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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 Techniques Summary 4

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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

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

Why PTM is necessary??? Stability of protein Biochemical activity (activity regulation) Protein targeting (protein localization) Protein signaling (protein-protein interaction,cascade amplification) 8

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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

Post-translational modification Modification Involving Peptide Bonds Modification of amino acids Subunit aggregation Protein folding and chaperones 11 Protein Splicing

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 peptidase Precursor protein mature protein (Insulin) Zymogen active enzyme Trypsinogen Trypsin Prohormone Hormone Modification Involving Peptide Bonds Cleavage (Limited Proteolysis) 13

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 isomerase Modification Involving Peptide Bond Isomerization (Intramolecular) 15

Modification of amino acids 16

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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

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 histones and thus reduces the interactions of histones with DNA. Ac BD H4 5/8 12/16 TAFII250 Ac BD 20

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

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 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 .

In vivo Protein folding in absence or presence of Chaperones Properly Folded proteins Improper folding and Aggregation Refolding Proteosome Degradation

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

Haemoglobin A 26

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 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 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 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???

Analogy to RNA Splicing

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 ???

DETECTION TECHNIQUES FOR PTM 1 Gel-based detection techniques 2 MS-based detection techniques 3 Microarray-based detection techniques

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 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 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 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 Kinase enzyme [g-33P] ATP ADP Ser Phosphorylated protein Ser Microarray-based Detection Techniques for PTMs Protein MicroarraysExample

Proteome array Washing Phosphorylated proteins Detection- Autoradiography film 33P 33P 33P 33P33P Developed image Radioactive emissions Protein Microarrays

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 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

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