Presentation is loading. Please wait.

Presentation is loading. Please wait.

Post-Translational Modifications: CrossTalk Robert Chalkley Chem 204.

Similar presentations


Presentation on theme: "Post-Translational Modifications: CrossTalk Robert Chalkley Chem 204."— Presentation transcript:

1 Post-Translational Modifications: CrossTalk Robert Chalkley Chem 204

2 A large variety of PTMs are used by the cell The different modifications do not act independent of each other Most studies analyze one PTM (or one site) at a time Can this reveal the biological control? Present examples why multiple PTM analysis important Show examples of how multiple PTM analysis can be performed Introduction

3 Multi-Site Phosphorylation Many examples where multiple phosphorylations required for protein activation / regulation Growth Factor receptors: Autophosphorylate (pY) to cause receptor dimerization Contain multiple phosphorylation sites (pY, pS and pT) Different phosphorylation sites represent binding sites for different proteins to start signaling cascades May be that phosphorylation of any one of several sites causes activation, but dephosphorylation of all sites required for inactivation. Cohen, P. Trends Biochem Sci (2000)

4 O-GlcNAcylation and Phosphorylation O-GlcNAc modified proteins are also potential phosphoproteins Many examples where the same or neighboring residues can be either GlcNAcylated or phosphorylated. Multiple experiments have shown the two modifications interact/effect each other Zeidan, Q. and Hart, G. W. J Cell Sci (2010)

5 O-GlcNAcylation and Phosphorylation: Ying-Yang Sites of Modification are sometimes the same Mutually exclusive and opposing effects? c-Myc: Proto-oncogene transcription factor Thr58 is a mutational ‘hotspot’ in lymphomas. Thr58 can be phosphorylated or O-GlcNAc modified O-GlcNAc in growth inhibited / starved cells Ser62 phosphorylation required for Thr58 phosphorylation Mutation of Ser62 increases O-GlcNAcylation at Thr58 Mutate Thr58: how do you know what PTM causes the effect? Chou, T-Y et al. J Biol Chem (1995)

6 Wang, Z. et al. Mol Cell Proteomics (2007) Effect of Inhibiting GSK-3 on O-GlcNAcylation GSK-3 inhibited using Lithium Quantitative study of the effect on O-GlcNAc modification

7 Wang, Z. et al. Mol Cell Proteomics (2007) O-GlcNAcylation Changes upon GSK-3 Inhibition Enriched for modified proteins by IP SILAC for quantifying changes Increase in O-GlcNAc: 10 proteins Decrease in O-GlcNAc: 19 proteins Is it safe to assume changes in protein levels after IP correspond to PTM level changes? Modifications play different roles Phosphorylation can lead to increases or decreases of O-GlcNAcylation

8 O-GlcNAc and Phosphorylation Co-Analysis Mouse Post-Synaptic Density GlcNAc- Enriched Fraction GlcNAc- Depleted Fraction Phospho- Enriched Fraction PTM Depleted Fraction LWAC TiO 2 High pH RPLC Digest LC/MS/MS

9 O-GlcNAcylation and Phosphorlyation Identification ≈2200 phosphorylated peptides ≈250 GlcNAcylated peptides ≈ 250 GlcNAcylated peptides => ≈ 200 O-GlcNAcylation sites on 80 different proteins For half of the O-GlcNAc modified proteins, phosphopeptides were also identified. 4 peptides both O-GlcNAcylated and phosphorylated. To understand relationship need quantitative data.

10 Bassoon is Heavily O-GlcNAcylated and Phosphorylated Bassoon is a major component of the cytomatrix in the presynaptic active zone. Involved in spatial and temporal control of neurotransmitter release. Phospho/GlcNAc sites (3) Why is this protein so heavily post-translationally modified? What are the modifications doing? Regulating protein-protein interactions?

11 OGT regulation by PTM Yang et al. Nature (2008)

12 Ubiquitination Reversible modification of lysine (or protein N-terminus) Can be addition of single Ub or chain of Ub can be built up i.e. Ubiquitin becomes ubiquitinated 7 different lysines in ubiquitin Site of linkage during polyubiquitination determines biological effect Kirkpatrick et al. Nat Cell Biol (2005)

13 Linkage-Specific Measurement Linkage-specific antibodies have also been developed.

14 Histones Four histones per nucleosome (H2A, H2B, H3, H4) + linker histone (H1) Heavily post-translationally modified: Acetylation Methylation, Dimethylation, Trimethylation Phosphorylation Ubiquitination GlcNAcylation Different combinations of PTMs interact to control gene expression – ‘Histone Code’ hypothesis 1 1 Jenuwein et al. Science (2001)

15 Abcam

16 Approaches for Analyzing Multiple PTMs on a Protein Mass spectrometry is the only practical way of monitoring multiple PTMs at the same time. Problem: Most proteomic analysis is of short peptides; e.g. tryptic If modifications occur on different peptides, how do you know if PTMs occur on same protein species? Solution: Analyze larger protein fragments More likely multiple PTMs will be present on the same fragment

17 AspN cleavage of Histone H4 produces 23 aa peptide. 74 Different Modified Versions of Histone H4 detected in single preparation. Phanstiel et al. PNAS (2008) Combinations of Modifications

18 Challenges of Intact Protein Analysis Dynamic range: Amount of protein with a given modification combination may be very low Multiple species with same modifications but on different residues Same mass – cannot differentiate at MS level Can only differentiate based on distinct fragment ions

19 Intact Protein Analysis Histone H2B

20 Challenges of Intact Protein Analysis Dynamic range: Amount of protein with a given modification combination may be very low Multiple species with same modifications but on different residues Same mass – cannot differentiate at MS level Can only differentiate based on distinct fragment ions Differently modified versions observed at similar m/z May not have sufficient resolution to isolate a single m/z for MSMS analysis

21 16+ Charge Envelope of Histone H3 Challenges of Intact Protein Analysis Dynamic range: Amount of protein with a given modification combination may be very low May be multiple species with same modifications but on different residues => same mass

22 Challenges of Intact Protein Analysis Dynamic range: Amount of protein with a given modification combination may be very low Multiple species with same modifications but on different residues Same mass – cannot differentiate at MS level Can only differentiate based on distinct fragment ions Differently modified versions observed at similar m/z May not have sufficient resolution to isolate a single m/z for MSMS analysis Proteins fragment at multiple peptide bonds in ECD and ETD + Can identify sites of modification - Precursor signal split between many peaks Need more sample The larger the protein, the more the problem

23 ECD Histones ECD Fragmentation of Histone H2B

24 + Me 3 +Me 2 and Me 3 + Me 3 Fragments from 90/134 bond cleavages observed: 67% Sequence Coverage on a 15kDa protein! 3 different PTMs observed Information on relative stoichiometry of modifications czcz czcz czcz Fragments observed by ECD Fragmentation of Histone H2B

25 Challenges of Intact Protein Analysis Resolution of protein chromatography is much lower than peptides Is it possible to resolve differentially modified proteins? Need to tailor your chromatography to the modification The bigger the protein, the more difficult to resolve Young, N.L. et al. Mol Cell Proteomics (2009)

26 Resolving Isobaric Protein Modification States

27 PTM Cross-Talk Between Proteins Modifications on one protein lead to modifications of different proteins Phosphorylation Cascades Histone PTM cross-talk: Histone H2B ubiquitination required for Histone H3 methylation of K4 and K79

28 Sin3 Transcriptional Regulation Complex Gene transcription regulated by complexes binding to gene promoter regions. Activator and repressor complexes PTMs on histones regulate binding of transcription complexes Acetylation => Activation De-Acetylation => Repression Sin3A OGT HDAC1 HDAC2 N-CoR OGase

29 Summary Proteins bear multiple PTMs simultaneously Strategies that only study one PTM miss important biological information To understand the interactions between modifications, you need: Information about co-occurrence on same molecule Quantitative information about changes upon stimulation Need to be able to distinguish between increased protein vs PTM levels Knowledge of protein complex composition and PTM cross-talk within complex


Download ppt "Post-Translational Modifications: CrossTalk Robert Chalkley Chem 204."

Similar presentations


Ads by Google