The role of TRIP br proteins in the regulation of mammalian gene transcription and cell cycle progression
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2003
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I wish to acknowledge the following kind individuals who made this work
possible.
First of all, I thank Dr. Stephen Hsu I-Hong (MD PhD), to whom I owe an
immense debt of gratitude for his support and encouragement, for his enlightening
teachings and guidance, and for his invaluable mentorship and friendship. He has
created a unique and yet conducive environment within which good science is learnt
and practiced and good spiritual values are nurtured and inculcated.
I am grateful to the members of my supervisory committee from IMCB,
Willian Chia, Paramjeet Singh and Hans Uli-Bernard for constantly monitoring my
progress and pointing me in the right research direction. I also wish to thank the
Medicine Faculty of NUS and IMCB for the special opportunity to undertake my
research training under a cross-faculty collaborative program.
No words can express my appreciation to all the beloved members of the
Laboratory of Molecular Nephrology and Gene Regulation, who made the lab such an
enjoyable place to work and learn. Special thanks to fellow colleagues Sharon Thio,
Christopher Yang, Shahidah, Jit Kong and Chui Sun for their moral and technical
supports, for reviewing my manuscripts and for many constructive and insightful
discussions. I also wish to acknowledge Olivia Chao, for her generous friendship and
help in obtaining reagents for some key experiments.
My utmost gratitude to my teachers, Charlie and Linda Lee, and all the great
members of the SOONYE
TM
organization, for opening my eyes to the true meaning of
iii
the proverb “It’s your attitude, not your aptitude that will determine your altitude in
life”.
To my beloved family – Mom and Khe Chai, endless thanks for your love and
support. Most importantly, I send all my love to my angel, my wife Ally, who made
everything possible through her love and spiritual support, who gave me the strength
and courage to carry on, and who gave me the reason to succeed.
Finally, I wish to convey my heartfelt gratitude to all those kind people whom
I neglect to mention by name.
KG, SIM
April, 2003
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Page
Title i
Acknowledgements ii
Table of Contents iv
List of Figures & Tables x
List of Abbreviations xvi
Presentations & Publications arising from PhD Thesis xviii
Abstract 1
Introduction
1. The Plant Homeodomain (PHD) zinc finger and the
bromodomain
1.1 PHD zinc fingers and bromodomains are evolutionarily
conserved secondary structural protein motifs
1.2 The PHD zinc fingers and the bromodomains: functional
implications
1.2.1 Protein motifs with biological significance
1.2.2 The role of PHD zinc fingers and the bromodomains
in the regulation of eukaryotic gene transcription
2. Transcriptional regulators interacting with the PHD zinc finger
and/or the bromodomain (TRIP-Br)
2.1 Historical perspective
2.2 The structural features of the TRIP-Br proteins
2.3 The functional properties of the TRIP-Br proteins
2.3.1 The unique ability to interact with the PHD zinc
finger and/or the bromodomain
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18
v
2.3.2 The TRIP-Br proteins possess potent acidic
transactivation domains
2.3.3 Co-regulation of the E2F-1/DP-1 transcriptional
activity
2.3.4 Functional significance of the PHD-bromodomain-
interacting potentials of the TRIP-Br proteins
2.4 The TRIP-Br proteins: a novel class of cell cycle regulator
2.4.1 The mammalian cell cycle
2.4.2 Functional relationships between the TRIP-Br
proteins and the E2F family of transcription factors
2.4.3 Functional interactions between the TRIP-Br
proteins and the cell cycle regulatory protein, cyclin
A
2.4.4 Cell cycle regulated expression of hTRIP-Br1
2.4.5 hTRIP-Br1, a Cdk4-interacting regulatory protein
2.4.6 The integrator model of TRIP-Br protein function in
cell cycle regulation
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Materials and Methods
1. Materials
1.1 Plasmid DNA and cDNA clones
1.2 Biochemical reagents
1.3 Synthetic peptides
1.4 Synthetic oligonucleotides
1.5 Bacterial strains
1.6 Tissue culture cell lines
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2. Methods
2.1 Maintenance and Handling of Tissue Culture Cells
2.2 Preparation of Competent Bacterial Cells
2.3 Transformation and Maintenance of Plasmid DNA
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vi
Clones
2.4 Mini- and Maxi-scale Preparation of Plasmid DNA
from Bacteria
2.5 Quantitation and Purity Assessment of DNA
2.6 Screening of transformants for positive clones
2.7 Gel electrophoresis of DNA products
2.8 Preparation of Protein by in vitro Translation
2.9 Preparation of Peptide Stock Solutions
2.10 Preparation of Proteins from Tissue Culture
2.11 Analysis of Proteins by Polyacrylamide Gel
Electrophoresis (PAGE)
2.12 Western Blot for Protein Detection
2.13 Electro-mobility Shift Assays (EMSA)
2.14 Treatment of Cells with Decoy Peptides
2.15 Confocal Microscopy
2.16 Measurement of Peptide Internalization Efficiency
by Fluorescence-activated Cell Sorting (FACS)
2.17 DNA Transfection and Sequential β-
Galactosidase/Luciferase Assays
2.18 DNA Enzyme Transfection
2.19 Semi-quantitative Reverse Transcription coupled to
Polymerase Chain Reaction (RT-PCR)
2.20 Cell Proliferation Assays
2.21 Determination of Cell Number
2.22 Colony Formation Assay
2.23 Cell Cycle Profile and TUNEL Staining Analyses by
Flow Cytometry
2.24 Cell Synchronization at the G2/M Boundary and
Cell Cycle Progression Analyses
2.25 Caspase Assay
2.26 Protein Decay Analysis
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2.27 Immunoprecipitation Assay (IP) 50
Results
1. Decoy peptides as molecular tools to probe the function of the
PHD-bromodomain-interacting domain of TRIP-Br proteins
1.1 Designing peptides that antagonize physical
interactions between TRIP-Br proteins and PHD
zinc fingers and/or bromodomain-containing
proteins
1.2 Evaluation of decoy peptide-mediated blocking
activity in vitro
1.3 Evaluation of the cell-penetrating properties and
blocking activity of the TRIP-Br decoy peptides in
vivo
51
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2. TRIP-Br decoy peptides reveal novel functions for TRIP-Br
proteins in the regulation of E2F-dependent transcriptional
activity
2.1 Decoy peptide treatment causes repression of an
artificial E2F-responsive reporter gene in vivo
2.2 The decoy peptides differentially down-regulate the
expression of endogenous E2F-responsive genes
64
67
3. TRIP-Br decoy peptides impose a proliferative block
3.1 The TRIP-Br decoy peptides inhibit DNA synthesis
as assessed by BrdU incorporation assay
3.2 The TRIP-Br decoy peptides suppress cellular
proliferation
70
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viii
4. The integrator function of the TRIP-Br proteins is implicated in
the regulation of cyclin E expression during cell cycle
progression
4.1 *Br1 or *Br2 perturbs the timing and the amplitude
of cyclin E protein expression
4.2 *Br1 or *Br2 does not alter the steady state level of
cyclin E mRNA transcript levels
4.3 *Br1 or *Br2 down-regulates the expression of
Fbxw7
4.4 Fbxw7 is a novel E2F-responsive and TRIP-Br co-
regulated gene
77
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5. Treatment with TRIP-Br decoy peptides is associated with sub-
diploidy
5.1 The TRIP-Br decoy peptides induce accumulation
of a sub-G1 population
5.2 Sub-diploidy induced by *Br1 Is biochemically
distinct from that triggered by *Br2
5.3 Sub-diploidy elicited by *Br1 or *Br2 is caspase-
independent
5.4 Incomplete DNA replication: an alternative
mechanism for sub-diploidization
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6. DNA enzymes targeting TRIP-Br mRNA inhibit serum-inducible
fibroblast proliferation
6.1 E-Br1 or E-Br2 DNA enzymes specifically “knock
down” serum-induced hTRIP-Br1 or hTRIP-Br2
gene transcription
6.2 E-Br inhibits serum-induced WI-38 cell proliferation
6.3 E-Br DNA enzymes prevent serum-induced S
phase entry
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6.4 E-Br DNA enzymes prevent serum-induced cyclin E
expression
122
Discussions 128
References 142
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FIGURE 1: Schematic illustration of the PHD zinc finger and
the bromodomain
5
FIGURE 2: Conservation of the PHD zinc finger across
evolutionary boundaries
7
FIGURE 3: Putative domain structure of human TRIP-Br
proteins
16
FIGURE 4: The mammalian cell cycle 22
FIGURE 5: The two possible mechanisms of pocket
protein/E2F-DP complex-mediated repression of
gene transcription in G1/G0 cells
26
FIGURE 6: Structural organization of mammalian E2F and DP
proteins
28
FIGURE 7: The integrator model of TRIP-Br Protein Function 33
FIGURE 8: Schematic illustration of the decoy peptide
antagonism strategy
52
FIGURE 9: Schematic representation of the TRIP-Br decoy
peptides
54
FIGURE 10: Electro-mobility shift analysis (EMSA)
demonstrating assembly of the DNA-GAL4/KRIP-
1/TRIP-Br super-shift complexes in vitro
57
xi
FIGURE 11A: EMSA showing blocking activity of the decoy
peptides
58
FIGURE 11B: Immunoprecipitation assay showing blocking
activity of the decoy peptides
59
FIGURE 12: Peptide internalization monitored by fluorescence
microscopy
61
FIGURE 13: Measurement of peptide internalization efficiency by
fluorescence activated cell sorting (FACS)
61
FIGURE 14: Antagonism of PHD-bromodomain interactions in
vivo
63
FIGURE 15A: Repression of E2F-responsive reporter by decoy
peptides
66
FIGURE 15B: Differential down-regulation of endogenous E2F-
responsive genes by decoy peptides
69
FIGURE 16A: Inhibition of DNA synthesis by decoy peptides 72
FIGURE 16B: Temporal growth profile of U2OS cells
73
FIGURE 16C: Inhibition of cell proliferation by decoy peptides
74
FIGURE 16D: Cell cycle analysis of U2OS cells after decoy peptide
treatment
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xii
FIGURE 17: Protein expression analysis of cell cycle regulators in
U2OS cells released from a nocodazole block with
or without 20 µM decoy peptide treatment
79
FIGURE 18A: TRIP-Br decoy peptide dose-response analysis in
asynchronous U2OS cells
82
FIGURE 18B: Dose-response effects of the TRIP-Br decoy peptides
on the expression levels of Fbxw7 in asynchronous
U2OS cells
83
FIGURE 18C: The 5’ flanking region of Fbxw7 contains several
putative E2F-binding elements
86
FIGURE 18D: Fbxw7 is a direct E2F target gene
87
FIGURE 18E: Fbxw7 is a novel E2F-responsive and TRIP-Br-
regulated gene
88
FIGURE 19A: Cell cycle analysis of U2OS cells at 48 hours post-
decoy peptide treatment
92
FIGURE 19B: Cell cycle analysis of U2OS cells at 72 hours post-
decoy peptide treatment
93
FIGURE 20A: TUNEL assay 94
FIGURE 20B: In vitro analysis of caspase activity in decoy peptide-
treated U2OS cells
97
FIGURE 20C: Western blot analysis of PARP cleavage by caspase-
3 in decoy peptide-treated U2OS cells
99
xiii
FIGURE 20D: Assay of DNA fragmentation in decoy peptide-
treated U2OS cells
99
FIGURE 21A: Control of DNA replication licensing in mammalian
cells
102
FIGURE 21B: Western blot analysis of Geminin expression in the
absence or presence of cyclin E/Cdk2 over-
expression during cell cycle progression
104
FIGURE 21C: Protein decay analysis of Geminin expression in the
absence or presence of cyclin E/Cdk2 over-
expression
105
FIGURE 21D: Protein decay analysis of Geminin expression in the
absence or presence of decoy peptide treatment
107
FIGURE 21E Protein decay analysis of Geminin expression in the
absence or presence of decoy peptide treatment, with
or without FBW7 rescue
108
FIGURE 21F Flow cytometry analysis of *Br1- or *Br2-induced
sub-diploidization in the absence or presence of
FBW7 rescue
109
FIGURE 22A: Schematic illustration of the general structural and
functional features of a DNA enzyme
111
FIGURE 22B: Post-transcriptional suppression of gene expression
by DNA enzymes
112
FIGURE 22C: DNA enzymes targeting TRIP-Br transcripts
113
FIGURE 23A: hTRIP-Br1 and hTRIP-Br2 expression is serum-
inducible
115
xiv
FIGURE 23B: E-Br1 and E-Br2 down-regulate TRIP-Br1 and
hTRIP-Br2 expression respectively
115
FIGURE 24A: E-Br1 or E-Br2 inhibits cell proliferation
117
FIGURE 24B: E-Br1 or E-Br2 suppresses colony formation
118
FIGURE 25A: E-Br1 or E-Br2 suppresses serum-inducible S phase
entry
120
FIGURE 25B: E-Br1 or E-Br2 does not influence E2F1/DP1-
and/or cyclin E/Cdk2-induced S phase entry
121
FIGURE 26A: E-Br1 and E-Br2 down-regulate serum-induced
cyclin E expression
124
FIGURE 26B: Post-translational regulatory events that contribute to
full activation of Cdk4 kinase activity
125
FIGURE 26C: E-Br1 and E-Br2 do not affect serum-induced cyclin
D1-Cdk4 interaction
126
FIGURE 26D: E-Br2, but not E-Br1 or E-SCR, abrogates Mat1-
Cdk7 interaction
127
FIGURE 27: A model for signal integration and transcriptional
regulation by TRIP-Br
130
FIGURE 28: TRIP-Br integrator function in the regulation of
E2F-dependent transcription and cell cycle
progression
140
xv
FIGURE 29: Regulation of serum-inducible cell cycle progression
by TRIP-Br
141
TABLE 1: Prominent examples of PHD zinc finger and/or
bromodomain-containing proteins.
6
TABLE 2: Descriptions of the E2F transcription factors 29
TABLE 3: Descriptions of six representative E2F-regulated
gene
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xvi
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AIRE Autoimmune Regulator
APC/C Anaphase Promoting Complex/Cyclosome
APECED Autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
ATRX
α-Thalassemia Mental Retardation, X-linked
BTM Basal Transcriptional Machinery
CAD Caspase-activated Deoxyribonuclease
CAK Cdk-activating Kinase
CBP CREB Binding Protein
Cdc Cell Division Cycle
Cdk Cyclin-dependent kinase
CHX Cycloheximide
Cki Cdk inhibitor
CREB Cyclic AMP Response Element Binding Protein
Cyp33 Cyclophilin-33
DFF DNA Fragmentation Factor
DHFR Dihydrofolate Reductase
DMSO Dimethyl Sulfoxide
EMSA Electromobility Shift Assay
ERK Extracellular Signal-regulated Kinase
Eto Etoposide
FAC-1 Fetal Alz50-reactive Clone-1
FACS Fluorescence-Activated Cell Sorting
FAT Factor Acetyltransferase
HAT Histone Acetyltransferase
HDAC Histone Deacetylase
ING-1 Inhibitor of Growth-1
IP Immunoprecipitation
KRAB Krüpple-type Associated Box
KRIP-1 KRAB Interacting Protein-1
LAP Leukemia-associated Protein
Mcm Minichromosome maintenance
MEKK1 Mitogen-activated protein kinase kinase Kinase-1
MLL Myeloid Lymphoid Leukemia
ORC Origin Recognition Complex
P/CAF p300/CBP-associated Factor
PAGE Polyacrylamide Gel Electrophoresis
PARP Poly(ADP-ribose) Polymerase
PCNA Proliferating Cell Nuclear Antigen
PHD zinc finger Plant Homeodomain zinc finger
xvii
pRB Retinoblastoma Tumor Suppressor
Pre-RC Pre-replication Complex
RBCC RING-B box-coiled coil
RT-PCR Reverse Transcription – Polymerase Chain Reaction
TAF
II
250 Transcription Activation Factor
TIF1 Transcriptional Intermediary Factor-1
TRIP-Br Transcriptional Regulator Interacting with PHD zinc finger and/or
bromodomain
TTC Trithorax Consensus
TUNEL Terminal dUTP Nucleotide End Labeling
WHSC1 Wolf-Hirschhorn Syndrome Complex-1
WSTF Williams Syndrome Transcription Factor
xviii
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Part of this PhD thesis work was presented in the following conferences
or meetings:
Year Conference/Meeting Nature of Participation
August 1999 3
rd
NUH-NUS Faculty of Medicine
Annual Scientific Meeting (ASM)
Poster presentation
May 2000 The Cell Cycle Meeting 2000 (Cold
Spring Harbor Laboratory, New
York)
Poster presentation
July 2000 4
th
NUH-NUS Faculty of Medicine
Annual Scientific Meeting (ASM)
NUH-NMRC Young
Scientist Award
Competition
July 2001 5
th
NUH-NUS Faculty of Medicine
Annual Scientific Meeting (ASM)
NUH-NMRC Young
Scientist Award
Competition; manuscript
short-listed for oral
presentation
October 2002 35
th
American Society of
Nephrology, Renal Week 2002,
Philadelphia
Short oral communication
Part of this PhD thesis work has been accepted for publication:
TRIP-Br Links E2F to Novel Functions in the Regulation of Cyclin E Expression
During Cell Cycle Progression and in the Maintenance of Genomic Stability
Khe Guan Sim, Zhijiang Zang, Christopher Maolin Yang, Joseph V. Bonventre and
Stephen I-Hong Hsu
Cell Cycle (Vol: 3; Issue: 10) In press.
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The TRIP-Br proteins (TRIP-Br1 and TRIP-Br2) are a novel family of
transcriptional regulators that have been proposed to function as “integrators” at E2F-
responsive promoters to integrate signals provided by PHD zinc finger- and/or
bromodomain-containing transcription factors. Two approaches were employed o
further probe the physiological function(s) of the TRIP-Br proteins, namely the decoy
peptide strategy and the DNA enzyme strategy.
Synthetic decoy peptides shown to antagonize the in vitro and in vivo
interaction between TRIP-Br1 (*Br1) or TRIP-Br2 (*Br2) and the PHD zinc finger
and/or bromodomain of other transcription factors, were introduced into U2OS cells
to further elucidate the TRIP-Br integrator function(s). Both the *Br1 and *Br2
peptides were found to differentially down-regulate the expression of endogenous
E2F-responsive genes in vivo, and impose a state of global cell cycle arrest. Thus, the
integrator function of TRIP-Br proteins is required for proper execution of E2F-
dependent mammalian cell cycle progression. Further analyses on synchronously
cycling U2OS cells showed that treatment with *Br1 or *Br2 caused deregulated
cyclin E protein accumulation during cell cycle progression. This was shown to be
associated with the down-regulation of the Fbxw7 gene, a novel E2F-responsive and
TRIP-Br co-regulated gene that encodes an ubiquitin ligase (E3) responsible for
targeting cyclin E for ubiquitin-mediated proteolysis. This finding suggests that the
regulation of ubiquitin-mediated proteolysis of cyclin E is one of the TRIP-Br
integrator functions required for proper execution of E2F-dependent cell cycle
progression. In addition, prolonged exposure of cells to the decoy peptides induced
2
massive caspase-independent cellular sub-diploidization. The process of decoy
peptide-induced sub-diploidization was associated with abnormal stabilization of
Geminin, which occurs through a mechanism involving cyclin E deregulation.
To study the involvement of the TRIP-Br proteins in the regulation of cellular
proliferation, DNA enzymes that specifically suppress the expression of endogenous
hTRIP-Br1 (E-Br1) or hTRIP-Br2 (E-Br2), were introduced into quiescent WI-38
human diploid fibroblasts. Both E-Br1 and E-Br2 were capable of efficiently
suppressing serum-inducible S phase entry and cellular proliferation in WI-38 cells,
consistent with a prior report that the TRIP-Br proteins are required for serum-
induced cell cycle progression.
3
I
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(1.1) PHD zinc fingers and bromodomains are evolutionarily conserved
secondary structural protein motifs
The Plant Homeodomain (PHD) zinc finger and the bromodomain are
evolutionarily conserved protein sequence motifs. The PHD zinc finger, also referred
to as TTC (trithorax consensus) or LAP (leukemia-associated protein) motif, is a
zinc-coordinating protein secondary structure that features seven cysteine residues
and a histidine residue spatially arranged in a cysteine4-histidine-cysteine3 (Cys4-
His-Cys3) consensus with intervening sequences varying in length and composition
(Figure 1, bottom panel)
1, 2
. In a PHD finger, two zinc atoms are coordinated by the
cysteine and histidine residues in a cross-brace fashion reminiscent of the zinc
coordination found in the RING finger
3
. In contrast, the bromodomain, which is a
~110-amino-acid structural module, has been shown to adopt an atypical left-handed
four-helix bundle (helices αZ, αA, αB and αC) (Figure 1, top panel)
4
.
The PHD zinc fingers and/or the bromodomains are structural features
characteristic of a host of nuclear proteins that function in gene transcriptional
regulation (Table 1). Proteins known to possess one or more PHD zinc fingers
include transcriptional activators such as HRX (ALL-1, MLL, Htrx), a human
homologue of Drosophila trithorax involved in chromosomal translocations in acute
leukemia
5
, as well as transcriptional repressors such as the Drosophila Polycomb
4
group proteins. Examples of bromodomain-containing proteins include integral
components of chromatin remodeling complexes (histone acetyltransferase [HAT]-
associated proteins), such as p300/CBP, P/CAF (p300/CBP-associated factor)
TAF
II
250, SNF2-SWI2 and GCN5
6
. PHD zinc fingers and bromodomains are also
found to co-exist in the transcriptional adaptor protein p300 as well as the co-
repressors KRIP-1 (KAP-1 or TIF1β) and TIF1α
1
.
5
Schematic illustration of the PHD zinc
finger and the bromodomain.
Top panel: The secondary structure of a bromodomain encoding helices Z, A,
B and C. Source of figure:
4
.
Bottom panel: The primary structure of a PHD zinc finger highlighting the
Cys4-His-Cys3 consensus. “X” represents any amino acid residue. Source of
figure:
3
.
6
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β
β
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β
(
(
K
K
A
A
P
P
-
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1
1
)
)
(
(
T
T
r
r
a
a
n
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s
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c
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r
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i
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p
p
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m
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F
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a
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c
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r
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1
1
β
β
)
)
a
a
n
n
d
d
T
T
I
I
F
F
1
1
α
αα
α
α
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C
C
o
o
m
m
p
p
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P
P
H
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D
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c
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r
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-
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b
b
r
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m
m
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d
d
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m
m
a
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T
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r
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p
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a
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c
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-
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r
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p
p
r
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r
r
s
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f
f
K
K
r
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ü
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p
p
p
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l
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-
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y
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p
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a
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s
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c
c
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x
(
(
K
K
R
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)
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m
m
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d
d
r
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p
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s
s
s
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i
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o
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n
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*
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M
M
i
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-
-
2
2
β
ββ
β
β
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P
P
H
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D
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c
c
f
f
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K
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w
w
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0
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r
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(
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)
;
;
p
p
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S
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1
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(
W
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H
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r
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c
c
h
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h
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d
d
r
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m
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C
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m
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p
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)
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c
c
f
f
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c
c
h
h
h
h
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r
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n
S
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d
d
r
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m
m
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(
(
c
c
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m
m
p
p
l
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x
x
d
d
y
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s
m
m
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r
p
p
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h
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,
m
m
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t
a
a
r
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d
a
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t
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)
)
*
*
W
W
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(
W
W
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l
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a
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)
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w
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t
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h
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t
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l
l
l
l
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a
m
m
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s
S
S
y
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n
n
d
d
r
r
o
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m
m
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c
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x
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d
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s
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m
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r
p
p
h
h
y
y
,
,
m
m
e
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n
n
t
t
a
a
l
l
r
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e
t
t
a
a
r
r
d
d
a
a
t
t
i
i
o
o
n
n
)
)
T
T
A
A
B
B
L
L
E
E
1
1
:
:
P
P
r
r
o
o
m
m
i
i
n
n
e
e
n
n
t
t
e
e
x
x
a
a
m
m
p
p
l
l
e
e
s
s
o
o
f
f
P
P
H
H
D
D
z
z
i
i
n
n
c
c
f
f
i
i
n
n
g
g
e
e
r
r
a
a
n
n
d
d
/
/
o
o
r
r
b
b
r
r
o
o
m
m
o
o
d
d
o
o
m
m
a
a
i
i
n
n
-
-
c
c
o
o
n
n
t
t
a
a
i
i
n
n
i
i
n
n
g
g
p
p
r
r
o
o
t
t
e
e
i
i
n
n
s
s
.
.
T
T
h
h
e
e
(
(
*
*
)
)
l
l
a
a
b
b
e
e
l
l
d
d
e
e
s
s
i
i
g
g
n
n
a
a
t
t
e
e
s
s
d
d
i
i
s
s
e
e
a
a
s
s
e
e
-
-
a
a
s
s
s
s
o
o
c
c
i
i
a
a
t
t
e
e
d
d
P
P
H
H
D
D
z
z
i
i
n
n
c
c
f
f
i
i
n
n
g
g
e
e
r
r
a
a
n
n
d
d
/
/
o
o
r
r
b
b
r
r
o
o
m
m
o
o
d
d
o
o
m
m
a
a
i
i
n
n
-
-
c
c
o
o
n
n
t
t
a
a
i
i
n
n
i
i
n
n
g
g
p
p
r
r
o
o
t
t
e
e
i
i
n
n
s
s
.
.
7
Conservation of the PHD zinc finger
across evolutionary boundaries.
Numbers denote amino acid residues. Residues in bold represent conserved
hydrophobic amino acids. Source of figure:
3
