An activity based probe for acyl protein thioesterase 1

1. An Activity-Based Probe for
Acyl Protein Thioesterases
Yiming Chen
Seto Lab Group

2. Palmitoylation Cycle
• Palmitoylation is covalent attachment of palmitic acid (C16)
to cysteine residues. Palmitoylation is reversible
• enhances hydrophobicity of proteins, anchor the protein
on cell membrane
• Depalmitoylation is mediated by acyl-protein thioesterases
(APTs)
• Remove palmitoyl group and releases proteins from
membrane
O APT1
S
O
+
OH HS

3. Rat sarcoma proteins
• Ras is signaling
proteins which control
cell proliferation and
survival
• Ras is a GTPase, can
hydrolyze GTP to GDP
• Mutations in Ras family
are found in 20% to
H-Ras PDB: 121p, 166 residues
30% human tumors

4. Ras Signaling Pathway
Schubbert, S.; Shannon, K.; Bollag, G. Nat. Rev. Cancer 2007, 7, 295–308.

5. F:farnesyl P: palmitoyl
• H/N-Ras is palmitoylated at the Golgi apparatus
• Transported to plasma membrane by vesicular transportation
• Depalmitoylation releases H/N-Ras to cytoplasm, allows it to return to Golgi
apparatus
• Localization regulates signaling activity
Cox, A. D. Nat. Chem. Biol. 2010, 6, 483–485.

6. Acyl Protein Thioesterases
• Two acyl protein thioesterases have been isolated
and characterized: APT1 and APT2
• APT1: originally isolated from rat liver as
lysophospholipase, promiscuous in substrate
specificity, deacylate proteins in vitro and vivo tests.
• APT2: 64% identity to APT1, has activity against
some lipid substrates in vitro. The biofunctions in
vivo is still unclear.

7. Acyl Protein Thioesterase I
• Quarternary structure
and sequence are well
characterized (25
kDa, 224 residues)
• The active site
contains a catalytic
t r i a d o f S e r- 1 1 4 ,
His-203, and Asp-169
Devedjiev,Y.; Dauter, Z.; Kuznetsov, S. R.; Jones, T. L.; Derewenda, Z. S. Structure 2000, 8, 1137–1146.

8. Acyl Protein Thioesterase I
ASP-169 His-203 Ser-114
O R
O O- N
H N H
O O
H
ASP-169 His-203 Ser-114
O
+ + R'-SH
O
O O- N H R OH
H N

9. Some Questions to Answer
• Are there any other enzymes hydrolyze thioester?
What are their biofunctions?
• Is APT1 involved in the progressions of cancers
induced by Ras mutation?
• How to study the specificities of APT1 inhibitors?

10. Activity-Based Probe
• Limitation of genomic study methods
• Level of transcription ≠level of expression
• Post-translational modification

11. Activity-Based Probe
• Activity-based probes (ABPs) are designed
to react with only the active form of the
enzyme , but not inactive form like
proenzyme; assay the activity of enzyme, but
not amino acids or genetic sequences
• Investigatehow enzyme activity differs
between normal cell vs. abnormal cell
• Discover and characterize new enzymes
which have specific catalytic activities
• Study the targets of enzyme inhibitor

12. gel
separation
LC/MS based probe-labeled protein profiling
Gel-based activity-based protein profiling across proteomes
Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Annu. Rev. Biochem. 2009, 77, 383–414.

13. Activity-Based Probe Design
Green: Substrate Red: Reactive group
Brown: “Clickable” reporter tag

14. Activity-Based Probe Mechanism
APTs
1,6-elimination
X-N3
click rxn
X=fluorescent marker, biotin

15. Potential Binding Site
Besides active site (Ser-114, Asp-169 and
His-203, in orange), some other potential
nucleophiles are marked.

16. Synthesis

17. Synthesis
OH
H
N O t-BuOK, 0 °C
O NH
O palmitoyl chloride, 47%
HO O
OH
H
N O DAST, CH2Cl2, 0 °C, 1 h, 72%
O O NH
O
O O
F
H
N O
O O NH
O
O O

20. Future Plans
• Validation of ABP design by kinetic assay
• ABP binding site investigation
• Novel thioesterases discovery
• Investigate APTs inhibitor specificity

21. Kinetic Assay of ABP
• Irreversible inhibition is also called “suicide
inhibition”
• Activity decreases in time-dependent
manner, follow exponential decay (pseudo
1st order reaction)
• Kinetics is investigated by incubating with
inhibitor and assaying the amount of activity
remaining over time

22. Kinetic Assay of ABP
k1 1 1 KI 1
= +
k2
E + ABP E-ABP P
k-1 kapp k2 k2 [ABP]
Lineweaver–Burk plot
•k appis apparent
inhibition rate
constant at specific
ABP concentration
• The x-axis intercept
is -1/KI, KI indicates
the affinity of ABP
KITZ, R.; WILSON, I. B. J. Biol. Chem. 1962, 237, 3245–3249.

23. Kinetic Assay of ABP
ABP
ABP p-nitrophenyl palmitate O2N
t
ABP O-
• Incubate APT1 with ABP at specific concentration
for time tx
• Transfer to p-nitrophenyl palmitate, monitor the
absorption at 410 nm. Because [PNP] >>[Ea], it’s
pseudo 0th order reaction, the slope is kx.
• Plot ln (kx) vs tx, slope is kapp at specific ABP
concentration
• Repeat these steps by changing [ABP], make
double-reciprocal plot of 1/kapp vs. 1/[ABP].

24. Binding Site Investigation
ABP Biotin Tag
ABP
1. Trypsin Peptide Fragment
ABP Biotin
2. Streptavidin Column ABP-Biotin
Mass Spectrometry Sequencing
Trypsin cleaves peptide chains mainly at the carboxyl side
of the amino acids lysine (K) or arginine (R), except when
either is followed by proline (P).

25. Peptide Sequencing by Mass
Spectrometry
http://www.ionsource.com
• De Novo: the most common ions in protein mass
spectrometry: a, b, y ions. Fragments information is
analyzed by software to obtain full sequence of
protein. B and Y type ions: 100%; A type ions: 20%
• Tandem mass spectrometry: more fragmentation
information from secondary mass spectrometry

26. Peptide Sequencing by Mass
Spectrometry
The partial-
overlapped jigsaw
puzzle can be
solved by software
like SEQUEST or
Mascot.

27. Peptide Sequencing by Mass
Spectrometry

28. Binding Site Investigation
Full sequence of APT1 protein
1 GAMDPEFMST PLPAIVPAAR KATAAVIFLH GLGDTGHGWA EAFAGIRSSH
51 IKYICPHAPV RPVTLNMNVA MPSWFDIIGL SPDSQEDESG IKQAAENIKA
101 LIDQEVKNGI PSNRIILGGF SQGGALSLYT ALTTQQKLAG VTALSCWLPL
151 RASFPQGPIG GANRDISILQ CHGDCDPLVP LMFGSLTVEK LKTLVNPANV
201 TFKTYEGMMH SSCQQEMMDV KQFIDKLLPP ID
Trypsin
GAMDPEFMSTPLPAIVPAAR M=2071 g/mol
ATAAVIFLHGLGDTGHGWAEAFAGIR M=2638 g/mol
PVTLNMNVAMPSWFDIIGLSPDSQEDESGIK M=3391 g/mol
IILGGFSQGGALSLYTALTTQQK M=2367 g/mol
.......
The molecular mass of enriched peptide is:
m(peptide) + m(H+) + m(ABP) – m(19F) + m(biotin
tag) = m(peptide) + 939 g/mol

29. Novel Thioesterases Discovery
• Some other thioesterases or lipases may
react with ABP:
• Palmitoyl protein thioesterases (PPTs)
• Palmitoyl coA hydrolayse
• Acetyl coA hydrolases (ACOTs)
• Some other lipases or hydrolases
• Use ABP to investigate the novel enzymes
in cell

30. Novel Thioesterases Discovery
ABP ABP biotinyl tag
click reaction
ABP
ABP affinity column ABP biotin
biotin biotin
ABP ABP biotin
Sequence BLAST
ABP biotin
Gel separation
Sequence BLAST
ABP biotin

31. Investigate APTs Inhibitor Specificity
O
O
O Palmostatin M (IC50 = 0.67 μM)
O
Dekker, F. J.; Rocks, O.;Vartak, N.; Menninger, S.; Hedberg, C.; Balamurugan, R.; Wetzel, S.; Renner, S.; Gerauer, M.; Schölermann, B.;
Rusch, M.; Kramer, J. W.; Rauh, D.; Coates, G. W.; Brunsveld, L.; Bastiaens, P. I. H.; Waldmann, H. Nat. Chem. Biol. 2010, 6, 449–456.
NH2
O
N
H
O benzodiazepinedione (IC50 = 27 μM)
N N
HN O
O S O O
S
O
Deck, P.; Pendzialek, D.; Biel, M.; Wagner, M.; Popkirova, B.; Ludolph, B.; Kragol, G.; Kuhlmann, J.; Giannis, A.; Waldmann, H. Angew. Chem.
Int. Ed. Engl. 2005, 44, 4975–4980.
• Have potential anti-tumor activities
• Mechanism or target is not clear
• Specificity is hard to investigate

32. Investigate APTs Inhibitor Specificity
• Apply the inhibitor to cell lysate, use the
untreated lysate without inhibitor as
negative control
No inhibitor
Inhibitor 1 1) Streptavidin Column
1) ABP
Inhibitor 2
2) Biotintag 2) Gel Separation
Inhibitor 3

33. Investigate APTs Inhibitor Specificity

34. Primary Amine Synthesis via Nitrone
O
M.S. N Si
+ H2N Si
40 °C, CH2Cl2
Cl Cl
UHP, 5% mol CH3ReO3 PhMgBr, Et2O
N Si
t-butanol, 40 °C, 30 min, 63% O- 0 °C, 1 hr, 75%
Cl
TBAF. CH2Cl2, rt
N Si NH2
OH
Cl Cl
+H2O
N Si F- N NH2
OH
Cl Cl Cl

35. Acknowledgements
Dr. Christopher Seto
Dr. Tun-Li Shen
Dr. Russell Hopson
Michael Zompa
Bennett Meier
Koki Nishimura

36. Thank you!

37. Supplemental Information

38. Ras Signaling Pathway
• Binding of growth factor to the receptor
introduces autophosphorylation and recruitment
of adaptor proteins that activate downstream
effectors
• Guanosine exchange factors (GEFs) like SOS1
promotes the activated Ras-GTP complex
• Gtpase activating proteins (GAPs) activates Ras’s
intrinsic phosphatase activity and turns activated
Ras-GTP complex to inactive Ras-GDP
• Activated Ras-GTP complex combines with
series of downstream effectors concerning cell
http://www.rcsb.org/
proliferation.

39. Azide Tag Synthesis
O
HN NH DIPC, NHS
H H + H2N Cl
DCM, RT, 67%
S COOH
O
HN NH NaN3, KI, 18-crown-6
H H
H
N Cl DMF, uW, 100 °C, 85%
S
O
O
HN NH
H H
H
N N3
S
O
Done by Koki Nishimura

40. Azide Tag Synthesis
COOH
CDI, NHS
+ H2N Cl
DMF, RT, 57%
N O N+
H H
N Cl N N3
O NaN3, KI, 18-crown-6 O
DMF, uW, 100 °C, 85%
N O N+ N O N+
Done by Koki Nishimura

41. Copper Catalyzed Azide-alkyne Huisgen
cycloaddition
CuLn
R1 H R1 CuLn
R1 H
LnCu2 R1
LnCu (LnCu)2 2
R2 N
N N N N
R2
R1 N R1
R1 N
R2 N R2 N L
N N Cu Cu
LnCu2 N L L
R1 N Cu Cu
R2 L

42. Kinetic Assay of ABP
[E0]: Total concentration of enzyme
[E]: Concentration of free enzyme
[E-ABP]: Concentration of intermediate
[Ea]: Concentration of active enzyme, [E]+[E-ABP]
[P]: Concentration of inactive enzyme
[ABP]: Concentration of ABP
k1, k-1, k2: rate constants; K1: equilibrium constant
kapp: apparent rate constant

43. Kinetic Assay of ABP
k1 k2
E + ABP E-ABP P
k-1
[E0] = [E]+[E-ABP]+[P] KI = [E][ABP]/[E-ABP]
[Ea] = [E]+[E-ABP] d[E a ]
− = k2 [E-ABP]
dt
If enzyme-inhibitor solution is extensively diluted, then
[E a ] −k2t
[E a ] −k2t[E-ABP] ln =
ln = [E 0 ] 1+ K I
[E 0 ] [E a ]
[ABP]
k2
kapp = [E a ]
If [ABP] >> E0, KI ln = −kappt
1+ [E 0 ]
[ABP]
KITZ, R.; WILSON, I. B. J. Biol. Chem. 1962, 237, 3245–3249.

44. Reactivity of Quinone Methide
with Amino Acids Residues
Jiang, J.; Zeng, D.; Li, S. Chembiochem 2009, 10, 635–638.

45. Alternative ABP Design
Green: Substrate Red: Reactive group
Brown: Reporter Tag

46. Alternative ABP Design
APT1

47. Alternative ABP Design
X-N3
click rxn
X=fluorescent marker, biotin

48. Tandem Mass Spectrometry
http://en.wikipedia.org/wiki/Tandem_mass_spectrometry

49. Tandem Mass Spectrometry
Ionization: Electrospray, MALDI, EI
Mass separarion: In space (transmission quadrupole,
sectors or TOF) or In time (ion trap or FTMS)
Fragmentation: In-source fragmentation or post-
s o u rc e f r a g m e n t a t i o n ( C o l l i s i o n - i n d u c e d
dissociation)

50. Lysophospholipase I
• APT1 is also named as Lysophospholipase
1 ( LY P L A 1 ) , w h i c h h y d r o l y z e s
lysophospholipid
• Lysophospholipid is phospholipid that
lacks one fatty acid chain
• Lysophospholipid hydrolysis mechanism is
almost identical.
O
O R1 OH
LYPLA1 O
HO H O HO H O +
O P O X O P O X R1 OH
O- O-
Wang, A.; Loo, R.; Chen, Z.; Dennis, E. A. J. Biol. Chem. 1997, 272, 22030–22036.

51. SEQUEST
• Basic Local Alignment Search Tool (BLAST):
algorithm for comparing primary biological
sequence information
• Compare the sequence of protein to the
known sequence in the database

52. Basic Local Alignment Search Tool
• Basic Local Alignment Search Tool (BLAST):
algorithm for comparing primary biological
sequence information
• Compare the sequence of protein to the
known sequence in the database