Post-translational Post-translational Modifications to Human Bile Modifications to Human Bile Acid CoA:Amino Acid N- Acid CoA:Amino Acid N- acyltransferase acyltransferase Erin Shonsey UAB Graduate Student September 12, 2006
Conjugation of Bile Acids Conjugation of Bile Acids OH SCoA C + + NH 3 CH 2 CH 2 SO 3 - O H O OH hBAT CoA-SH HO OH C NHCH 2 CH 2 SO 3 - OH C O O H O OH (Amidate, N-Cholyltaurine) O H H O (CA)
Catalytic Triad of BAT Catalytic Triad of BAT • Various mutation and other studies performed previously led to the hypothesis that BAT works through a catalytic triad • The members of this catalytic triad are believed to be Cys235, His362, and Asp328
Charge Relay Mechanism shared by hBAT, Charge Relay Mechanism shared by hBAT, thioesterases, and a large group of hydrolases thioesterases, and a large group of hydrolases His 362 Cys 235 His 362 Cys 235 S S . H + - . 1 O - - N - N - - O C H - NH O --- Nucleophilic attack - NH - O - C --- - - S … … C Asp 328 - … Bile acid … S CoA C Asp 328 Bile acid CoA O O Tetrahedral intermediate Michaelis complex 2 His 362 Cys 235 (Ser235) Cys 235 CH 2 Gly or Tau CH 2 S N ---- R 3 H S H NH C O --- R CoA … - C O … N C Asp 328 S Gly or Tau O CoA O (H 2 O) 4 Acyl-enzyme intermediate His 362 Cys 235 O Cys 235 5 CH 2 S C N H - - - - N S + Gly or Tau R Bile acid NH --- C O N - … Gly or Tau … - C O Asp 328 Active enzyme Bile acid conjugate O
MALDI-TOF MS Analysis of hBAT-Avi/cholic acid intermediate MALDI-TOF MS Analysis of hBAT-Avi/cholic acid intermediate 49729.45 50430.89 51477.16 45000 48000 51000 54000 57000 60000 49516.04 50549.36 57314.61 45000 48000 51000 54000 57000 60000 Mass (m/z)
Deconvoluted Q-TOF MS Analysis of hBAT- Deconvoluted Q-TOF MS Analysis of hBAT- Avi/cholic acid intermediate Avi/cholic acid intermediate 49504 49894 + 390 Da 49504 mass 49000 49100 49200 49300 49400 49500 49600 49700 49800 49900 50000 50100 50200
Amino Acids Modified by 4HNE Amino Acids Modified by 4HNE OH CHO N N H N OH N N H H N O O H O 4HNE-Modified Lysine 4HNE-Modified Lysine 4HNE-Modified Lysine Michael Addition Schiff Base Adduct 2-Pentylpyrrole Adduct OH OH Adduct Mol wt CHO CHO S N Michael 156 N Schiff base 138 N H N O Pentylpyrrole 120 H O 4HNE-Modified Cysteine 4HNE-Modified Histidine J Am Soc Mass Spectrom. 2004 Michael Addition Aug;15(8):1136-47. Michael Addition
Potential 4HNE targets in hBAT Potential 4HNE targets in hBAT MIQLTATPVSALVDEPVHIRATGLIPFQMVSFQASLEDENGDMF YSQAHYRANEFGEVDLNHASSLGGDYMGVHPMGLFWSLKPEKLL TRLLKRDVMNRPFQVQVKLYDLELIVNNKVASAPKASLTLERWY VAPGVTRIKVREGRLRGALFLPPGEGLFPGVIDLFGGLGGLLEF RASLLASRGFASLALAYHNYEDLPRKPEVTDLEYFEEAANFLLR HPKVFGSGVGVVSVCQGVQIGLSMAIYLKQVTATVLINGTNFPF GIPQVYHGQIHQPLPHSAQLISTNALGLLELYRTFETTQVGASQ YLFPIEEAQGQFLFIVGEGDKTINSKAHAEQAIGQLKRHGKNNW TLLSYPGAGHLIEPPYSPLCCASTTHDLRLHWGGEVIPHAAAQE HAWKEIQRFLRKHLIPDVTSQL 17 His 16 Lys 19 Arg 3 Cys
In vitro Modification of hBAT with Modification of hBAT with In vitro HNE HNE + Incubate at 4°C for 1 hour HNE hBAT HNE treated hBAT + HNE modified HNE treated hBAT hBAT 100 Incubate at 37°C overnight % Intensity Mix with matrix and spot on MALDI plate 0 900 1520 2140 2760 3380 4000 Mass (m/z)
Inactivation of hBAT by HNE Inactivation of hBAT by HNE 120 % Residual Activity 1.6 µ M 100 hBAT 80 60 40 20 0 control 8 16 32 64 128 HNE Concentration ( µ M)
MALDI-TOF MS of Chymotrypsin Digest MALDI-TOF MS of Chymotrypsin Digest of HNE modified hBAT of HNE modified hBAT 1524.83 1009.62 1163.60 Native hBAT 1599.91 1029.58 1039.64 2405.38 1664.94 1487.73 1758.86 1739.92 1563.78 1178.61 1883.10 1395.66 970.59 1936.13 2151.00 1009.62 1523.82 Modified hBAT 1039.55 1163.60 1487.71 1758.86 1320.71 1077.57 1195.67 1719.95 1890.00 970.62 1395.87 1563.79 1914.95 2152.00 900 1220 1540 1860 2180 2500 Mass (m/z)
Histidine Residues Modified by 1 mM Histidine Residues Modified by 1 mM HNE Found by QTOF HNE Found by QTOF Residue Sequence Unmodified Modified Mass Shift Numbers Mass Mass 185-201 GFASLALAYHNYEDLPR 1937 2093 156 271-283 HGQIHQPLPHSAQ 1563 1719 156 271-283 HGQIHQPLPHSAQ 1563 1875 312 336-345 AHAEQAIGQLK 1164 1320 156 382-400 LHWGGEVIPHAAAQEHAWK 2136 2292 156
MS/MS Spectra Using CID of [MH] + + 2362.3051 2362.3051 MS/MS Spectra Using CID of [MH] M I Q L T A T P V S A L V D E P V H I R [M+2H] 2+ - HNE Michael adduct y 8 y 10 b 14 b 13 b 9 b 12 y 11 y 7 y 13 b 15 b 5 b 7 y 5 y 6 y 9 y 15 b 17 b 3 y 12 y 14 y 16 b 6 400 600 800 1000 1200 1400 1600 1800 m/z
Electron Capture Dissociation Electron Capture Dissociation Curr Opin Biotechnol. 2004 Feb;15(1):12-6.
ECD Fragmentation ECD Fragmentation y 3 z 3 z 2 z 1 + H R1 O R2 O R3 O R4 O H N C C N C C N C C N C C OH H H H H H H H H b 1 c 1 c 2 c 3
MS/MS Spectra Using ECD of [MH]+ 1460.8408 MS/MS Spectra Using ECD of [MH]+ 1460.8408 A H A E Q A I G Q L K R [M+3H] 3+ [M+2H] 2+ c 5 z 8 ~ x 5 c 3 c 4 c 8 z 7 c 2 c 11 z 1 z 3 z 4 c 6 c 7 c 9 c 10 z 2 200 400 600 800 1000 1200 1400 m/z
Sequence Coverage Using FT-ICR/MS Sequence Coverage Using FT-ICR/MS MIQLTATPVSALVDEPVHIRATGLIPFQMVSFQASL EDENGDMFYSQAHYRANEFGEVDLNHASSLGGDYMG VHPMGLFWSLKPEKLLTRLLKRDVMNRPFQVQVKLY DLELIVNNKVASAPKASLTLERWYVAPGVTRIKVRE GRLRGALFLPPGEGLFPGVIDLFGGLGGLLEFRASL LASRGFASLALAYHNYEDLPRKPEVTDLEYFEEAAN FLLRHPKVFGSGVGVVSVCQGVQIGLSMAIYLKQVT ATVLINGTNFPFGIPQVYHGQIHQPLPHSAQLISTN ALGLLELYRTFETTQVGASQYLFPIEEAQGQFLFIV GEGDKTINSKAHAEQAIGQLKRHGKNNWTLLSYPGA GHLIEPPYSPLCCASTTHDLRLHWGGEVIPHAAAQE HAWKEIQRFLRKHLIPDVTSQL 70.1% Sequence Coverage
Amino Acids Modified by HNE Using Amino Acids Modified by HNE Using FT-ICR/MS FT-ICR/MS Modified Amino Acid 128 µ M Peptide HNE 64 µ M HNE 32 µ M HNE 16 µ M HNE 8 µ M HNE AHAEQAIGQLKR H336 H336 H336 H336 RLHWGGEVIPHAAAQEHAWK H397 H397 H397 H397 K329, K329, K329, AQGQFLFIVGEGDKTINSK K329, K334 K334 K334 K334 K329, K334 MIQLTATPVSALVDEPVHIR H18 H18 RANEFGEVDLNHASSLGGDYMGVHPMGLFWSLKPEK 1*156 H362, C372, NNWTLLSYPGAGHLIEPPYSPLCCASTTHDLR C373, H378
Summary Summary • A covalent enzyme intermediate was identified between hBAT and cholic acid • LC-ESI-MS/MS on the Q-tof identified 6 modifications at 1 mM HNE • LC-ESI-FT-ICR/MS identified up to 10 modifications at 128 µM, down to 3 modifications at 8 µM • 4HNE may first alter sites in the region of the catalytic triad
Acknowledgements Acknowledgements FT-ICR/MS Lab for Barnes Lab: Biomedical Research: Stephen Barnes Matthew Renfrow Tracy D’Alessandro Monica Stinnett Kim Lab: Mass Spec Shared Helen Kim Facility: Shannon Eliuk Marion Kirk Landon Wilson
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