Helen Kim, Ph.D. and John Cutts. Dept of Pharmacology & Toxicology University of Alabama at Birmingham

Size: px

Start display at page:

Download "Helen Kim, Ph.D. and John Cutts. Dept of Pharmacology & Toxicology University of Alabama at Birmingham"

Alexandra Welch
5 years ago
Views:

1 Understanding the actions of a dietary anti-oxidant at the protein and small molecule level using top-down proteomics, enzyme assays and mass spectrometry elen Kim, Ph.D. and John Cutts Mar 9, 2012 UAB BMG744 Dept of Pharmacology & Toxicology University of Alabama at Birmingham helenkim@uab.edu elenkim/uab/pharmtox UTLIE I. Top-down proteomic analysis of actions of a dietary supplement, grape seed extract (GSE), in mammalian brain; II. FT-ICR MS mapping of reactive aldehyde 4E adduct sites on recombinant creatine kinase (CK- BB); III. Enzyme assays: E stoichiometrically poisons CK- BB; elenkim/uab/pharmtox 2 1

2 ur principal goal: to understand the molecular basis of human chronic conditions/diseases, to develop prevention or therapies. Strategy: a proteomics approach ypothesis: Actions of beneficial agents such as dietary antioxidants in normal and disease tissue will reveal subproteomes of proteins at risk for disease-relevant changes. elenkim/uab/pharmtox 3 PLYPELS: similar structures among themselves, and with 17b-estradiol GRAPE: resveratrol TEA (grapes): catechin SY: genistein 17b-estradiol elenkim/uab/pharmtox 4 2

The experiment: give grapeseed powder in rat diet; examine brain proteomes AI-76A diet; normal adult rats (n = 5) AI-76A + 5% GSE; normal adult rats (n = 5) whole brain homogenates, triplicate 2D

3 The experiment: give grapeseed powder in rat diet; examine brain proteomes AI-76A diet; normal adult rats (n = 5) AI-76A + 5% GSE; normal adult rats (n = 5) whole brain homogenates, triplicate 2D gels elenkim/uab/pharmtox 5 Image analysis indicated rat brain proteins were affected by GSE. Creatine kinase GSE Control Different in intensity Different in horizontal position elenkim/uab/pharmtox 6 3

4 Database of protein differences in GSE vs CT brains Protein ame Mitochondrial matrix proteinprecursorp60 Creatine Kinase BB chain #matched pep Accession # MWSE bs m.w. Pred m.w. bs pi Pred pi 10 P E ature of change in GSE brains 12 P E Translocation to Acidic p Actin 8 P E Less complex GFAP 20 P E epsilon 10 P E Alpha Enolase 9 P E Less complex Gamma Enolase 10 P Less complex RIKE cda (M ) 9 P SC gi SC gi eurofilament L 14 gi Triplet protein eurofilament M 19 gi triplet protein Vimentin 10 gi elenkim/uab/pharmtox 7 (Deshane et al., J. Agric. Food Chem.) Initial conclusion: GSE is neuroprotective, since its effects on proteins are counter to the directions of change for the same proteins in disease. Mitochondrial matrix protein precursor SP epsilon protein eat shock cognate 70 eat shock cognate 71 GREE: folding/ stress response eurofilament triplet protein L PURPLE : energy eurofilament triplet protein M BLUE: new GFAP Vimentin RAGE: cytoskeleton Creatine kinase Brain isoform Gamma enolase Alpha enolase Actin RIKE cda M (Schonberger et al., 2001) (Tilleman et al., 2002a) (Tilleman et al., 2002b) elenkim/uab/pharmtox 8 (Deshane et al., J. Agric. Food Chem.) 4

5 Densitometry units 3/8/2012 Western blot analysis corroborated 2D gel image analysis quantitation A. Stained gel for SP-60 B. Western Blots CT GSE 50 kda 50 kda C. Quantitative Densitometry CT GSE (Deshane et al., J. Agric. Food Chem.) elenkim/uab/pharmtox 9 Use chemistry to determine whether GSE affects oxidations elenkim/uab/pharmtox 10 5

kda kda 3/8/2012 Preliminary experiments indicated GSE affected oxidations of abundant proteins 4 pi 7 250 100

blot 11 4-hydroxy-2-nonenal (4E) A reactive aldehyde resulting from arachidonic acid Reacts with K, C, and S C

6 kda kda 3/8/2012 Preliminary experiments indicated GSE affected oxidations of abundant proteins 4 pi A Coomassie stained gel A Creatine kinase Anti-DP Western elenkim/uab/pharmtox blot 11 4-hydroxy-2-nonenal (4E) A reactive aldehyde resulting from arachidonic acid Reacts with K, C, and S C C 4E-Modified Lysine 4E-Modified Cysteine 4E-Modified istidine Schiff Base Adduct Michael Adduct Michael Adduct 6

elenkim/uab/pharmtox 13 Modified Amino Concentration of 4E (_M) Acid 5000 300 100 30 10 5 7 M,S M,S M,S S 26 M M M M 29 M M K 45 M 66 M M K 86 M M 97 M M M K 101 M M C 141 M M M M C 145 M M M M K 177

7 elenkim/uab/pharmtox 13 Modified Amino Concentration of 4E (_M) Acid M,S M,S M,S S 26 M M M M 29 M M K 45 M 66 M M K 86 M M 97 M M M K 101 M M C 141 M M M M C 145 M M M M K 177 M 191 M M M 219 M,S M,S M 234 M,S M,S S K 247 M,S C 254 M,S M,S M,S M,S M M 276 M M C 283 M M M M M 296 M,S M,S S 305 M M K 313 M K 358 M elenkim/uab/pharmtox 14 K 381 M Sites of Eadducts on CKBB over a range of concentrations; RED indicates active-site residues 7

% of unmodified hck-bb activity 3/8/2012 Crystal structure of CK-BB showing the E adducts at active-site and non-active-site residues detected at 30 M 4E.

8 % of unmodified hck-bb activity 3/8/2012 Crystal structure of CK-BB showing the E adducts at active-site and non-active-site residues detected at 30 M 4E. C C 254 C 141 C Active site 4E -modifications on-active site modifications (crystal structure adapted from Eder et al., 1999) elenkim/uab/pharmtox 15 Reduction of CK-BB activity correlated with increased E-adducts formed at active-site residues µm hck-bb = active site modification E concentration (µm) 8

9 elenkim/uab/pharmtox 17 elenkim/uab/pharmtox 18 9

10 elenkim/uab/pharmtox 19 Modifications of CKBB in both AD and CT brain: elenkim/uab/pharmtox 20 10

11 Modifications on CKBB detected by MS/MS analysis Peptide Sequence Modified Age-matched Modification Mass Shift AD Amino Acid Control FPAEDEFPDLSAMAK M30 xidation ü ü FPAEDEFPDLSAMAK M30 Di-oxidation ü ü GIWDK W218 xidation ü ü TFLVWVEEDLR W228 Kynurenine ü ü TFLVWVEEDLR W228 xidation ü ü TFLVWVEEDLR W228 Di-oxidation ü ü SKDYEFMWP M272 xidation ü ü LGFSEVELVQMVVDGVK M352 xidation ü ü LLIEMEQR M363 xidation ü ü LEQGQAIDDLMPAQK M377 xidation ü ü Detected only by GPMS S 3 + Methionine Tryptophan Kynurenine Structures courtesy of D. Stella Conclusions regarding the grapeseed studies GSE has pleiotropic effects in the brain: gene expression/protein turnover; protein oxidations; These actions may be consistent with neuroprotection, since the majority are in the opposite direrction to changes affecting these proteins in AD or models of dementia. These were the first studies to identify specific proteins affected by dietary intake of a complex botanical mixture. elenkim/uab/pharmtox 22 11

12 Conclusions from the studies with CKBB Incubation with E stoichiometrically inhibited hckbb activity. this was correlated with increased E adducts on CKBB, revealed by FT-ICR-MS. At the lowest activity, all four active-site residues of CKBB were E-modified. Thus, a combination of state of the art mass spectrometry and conventional biochemistry was optimal in determining the role of E adducts on CKBB function. elenkim/uab/pharmtox 23 More conclusions from studies of CKBB In AD brain, CKBB was not detectably modified with E; Rather, oxidative modifications on CKBB were similar between AD and CT brains. These non-differences correlated with a lack of difference in specific activity of CKBB between AD and CT. elenkim/uab/pharmtox 24 12

Section 1 Proteins and Proteomics

Section 1 Proteins and Proteomics Learning Objectives At the end of this assignment, you should be able to: 1. Draw the chemical structure of an amino acid and small peptide. 2. Describe the difference