE OF INVENTION: Synducin Mediated Modulation of
Tieaue Repair
(iii) NUMBRR OF SEQUENCES: 4
(iv) CORRRSPONDENCB ADDRESS:
(A) ADDRESSER: Patrea L. Pabst
(B) STREET: 2800 One Atlantic Center
1201 West Peachtree
(C) CITY Atlanta
(D) STATE: Georgia
(E) COONTRY: D8A
(F) ZIP: 30309-3450
( v ) COMT''LJTER READABLE FORM
(A) MEDIUM TYPE: Floppy disk
(B) COMh'~TER: IHM PC Compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PateritIn Release t#1.0, Version #1.25
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (404)-873-8794
(B) TELEFAX: (404)-815-8795
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CfiARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYFOTHBTICAL: NO
(iv) ANTI-SENSE: ND
(x) PUBLICATION INFbRMATION:
(A) AUTHORS: Lee, Jong-Youn
Boma.n , H us s G .
Mutt, Viktor
Jornvall, Mane
(B) TITLE: Novel Polypeptidee And Their Uee
(C) JOQRNAL: PCT WO 92/22578
(G) DATE: 12/23/92
(K) RELEVANT RESIDU$S IN SEQ ID NO:1: FROM 1 TO 39
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Pro
1 5 10
Phe Phc Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe
Pro Pro
25 30
Arg Phe Pro Pro Arg Phe Pro
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24
1 Synthesis
The PR-39 peptide can be synthesized using standard amino acid synthetic
techniques. An example is the conventionally used solid phase synthesis
[Merrifield, J., J. Am. hem ~ $S: 2149 (1964)] described in U.S. Patent No.
4,244,946, wherein a protected alpha-amino acid is coupled to a suitable
resin, to
6 initiate synthesis of a peptide starting from the C-terminus of the peptide.
Other
methods of peptide synthesis are described in U.S. Patent Nos. 4,305,872 and
4,316,891, the teachings of which are incorporated herein. These methods can
be
used to synthesize peptides having identity with the native PR-39 peptide
amino acid
sequence described herein, or to construct desired substitutions or additions
of
11 specific amino acids, which can be screened for content and evaluated for
activity.
PR-39 can also be commercially obtained from Magainin, Inc. (Plymouth Meeting,
PA) .
Pharmaceutical Formats
16 After synthesis or purchase, the PR-39 peptides (as a family of homologs
and
analogs with substituted amino acid residues) can be introduced as a peptide-
containing preparation in a pharmaceutically acceptable format.
The PR-39 can be administered and introduced in-vivo systemically,
topically, or locally. The peptide can be administered as the peptide or as. a
21 pharmaceutically acceptable acid- or base-addition salt, formed by reaction
with an
inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid,
nitric
acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic
acid
(such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid,
pyruvic
acid, oxalic acid, malonic acid, succinic acid, malefic acid, and fumaric
acid); or by
26 reaction with an inorganic base (such as sodium hydroxide, ammonium
hydroxide,
potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl
and aryl
amines and substituted ethanolamines).
PR-39 peptide and any of the PR-39 derived oligopeptide family members
may also be conjugated to sugars, lipids, other polypeptides, nucleic acids
and
31 PNA; and function in-situ as a conjugate or be released locally after
reaching a
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1 targeted tissue or organ. The PR-39 family of peptides may also be linked to
targeting compounds for attachment in-situ to a specific cell type, tissue or
organ.
IV . Means For Introduction Of PR-39 Peptide And/Or Its Shorter-Length
6 Derived Homologs
DNA Fragments and ExprP~~;~n Vectors
A variety of means and methods are conventionally known and presently
available to the user or practitioner of the present invention in order to
introduce
11 PR-39 peptide (or a derived oligopeptide family member) to living cells and
tissues.
One desirable means uses a prepared DNA sequence fragment encoding the PR-39
peptide (or a shorter-length homology in a suitable vector as the means of
introduction to the intended target in-situ. These means for delivery envision
and
include in-vivo use circumstances; ex-vivo specimens and conditions; and in-
vitro
16 cultures. In addition, the present invention intends and expects that the
prepared
DNA sequence fragment coding for PR-39 peptide (or shorter-length homologs)
has
been inserted in a suitable expression vector and will be used in a route of
administration for delivery to living tissues comprising endothelial cells,
and
typically vascular endothelial cells which constitute the basal layer of cells
within
21 capillaries and blood vessels generally. Clearly, the cell recipients
themselves are
thus eukarytoic in origin, typically mammalian cells from human and animal
sources; and most typically would include the higher orders of mammals such as
humans and domesticated mammalian animals kept as pets or sources of food
intended for future consumption. Accordingly, the range of animals includes
all
26 domesticated varieties involved in nutrition including cattle, sheep, pigs
and the
like; as well as those animals typically used as pets or raised for commercial
purposes including horses, dogs, cats, and other living mammals typically
living
with and around humans.
Clearly, the expression vectors must be suitable for transfection of
31 endothelial cells in living tissues of mammalian origin and thus be
compatible with
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26
1 that type and condition of cells under both in-vivo and/or in-vitro
conditions. The
expression vectors thus typically include plasmids and viruses as expression
vectors.
Also, both the plasmid based vectors and the viral expression vectors
constitute conventionally known means and methods of introduction which are
conventionally recognized today as "gene therapy" modes of delivery. However,
6 this overall approach is not the only means and method of delivery available
for the
present invention.
Direct Introduction of Previou~~Svnthesized PR-39 Peptides or a PR-39 Derived
Oligopeptide Familv Member
11 PR-39 peptide or an oligopeptide family member can be introduced directly
as a synthesized compound to living cells and tissues via a range of different
delivery means. These include the following.
1. Intracoronary delivery is accomplished using catheter-based deliveries of
16 synthesized PR-39 peptide (or homolog member) suspended in a suitable
buffer
(such as saline) which can be injected locally (i.e., by injecting into the
myocardium
through the vessel wall) in the coronary artery using a suitable local
delivery
catheter such as a lOmm InfusaSleeve catheter (Local Med, Palo Alto, CA)
loaded
over a 3.Omm x 20mm angioplasty balloon, delivered over a 0.014 inch
angioplasty
21 guidewire. Delivery is typically accomplished by first inflating the
angioplasty
balloon to 30 psi, and then delivering the protein through the local delivery
catheter
at 80 psi over 30 seconds (this can be modified to suit the delivery
catheter).
2. Intracoronary bolus infusion of PR-39 peptide (or a short-length homology
26 synthesized previously can be accomplished by a manual injection of the
substance
through an Ultrafuse-X dual lumen catheter (SciMed, Minneapolis, MN) or
another
suitable device into proximal orifices of coronary arteries over 10 minutes.
3. Pericardial delivery of synthesized PR-39 peptide (or a shorter-length
31 homology is typically accomplished by instillation of the peptide-
containing solution
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27
1 into the pericardial sac. The pericardium is accessed via a right atrial
puncture,
transthoracic puncture or via a direct surgical approach. Once the access is
established, the peptide material is infused into the pericardial cavity and
the
catheter is withdrawn. Alternatively, the delivery is accomplished via the aid
of
slow-release polymers such as heparin-alginate or ethylene vinyl acetate
(EVAc). In
6 both cases, once the PR-39 peptide (or homology is integrated into the
polymer, the
desired amount of PR-39/polymer is inserted under the epicardial fat or
secured to
the myocardial surface using, for example, sutures. In addition, the PR-
39/polymer
can be positioned along the adventitial surface of coronary vessels.
11 4. Intramyocardial delivery of synthesized PR-39 peptide (or a shorter-
length
homology can be accomplished either under direct vision following thoracotomy
or
using thoracoscope or via a catheter. In either case, the peptide containing
solution
is injected using a syringe or other suitable device directly into the
myocardium.
Up to 2 cc of volume can be injected into any given spot and multiple
locations (up
16 to 30 injections) can be done in each patient. Catheter-based injections
are carried
out under fluoroscopic, ultrasound or Biosense NOGA guidance. In all cases
after
catheter introduction into the left ventricle the desired area of the
myocardium is
injected using a catheter that allows for controlled local delivery of the
material.
21 Pharmaceutical Carriers Of PR-39 Peptides or a PR-39 Derived Olig-opeptide
Family Member
A range of suitable pharmaceutical carriers and vehicles are known
conventionally to those skilled in the art. Thus, for parenteral
administration, the
compound will typically be dissolved or suspended in sterile water or saline.
26 For enteral administration, the PR-39 peptide or homologous oligopeptide of
choice will be typically incorporated into an inert carrier in tablet, liquid,
or
capsular form. Some suitable carriers are starches and sugars; and often
include
lubricants, flavorings, binders, and other materials desirable in tablet
making
procedures.
31
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28
1 The PR-39 peptide and oligopeptide family of compounds can also be
administered topically by application of a solution, cream, gel, or polymeric
material (for example, a PluronicTM, BASF).
As an alternative, the chosen peptide can be administered in liposomes or
microspheres (or microparticles), which can be injected for local or systemic
6 delivery. Methods for preparing liposomes and microspheres for
administration to a
patient are conventionally known to those skilled in the art. For example,
U.S.
Patent No. 4,789,734 describes methods for encapsulating biological materials
in
liposomes. Essentially, the material is dissolved in an aqueous solution, the
appropriate phospholipids and lipids added, along with surfactants if
required, and
11 the material dialyzed or sonicated, as necessary. See also, G. Gregoriadis,
Chapter
14, "Liposomes", Drug Carriers in Biolog" and Medicine, chap. 14, pp. 287-341
(1979). Microspheres formed of polymers or proteins are well known to those
skilled in the art, and can be tailored for passage through the
gastrointestinal tract
directly into the bloodstream. Alternatively, the compound can be incorporated
and
16 the microspheres, or composite of microspheres, implanted from days to
months.
See, for example, U.S. Patent Nos. 4,906,474; 4,925,673; and 3,625,214.
Examplary Introductions And Preferred Routes of Administration
A variety of approaches, routes of administration, and delivery methods have
21 been identified herein and are available for introduction of PR-39 peptide
and the
derived family of oligopeptides. It is envisioned, however, that a majority of
the
approaches and routes of administration described herein will be medical
applications and specific clinical approaches intended for use with individual
human
patients having specified medical problems and diagnosed pathologies. It is
26 expected, accordingly, that the reader is familiar generally with the
typical clinical
human problem, pathology, and medical conditions described herein; and
therefore
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29
will be able to follow and easily understand the nature of the intervention
clinically
using the present invention and the intended outcome and result of the
clinical
treatment - particularly as pertains to the stimulation of angiogenesis under
in-vivo
treatment conditions. A representative listing of preferred clinical
approaches is
given by Table 5 below.
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Table S
Preferred Routes of Administration
Catheter-based (intracoronary) injections and infusions;
Direct myocardial injection
(intramyocardial guided);
Direct myocardial injection
(direct vision-epicardial-open chest or under thorascope guidance);
Local intravascular delivery;
Liposome-based delivery;
Delivery in association with receptor-specific peptides;
Oral delivery;
In instances of peripheral vascular disease:
intramuscular injection
intraarterial injection and/or infusion.
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1 V. Experiments and Empirical Data
To demonstrate the merits and value of the present invention, a series of
planned experiments and empirical data are presented below. It will be
expressly
understood, however, that the experiments described and the results provided
are
6 merely the best evidence of the subject matter as a whole which is the
invention; and
that the empirical data, while limited in content, is only illustrative of the
scope of
the invention envisioned and claimed.
Introduction
11 Proteolytic degradation in mammalian cells is known to proceed via two
distinct pathways: lysosome-dependent degradation and proteasome-dependent.
The proteasome in its pathway plays a key role in proteolysis of intracellular
proteins which are marked for degradation by the ubiquitin system. The
multienzyme complex involved in these events, the 26S proteasome, consists of
a
16 20S catalytic proteasome "core" and two 19S caps that bind uniquitylated
proteins,
as has been described in detail previously herein. Proteasome-mediated
proteolysis
is a principal event quantitatively controlling intracellular levels of a
number of
different proteins including hypoxia-inducing factor (HIF)-la, heat shock
protein
HSP70, protooncogenes c-Fos, c-Jun and c-Mos, NFxB inhibitor IKBa, and various
21 cyclins. In addition, the proteasome also is known to play a critical role
in the
specific processing and presentation of major histocompatibility complex (MHC)
class I-restricted antigens as well as provide partial proteolytic cleavage of
p105
NFxB to the active p50 subunit.
The PR-39 peptide, belonging to the cathelin family of proteins, plays an
26 important role in several inflammatory events including wound healing and
myocardial infarction. The PR-39 peptide typically is rapidly taken up by a
number
of different cell types including endothelial cells; and prolonged treatment
with PR-
39 peptide leads to increased cell growth and angiogenesis. However, the
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32
1 mechanism of action for this peptide activity has yet to be understood or
defined.
The experiments and data presented below reveal for the first time the nature
and
detailed intracellular actions exerted by the PR-39 peptide.
Methods and Materials:
6
Yeast two-hybrid screening
Two-hybrid screening was carried out using MATCHMAKER GAL4 System
2 (Clontech) with exon 4 of the porcine PR-39 gene as a bait to screen the
mouse
embryo 3T3 cDNA library in yeast CG1945.
11
Cell culture studies
U937 cells (ATCC) grown in RPMI medium 1640 with 10 % FBS (Gibco-
BRL) and ECV cells were treated with synthetic PR-39, lactacystin (CalBiochem,
426100) or MG132 (CalBiochem, 474790) at concentration indicated in the
presence
16 of 100 mM cyclohexamide 20 mM chloroquine [Merin et al. , J. Biol. Chem.
6373-6379 (1995)]. After 45 min. of incubation, TNFa (1 ng/ml) was added.
After
min of 37°C incubation, the cells were lysed in SDS-PAGE loading
buffer.
Following SDS-PAGE of the total protein extract, IxB-a and NF-xB p105 p50
expressions were determined by Western blotting with anti-human antibodies
(Santa
21 Cruz, sc-203, sc114G). For studies of HIF-la and VEGF expression, ECV cells
were cultured in a hypoxia chamber (5 % COZ/95 % NZ) at 37°C for 16 hr.
HIF-1 a
was immunoprecipitated with anti-HIF-la mAb (0Z12 1:5) in RIPA buffer and
Western blotting with anti-HIF-la mAb (0Z15 1:10) (courtesy of Dr. A. King,
DFCI, Boston). VEGF expression was shown by Western blotting of hypoxia
26 treated ECV cell lysate with anti-human VEGF antibody (Santa Cruz, sc-152).
For
HSP70 expression, U937 was treated for 3 hr, harvest with SDS-PAGE loading
buffer, Western blotting with anti-human HSP70 polyclonal antibody (Santa
Cruz,
sc-1060).
31
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33
1 In-vitro proteasome activity assays
Rabbit muscle 20S proteasome preparation (courtesy of Dr. M. Sherman,
BBRI, Boston) was used for all studies. For determination of proteasome
activity,
~,1 of 1:10 diluted proteasome preparation was incubated at room temperature
in
eukaryotic proteasome assay buffer (20 mM Tris-HCl pH 8.0, 0.5 mM EDTA and
6 0.01 % SDS) with 20 ~M proteasome substrates (CalBiochem, 539140-3) and PR-
39
or other proteasome inhibitor at indicated concentration [Rock etet al.,
~gll~: 761-
771 (1994)]. The extent of substrate degradation was monitored continuously by
fluorescence spectrophotometry (380 nm excitation, 460nm emission Hitachi
F-2000) for 10 min.
11
Experiment 1:
This experiment was designed to reveal the ability of PR-39 peptide to affect
proteasome function. To test this capability, the effect of PR-39
administration
upon a7 subunit processing was empirically determined. The results are
illustrated
16 by Figs. lA-1D respectively.
Experimentally, a peptide corresponding to the 4th exon porcine PR-39 gene
sequence was used to generate a rabbit polyclonal antibody RPE4. Full length
porcine cDNA (containing leader sequence) and a sequence corresponding to the
4th
exon of porcine PR-39 gene were cloned into eukaryotic expression vector pGRES-
2
21 (USB). These expression constructs were then used to stably transfect an
immortalized human endothelial cell line (ECV304, ATCC). For co-
immunoprecipitation, wild type ECV, full length PR-39 (ECV-PR39) and exon 4
PR39 and exon 4 PR39 (ECV-E4) transfected cells were cultured in Medium 199
with 10 % fetal bovine serum (FBS) and penicillin/streptomycin. Cells were
lysed
26 with RIPA buffer; immunoprecipitated with 10 ~cg affinity purified rabbit
anti-PR39
antibody; and following Protein A-Sepharose purification and SDS-PAGE,
subjected
to immunoblotting with 1:1000 mouse anti-HC8 mAb (Affiniti Research Products
Limited UK, PW8110).
Figs. lA-1D show the interactions of PR-39 peptide and the a7 subunit of
31 proteasomes. Fig. 1A recites the cDNA sequence of cloned mouse a7 subunit
(top;
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34
1 GeneBank accession number AF055983) and corresponding human HC8 subunit of
20S proteasome. Fig. 1B shows the sequence alignment of C-terminal tails mouse
a
subunits of 20S proteasome. Fig. 1C shows a deletion analysis of a7-PR39
binding.
Deletion mutants of the mouse a7 subunit were cloned into an yeast-two hybrid
vector and the extent of growth of lacZ+ colonies on selective medium
following co-
b transformation with PR-39 construct in the yeast CG1945 was determined. It
is
noted that only full length a7 construct was able to bind to PR-39. Finally,
Fig. 1D
shows the co-immunoprecipitation of PR-39 and a7 subunit in ECV cells.
It will be noted also that Fig. 1 represents the evidence of four clones
growing on selective media and demonstrating lacZ staining. All four clones
11 encoded overlapping identical cDNA sequences highly homologous to the human
sequence of a7 (HC8) subunit of proteasome (Fig. 1A). Similar to all a
subunits of
the 20S proteasome, the cloned mouse protein possesses a highly conserved N-
terminal region; in addition it demonstrated the presence of 16 amino acid
long C-
terminal sequence found in some but not all a subunits (Fig. 1B). Deletion
analysis
16 showed that the presence of both C-terminal as well as N-terminal amino
acids
sequences was required for PR-39 binding (Fig. 1C). In order to confirm the
PR39-
a7 subunit interaction in-vivo, anti-PR39 antibody was used to
immunoprecipitate
PR39 protein from ECV-PR39, ECV-E4 and mock-transfected ECV cells. Western
blotting of the immunoprecipitate from ECV-PR39 and ECV-E4 but not wild type
21 ECV cells with anti-a7 subunit antibody demonstrated the presence of a
29kDa band
corresponding to the known size of a7 subunit protein (Fig. 1D). The evidence
therefore reveals that PR39 peptide interacts with a7 subunit of proteasome in
ECV
cells.
26 Experiment 2:
To test the ability of PR-39 peptide to affect proteasome function in-vivo,
the
effect of PR-39 peptide administration on IxBa processing was assessed. The
results are illustrated by Figs. 2A-2D respectively.
Fig. 2A shows a Western analysis of IxBa expression in ECV cells. The
31 results show that pretreatment of cultured ECV cells with lactacystin (10
~,M, 4th
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1 lane) or stable expression of full length (ECV-PR39) or PR39 exon 4 (ECV-E4)
constructs inhibited TNF-a-induced degradation of IKB.
Thus, tumor necrosis factor (TNF)-a induces rapid degradation of IxBa - a
function that is blocked by the proteasome inhibitor lactacystin. However,
Western
analysis of IxBa levels after TNF-a treatment demonstrated comparable levels
of
6 IKBa expression in both ECV-PR39 and ECV-E4 cells to that seen in ECV cells
pre-treated with lactacystin.
Fig. 2B shows the effect of PR39, MG132 and lactacystin pretreatment on
IKBa expression in U937 cells following TNF-a treatment. Note similar extent
of
inhibition of IxBa degradation by TNF-a following pretreatment with PR39,
11 MG132 or lactacystin. Thus, it is clear that pretreatment of U937 cells
with PR39
blocked TNF-a induced IxBa degradation in a manner that was similar to the
degree
of inhibition seen with MG132 and lactacystin.
Fig. 2C demonstrates the reversibility of PR39 inhibition of proteasome
activity. U937 cells were pretreated with PR39, MG132 or lactacystin for 45
min.
16 After that time, the cells were extensively washed with fresh medium. 45
min later
TNF-a (1 ng/ml) was added to the medium and the extent of IKBa degradation was
determined 10 min later by Western blotting. Note preservation of IxBa in
lactacystin-treated cells but not PR39-treated cells. Thus, unlike lactacystin
but
similar to MG132, PR-39 peptide mediated inhibition of IKBa degradation was
21 rapidly reversible.
Finally, to show that PR-39 inhibition of IxBa degradation affected NFKB-
dependent transcription, ECV cells were transiently transfected with a NFKB-
Luc
reporter construct containing a tandem of four NFxB binding sites in front of
luciferase cDNA. The results of Fig. 2D show that stimulation with TNF-a
induced
26 a significant increase in luciferase activity that was completely inhibited
by
pretreatment with PR39.
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1 Accordingly, the true functional significance of PR39-mediated inhibition of
IKBa degradation in ECV cells transiently transfected with pNFxB-Luc reporter
vector (Clontech) is clearly shown by Fig. 2D. Pre-treatment with PR39
completely
inhibited TNF-a-induced increase in luciferase activity. *p < 0.01 vs. control
(Luc
activity in the absence of TNF-a).
6
Experiment 3:
To demonstrate directly the ability of PR-39 peptide to inhibit proteasome-
mediated protein degradation, preparations of eukaryotic 20S proteasomes were
tested for their ability to induce proteolysis of various synthetic peptides
in-vitro.
11 The results are graphically illustrated by Figs. 3A-3D respectively.
For determination of proteasome activity, 5 ~,1 of 1:10 diluted rabbit muscle
20S proteasome preparation (courtesy of Dr. M. Sherman, BBRI, Boston) was
incubated at room temperature in an assay buffer (20 mM Tris-HCl pH 8.0, 0.5
mM
EDTA and 0.01 % SDS) with 20 p,M of four different proteasome substrates
16 (CalBiochem, 539140-3) and PR39 or other proteasome inhibitor at indicated
concentration. The extent of substrate degradation was monitored continuously
by
fluorescence spectrophotometry (380 nm excitation, 460 nm emission Hitachi F-
2000) for 10 min.
Figs. 3A-3D reveal that the PR-39 peptide inhibited, in a dose-dependent
21 manner, degradation of all 4 peptides tested. PR-39 peptide was as potent
as
lactacystin or MG132 in inhibiting degradation in three of the four peptides
tested
and was considerably more potent in inhibiting degradation of the Z-Leu-Leu-
Glu-
AMC peptide.
26 Experiment 4:
To test the effect of PR39 treatment on cellular levels of other proteasome-
dependent proteins, the in-vivo expression of p105 and p50 NFKB, HSP70 and HIF-
la within transfected and wild type ECV cells was determined. The results are
illustrated by Figs. 4A-4C respectively.
31
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37
1 Fig. 4A shows the results of a Western blot analysis of HIF-la, p50 and
p105 NFKB expression in wild type ECV cells and ECV-E4 and ECV-PR39 clones.
It is noted that an increase in HIF-la expression occurs (but not p105 or p50
NFKB
expression) in ECV-E4 and ECV-PR 39 clones. Thus, while there was a
significant
increase in expression of HIF-la and IKB in PR39 transfected or treated
compared
6 to wild type cells, there was no significant change in expression of either
HSP70 or
NFxB - demonstrating that effects of PR39-proteasome interaction are
selective.
Since increased expression of HIF-la is known to result in increased
transcription of a number of angiogenesis-related molecules including VEGF,
Northern analysis of VEGF mRNA levels in wild type and PR39 transfected ECV
11 cells was performed. The results are shown by Fig. 4B. As expected, there
was a
significant increase in expression of both of these genes in ECV-PR39 and ECV-
E4
cells compared to ECV controls.
Finally, exposure to proteasome inhibitor lactacystin is known to induce
rapid cell death. To test the effect of PR39 on cell survival, growth rates of
ECV
16 cells treated with PR39 peptide were assessed. The results are shown by
Fig. 4C.
50,000 ECV cells were cultured in 10% FBS-M199 in the absence (control) or
presence of 10 ~.M of PR39, lactacystin or MG132. Note that while exposure to
PR39 did not affect cell growth, exposure to lactacystin or MG132
substantially
inhibited cell growth. Thus, following 3 days of PR39 exposure, treated cells
21 demonstrated normal growth compared to controls while those cells exposed
to
lactacystin demonstrated markedly reduced survival.
Experiment 5:
To demonstrate the stimulation of angiogenesis directly in living cells and
26 tissues via the introduction of PR-39 peptide, a mice matrigel assay system
was
employed. Growth factor-depleted Matrigel pellets containing 5 ~cg of PR39, 50
ng
of FGF2 or saline (control) were inserted intraperitoneally into C57BL/6 mice.
Ten
days later the pellets were removed, sectioned and stained with anti-CD31
antibody.
The number of vessels was determined in multiple sections using a digital
camera
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38
1 and Optimas 5.0 software. The results are shown by Figs. SA-5C and Fig. 6
respectively.
Figs. SA-SC are representative sections from control, PR39 and FGF2
impregnated Matrigel pellets and Fig. 6 provides a quantitative analysis of
vascularity. Clearly, the results of the representative sections and the
graphic
6 quantitative evaluations demonstrate that insertion of growth-factor
depleted
Matrigel pellet containing PR-39 peptide induced intense vessel growth that
exceeded that seen with implantation of pellets containing 50 ng/ml of bFGF.
Experiment 6:
11 To demonstrate the efficiency of shorter-length peptides which collectively
are members of the PR-39 derived oligopeptide family in stimulating
angiogenesis
in-vivo, a novel peptide, PR11, composed of the first 11 amino acid residues
[N-
terminal end] of the native PR-39 sequence was purposely synthesized. The
amino
acid sequence of PR11 is as follows:
16
1 2 3 4 5 6 7 8 9 10 11
Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg
21 To introduce the short-length PR11 peptide in-vivo, a mouse Matrigel assay
system was utilized. In sum, either S ~.g/ml of PR11 peptide or 5 ~cg/ml of
native
PR-39 peptide were individually placed into a growth factor-depleted Matrigel
pellet; and then each prepared Matrigel pellet was inserted into the
peritoneal cavity
of a mouse. After 14 days intraperitoneal placement, each pellet was removed
from
26 its living host; and each pellet was examined for evidence of new
vascularity. The
results are graphically presented by Fig. 7. Note that the bar graph of Fig. 7
shows
the number of blood vessels [mean~SD] per 10 high power fields (HPF)
As evidenced by Fig. 7, the analysis of Matrigel pellet vascularity after 14
days incubation in-vivo demonstrated significant induction of angiogenesis in
both
31 the PR11 and the native PR-39 pellets. The control Matrigel pellets,
however,
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39
1 showed no evidence of angiogenesis as such. Clearly therefore, the short-
length
PR11 peptide is fully efficacious and effective in stimulating angiogenesis in-
vivo.
Conclusions:
(1) The described experiments and empirical data have demonstrated that PR-39
6 peptide has the ability to selectively alter activity of 20S proteasome in
human
endothelial cells by interacting with proteasomes in a reversible manner. This
interaction leads to suppression of IxB and HIF-la degradation while not
affecting
expression of other proteasome-dependent proteins such as p105 NFxB or HSP70.
Unlike other proteasome inhibitors, treatment with PR39 is not associated with
any
11 cellular cytotoxicity. Thus, PR39 and its related peptides provide a unique
and
unforeseen means of regulating cellular function and stimulating angiogenesis.
(2) Several observations also set PR39 apart from the conventionally known
proteasome inhibitors. First, PR39-mediated inhibition of IxBa degradation is
16 demonstrably reversible, unlike that of lactacystin. Second, long-term
exposure of
several cell types to PR39 did not result in any cytotoxicity, in contrast to
the rapid
cell death typically observed following cell treatment with lactacystin or
MG132.
This observation shows that PR39 peptide differentially affects processing of
various
and different intracellular proteins. Also supporting this view is the
observation that
21 while increasing HIF-la expression, PR39 administration had no meaningful
effect
on the expression of either NFxB or HSP70. Third, PR-39 peptide modulation of
proteasome activity plays a functional role since the observed increased
expression
of HIF-la was directly associated with an increased expression of its target
genes,
VEGF and flt-1.
26
The present invention is not to be limited in form nor restricted in scope
except by the claims appended hereto.
