Protein evolution The role of domains

1. Protein evolution The role of domains Alice Skoumalová

2. Definition of a domain • an independent structural, functional and evolutionary unit • Structural unit Self-stabilizing locally folded region of tertiary structure Combination of motifs α-helix andβ-sheet Most proteins have 2 and more domains 2.Functional unit Various functions Ligands binding, membrane transit, catalytic activity, DNA binding, protein-protein interaction, etc. An independent function, cooperation with other domains 3. Evolutionary unit The relationship of proteins(superfamilies formation) The „family tree“ SCOP (Structural Classification of Proteins) with 1200 protein superfamilies

3. The creation of new proteins • duplication, divergence and recombination of domains • new function (sequence divergence, combining with other domains) • This mechanism facilitates the creation of proteins from different protein domains (no need of new genes for the formation of new proteins)

4. The recombination of domains Two generic principles: A domain can perform the same function, but in different protein contexts (with different partner domains) Syntactical shift A domain can diverge and aquire a novel or modified function Semantic shift

5. Transcription factor FadR Human methionine aminopeptidase WHD Oligomerisation/CoA-binding domain Creatinase/aminopeptidase domain Restriction endonuclease Fokl WHD (Winged helix domain) WHD Catalytic domain

6. The creation of new proteins by the domain recombination (an example) Proteins that participate in the haemostasis • form the superfamily of the related proteins (duplication, reception or deletion of the specific domains) • contain a domain that is homologous to trypsin (they have a common ancestor with trypsin) • the family tree can be generated with 7 gene modules

7. Ancestral protein Trypsin-like serine protease P A modul which codes the structure called kringle Kringle addition K P Parent of all proteins

8. P EGF domain addition K P Urokinase E K P Fibronectin domain 2 addition F2 E K P Fibronectin domain 1 addition Kringle duplication F1 F2 E K P F2 E K K P EGF domain duplication t-PA F1 E F2 E K P Factor XII

9. P K P Propeptide addition Urokinase E K P K P Pr Calcium binding domain addition F2 E K P C K P Pr Kringle duplication Kringle deletion Pr C P F1 F2 E K P 2 EGF domains addition C K K P Pr F2 E K K P Prothrombin t-PA C E E Pr P Factors VII, IX, X Protein C F1 E F2 E K P Factor XII

10. P K P Repeat kringle duplication Urokinase E K P K P Pr Hepatocyte growth factor F2 E K P K K K K P C K P Pr Kringle duplication K K K K K P Pr C P F1 F2 E K P Plasminogen C K K P Pr F2 E K K P Prothrombin K K K K K K P t-PA C E E Pr P Apolipoprotein (a) Constits of 40 kringles Factors VII, IX, X Protein C F1 E F2 E K P Factor XII

11. From the example above we can deduce: • The relationship of the haemostatic proteins is an example of the universal principle of the new protein creation • Simple arithmetic operations with gene modules facilitate the creation of new proteins with different properties

12. Summary • There is no simple relation: 1 gene - 1 protein • One gene can produces more proteins (various conformations, various domain recombination) • Duplication, divergence and recombination of domains are crucial for the protein creation (there si no need of new genes for the new proteins formation) • An example of relationship of proteins participating in the haemostasis

13. Proteomics

14. Proteomics Genomics What is proteomics? The large-scale study of proteins PROTEin+genOME Expression Genom Proteom +posttranslational modification +alternative splicing +alternative folding All genes in DNA of an organism The human genome contains 20-25000 genes The genom is a constant entity All proteins produced by an organism The human body contains millions proteins One organism has different protein expression in different parts of its body, stages of its life cycle and environmental conditions

15. Increase in protein diversity • Posttranslational modification • Alternative splicing • Alternative folding Primary transcript • mRNA before the posttranscriptional modification Alternative splicing Posttranslational modification Alternative folding

16. Posttranslational modification • The chemical modification of a protein after its translation • Addition of functional groups (acetate, phosphate, lipids, carbohydrates) • Modification of amino acids • Structural changes ( the formation of disulfide bridges, proteolytic cleavage)

17. Alternative splicing of a pre-mRNA transcribed from one gene can lead to different mature mRNA molecules and therefore to different proteins

18. Alternative folding The protein folding proceeds from a disordered state to progressively more ordered conformations corresponding to lower energy levels Local minimum (alternative conformation) Global minimum (native state)

19. Basic proteomic analysis scheme Protein mixture 1. Separation 2D-PAGE Individual proteins 2. Spot cutting Trypsin digestion Peptides 4. Sequence analysis Peptide fragmentation 3. Mass analysis Mass spectroscopy Sequence information Peptide mass 5. Database search Protein identification

20. 2D gel electrophoresis The synchronous analysis of hundreds or even thousands of proteins Proteins spread out on the surface

21. The role of proteins in the pathogenesis of diseases Protein expression in diseases Application of proteomics in medicine (disease proteomics) Using specific protein biomarkers to diagnose disease Alzheimer disease (amyloid β) Heart disease (interleukin-6 and 8, serum amyloid A, fibrinogen, troponins) Renal cell carcinoma (carbonic anhydrase IX) Biomarkers of diseases Design of new drugs Information about proteins causing diseases is used for the identification of potential new drugs

22. Biomarkers of diseases • Proteome-based plasma biomarkers for AD • Diagnosis of AD • On clinical grounds+post mortem (histology) • There is no reliable diagnostic test • Plasma may offer a rich source of disease biomarkers • Identification of diagnostic biomarkers in the blood by proteomics • Plasma samples of patients and control were analysed by 2D gel electrophoresis • Spots that were significantly different between case and control groups were excised and analysed by mass spectroscopy

23. Results • 15 spots were significantly different between patients and controls • MS analysis: 2-macroglobulin, complement factor H, …

24. Virtual ligand screening The identification of new drugs to target and inactivate the HIV-1 protease (cleaves a very large HIV protein into smaller, functional proteins; virus cannot survive without this enzyme; it is one of the most effective protein targets for killing HIV)

25. Summary • Proteomics studies proteins, particularly their structure, function and interaction • The genome has already been analysed, now scientists are interested in the human proteome (millions of proteins) • Key technologies used in proteomics are 2D gel electrophoresis and mass spectrometry • Proteins play a central role in the life of an organism, their malfunction startes diseases; proteomics is instrumental in discovery of pathogenesis of disease, biomarkers and potential therapetic agents

26. Questions • Definition of a domain (3 aspects), mechanisms of the new protein creation (in general), syntactical and semantic shift (the principle) • Increase in protein diversity compared to genom • Identification of the renal carcinoma biomarkers in the plasma • Using of computer sofware for the development of new drugs