Posttranslational Modifications The challenge of the next decade

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Post-translational ModificationsThe challenge
of the next decade

• Kathy Schegg
• November 3, 2006

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Definitions

• General definition Any modification to the
  translated form of a protein
• Disulfide bonds, oxidation, cleavage
• My working definition Covalent additions that
  regulate protein function, determining their
  activity state, cellular location and dynamic
  interactions with other proteins

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General Characteristics

• Are gt 200 known types
• Present on at least 80 of eukaryotic proteins
• Found in all species
• Located at specific sequence motifs (sequons)
• Often transient
• Usually present at substoichiometric levels
• Often act together

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Jensen Nature Reviews Molecular Cell Biology 7,
391-403 (June 2006) doi10.1038/nrm1939
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Additional PTMs

• Nitrosylation
• Glycation
• ADP-ribosylation
• O-GlcNAc

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Analysis of PTMs

• Ideal method Mass Spectometry
• PTMs cause a predictable mass change
• Give characteristic m/z peaks during ms/ms
  (neutral loss or precursor ions)
• Antibodies against specific motifs
• Stains

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Analysis is very challenging

• Global proteomics methods do not work to find
  PTMs
• A few modifications can be programmed into search
  programs destroy MS speed and accuracy
• PTMs are usually present at too low a
  concentration. Only the unmodified version will
  be seen
• Affinity enrichment techniques are required

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Affinity enrichment

• Affinity columns (eg., lectin cloumns, IMAC)
• Antibodies
• Tags (e.g., biotin)

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Phosphorylation

• HPO3 is added to Ser, Thr or Tyr
• One of the most important and abundant PTMs
• gt30 of all proteins are phosphorylated
• Many multiply phosphorylated
• Functions
• Ser, Thr association activation of kinases
• Tyr receptor dimerization, activation

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Analysis of Phosphoproteins

• 80 is added to mass of a peptide
• On MS/MS, m/z peaks of 98 (H3PO4) or 80 (HPO3)
  can be detected
• Phosphopeptides must be purified first
• Tyr phosTyr antibodies
• Ser, Thr IMAC, SAX
• Isolate phosphoproteins, cleave, isolate
  phosphopeptides, MS/MS
• Beta-elimination and tagging (e.g., biotin)
• Need lots of protein

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Human p53

• MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDI
  EQWFTEDPGPDEAPRMPEAAPPVAPAPAAP 80
  TPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPAL
  NKMFCQLAKTCPVQLWVDSTPPPGTRVRAM 160
  AIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRN
  TFRHSVVVPYEPPEVGSDCTTIHYNYMCNS 240
  SCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLR
  KKGEPHHELPPGSTKRALPNNTSSSPQPKK 320
  KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLK
  SKKGQSTSRHKKLMFKTEGPDSD 400 ........S.....S.......
  .......................S..........................
  ........ 80 T.................S...................
  ..S............................T....T..... 160
  ..Y...................S...........................
  T...S......................... 240
  ...................S........S..............T......
  ............ST..........S..... 320
  .............................................S....
  S....ST..............S. 400
• Phosphorylation sites predicted Ser 15 Thr 7
  Tyr 1

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Ubiquitin

• A 76 aa protein
• Targets proteins for degradation by the 26S
  proteosome
• Found in all eukaryotes
• Forms an isopeptide bond between the C-terminal
  Gly of Ub and the NH2 of a Lys sidechain on the
  target
• Polyubiquitins are formed

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Analysis of Ubiquitin Attachment

• Genetic tagging of Ubiquitin
• Affinity purification
• Antibody precipitation

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SUMO, etc.

• Ever more ubiquitin-like proteins are being
  discovered
• Functions transcriptional regulation, cellular
  localization, control of E3
• Pathway is similar to ubiquitin
• Have characteristic ubiquitin-fold tertiary
  structure

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SUMO, etc.

• SUMO (Small ubiquitin-like modifier)
• 100 amino acid protein
• 60 known target proteins
• 3 isoforms
• NEDD8
• regulates E3

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Glycosylation

• Most common PTM
• 50 of all mammalian proteins are glycosylated
• CHO structures are very heterogeneous
• In mammals, 9 different sugars are used
• 2 types N-linked and O-linked

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N-Linked Glycosylation

• Always hooked to Asn
• Sequon Asn-X-Ser/Thr/Cys (X is not Pro)
• Can be completely removed by PNGase F

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O-linked Glycosylation

• Hooked to Ser or Thr
• No known sequon
• No easy removal method
• Beta-elimination works to remove CHOs, but amino
  acids are modified

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Analysis of Glycoproteins

• Purified by binding to lectin columns
• Can be done at level of proteins, trypsin
  peptides or both
• Glycans released by PNGase F or beta-elimination
  can be separated by HPLC and analyzed by MS
• The location of the glycans can be identified by
  18O labelling

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O-GlcNAc

• N-acetylglucosamine is O-linked to Ser and Thr
• On nuclear and cytoplasmic proteins
• Reflects nutritional status of cell (glucose)
• Changes rapidly and dynamically
• In equilibrium with phoshorylation

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S-Nitrosylation

• NO couples to cysteine thiol
• gt100 substrates, numerous signalling pathways
• NO is produced by NO synthetase and from food
  bacterial sources
• Various enzymes nitrosylate the substrates - SOD
  acts on hemoglobin

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Lipidation

• Five major forms - addition of
• Palmitic acid (C160) - intracellular proteins
• attached to Cys
• Myristic acid (C140) - cytosolic proteins
• attaches to NH2-terminal Gly
• consensus sequence Met-Gly X-X-X-Ser/Thr
• Glycophosphatidyl inositol (GPI) - membrane
  proteins
• 0.5 of proteins in eukaryotic cells are GPI
  anchored

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Lipidation

• Prenyl groups
• Farnesylation
• Geranylgeranylation
• attached to Cys at or near C-terminus
• targets proteins to membranes
• Cholesterol
• attaches to C-terminal Gly
• firmly anchors proteins in membranes

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Analysis of Lipidation

• Mass Spec
• Myristylation was detected in a peptide by
  m/z212
• Farnesylated peptides were subjected to
  beta-elimination and reaction with a biotin
  derivative

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Sulfation

• Tyr is sulfated by tyrosylprotein
  sulfotransferase using phosphoadenosine
  -5-phosphosulfate
• 1 of all Tyrs may be sulfated
• Modulator of protein-protein interactions of
  secreted and membrane proteins
• Analysis
• 35S-labelling
• degrades rapidly in MS (-80)

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Acetylation and Methylation

• Lys or N-terminus may be acetylated
• Arg may be mono-, di- or tri- methylated
• Are important mechanisms in histone regulation
• Easily detected by MS