Telomerase: a promising target for cancer Scott Cohen Sydney - - PowerPoint PPT Presentation

Telomerase: a promising target for cancer

Scott Cohen

Sydney Australia

linear chromosomes have ends!

prokaryotic chromosome (circular) eukaryotic chromosome (linear) presence of chromosome “ends” posed new challenges to cellular biology

telomeres: distinguishing “end” from “break”

how can the cell distinguish the chromosome end from a deleterious internal DNA break that requires repair?

telomeres:

the unique DNA/protein complexes at the ends of linear chromosomes

telomeres: unique DNA/protein complex

human telomeres consist of tandem repeats of 5’-TTAGGG-3’: ~5-15 kb double-stranded DNA ~300-500 nt single-stranded TTAGGG 3’-overhang

5’-TTAGGGTTAGGGTTAGGG(TTAGGG)nTTAGGG-3’ 3’-A A T C C C A A T C C C centromere

5’-TTAGGGTTAGGGTTAGGG(TTAGGG)nTTAGGG-3’ 3’-A A T C C C A A T C C C centromere

telomeric DNA coated with sequence-specific DNA binding proteins

Telomeric Repeat Factors 1,2 (TRF1, TRF2) Protection of Telomeres 1 (POT1)

human telomeres consist of tandem repeats of 5’-TTAGGG-3’:

telomeres: unique DNA/protein complex

5’-TTAGGGTTAGGGTTAGGG(TTAGGG)nTTAGGG-3’ 3’-A A T C C C A A T C C C centromere

this unique DNA/protein structure distinguishes the telomere from an internal double-strand DNA break human telomeres consist of tandem repeats of 5’-TTAGGG-3’:

telomeres: unique DNA/protein complex

telomeres: a conserved structure in eukaryotes

5’-TTAGGG-3’ 5’-TTGGGG-3’

human Tetrahymena

the telomere: •a “cap” protecting the end of the chromosome, essential for genomic stability

• a conserved structure in eukaryotic evolution

telomere shortening and cellular lifespan

20 40 60 80 100

population doublings

4 5 6 7 8 9

telomere length (kb)

Rufer et al., (1999) J. Exp. Med. 190: 157-167.

telomere shortening and cellular lifespan

age (yrs)

0 10 20 30 40 50 60 70 80 90

telomere length (arbitrary units) lymphocytes

telomere shortening and cellular lifespan

senescence: non-proliferative cellular state – permanent withdrawal from cell cycle senescence evolved as requisite barrier to unlimited cellular proliferation…..cancer

marginal critical

senescence telomere length population doublings

telomere shortening and cellular lifespan

Telomerase

marginal critical

genetic mutations  activation of a telomere maintenance mechanism: immortalisation senescence population doublings telomere length

specific association of telomerase activity and cancer

cancer % telomerase positive breast carcinoma 88 colorectal carcinoma 86 small cell lung carcinoma 100

• varian carcinoma

91 prostate carcinoma 90

Kim et al., (1994) Science 266: 2011-2015.

telomerase NOT detected in corresponding normal tissue!!!

Telomerase: a specialised reverse transcriptase

TERT motifs RT motifs

hTERT

hTR A A U C C C A A U C

3’

catalytic protein component: Telomerase Reverse Transcriptase (hTERT)

Lingner et al., (1997) Science 276: 561-567. human

TERT motifs RT motifs

hTERT

hTR A A U C C C A A U C

3’ Tetrahymena human

Telomerase: a specialised reverse transcriptase

catalytic protein component: Telomerase Reverse Transcriptase (hTERT)

Lingner et al., (1997) Science 276: 561-567. Bryan et al., (1998) Proc. Natl. Acad. Sci. USA 95: 8479-8484.

hTERT

hTR A A U C C C A A U C

3’

Telomerase RNA (hTR) provides an internal template complementary to the telomeric DNA sequence: 5’-TTAGGG-3’

TERT motifs RT motifs

Telomerase: a specialised reverse transcriptase

catalytic protein component: Telomerase Reverse Transcriptase (hTERT)

hTERT

hTR A A U C C C A A U C

3’

extension of telomere by telomerase: 1) binding to 3’-end

TTAGGGTTAGGG T T A G-3’ centromere

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G-3’ centromere

extension of telomere by telomerase: 1) binding to 3’-end

extension of telomere by telomerase: 2) nucleotide addition

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G-3’ centromere

extension of telomere by telomerase: 2) nucleotide addition

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G-3’ centromere

extension of telomere by telomerase: 2) nucleotide addition

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G-3’ centromere

extension of telomere by telomerase: 2) nucleotide addition

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T-3’ centromere

extension of telomere by telomerase: 2) nucleotide addition

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T T-3’ centromere

extension of telomere by telomerase: 2) nucleotide addition

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T T A-3’ centromere

extension of telomere by telomerase: 2) nucleotide addition

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T T A G-3’ centromere

extension of telomere by telomerase: 3) translocation

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T T A G-3’ centromere

extension of telomere by telomerase: 3) translocation

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T T A G-3’ centromere

hTERT

hTR A A U C C C A A U C

3’ 5’-TTAGGGTTAGGG T T A G G G

extension of a telomeric primer with dTTP, dATP, α-32P-dGTP

dTTP dATP α-32P-dGTP

direct activity assay for human telomerase

hTERT

hTR A A U C C C A A U C

3’ 5’-TTAGGGTTAGGG T T A G G G

extension of a telomeric primer with dTTP, dATP, α-32P-dGTP

dTTP dATP α-32P-dGTP 5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG 5’-TTAGGGTTAGGGTTAGGGTTAG 5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAG

direct activity assay for human telomerase

the precise composition of the core human telomerase enzyme complex remained undefined

my research objective in 2003

• size measurements indicated complex of ~0.7 – 1 MDa:
• 35 distinct proteins proposed to associate with telomerase:

total mass ~ 2.5 MDa hTERT (1132 aa) hTR (451 nt) = 127 kDa = 153 kDa = 280 kDa

PURIFICATION

my research objective in 2003

• size measurements indicated complex of ~0.7 – 1 MDa:
• 35 distinct proteins proposed to associate with telomerase:

total mass ~ 2.5 MDa hTERT (1132 aa) hTR (451 nt) = 127 kDa = 153 kDa = 280 kDa

telomerase: the rarest enzyme in the human cell?

~20 – 50 molecules per cell ~1 part in 109 ~1 ng per 300,000,000 HEK293 cells

telomerase: a collaborative effort of CMRI & CSIRO

George Lovrecz Tram Phan Louis Lu

telomere-substrate affinity purification

hTERT

hTR A A U C C C A A U C

3’ Avidin bead TTAGGGTTAGGG T T A G G G

t1/2 > 10 h

5’-TTAGGGTTAGGGTTAGGG 5’-TTAGGGTTAGGGTTAGGGT 5’-TTAGGGTTAGGGTTAGGGTT 5’-TTAGGGTTAGGGTTAGGGTTA 5’-TTAGGGTTAGGGTTAGGGTTAG 5’-TTAGGGTTAGGGTTAGGGTTAGG

Kd and koff

between enzyme and substrate change as a function of the nucleotide at the 3’-end

dissociation of telomeric DNA substrate

5’-TTAGGGTTAGGGTTAGGG 5’-TTAGGGTTAGGGTTAGGGTTA

Kd and koff

between enzyme and substrate change as a function of the nucleotide at the 3’-end

dissociation of telomeric DNA substrate

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G

t1/2 > 10 h

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T T A

t1/2 < 5 min

activity-dependent elution of telomerase

activity-dependent elution of telomerase

t1/2 > 10 h

hTERT

hTR A A U C C C A A U C

3’ Avidin bead TTAGGGTTAGGG T T A G G G

t1/2 > 10 h t1/2 < 5 min

hTERT

hTR A A U C C C A A U C

3’ TTAGGGTTAGGG T T A G G G T T A Avidin bead hTERT

hTR A A U C C C A A U C

3’ Avidin bead TTAGGGTTAGGG T T A G G G dTTP dATP

activity-dependent elution of telomerase

t (min) relative activity

10 20 30 40 50 60 70 80 90 100 110 10 20 30 40 50 60

• dNTP

+dNTP

k = 0.25 ± 0.05 min-1 k = < 0.002 min-1

activity-dependent elution of telomerase

hTERT:

TPVGQGSWAHPGR RVGDDVLVHLLAR HRVTYVPLLGSLR RPGLLGASVLGLDDIHR LVDDFLLVTPHLTHAK AWNHSVR RSPGVGCVPAAEHR ARPAEEATSLEGALSGTR ARRPGLLGASVLGLDDIHR RGAAPEPERTPVGQGSWAHPGR PERTPVGQGSWAHPGR

168 amino acids = 15%

dyskerin:

HGKPTDSTPATWK TTHYTPLACGSNPLKR QGLLDKHGKPTDSTPATWK KSLPEEDVAEIQHAEEFLIKPESK KWGLGPK LLTSHKR SQQSAGKEYVGIVR

100 amino acids = 19%

mass spectrometry

hTERT: dyskerin: hTR: total mass: 127 kD 58 kD 153 kD 338 kD

core human telomerase enzyme complex

• bserved size: ~670 kD

2 x hTERT: 2 x dyskerin: 2 x hTR: total mass: 254 kD 116 kD 306 kD 676 kD

core human telomerase enzyme complex

• bserved size: ~670 kD

2 x hTERT 2 x hTR 2 x dyskerin core human telomerase enzyme complex

315: 1850-1853 (2007)

telomerase over-expression in 293T cells

• transient >>> stable
• 293T >>> 293, 293F
• ~500-fold activity over endogenous
• scalable to 20-L WAVE (Tram, Louis)

hTR dyskerin hTERT

telomerase over-expression in 293T cells

pcDNA3.1 5428 bp U3-hTR

NruI

dyskerin

(mcs)

SV40ori U3: snoRNA Polymerase II promoter

telomerase research at CMRI structure

enzymology

HTS

telomerase-DNA binding mechanism

6 KDs 6 koffs

Chris Tomlinson Tracy Bryan

6 12 18 24 30

1 2 3 4 5 6 koff

KD

DNA primer

koff (min-1 x 103)

[enzyme·DNA] KD (nM) 15 9 3 6 12

NO correlation between KD and koff

telomerase-DNA binding mechanism

telomerase-DNA binding mechanism

• TWO-STEP binding mechanism!
• six DISTINCT sets of binding interactions?!?

HTS for telomerase inhibitors

in collaboration with Greg Arndt

• 115,000 compound library
• automated 384-well format

chemiluminescent 96-well direct activity assay

chemiluminescent 96-well direct activity assay

negative-stain EM of human telomerase

Rosalba Rothnagel Ben Hankamer 1) adsorb on EM grid 2) stain with uranyl formate

negative-stain EM of human telomerase

negative-stain EM of human telomerase

~300 Angstrom

negative-stain EM of human telomerase

• Nat. Struct. Mol. Biol.

20: 454-460 (2013)

~300 Angstrom

next aim: high-resolution cryo-EM watch this space...

Thank You!

Tracy Bryan Roger Reddel George Lovrecz Tim Adams Tram Phan Louis Lu Rosalba Rothnagel Ben Hankamer