An activity based probe for acyl protein thioesterase 1
An Activity-Based Probe forAcyl Protein Thioesterases Yiming Chen Seto Lab Group
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
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% toH-Ras PDB: 121p, 166 residues 30% human tumors
Ras Signaling Pathway Schubbert, S.; Shannon, K.; Bollag, G. Nat. Rev. Cancer 2007, 7, 295–308.
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.
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.
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.
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
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?
Activity-Based Probe• Limitation of genomic study methods • Level of transcription ≠level of expression • Post-translational modiﬁcation
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
gel separation LC/MS based probe-labeled protein proﬁling Gel-based activity-based protein proﬁling across proteomesCravatt, B. F.; Wright, A. T.; Kozarich, J. W. Annu. Rev. Biochem. 2009, 77, 383–414.
Activity-Based Probe DesignGreen: Substrate Red: Reactive group Brown: “Clickable” reporter tag
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
Future Plans• Validation of ABP design by kinetic assay• ABP binding site investigation• Novel thioesterases discovery• Investigate APTs inhibitor speciﬁcity
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
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 ABPKITZ, R.; WILSON, I. B. J. Biol. Chem. 1962, 237, 3245–3249.
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].
Binding Site Investigation ABP Biotin Tag ABP 1. Trypsin Peptide Fragment ABP Biotin 2. Streptavidin Column ABP-Biotin Mass Spectrometry SequencingTrypsin cleaves peptide chains mainly at the carboxyl sideof the amino acids lysine (K) or arginine (R), except wheneither is followed by proline (P).
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
Peptide Sequencing by Mass Spectrometry The partial- overlapped jigsaw puzzle can be solved by software like SEQUEST or Mascot.
Binding Site Investigation Full sequence of APT1 protein1 GAMDPEFMST PLPAIVPAAR KATAAVIFLH GLGDTGHGWA EAFAGIRSSH51 IKYICPHAPV RPVTLNMNVA MPSWFDIIGL SPDSQEDESG IKQAAENIKA101 LIDQEVKNGI PSNRIILGGF SQGGALSLYT ALTTQQKLAG VTALSCWLPL151 RASFPQGPIG GANRDISILQ CHGDCDPLVP LMFGSLTVEK LKTLVNPANV201 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
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
Investigate APTs Inhibitor Speciﬁcity O O O Palmostatin M (IC50 = 0.67 μM) ODekker, 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 ODeck, 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
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
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 OHCl Cl Cl
AcknowledgementsDr. Christopher SetoDr. Tun-Li ShenDr. Russell HopsonMichael ZompaBennett MeierKoki Nishimura
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 cellhttp://www.rcsb.org/ proliferation.
Azide Tag Synthesis OHN NH DIPC, NHSH H + H2N Cl DCM, RT, 67% S COOH OHN NH NaN3, KI, 18-crown-6H H H N Cl DMF, uW, 100 °C, 85% S O OHN NHH H H N N3 S O Done by Koki Nishimura
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
Copper Catalyzed Azide-alkyne Huisgen cycloaddition CuLn R1 H R1 CuLn R1 H LnCu2 R1 LnCu (LnCu)2 2R2 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
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 ABPk1, k-1, k2: rate constants; K1: equilibrium constantkapp: apparent rate constant
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.
Reactivity of Quinone Methide with Amino Acids Residues Jiang, J.; Zeng, D.; Li, S. Chembiochem 2009, 10, 635–638.
Alternative ABP Design Green: Substrate Red: Reactive group Brown: Reporter Tag
Alternative ABP Design X-N3 click rxn X=ﬂuorescent marker, biotin
Tandem Mass Spectrometry http://en.wikipedia.org/wiki/Tandem_mass_spectrometry
Tandem Mass SpectrometryIonization: Electrospray, MALDI, EIMass 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 ddissociation)
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 OHO 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.
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
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