Post translational modifications
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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
- 2. Submitted To: Dr. Uzma Qaiser 2
- 3. 21 June 2014 Presented By: Saadia Aslam Nasira Rafi Hamna Imtiaz Hira Butt Mehwish Iqra Almas 3
- 4. Contents Introduction . Modifications • Effects of PTMs • Types of PTMs • Protein Splicing Applications Detection Techniques Summary 4
- 5. 5
- 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. 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. 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
- 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. Post-translational modification Modification Involving Peptide Bonds Modification of amino acids Subunit aggregation Protein folding and chaperones 11 Protein Splicing
- 12. Modification Involving Peptide Bonds 1 • Peptide Bonds Cleavage 2 • Peptide Bond Isomerization 12
- 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 iIn vivo conversion of preproinsulin to Insulin 14 (103 amino acids) (51 amino acid)
- 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
- 17. 17
- 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. Histone Modification • Different modifications occur on specific residues to perform specific regulatory functions. 19
- 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. • 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. 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 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. In vivo Protein folding in absence or presence of Chaperones Properly Folded proteins Improper folding and Aggregation Refolding Proteosome Degradation
- 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. Haemoglobin A 26
- 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 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 How Inteins look like??? Split intein Mini intein Maxi intein
- 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. Analogy to RNA Splicing
- 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. DETECTION TECHNIQUES FOR PTM 1 • Gel-based detection techniques 2 • MS-based detection techniques 3 • Microarray-based detection techniques
- 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 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 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 Matrix & analyte Target plate Detector Flight tube MALDI Laser Ionization and Detection
- 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. Proteome array Washing Phosphorylated proteins Detection- Autoradiography film 33P 33P 33P 33P33P Developed image Radioactive emissions Protein Microarrays
- 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 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
- 43. 6/21/2014 43
Editor's Notes
- Protein translation: The process by which the mRNA template is read by ribosomes to synthesize the corresponding protein molecule on the basis of the three letter codons, which code for specific amino acids.
- SRP: Signal Recognition Particle. N terminal signal peptide is also known as Leader sequence. It is hydrophobic amino acid chain present in nascent state configuration of proteins. After formation, a protein moves from ribosome to ER where SRP recognizes the signal peptide. Protein passes through a channel of proteins called TRANSLOCON present on the ER membrane and in prokaryotes it is present on plasma membrane. Here in ER the signal peptide is cleaved by signal peptidase.
- Stability of protein is done mainly by folding process in which nascent state is converted into native state of protein. Biochemical activity regulation means proteins are activated inside the cell whenever they are required otherwise they remain inactive. This is only possible by post translational modifications.
- N-glycosylation is the addition of carbohydrate occurs on amino group of amino acids. O-glycosylation is the addition of carbohydrate occurs on hydroxyl group of amino acids. GPI: glycosyl phosphatidyl inositol
- Enzymatic Cleavage by the use of enzymes i.e. Trypsin, Chymotrypsin Non-enzymatic cleavage by the use of chemicals or heat i.e Cyanogen Bromide is often used to cleave the peptide bond after a methionine.
- 1st step: disulphide bond formation 2nd step: Removal of leader sequence by signal peptidase. 3rd step: Cleavage of C peptide (C for connecting) and formation of active fragment insulin.
- The peptide bond of Proline with other amino acids is in cis form. During folding this cis bond is converted to trans form by an enzyme prolyl isomerase.
- Prenylation: Addition of isoprenoid units. It is also called lipidation. ADP Ribosylation: Addition of one or more ADP ribose moieties to a protein.Cholera toxin ADP-ribosylates G proteins causing massive fluid secretion from the lining of the small intestine, resulting in life-threatening diarrhea.
- Phosphorylation is done by protein kinases and it mainly activates the proteins like in case of signaling proteins. While phosphatases dephosphorylate the protein mainly resulting in inactivation of protein. Ubiquitin is small 8.5 kDa protein that attaches preferably at lusine residue by help of E3 ligase. i.e. ubiquitination of histone protein
- PAD: peptidyl arginine deiminase
- There are two classes of chaperons: Molecular chaperones that only prevent improper folding. Chaperonins or Heat shock proteins which are also involved in proper folding of a protein.
- The DNA with coding regions for inteins are transcribed into mRNA and translated into a premature protein. A protein is not fully mature until a intein is excised from the protein.
- DOD = dodecapeptide The type of the splicing proteins is categorized into four classes: maxi-intein, mini-intein, trans-splicing intein, and alanine intein. The maxi-inteins are N- and C-terminal splicing domains containing an endonuclease domain. The mini-inteins are typical N- and C-terminal splicing domains; however, the endonuclease domain is not present. The trans-splicing inteins are split inteins which are divided into N-termini and C-termini. Alanine inteins have the splicing junction of an alanine instead of a cysteine or a serine, in both of which the protein splicing occurs.
- Like introns within an RNA precursor, inteins are found within a mRNA strand. Just as introns are connected to RNA splicing, inteins are connected to protein splicing.
- One of the best understood posttranslational modifications is the reversible phosphorylation of proteins at serine, threonine, or tyrosine residues. These phosphoamino acids are relatively stable in acidic solutions.
- 2. Immunoblotting: This process, also known as Western blotting, is a commonly used analytical technique for detection of specific proteins in a given mixture by means of specific antibodies to the given target protein. Protein mixture containing phosphorylated as well as other unmodified proteins can be separated by a suitable electrophoresis technique. SDS-PAGE and two dimensional gel electrophoresis are most commonly used for protein separation. Two-dimensional gel electrophoresis (2-D electrophoresis) separate proteins in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW).
- The separated protein bands are then blotted onto a nitrocellulose membrane. These membranes are then probed by means of specific anti-phospho-amino acid antibodies that specifically bind to proteins having phosphorylation at a particular amino acid residue. This binding interaction can then be detected by means of suitably labeled secondary antibodies or by autoradiography using a radioactive probe.
- MALDI-TOF-MS: A mass spectrometry instrument that produces charged molecular species in vacuum, separates them by means of electric and magnetic fields and measures the mass-to-charge ratios and relative abundances of the ions thus produced. In MALDI-TOF-MS, the ion source used is MALDI. The Time-of-Flight mass analyzer correlates the flight time of the ion from the source to the detector with the m/z of the ion. The modified protein of interest is digested into smaller peptide fragments using a suitable enzyme like trypsin. This digest is then mixed with a suitable organic matrix such as a-cyano-4-hydroxycinnamic acid, sinapinic acid etc. and then spotted on to a MALDI plate.
- The target plate containing the spotted matrix and analyte is placed in a vacuum chamber with high voltage . The laser energy gets absorbed by the matrix and is transferred to the analyte molecules which undergo rapid sublimation resulting in gas phase ions. These ions are accelerated and travel through the flight tube at different rates. The lighter ions move rapidly and reach the detector first while the heavier ions migrate slowly. The ions are resolved and detected on the basis of their m/z ratios and a mass spectrum is generated.
- Protein microarrays: These are miniaturized arrays normally made of glass, onto which small quantities of many proteins can be simultaneously immobilized and analyzed. Potential substrates for protein phosphorylation are immobilized on a suitably coated array surface. To this, kinase enzyme and gamma P-32 labeled ATP are then added and the array is incubated at 30oC. The phosphorylation reaction occurs at those sites containing proteins that can be modified.
- After sufficient incubation, excess unbound ATP and enzyme are washed off the array surface. Detection is carried out by means of autoradiography. The radioactive emissions from the phosphate label present at the phosphorylated protein sites strike the film. Upon development, the positions at which phosphorylation has occurred can be clearly determined.
- “Classical system” utilizes NTPs as phosphoryl donors and leads to protein modification at Ser/Thr or Tyr residues. “Two-component system” requires first a sensor kinase which autophosphorylates at a His residue at the expense of ATP, then in turn a response regulator is modified at an Asp residue and thereafter induces a metabolic change within cell. “PTS system” use PEP to generate a phosphoryl group which is passed down a chain of several proteins and finally transferred to a sugar. Cleavages of N-terminal f- methionine residues: the formyl group of f-met is removed by deformylase. The initiating met residue of some polypeptides may also be removed by methionine aminopeptidase.