In a different experiment, stable transfection of ID2 in U2OS, a human osteosarcoma cell line, resulted in an increase of cells in S phase detected by flow cytometry Iavarone, et al., 19
Trang 1the E-proteins, ID1 was shown to positively regulate the cell cycle by inhibition of an E-protein transcribed gene, the cyclin dependent kinase (CDK) inhibitor, p21
Downregulation of p21 caused a cascade of signaling events that ultimately led to the activation of genes required for S phase progression (Prabhu, et al., 1997) In a different experiment, stable transfection of ID2 in U2OS, a human osteosarcoma cell line, resulted in an increase of cells in S phase detected by flow cytometry (Iavarone,
et al., 1994)
Constitutively expressed ID genes in immortalized fibroblast cells was shown to cause cytoskeletal disorganization and loss of adhesion (Deed, et al., 1993) ID genes had also been shown to immortalize primary mouse fibroblasts when co-transfected with Bcl2 (Norton, et al., 1998) and in particular, ID1 was able to
immortalize primary human keratinocytes leading to the activation of telomerase and inhibition of pRb, a known tumour suppressor (Alani, et al., 1999)
Best illustrated in breast cancer, overexpression of ID1 caused mammary
epithelial cells to invade the basement membrane and had been shown to be highly associated with more aggressive tumours (Desprez, et al., 1998) Constitutive
expression of ID1 in a non-invasive breast cancer cell line produced uncontrolled growth and increased invasion (Lin, et al., 2000) In addition, ID1 was shown to be involved in the regulation of steroid-hormone-responsive growth in breast cancer cells, a loss of which led to uncontrolled growth of breast cancer cells
1.8 Properties and roles of ID2
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ID2 was first cloned in 1991 and functioned to inhibit bHLH-domain containing transcription factors in a similar capacity as the other IDs (Langlands, et al., 1997, Sun, et al., 1991) Full-length monomeric ID2 has 134 residues and a calculated molecular weight of 15kDa The HLH domain of ID2 predicted by Pfam centered around residues 24-76 Expression of ID2 was prevalent in early development in
Trang 2many different cell types (Biggs, et al., 1992, Sun, et al., 1991) but had been most studied in the developing muscle and nervous systems (Neuman, et al., 1993, Zhu,
et al., 1995) Recently, it was also found to be the earliest marker of trophectoderm cell fate in mouse pre-implantation embryos (Guo, et al., 2010)
Besides inhibition of bHLH-containing proteins, ID2, unlike ID1 and ID3 had the ability to bind a non-HLH tumour suppressor, the retinoblastoma protein (pRb), a nuclear phosphoprotein that blocked cell cycle progression by complexing with E2F transcription factors (Sidle, et al., 1996) E2F transcription factors acted to transcribe genes involved in the G1-S transition as well as the S phase of the cell cycle pRb bound E2F proteins to inhibit their function by blocking cell cycle progression
Sequestering of pRb by ID2 therefore promoted cell cycle progression (Iavarone, et al., 1994, Lasorella, et al., 1996, Toma, et al., 2000) Introduction of pRb in pRb-null SAOS2 human osteosarcoma cells showed a reduction in proliferation When these cells were co-transfected with both pRb and ID2, the proliferative inhibition was
mitigated by the binding of ID2 HLH to Rb (Iavarone, et al., 1994) Owing to this property, an increased level of ID2 in some tumour cells was shown to lead to cellular transformation and tumourigenesis (Gabellini, et al., 2006, Perk, et al., 2005) This made ID2 a promising therapeutic target for the treatment of some cancers (Fong, et al., 2004, Gray, et al., 2008) Therefore, biochemical and structural studies would be useful in understanding ID2’s mechanism of action for developing compounds to block the ID2-pRb interaction
Sequence conservation to the other IDs within the HLH domain averaged at 85% identity whilst at the N and C-termini, the identity dropped to an average of 0-40% over 70-80 residues As the HLH domain was found to be key for dimerization (Pesce,
et al., 1993), the high amount of identity suggested that subtle structural differences between ID2 and other IDs as well as the other bHLHs at the dimer interface could
Trang 3be responsible for these binding preferences Extrapolating the ID3 homology model
to ID2, it was expected that the predicted homo- and heterodimeric interactions would be very similar At a sequence level, the modeled ID3 and ID2 homodimers shared conserved hydrogen bonds at Y44, L50, Y72, Q77 as well as core
hydrophobic residues M39, L46, L49, M62, I69, I72, L75 At a structural level, the predicted ID-HLH topology was the same as other bHLH-containing proteins so it was expected that they would bind to all bHLH-containing proteins of the same structure
However, studies showed that ID2 did not form heterodimers with all
bHLH-containing proteins; rather, it selectively interacted with the Group A HLH-bHLH-containing proteins E47 and E12 as well as MYOD1 but not the Group B USF1 (Sun, et al., 1991) nor the bHLH-z structures like MYC and MAX (Figure 4) When ID2 was
cloned, the authors wrote that it did not homodimerize well (Sun, et al., 1991) Others reported ID2 homodimer to be insoluble and tending to aggregate, especially at high concentrations (Colombo, et al., 2006) This could be a reason for the sparse
structural information on ID2
Trang 41.9 Aim and Scope of Project
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From previous studies, it was clear that the bHLH-containing proteins played crucial roles in early development, neurogenesis, myogenesis and cancer The HLH domain was found to be well conserved throughout evolution with ID2 having an ortholog in Drosophila (emc gene) A special class of HLH-containing proteins, the IDs were especially interesting due to their lack of a basic domain along with their availability in almost all eukaryotic cells Members of this family have been known to regulate other Group A bHLH-containing proteins such as E47 (TCF3) and MyoD (MYOD1) but very little was known about why they were so specific in their
interactions given the structural similarities to each other and to all the different groups of HLH-containing proteins Compounded with this was the fact that IDs were short-lived proteins, as they functioned to regulate cell fate and were required to disengage once their roles were complete This caused problems in studying these proteins as they tended to be highly unstable
ID2 was chosen to represent the Group D HLH-containing proteins in order to find
a way in which to stabilize the protein enough for expression and crystallization without compromising its functionality An ID2 structure would provide a means to better understand how this group differed structurally from other HLH-containing proteins as well as to its paralog ID3 Finally, mutations at key residues based on the structural analysis of ID would help to explain differences in binding affinities
Hence, the specific aims of this study were to:
1) Clone, express, purify and crystallize ID2
2) Solve the crystal structure of ID2
3) Analyze the structure of ID2 and look at similarities and differences to other HLH-containing proteins including ID3
Trang 54) Determine differences in binding between ID1, ID2 and ID3 to E47, MyoD and MASH1 through electrophoretic mobility shift assays and mutagenesis experiments
of specific residues based on the structural analysis
Trang 6CHAPTER 2: MATERIALS and METHODS
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2.1 Cloning
ID2 constructs detailed in Table 2 were cloned from full-length cDNA (a gift from Scripps) using Gateway (Invitrogen) cloning technology Inserts were amplified by PCR using custom attB-containing primers shown in Table 3 and Table 4 PCR
products were recombined with entry vector pDONR221 (Invitrogen) to yield an entry
clone that was transformed into OneShot competent Escherichia coli (DE3) cells
(Invitrogen) and plated on LB agar plates containing 100 µg/ml kanamycin Single colonies were used to inoculate 5 ml Luria Broth (LB) containing 50 µg/ml kanamycin and allowed to grow shaking overnight at 37°C The overnight culture was
centrifuged at 720 g in an Eppendorf 5804R with A-4-44 swing-bucket rotor and the
pellet used for plasmid isolation using QiaPrep Spin Plasmid Miniprep Kit The entry clone was subsequently subcloned via the Gateway LR reaction (Invitrogen)
according to the manufacturer’s protocols into several expression vectors containing sequences for different affinity and solubility tags namely, pDest-17 (His6), pETG-20A (His6-TrxA), pDest-565 (His6-GST), pDest-HisMBP (His6-MBP), pETG-60A (His6-NusA) The expression clones were transformed into BL21 (DE3) Competent E
coli cells (Invitrogen) and plated on LB agar plates containing 100 µg/ml Ampicillin Single colonies were isolated and grown in 5 ml LB + 100 µg/ml Ampicillin and
allowed to grow overnight at 37°C, shaking The same protocol used to isolate the entry clones was used for the expression plasmids Inserts were confirmed by
sequencing (1st base, http://www.base-asia.com) In addition, 2 ml glycerol stocks of the expression clones were stored (1 ml 70% glycerol + 1 ml overnight culture) at -80°C for future use
Trang 7Table 2: ID2 constructs and their theoretical biochemical properties estimated by ProtParam (Wilkins, et al., 1999) Constructs described in detail (yellow highlight)
Construct cDNA
(bp)
AA start
AA end #AA pI
MW (kDa)
Extinction Coefficient (M -1 cm -1 )
Table 3: Primer base for BP cloning (Invitrogen) to create the entry clone for Gateway LR reaction (Invitrogen) attB sites (italics), sequence transferred into pDonr vector during BP reaction (bold), protease sites (underlined) Final selected protease is highlighted in yellow
Primer
Type
Protease
Site
Primer base sequence
Forward
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCTGG
AAGTGCTGTTTCAGGGCCCG Forward
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAA
ACCTGTATTTTCAGGGC
Forward
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCTGG
TGCCGCGTGGCAGC Reverse
ggggaccactttgtacaagaaagctgggttTTA
Table 4: Sequences for each construct were added to the primer base in Table 3 to complete the primer sequences used for BP cloning
Construct Forward 5’ Reverse 3’
GAGG
TCAGCCACACAGTGCTTTGC TGTC
ACGAC
ATGCGAGTCCAGGGCGATCT GCA
GAGG
ATGCGAGTCCAGGGCGATCT GCA
GAGG
ACAGGATGCTGATATCCGTG TTGAG
ACGAC
GATGCGAGTCCAGGGCGATC
TGCA
GAGG
GATGCGAGTCCAGGGCGATC
TGCA
Trang 82.2 Site directed mutagenesis
Site-directed mutagenesis (QuickChange Kit, Stratagene) was performed as per manufacturer’s instructions ID2 helix-1 single mutants Y37D, D41G, D41H, K47R and the double mutant Y37D_D41H were created using specific primers The same was done for ID2 helix-2 mutants Y71A, Y71F, Q76A, Q76D and double mutant Y71A_Q76A Mutants to interrogate the loop region of ID2 were Q55A, Q55R, K61A, K61Q and the double mutant Q55A_K61A The equivalent ID3 loop mutants were R60A, R60Q, Q66A and Q66K Primers are listed in Table 5 and were ordered as HPLC grade to ensure purity for the increased success of the mutagenesis
experiment
Table 5: Mutagenesis primers Mutation shown after first underscore and changed residue denoted by red bold letter Forward and reverse primers denoted by _F and _R respectively Changed nucleotide (s) denoted by grey highlight
Hydrogen-bond mutants (helix-2)
Residue 66-76
ID2_Y71A_F
ID2_Y71A_R
Q H V I D A I L D L Q
CAG CAC GTC ATC GAC GCC ATC TTG GAC CTG CAG CTG CAG GTC CAA GAT GGC GTC GAT GAC GTG CTG
Residue 71-81
ID2_Q76A_F
ID2_Q76A_R
Y I L D L A I A L D S
TAC ATC TTG GAC CTG GCG ATC GCC CTG GAC TCG CGA GTC CAG GGC GAT CGC CAG GTC CAA GAT GTA
Residue 68-79
ID2_Y71A_Q76A_F
ID2_Y71A_Q76A_R
V I D A I L D L A I A L
GTC ATC GAC GCC ATC TTG GAC CTG GCG ATC GCC CTG
CAG GGC GAT CGC CAG GTC CAA GAT GGC GTC GAT GAC
Residue 71-81
ID2_Q76D_F
ID2_Q76D_R
Y I L D L D I A L D S
TAC ATC TTG GAC CTG GAT ATC GCC CTG GAC TCG CAG CGA GTC CAG GGC GAT ATC CAG GTC CAA GAT
Residue 65-75
ID2_Y71F_F
ID2_Y71F_R
L Q H V I D F I L D L
CTG CAG CAC GTC ATC GAC TTC ATC TTG GAC CTG CAG GTC CAA GAT GAA GTC GAT GAC GTG CTG CAG
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Trang 9!
Table 5: Mutagenesis primers (continued from above)
N-terminal helix-1 binding specificity mutants
Residue 32-43
ID2_Y37D_F
ID2_Y37D_R
-P M S L L D N M N D C Y-
CCGATGAGCCTGCTAGACAACATGAACGACTGCTAC GTAGCAGTCGTTCATGTTGTCTAGCAGGCTCATCGG
Residue 35-47
ID2_D41G_F
ID2_D41G_R
-L L Y N M N G C Y S K L K-
CTGCTATACAACATGAACGGCTGCTACTCCAAGCTCAAG CTTGAGCTTGGAGTAGCAGCCGTTCATGTTGTATAGCAG
Residue 35-47
ID2_D41H_F
ID2_D41H_R
-L L Y N M N H C Y S K L K-
CTGCTATACAACATGAACCACTGCTACTCCAAGCTCAAG CTTGAGCTTGGAGTAGCAGTGGTTCATGTTGTATAGCAG
Residue 42-52
ID2_K47R_F
ID2_K47R_R
-C Y S K L R E L V P S-
TGCTACTCCAAGCTCAGGGAGCTGGTGCCCAGC GCTGGGCACCAGCTCCCTGAGCTTGGAGTAGCA
Residue 35-47
ID2_Y37D_D41G_F
ID2_Y37D_D41G_R
-L L D N M N G C Y S K L K-
CTGCTAGACAACATGAACGGCTGCTACTCCAAGCTCAAG CTTGAGCTTGGAGTAGCAGCCGTTCATGTTGTCTAGCAG
Residue 35-47
ID2_Y37D_D41H_F
ID2_Y37D_D41H_R
-L L D N M N H C Y S K L K-
CTGCTAGACAACATGAACCACTGCTACTCCAAGCTCAAG CTTGAGCTTGGAGTAGCAGTGGTTCATGTTGTCTAGCAG
ID2 loop region mutants
Residue 50-60
ID2_Q55A_F
ID2_Q55A_R
N K K V S-GTGCCCAGCATCCCCGCGAACAAGAAGGTGAGC
GCTCACCTTCTTGTTCGCGGGGATGCTGGGCAC
Residue 50-60
ID2_Q55R _F
ID2_Q55R _R
N K K V S-GTGCCCAGCATCCCCCGGAACAAGAAGGTGAGC
GCTCACCTTCTTGTTCCGGGGGATGCTGGGCAC
Residue 56-66
ID2_K61A_F
ID2_K61A_R
M E I L Q-AACAAGAAGGTGAGCGCGATGGAAATCCTGCAG
CTGCAGGATTTCCATCGCGCTCACCTTCTTGTT
Residue 56-66
ID2_K61Q_F
ID2_K61Q_R
M E I L Q-AACAAGAAGGTGAGCCAGATGGAAATCCTGCAG
CTGCAGGATTTCCATCTGGCTCACCTTCTTGTT
Residue 52-64
ID2_Q55A_K61A_F
ID2_Q55A_K61A_R
M E I-AGCATCCCCGCGAACAAGAAGGTGAGCGCGATGGAAATC
GATTTCCATCGCGCTCACCTTCTTGTTCGCGGGGATGCT
ID3 loop region mutants
Residue 55-65
ID3_R60A_F
ID3_R60A_R
G T Q L S-GTACCCGGAGTCCCGGCAGGCACTCAGCTTAGC
GCTAAGCTGAGTGCCTGCCGGGACTCCGGGTAC
Residue 55-65
ID3_R60Q_F
ID3_R60Q_R
G T Q L S-GTACCCGGAGTCCCGCAAGGCACTCAGCTTAGC
GCTAAGCTGAGTGCCTTGCGGGACTCCGGGTAC
Residue 61-71
ID3_Q66A_F
ID3_Q66A_R
V E I L Q-GGCACTCAGCTTAGCGCGGTGGAAATCCTACAG
CTGTAGGATTTCCACCGCGCTAAGCTGAGTGCC
Residue 61-71
ID3_Q66K_F
ID3_Q66K_R
V E I L Q-GGCACTCAGCTTAGCAAGGTGGAAATCCTACAG
CTGTAGGATTTCCACCTTGCTAAGCTGAGTGCC
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Trang 102.3 Protein expression optimization
To test for protein expression and solubility of the different expression clones, factors known to affect protein expression were varied Experiments were performed
in 5 ml small-scale experiments Variable factors included media (Luria Broth (LB) and Terrific Broth (TB)), induction temperatures and times (17°C for 18 hrs, 25°C for
5 hrs, 30°C for 3 hrs), IPTG concentrations (0.2 mM – 1 mM) and solubility tags Glycerol stock scrapes were used to inoculate 5 ml LB overnight at 37°C 2% overnight inoculums were added to 5 ml fresh LB or TB and grown shaking at 37°C till an OD600 of 0.6 was reached 500 µl samples were taken before induction,
pelleted down for 10 min at 15,700 g in an Eppendorf 5415R mini-centrifuge with
F45-24-11 rotor at 4°C, and stored at -20°C for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Each tube was then induced with varying concentrations of Isopropyl-β-D-thio-galactoside (IPTG) and grown at each of the temperatures mentioned previously At the end of the induction times, the
cultures were centrifuged at 4,225 g for 10 min at 4°C in a Sorvall SLA-3000 rotor
and the supernatant discarded Pellets were resuspended in 500 µl lysis buffer (50
mM Tris-HCL pH 8.0, 300 mM NaCl), transferred to a 1.5 ml eppendorf tube and sonicated on ice for 10s at 35% amplitude (1s on, 1s off) The sonicate was
centrifuged for 10min at 15,700 g in an Eppendorf 5415R mini-centrifuge with
F45-24-11 rotor at 4°C and samples of the supernatant and pellet together with the uninduced sample were evaluated by SDS-PAGE on 12% SDS-Tris-Glycine gels run
at 200V for 40 min
2.4 Native protein expression
Glycerol stock scrapes of HLH24-82-L and N-HLH82-L pDest-565/TEV constructs were grown overnight in 200 ml LB at 37°C 10 ml overnight inoculums were cultured
in 5L of LB containing 100 µg/ml Ampicillin separated equally into 10 2L flasks in a