Download
role of proteolysis in physiological and pathological processes n.
Skip this Video
Loading SlideShow in 5 Seconds..
Role of proteolysis in physiological and pathological processes PowerPoint Presentation
Download Presentation
Role of proteolysis in physiological and pathological processes

Role of proteolysis in physiological and pathological processes

156 Vues Download Presentation
Télécharger la présentation

Role of proteolysis in physiological and pathological processes

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Role of proteolysis in physiological and pathological processes Zdenka Kučerová

  2. Proteolyticenzymes : • cleave peptide bonds ( proteinsorpeptides) • control of the cell cycle • key roles in many pathophysiological processes • targets for therapeutic strategies • speed of the cleavage depends on the strategically located positions of cleaved bonds

  3. Activity of proteolytic enzymes • determined by many factors at the post-translational level • allosteric nature  physiologically important interactions •  explains why the enzymes carry a large body of structural and functional information • degradom

  4. Regulation by proteolytic enzymes • limited proteolysis or by proteinase inhibitors • Limited proteolysis: • post-translational modification • conversion of an inactive precursor into an active form • Proteinase inhibitors: • regulate:coagulation •  fibrinolysis •  connective tissue turnover • complement activation • concentration changes during physiological or pathological processes

  5. Proteolysis • complete degradation of proteins to free amino acids • requires series of enzymes with different specificity • not only in the digestive tract, but also in every cell in lysosomes, in cytoplasm and other parts of the cell • Division of proteolytic enzymes peptidasesendopeptidases and exopeptidases (position of the cleavage site within the substrate molecule) • endopeptidases(proteinases)  groups according to similar reaction mechanisms (e.g. serine or cysteine proteinases)

  6. Mechnism of interaction • enzymes recognise and bind to a short amino acid sequences and then specifically hydrolyse bonds between particular aminoacid residues

  7. Classification of proteinases A) endopeptidases – hydrolysis within the peptide chain 1)serine proteinases – serine or histidine residues in active sites, pH optimum 7 – 9 trypsin, chymotrypsin, thrombin, plasmin 2)cysteine proteinases – cysteine residue in active site, pH optimum 4 – 7 calpain, caspases 3)aspartic proteinases – two aspartic residues in active site, pH optimum below 5 pepsin, gastricsin, rennin, HIV proteinase 4)metaloproteinases – metal ion important for catalysis, pH opt. 7 – 9 matrix metaloproteinases, collagenase, gelatinase 

  8. Homology of various proteinases

  9. B)exopeptidases - cleave off single amino acid one after another • 1)aminopeptidases – cleave off a single amino acid from amino terminus • 2)dipeptidyldipeptidases – cleave off dipeptide from amino terminus • 3)carboxypeptidases – cleave off a single amino acid from carboxy terminus • 4)dipeptidases – hydrolyse dipeptides in two single amino acids • Individual proteinases differ in their substrate specifity • For complete degradation of a protein, enzymes from each of the above-named groups are required

  10. Characterization of some proteinases enzym m.wt isoelektric point (pH) chymotrypsino- gen 25.000 8.1 trypsin 24.000 10.8 plasmin 75.000 - elastase 26.000 9.5 papain 21.000 8.7 pepsin 33.000 1.0 carboxypepti- 34.000 6.0 dase A

  11. Inhibitors • differ according to individual groups of proteinases • Serine proteinases…..chlormethylketons, • Cysteine proteinases…. dithiothreitol, mercaptans, • Aspartic proteinasess… epoxy- and diazo- compounds, pepstatin • Metalloproteinases….. chelates

  12. Serine proteinases • Proteinases of digestive tract – trypsin, chymotrypsin • important for: • fibrinolysis • blood coagulation • complement activation • produced as inactive zymogens • activated by limited proteolysis by another proteinase

  13. Blood clotting • enzymatic conversion of a soluble plasmatic fibrinogen into a fibrous network of insoluble fibrin polymers • fibrinogen cleaved by thrombin and fibrin aggregate into insoluble fibrin polymer • most of the coagulation factors are proteinases

  14. Initiation of blood clotting • two different ways • extravascular pathway- triggered by an injury of tissue • intravascular pathway- initiated by contact with a damaged inner surface of blood vessel • cascade of proteolytic cleavages • requires calcium and phospholipids • conversion of inactive prothrombin to active thrombin • series of zymogens of serine proteinases are sequentially activated (active serine proteinases arise from inactive precursors, the active enzymes then activate other inactive proteins)

  15. Unique aspects of coagulation • requirement for a protein or a lipoprotein cofactor for optimal reaction rates • requirement for a membrane surface • metal ion requirement • uniqueness of initiating reactions in comparison to those which activate the pancreatic zymogens

  16. Blood coagulation cascade • ended by dissociation of the membrane complex • regulated by activators and inhibitors in dynamic equilibrium • Antithrombin III • inhibits activity of thrombin an anticoagulant factor • anticoagulant therapy reduction of formation or reduction of function of thrombin and other serine proteinases • Protein C • Serine proteinase…inactivation of factor Va and VIIIa

  17. Fibrinolysis • fibrin networks are degraded by plasmin • plasmin is serine proteinase • plasmin evolves from precursor plasminogenafter activation by plasminogen activator • plasmin activity inhibited by α-2 antiplasmin • therapy by fibrinolytic proteinases  thrombus degradation

  18. Characterization of plasmin, plasminogen and plasminogen activator Plasminogen is one-chain glykoprotein hydrolyse bounds Arg67-Met68, Lys76-Lys77, Lys77-Val78 Plasmin is two chain serine protease (with disulfidic bound) active site His-603, Asp-646, Ser-741 its main target is fibrin Human plasminogen activator [PA] is serine protease (tissue and urokinase) one polypeptide chain ( single chain-ScuPA) hydrolyse bounds Arg275-Ileu276(two chain-TctPA)

  19. plasmin formation is dependent on: • plasmatic concentration of plasminogen • availability of activators of plasminogen in the plasma • surrounding tissue environment • concentration of naturally present inhibitors • concentration of fibrin

  20. Pathological fibrinolysis • abnormal fibrinolysis, bleeding or thrombus formation • Higher fibrinolysis • increased level of plasminogen activator (PA) • deficiency of plasminogen activator inhibitor (PAI) • deficiency of antiplasmin • Thrombus formation • defective synthesis of plasmin or PA • PA used to pharmacological digestion of thrombus • Malignant tumors • disorders of hemostasis - ability of the tumor to alter the coagulation system

  21. Tumor malignanciesconnected with invasive growth and metastasisenzymatic breakdown of two proteolytic systems (fibrinolytic s. and matrix metaloproteinases s.) • Methastatic disease • disintegrated regulation • irreversible progression of cell cycle • resistance to a cell signalling • understanding of the mechanismpreventing tumor cell spread • proteases have proteolytic activity that may affect cancer progression

  22. Proteinases - Biomarkers • correspond to changes in the whole organism • changes: - biochemical • - histological • - morphological • - physiological • very important for early diagnosis

  23. Complement • This system collaborates in recognition and elimination of pathogens as a part of both the innate and acquired immune systems. • The activation of complement pathway - through attached serine proteases • the complement system is one of the most highly organized innate immune systems • serine proteinases are responsible for regulation of the early event of complement

  24. Kallikrein • plasmatic serine proteinases (15 ) • kalikrein - kinin is a complex system produced in various organs • the kallikreins must now be considered as important 'hormonal' regulators of tissue function • important for initiation of cellular fibrinolysis, which is independent on plasmin, fibrin and tissue plasminogen activator • predictive marker for cancer

  25. Cysteine proteinases • lysosomal- cysteine cathepsins • implicated in tumor spread and metastasis • prognostic factors for tumor recurrence in human breast cancer • non-lysosomal – calpain • cleave cell-cycle proteins, cytoskeletal and myofibrilar proteins • cytoplasmatic enzyme

  26. Apoptosis • evolutionary conserved form of cell suicide • requires a proteolytic system - mainly caspases • caspase family of proteases can be divided into pro-apoptotic and pro-inflammatory members based on their substrate specificity and participation in separate signalling cascades • caspase regulation therapeutic purposes • caspases -minimally requires a tetrapeptide substrate in which Asp is in absolute requirement in P1 position, the P4 substrate residues is unique to each homologue.

  27. defective control of apoptosis  pathogenesis of diseases • cancer chemotherapy and radiation  cancer cell death by apoptosis • Caspases are responsible for crucial aspects of inflammation and immune-cell death that are disrupted in a number of genetic autoimmune and autoinflammatory diseases. • caspases  proteolytic devitalizing and remodelling of tumor cellssuppress tumor cell growth invasion and metastasis

  28. Aspartic proteinases • Acid proteinases from mammalian gastric mucosa pepsins • Proteinases associated with limited proteolysis renin blood pressure cathepsins D,E lysosomal enzyme proteinase HIV

  29. Renin • aspartic proteinase • formed as a precursor (prorenin) in kidneys • active form (renin) released into the blood • renin cleaves angiotensinogen to form angiotensin I • angiotensin-converting enzyme (ACE) then converts angiotensin I to angiotensin II

  30. Renin • highly specific • synthesized in response to decreasing levels of sodium ions and declining blood pressure • renin  angiotensin II  affects:- kidney • - brain stem • - hypophysis • - adrenal cortex • - blood vessel walls • - heart

  31. Cathepsins D and E • Cathepsin D • aspartic proteinase localized in lysosomes • in most tissues and cells of human organism • widely distributed among biological species • influences:- intracellular protein catabolism • - hormone and antigen processing • - pathological processes (neoplasia and • neurodegenerative changes) • Cathepsin E • in human stomach cells (another stomach cells than pepsinogen and progastricsin) • Both is over- expressed by cancer cells

  32. Proteinase HIV • AIDS  retrovirus (human immunodeficiency virus) • retroviral aspartic proteinases  deeply studied proteins in today molecular biology chemistry • three-dimensional structure of active site is similar to that of eucariotic proteinases • proteinase (and other enzymes)design of novel drugs for the treatment of AIDS • fast development of resistance of virus towards synthetic inhibitors and low bioavailability of these inhibitors • resistance results primarily from multiple mutations of the proteinase

  33. drug-resistant HIV proteinase →altered substrate specifity • altered substrate specifity understanding→valuable in the design of new protease inhibitors • active antiretroviral therapy (HAART) combining potent drugs that can inhibit reverse transcriptase, integrase and protease activities

  34. Metaloproteinases • matrix metaloproteinases (MMPs) • zinc-dependent enzymes • regulate tissue remodelling in physiological and pathological conditions • MMPs are key enzymes for tumor progression cell carcinoma • individual MMPs found in most tumor types • presence of specific MMPs has been shown to be a prognostic marker at tumor invasion • tumor invasion and metastasis→ degradation of the extracellular matrix around tumor cells and cell migration

  35. cancer metastasis →imbalance between MMPs and its inhibitor • MMPs→ regulatory effects on both primary and secondary tumors • inhibitors of MMPs were assessed for anticancer properties • proteolysis has a central role for cancer metastasis

  36. Exopeptidases • Peptidyl dipeptidases • angiotensin-converting enzyme in the control of blood pressure • Human prolylcarboxypeptidasealso known as angiotensinase C inactivates angiotensin II • Inhibitors of dipeptidyl peptidase IV (DPP IV) provide a strategy for the treatment of type 2 diabetes • Methionine aminopeptidase (MetAP) is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis • inhibitors of all peptidases have therapeutic effect

  37. Human gastric proteinases • digestive proteinases • adult mammalian gastric mucosa contains the highest concentration and greatest diversity of gastric proteinases • human gastric mucosa contains four aspartic proteinases: - pepsinogen A (PGA) • - pepsinogen C (PGC) • -chymosin • - cathepsin E • common biological properties (the ability to hydrolyze proteins at acid pH) but each is distinguishable by immunologic technique • serum contains both PGA and PGC, but only PGA is detectable in urine

  38. Schematic view of pepsinogen A

  39. Pepsinogen A and pepsinogen C • aspartic proteinases • differ in charge → distinguishable by electrophoresis • heterogeneity of pepsinogens → multiple genes • →posttranslational modifications of the primary gene product

  40. Duodenal ulcer • high level of PGA → subclinical marker of duodenal ulcer • Superficial gastritis • acute or chronic superficial gastritis → high serum PGA and PGC levels • inflammation of the mucosa→ increased rates of release of both zymogens into the circulation → elevated levels of PGA and PGC • level of PGC increased much more than level of PGA

  41. Pepsinogen A / pepsinogen C ratio in peptic ulcer, gastric cancer and control

  42. Gastric ulcer • changed levels of PGA and PGC • gastric ulcer usually develops within areas of chronic gastritis • pepsinogen A / pepsinogen C ration decreases

  43. Gastric cancer • very low serum levels of PGA • low serum PGA level → subclinical marker of increased risk for gastric cancer→ it is possible to identify high-risk subjects→ more effective approaches to diagnosis and treatment • serum PGA and PGC levels → subclinical markers of disorders of gastric mucosal structure and function