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1
Submitted To:
Dr. Uzma Qaiser
2
21 June 2014
Presented By:
Saadia Aslam
Nasira Rafi
Hamna
Imtiaz
Hira Butt
Mehwish
Iqra Almas
3
Contents
Introduction
. Modifications
• Effects of PTMs
• Types of PTMs
• Protein Splicing
Applications
Detection Techniqu...
5
A LITTLE BACKGROUND: PROTEINS
Plays a very significant role in the
structural and functional organization of
any cell.
Tra...
WHAT THE POST TRANSLATIONAL
MODIFICATION IS???
Post translational modification (PTM) is the
chemical modification of a pro...
Why PTM is necessary???
 Stability of protein
 Biochemical activity (activity regulation)
 Protein targeting (protein l...
9
1,000,000 proteins out of only 30,000
genes???
•Splicing Variants
In eukaryotic cells, likely 6-8
proteins/gene.
• Post-tr...
Post-translational modification
Modification Involving Peptide
Bonds
Modification of amino acids
Subunit aggregation
Prote...
Modification Involving Peptide Bonds
1 • Peptide Bonds Cleavage
2 • Peptide Bond Isomerization
12
13
• Specific and well-regulated
• Enzymatic and Non-Enzymatic
• Examples:
Removal of signal leader peptide by signal pep...
14
iIn vivo conversion of preproinsulin to Insulin
14
(103 amino acids)
(51 amino acid)
15
• Ser → esters
• Cys → thioesters
• Asp or Asn → isoaspartate
• Prolyl peptide cis-trans isomerization by prolyl
isomer...
Modification of
amino acids
16
17
Different types of PTMs & their
modification sites
Phosphorylation
Glycosylation
Acylation
Alkylation
Hydroxylation
Pro, L...
Histone Modification
• Different modifications occur on specific residues to perform
specific regulatory functions.
19
How does Histone Acetylation
promote Transcription
• Acetylation neutralizes the positively charged Lys residues on histon...
• Citrullination:
The conversion of arginine to citrulline.
Arginine Citrulline
• Deamination:
The conversion of glutamine...
PROTEIN
FOLDING
• Physical process leading from an unfolded
polypeptide chain to a functional protein with a
definite stru...
23
Protein Folding
The folding process
depends on the solvent , the
salts concentration , the pH,
the temperature and
mole...
In vivo Protein folding in absence or presence of
Chaperones
Properly Folded
proteins
Improper folding
and Aggregation
Ref...
SUBUNIT
AGGREGATION
Multimeric proteins are assembled in
the ER.
Some folded protein chains (subunits)
must aggregate with...
Haemoglobin A
26
• Intramolecular process catalyzed entirely
by amino acid residues contained in the
intein.
• No coenzymes or sources of m...
28
What the INTEINS are
• An intein is a segment of
a protein that is able to
excise itself and join the
remaining portion...
29
How Inteins look like???
Split
intein
Mini
intein
Maxi
intein
30
Step 1
Formation of a linear ester
intermediate by NO or NS
acyl rearrangement
involving the first
nucleophilic amino a...
Analogy to RNA Splicing
 Regulation of :
1. Enzymatic activity
2. Half life of proteins
3. Interaction with other molecules
4. Subcellular locali...
DETECTION TECHNIQUES FOR PTM
1
• Gel-based detection
techniques
2
• MS-based detection
techniques
3
• Microarray-based
det...
Direction of
migration
Anode
Cathode
-
+
Buffer
Acrylamide
gel
Sample loading
Protein
mixture
SDS-PAGE
2-D Electrophoresis...
35
Completed
gels
Nitrocellulose
sheetBlotting
Specific phospho-
tyrosine
antibodies added
Detection using
labeled seconda...
36
PTM modified
protein of
interest
Trypsin
digestion
Protein
Matrix
196 –well MALDI Plate
Digested
protein
MS-based Detec...
37
Matrix & analyte
Target plate
Detector
Flight tube
MALDI
Laser
Ionization and Detection
Proteome array containing
potential substrates for
phosphorylation
Kinase
enzyme
[g-33P] ATP
solution
Protein
substrate
Ki...
Proteome array
Washing
Phosphorylated
proteins
Detection-
Autoradiography
film
33P
33P
33P
33P33P
Developed image
Radioact...
40
Does Post-translational Modification Occurs
in Prokaryotes???
Chemical modifications e.g. Phosphorylation
 Classical s...
41
SUMMARY
• PTM is the chemical modification of a protein after its translation.
• PTMs are key mechanisms to increase pr...
42
6/21/2014 43
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a very brief and informative presentation on PTM......... presented in CEMB by Saadia Aslam, Punjab University Lahore.

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Post translational modifications

  1. 1. 1
  2. 2. Submitted To: Dr. Uzma Qaiser 2
  3. 3. 21 June 2014 Presented By: Saadia Aslam Nasira Rafi Hamna Imtiaz Hira Butt Mehwish Iqra Almas 3
  4. 4. Contents Introduction . Modifications • Effects of PTMs • Types of PTMs • Protein Splicing Applications Detection Techniques Summary 4
  5. 5. 5
  6. 6. A LITTLE BACKGROUND: PROTEINS Plays a very significant role in the structural and functional organization of any cell. Translation is the final stage of gene expression and synthesize the immature protein. Many transmembrane or secretory preproteins have an N-terminal signal peptide. A signal peptide is recognized by a SRP that binds the translocon. 6
  7. 7. WHAT THE POST TRANSLATIONAL MODIFICATION IS??? Post translational modification (PTM) is the chemical modification of a protein after its translation. OR The chemical modifications that take place at certain amino acid residues after the protein is synthesized by translation are known as post-translational modifications. These are essential for normal functioning of the protein. PTMS occur mostly in E.R and golgi apparatus. 7
  8. 8. Why PTM is necessary???  Stability of protein  Biochemical activity (activity regulation)  Protein targeting (protein localization)  Protein signaling (protein-protein interaction,cascade amplification) 8
  9. 9. 9
  10. 10. 1,000,000 proteins out of only 30,000 genes??? •Splicing Variants In eukaryotic cells, likely 6-8 proteins/gene. • Post-translational modification 22 different forms of antitrypsin observed in human plasma. Post-translational modifications are key mechanisms to increase proteomic diversity and regulate cellular activity. 10
  11. 11. Post-translational modification Modification Involving Peptide Bonds Modification of amino acids Subunit aggregation Protein folding and chaperones 11 Protein Splicing
  12. 12. Modification Involving Peptide Bonds 1 • Peptide Bonds Cleavage 2 • Peptide Bond Isomerization 12
  13. 13. 13 • Specific and well-regulated • Enzymatic and Non-Enzymatic • Examples: Removal of signal leader peptide by signal peptidase  Precursor protein → mature protein (Insulin) Zymogen → active enzyme Trypsinogen → Trypsin Prohormone → Hormone Modification Involving Peptide Bonds Cleavage (Limited Proteolysis) 13
  14. 14. 14 iIn vivo conversion of preproinsulin to Insulin 14 (103 amino acids) (51 amino acid)
  15. 15. 15 • Ser → esters • Cys → thioesters • Asp or Asn → isoaspartate • Prolyl peptide cis-trans isomerization by prolyl isomerase Modification Involving Peptide Bond Isomerization (Intramolecular) 15
  16. 16. Modification of amino acids 16
  17. 17. 17
  18. 18. Different types of PTMs & their modification sites Phosphorylation Glycosylation Acylation Alkylation Hydroxylation Pro, Lys Ser, Thr, Tyr Asn, Ser, Thr Asn, Gln, Lys Lys, Arg 18
  19. 19. Histone Modification • Different modifications occur on specific residues to perform specific regulatory functions. 19
  20. 20. How does Histone Acetylation promote Transcription • Acetylation neutralizes the positively charged Lys residues on histones and thus reduces the interactions of histones with DNA. Ac BD H4 5/8 12/16 TAFII250 Ac BD 20
  21. 21. • Citrullination: The conversion of arginine to citrulline. Arginine Citrulline • Deamination: The conversion of glutamine to glutamic acid or asparagine to aspartic acid. PTMs involving changing the chemical nature of amino acids 21 PAD
  22. 22. PROTEIN FOLDING • Physical process leading from an unfolded polypeptide chain to a functional protein with a definite structure. • Minimizing the number of hydrophobic side- chains exposed to water is an important driving force. • The native state is the most stably folded form. POST TRANSLATION MODIFICATION
  23. 23. 23 Protein Folding The folding process depends on the solvent , the salts concentration , the pH, the temperature and molecular chaperones. Chaperones are proteins that facilitate the folding of other proteins without being part of assembled complex .
  24. 24. In vivo Protein folding in absence or presence of Chaperones Properly Folded proteins Improper folding and Aggregation Refolding Proteosome Degradation
  25. 25. SUBUNIT AGGREGATION Multimeric proteins are assembled in the ER. Some folded protein chains (subunits) must aggregate with other subunits to form quaternary structure. Such multi-subunit proteins include many of the most important enzymes and transport proteins in the cell. 25
  26. 26. Haemoglobin A 26
  27. 27. • Intramolecular process catalyzed entirely by amino acid residues contained in the intein. • No coenzymes or sources of metabolic energy. • Involves bond rearrangements rather than bond cleavage followed by resynthesis. • Converts Inactive protein precursor to biologically active protein. PROTEIN SPLICING
  28. 28. 28 What the INTEINS are • An intein is a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond. • Found in bacteria eukaryotes, archaea and viruses.
  29. 29. 29 How Inteins look like??? Split intein Mini intein Maxi intein
  30. 30. 30 Step 1 Formation of a linear ester intermediate by NO or NS acyl rearrangement involving the first nucleophilic amino acid residue at the N-terminal splice junction and the final residue of the N-extein. Step 2 Formation of a branched ester intermediate by the attack of the first nucleophilic residue of the C-extein on the linear ester intermediate. Step 3 Cyclization of the last residue(Asn) of the intein, cleaves apart the peptide bond between the intein and the C-extein, resulting in a free intein segment with a terminal cyclic imide. Step 4 Spontaneous rearrangement of the ester linkage between the ligated exteins to the more stable amide bond. The last step is spontaneous and irreversible. The first three steps are catalyzed by the intein. What actually the Mechahanism of Splicing is???
  31. 31. Analogy to RNA Splicing
  32. 32.  Regulation of : 1. Enzymatic activity 2. Half life of proteins 3. Interaction with other molecules 4. Subcellular localization of proteins  Rapid Purification of Target proteins. In vitro Intein mediated Protein Purification What are the Applications ???
  33. 33. DETECTION TECHNIQUES FOR PTM 1 • Gel-based detection techniques 2 • MS-based detection techniques 3 • Microarray-based detection techniques
  34. 34. Direction of migration Anode Cathode - + Buffer Acrylamide gel Sample loading Protein mixture SDS-PAGE 2-D Electrophoresis Proteins focused on IPG strip Direction of migration Completed stained gels Gel-based Detection Techniques • ImmunoblottingExample
  35. 35. 35 Completed gels Nitrocellulose sheetBlotting Specific phospho- tyrosine antibodies added Detection using labeled secondary antibodies Proteins phosphorylated at Tyr residues Proteins phosphorylated at Tyr residues Immunoblotting
  36. 36. 36 PTM modified protein of interest Trypsin digestion Protein Matrix 196 –well MALDI Plate Digested protein MS-based Detection Techniques for PTMs • MALDI-TOF-Mass SpectrometryExample Digestion and Sample Spotting
  37. 37. 37 Matrix & analyte Target plate Detector Flight tube MALDI Laser Ionization and Detection
  38. 38. Proteome array containing potential substrates for phosphorylation Kinase enzyme [g-33P] ATP solution Protein substrate Kinase enzyme [g-33P] ATP ADP Ser Phosphorylated protein Ser Microarray-based Detection Techniques for PTMs • Protein MicroarraysExample
  39. 39. Proteome array Washing Phosphorylated proteins Detection- Autoradiography film 33P 33P 33P 33P33P Developed image Radioactive emissions Protein Microarrays
  40. 40. 40 Does Post-translational Modification Occurs in Prokaryotes??? Chemical modifications e.g. Phosphorylation  Classical system  Two-component system  PTS system Signal peptide cleavages Cleavages of N-terminal f- methionine residues Protein Splicing
  41. 41. 41 SUMMARY • PTM is the chemical modification of a protein after its translation. • PTMs are key mechanisms to increase proteomic diversity and regulate cellular activity. • PTMs include modifications of peptide bonds, amino acids, subunit aggregation and protein folding. • Protein splicing is intramolecular process catalyzed entirely by amino acid residues contained in the intein. • PTMs can be detected by Gel based detection techniques, MS techniques and Microarray based detection techniques. 41
  42. 42. 42
  43. 43. 6/21/2014 43
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a very brief and informative presentation on PTM......... presented in CEMB by Saadia Aslam, Punjab University Lahore.

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