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Structure function relationship of clinically important peptides

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A comprehensive presentation on Structure-function relationship of clinically important Peptides for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.

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Structure function relationship of clinically important peptides

  1. 1. Structure-function relationship of clinically important Peptides Dr. Rohini C Sane
  2. 2. Protein structure-function relationship • The overall conformation of a protein , the particular position of amino acid side chains in three-dimensional space determines the function of the protein. • The diversity of protein structure and its correlation with function can be explained by the following clinically important proteins. Protein Function Carbonic anhydrase Formation and degradation of carbonic acid Myoglobin(Mb) Stores oxygen in muscle cells Hemoglobin (Hb) Transports oxygen from lungs to the tissue Collagen Providestensile strengthtomanytissueinthebody Elastin(withrubberlikeproperties) Occur in distensible structure
  3. 3. Quaternary structure of enzyme regulates its functions Structural conformation of the active site the enzyme precisely oriented for the substrate binding Binding of enzyme to the substrate . enzyme catalysis →product + enzyme set free for substrate binding again
  4. 4. Disordered region of enzyme and its Importance Schematic diagram Many Disordered region become ordered region when a specific ligand is bound . Disordered region gives flexibility and performs a vital biological role. Disordered regions of enzyme- catalytic site ordered regions of enzyme Substrate added to enzyme catalyzed reaction mixture Enzyme with Disordered regions set free at the end of reaction Product Enzyme-substrate complex –induced fit +
  5. 5. QuaternarystructureofenzymeCarbonicanhydraseregulatesitsfunctions ❖Function of Carbonic anhydrase : catalyzes the reversible hydration of carbon dioxide. H2O + CO2  H2CO3(Carbonic acid) 1.H2O binds to Zinc ion (located in deep cleft of Carbonic anhydrase and is coordinated to Histidine residue present at its active site). Binding of CO2 to histidine residue is close to Zinc ion. 2.H2O→ H+ + OH- (Ionization of water to hydroxyl ion) 3.OH- +CO2(located proximally to OH- ions)→HCO- 3 Carbonic anhydrase facilitates the precise positioning of CO2 molecule and hydroxyl ion OH- for the formation of bicarbonate ion HCO- 3. 4.HCO- 3 + H+ → H2CO3(Carbonic acid) Thus , two substrates CO2 and H2O are brought in close proximity for reaction to proceed and to facilitate the function of Carbonic anhydrase .
  6. 6. QuaternarystructureofenzymeCarbonicanhydrase
  7. 7. QuaternarystructureofenzymeCarbonicanhydraseregulatesitsfunctions
  8. 8. StepsofenzymecatalysisbyCarbonicanhydrase 1.H2O binds to Zinc ion (located in a deep cleft of Carbonic anhydrase and is coordinated to Histidine residue present at an active site of enzyme ) 2.H2O→ H+ + OH- (Ionization of water) 3. Binding of CO2 toHistidine residue close to Zinc ion of Carbonic anhydrase 4.OH- + CO2( located proximally to OH- ions)→HCO- 3 5.HCO- 3 + H+ →H2CO3(Carbonic acid)
  9. 9. Structure of Myoglobin ❖StructureofMyoglobin: ❖ Oxygenbindingcytosolicmetalloprotein functionalwithinskeletal&cardiacmuscle. • hasasinglepolypeptidechainwith153aminoacidsanditcontainsonehemegroup(iron containingporphyrinringsooneFe2+ ionwhichimpartsredcolortomyoglobin– chromoprotein).Itisaglobularproteinwithlowmolecularweight(16700Dalton). • 80%ofitspolypeptidechain isfoldedinto8-helices,whicharelabeledas AtoH. -helicalregionsareterminatedbypresenceofProline(5-memberedringofProcannot beaccommodatedinalpha-helix→Proishelixdestabilizingaminoacid).Restofthe polypeptidechainformsturnsandloopsbetweenhelices. • Thehelicescreatehydrophobicoxygen(O2)bindingpocketcontainingtightlybound hemewithanironatom(Fe2+)initscenter.WithinthepocketofMyoglobin,O2 binds directlytotheFe2+ ionoftheporphyrinring. • hasnobetasheets.Thisstructureisunusual fortheglobularproteins.
  10. 10. Myoglobincontentofmuscles Type of muscle Myoglobincontentofmusclesin gm/100gmof muscles Skeletal 2.5 Cardiac 1.4 Smooth 0.3
  11. 11. Structure of Myoglobin Structure of Myoglobin(Mb) :hasasinglepolypeptidechainwith153aminoacidsand containsonehemegroup(ironcontainingporphyrinringsooneFe2+ ion-impartingred colortoMyoglobin).Itis a globularproteinwithmolecularweight(16700Dalton) andhas cytosoliclocation.ThisOxygenbindingmetalloproteinproteinfunctionalwithinskeletal& cardiacmuscles.Onemoleculeof(Mb)containsoneoxygenmolecule. JohnKendrewandMaxPerutz(Noble1962):determinedthestructureofMyoglobinby highresolutionX-crystallography.
  12. 12. Tertiary Structure of Myoglobin(Mb) Myoglobin:PrimarystructuresimilartosinglemonomericunitofHemoglobin withasinglepolypeptidechainhaving 153aminoacids(molecularweight16700Dalton).It haseightalpha–helices(AtoH)andonehemegroup(iron containing porphyrin)tofacilitateitsfunctionofoxygenstorageincardiacandskeletalmusclesinhumanbody,Whales andSeals.
  13. 13. Tertiary Structure of Hemoglobin (Hb) Hemoglobin:Tetramericwith4hemegroups.Eachpolypeptidechainhassimilarstructureto single polypeptidechainofMyoglobin.Ithasaloweraffinity foroxygenthanMyoglobin.FoursubunitsofHb functioncooperatively.TetramericstructureofhemoglobinfacilitatessaturationwithO2inthelungand releaseofoxygenasittravelsthroughthecapillarybed.
  14. 14. Functions of Myoglobin ❖Functions of Myoglobin(Mb): acts as storage and transport protein for oxygen in skeletal&cardiacmuscles. In resting muscle cells ,myoglobin binds oxygen (O2)that has been released by Hemoglobin(Hb) under the conditions of oxygen deprivation (in severe exercise for use O2 by muscle). ❖HbO2 (oxyhemoglobin) → → → Mb(Myoglobin) →MbO2(oxymyoglobin)→ tissue → Respiration Oxygen (O2) transport
  15. 15. Oxygen dissociation curve of Myoglobin and hemoglobin When the amount oxygen (O2)bound to the Hemoglobin and Myoglobin is plotted against the partial pressure of oxygen (pO2), a hyperbolic curve is obtained for Myoglobin whereas that for Hemoglobin is sigmoidal. When the (pO2) is high both Myoglobin and Hemoglobin are saturated with oxygen. At lower levels of (pO2), however , Myoglobin contains more oxygen O2 than Hemoglobin . Bohr’s effect ,co-operative effect and 2,3 BPG effect are absent for Myoglobin . Mb has higher affinity for Oxygen (O2) than Hb. At PO2 in tissue (30mmHg): Mb is 90% saturated, Hb is 50% saturated . Physical exercise: PO2 in tissue is (5mmHg), when Mb releases all bound Oxygen (O2).
  16. 16. Clinical aspects associated with Myoglobin ❖Myoglobin : under physiological conditions , Myoglobin( being a small molecular weight protein)is filtered and excreted in urine. • is a sensitive maker for muscleinjury,makingitpotential cardiacmarkerfor myocardialinfarctioninpatientswithchestpain. • rises rapidly after the chest pain at about the same rate as CK-MB. • is non-specific ,since it is raised following any form of muscle damage. • Estimation ofMyoglobin–usingmonoclonalantibodyforbyRIA/ELIZA/ chemiluminescence • Cost of the analysis has prevented its widespread use. • TemporalpatternofserumMyoglobin&Creatinekinase-2inpatientswithmyocardial infarctionisdepictedinadiagram.
  17. 17. SerumMyoglobin&Creatinekinase-2(CK-MB)inpatientswithmyocardialinfarction • The cellular release Myoglobin is often associated with an increase in Creatine kinase (CPK), Aldolase , Lactate dehydrogenase(LDH) , Serum Glutamate Pyruvate Transaminase(SGPT) in patients with myocardial infarction.
  18. 18. Myoglobinuria ❖Myoglobinuria : under physiological conditions , Myoglobin being low molecular weight protein, it is filtered and excreted in urine. Presence of excessive Myoglobin in the urine usually associated with muscle destruction or rhabdomyolysis . Urine color becomes dark brown. ❖Damage to muscle( crush injury), releases Myoglobin to the circulation and filtered by the kidney. If too much of Myoglobin is released into circulation , Myoglobin precipitate and obstruct the renal filtration system results in acute renal failure. ❖Myoglobin is released from myocardium during Myocardial infarction (MI) and is elevated in serum(cardiac marker)followed by its excretion in urine.
  19. 19. ClinicalconditionassociatedwithMyoglobinuria ❖ClinicalconditionassociatedwithMyoglobinuriainadultinclude: 1. Extremeexercise(mostcommoncauseofrhabdomyolysisinadolescents):excessive physicalactivitycanproduceimbalancebetweenenergyconsumptionand productionresultinginmuscledestruction(duetooxidativestress) 2. Trauma/Vascularproblems 3. Prolongedethanol/Alcoholconsumption 4. Drugabuse 5. ToxicityofVenoms
  20. 20. Myoglobinuria
  21. 21. Rhabdomyolysis
  22. 22. Quaternary structure of Hemoglobin Hemoglobin (HbA1)has 4 Polypeptides chains ( tetramer) associated by non-covalent bonds : 2 Alpha() chains + 2 Beta( )chains It possesses Quaternary structure(oligomeric). Inthis, Rgroupcontactsare presentbetweensimilarside chainsandthereisverylittle contactbetweendissimilar side chains. HbA:Tetramer22 Each chain/subunit has one heme group and so one Fe2+ ion similar to that of myoglobin. Hb is found exclusively in RBC
  23. 23. Structure of normal Hemoglobin Hemoglobin variant Structure of normal Hemoglobin Abbreviation Hemoglobin (HbA 1) 2 Alpha() chains and 2 Beta( )chains 22 Hemoglobin (HbA 2) 2 Alpha() chains and 2 delta( )chains 22 Fetal Hemoglobin (HbF) 2 Alpha() chains and 2 gamma ( )chains 22
  24. 24. Functions of Hemoglobin ❖Functions of Hemoglobin: • Hemoglobin is found exclusively in RBC .It transports four molecules oxygen(O2) from lungs(high pO2) to the tissues (low pO2) . Myoglobin is still very saturated with oxygen at the (pO2) of tissue. It can transport H + and carbon dioxide (CO2) from peripheral tissues to lungs for its excretion in air. oxygenation Deoxyhemoglobin + 4O2 oxyhemoglobin (in lungs) Deoxygenation is the reverse process by which O2 is liberated. deoxygenation Oxyhemoglobin 4O2+ Deoxyhemoglobin • Hemoglobin is rich in amino acid Histidine ,helps to maintain pH of blood (acts as buffer). • Transports oxygen(O2)by Hb is regulated by 2-3-bisphosphate glycerate(2,3BPG).
  25. 25. Functions of Hemoglobin as a transporter of oxygen(O2) from lungs(high pO2) to the tissues (low pO2)
  26. 26. Quaternary structure of Hemoglobin favors its functions ❖ Hemoglobin found exclusively in red cells and functions as transporter of oxygen(O2) from lungs to tissue . Each molecule carry four oxygen(O2)molecules from lungs to the cells .This function is favored by presence of one heme unit one each in monomer (4 heme→4 oxygen(O2)/ hemoglobin). ➢Binding of oxygen(O2) to one heme unit of tetramer facilitates oxygen binding by other subunits. Each subunit has a heme binding pocket similar to that of myoglobin . ➢It can transport H+ and CO2 from the tissue to the lung. ➢Binding of H+ and CO2 promotes release of oxygen(O2) from hemoglobin . ➢This allosteric interaction is physiologically important and as Bohr’s effect . ➢Even a single amino acid substitution alters the structure and thereby functions.
  27. 27. Co-operative binding of oxygen to hemoglobin • Binding of oxygen to Heme will increase binding of oxygen to other heme • Affinity of oxygen for hemoglobin • Last oxygen binds with affinity 100 time greater than first oxygen • Heme –Heme interaction ( co-operative binding of oxygen to Heme ) • Release oxygen from one Heme will release oxygen from other • As there is communication between Heme groups of hemoglobin • Myoglobin is reservoir (transient )& supplier of oxygen Lung Tissue Oxygen concentration high Oxygen concentration low Oxygen binds to hemoglobin Oxygen is released to tissue
  28. 28. Structural changes in hemoglobin on oxygen binding • Studied using x ray crystallographic • Homotropic effect→ binding of oxygen to hemoglobin • Heterotrophic effect → binding of 2,3BPG to hemoglobin • Distance between two Beta-chain decreases oxygenation from 4nm to 2nm • Increasing affinity for oxygen with addition every molecule of oxygen • On oxygenation iron moves in plane of Heme • Decrease in diameter of iron (movement of iron accompanied by pulling of proximal Histidine • affinity of hemoglobin for last oxygen > first oxygen ( 100 times greater ) • Cooperative binding of oxygen to hemoglobin or Heme →Heme interaction • Release of oxygen from one Heme → Release of oxygen from other Heme ❖Therefore communication between Heme groups of hemoglobin.
  29. 29. On oxygenation iron moves in plane of Heme → decrease in diameter of Iron →movement of Fe Is accompanied by pulling of proximal site →primary event of Heme –Heme interaction of Hemoglobin Structural changes in Hemoglobin on its Oxygenation and deoxygenation
  30. 30. Deoxy –Hb Hb O₂ Hb O₄ Hb O₆ Hb O₈ T form ↓ ↓ ↓ ↓ ↓ ↑ R form Oxygenation of hemoglobin Quaternary structure of Hemoglobin favors its functions :Binding of oxygen(O2) to one heme unit facilitates oxygen binding by other subunits.
  31. 31. Structural changes in Hb on oxygen binding • Structural change in one subunit of hemoglobin on oxygenation is communicated to other subunits • Binding of oxygen to one Heme distorts globin chain to which it is attached → distortion in neighboring chain →oxygen binds more easily.
  32. 32. Effect of 2,3 BPG on oxygen affinity of Hb • 2,3 BPG :Most abundant phosphate in RBC • Molar concentration of 2,3 BPG = Molar concentration of Hb • Synthesis ( synthesis through Rapport Leu Bering cycle) 2, 3 BPG mutase ( Glycolysis ) 1,3 BPG 2,3 BPG • Reinhold's & Ruth Benesch’s (1967 )→ 2,3 BPG decreases affinity of Oxygen to Hemoglobin • 2,3 BPG regulates the binding of oxygen • 1mole of 2,3 BPG binds to 1mole of Deoxy Hb not to oxy –Hb • Molecular concentration 2,3 BPG = Molecular concentration of hemoglobin • At partial pressure of oxygen(O₂) in tissue: HbO₂+ 2,3 BPG→Hb 2,3 BPG +O₂ (release of O₂) (Oxy–Hb) (De-oxy Hb) • In tissue → 2,3 BPG shift curve towards right
  33. 33. 1mole of 2,3 BPG binds to 1mole of Deoxy Hb not to oxy –Hb Regulation of oxygen binding by 2,3 BPG
  34. 34. Effect of 2,3 BPG on oxygen affinity of Hb 2,3 BPG decreases affinity of Oxygen to Hemoglobin. In tissue → 2,3 BPG shift curve towards right At partial pressure of O₂ in tissue: HbO₂+ 2,3 BPG→ Hb 2,3 BPG +O₂ (release of O₂) (Oxy –Hb) (De-oxy Hb)
  35. 35. 2,3 BPG shift oxygen dissociation curve of hemoglobin towards right to release of oxygen (O₂) in hypoxic conditions ,anemia ,hyperthermia. Effect of 2,3 BPG shift oxygen dissociation curve of hemoglobin
  36. 36. Clinical significance of 2,3 BPG:1 ❖Function of 2,3BPG : Release of oxygen to tissue ( supply of oxygen to tissue ) to cope with oxygen demand → varied concentration of 2,3 BPG 1. Hypoxia : concentration of 2,3BPG in RBC increases during chronic hypoxic conditions e.g. a. Adaptation to high altitude b. Obstruction to pulmonary odema ( air flow in bronchial blocked ) 2. Anemia : concentration of 2,3 BPG in RBC increases in chronic anemic conditions → to cope with oxygen (O₂) demand of body even at low Hb concentration.
  37. 37. Clinical significance of 2,3 BPG:2 3. Blood Transfusion : storage of blood in acid citrate dextrose → decrease in concentration of 2,3, BPG ( O₂ remains bound to Hb ) • Blood stored in ACD fails to supply O₂ to tissue→ with 24-48 hr. until 2,3 BPG restored • O₂ supply /tissue O₂ demand met adequately after 24-48 hrs. • Blood with (ACD )+ Inosine ( Hypoxanthine Ribose )→ prevent decrease in 2,3, BPG • Inosine→ phosphorylation of tissue→ entry into HMP shunt → get converted to 2,3BPG →increase in Conc in 2,3 BPG → release of oxygen 4.Fetal hemoglobin(HbF) : the binding of 2,3BPG to fetal hemoglobin is very weak. Therefore, HbF has higher affinity for oxygen compared to adult hemoglobin (HbA). This is needed for transfer of oxygen from the maternal blood to fetus.
  38. 38. Protein structure-function relationship in Adult Hemoglobin(HbA1) and Fetal Hemoglobin (HbF) • Function of Fetal Hemoglobin (HbF) : The transfer of oxygen from the mother to the fetus. • This function of Fetal Hemoglobin (HbF) is facilitated by structural differences in between the hemoglobin molecule of the mother and that of fetus (HbF). • Adult Hemoglobin(HbA1) : 2 Alpha() chains and 2 Beta()chains (22) • Fetal Hemoglobin (HbF) : consist of 2 Alpha() chains and 2gamma () chains- 22 • The difference in amino acid composition between the beta chain of HbA1 and gamma ( )chains of (HbF) results in structural changes that causes HbF to have lower affinity for 2,3- biphosphoglycerate (2,3 BPG) than HbA1 and thus greater affinity for oxygen. ➢Therefore , the oxygen released from the mother’s HbA1 is readily bound by HbF in the fetus.
  39. 39. Protein structure-function relationship in Myoglobin and adult Hemoglobin(HbA1):1 Criteria Myoglobin Hemoglobin(HbA1) Primarystructure SimilartosinglemonomericunitofHemoglobin withasinglepolypeptidechainhaving 153 aminoacids(molecularweight16700)andone hemegroup(ironcontaining porphyrin). Tetramericwith4hemegroups.Each polypeptidechainhassimilarstructure to singlepolypeptidechainof Myoglobin. Affinityforoxygenof oxygenbinding protein Hasahigheraffinity foroxygenthan hemoglobin.ThebindingofO2 tohemegroup ofeachmoleculeisindependentofanother moleculebecauseitcontains onlyoneheme group(onepolypeptidechain). Hasaloweraffinity foroxygenthan myoglobin.Foursubunits of Hb functioncooperatively. Function of metalloprotein Astoragereserve foroxygen,releasesoxygen theboundoxygenforcellularusewhenoxygen supplyisreduced.Storeofoxygenindeep- divingmammals. Allowsefficienttransferofoxygenfrom lungtotissue.Tetramericstructureof hemoglobinfacilitatessaturationwith O2inthelungandreleaseofoxygenas ittravelsthroughthecapillarybed.
  40. 40. Structure of Myoglobin and adult Hemoglobin(HbA1) PrimarystructureofMyoglobin:SimilartosinglemonomericunitofHemoglobin withasinglepolypeptide chainhaving 153aminoacids(molecularweight16700)andonehemegroup(ironcontaining porphyrin). PrimarystructureofadultHemoglobin(HbA1):Tetramericwith4hemegroups.Eachpolypeptidechainhas similarstructureto singlepolypeptidechainofMyoglobin.
  41. 41. Functions ofMyoglobin and adult Hemoglobin(HbA1) Functionofmyoglobin:Astoragereserve foroxygen,releasesoxygentheboundoxygenforcellularuse whenoxygensupplyisreduced.Storeofoxygenindeep-divingmammals. Functionofadulthemoglobin(hba1):allowsefficienttransferofoxygenfromlungtotissue.Tetrameric structureofhemoglobinfacilitatessaturationwith O2inthelungandreleaseofoxygenasittravelsthrough
  42. 42. Protein structure-function relationship of Myoglobin and adult Hemoglobin(HbA1):2 Condition Myoglobin Hemoglobin(HbA1) Highpartialpressureof oxygen(pO2) Saturated saturated Lowpartialpressureof oxygen(pO2 ) ContainsmoreO2thanhemoglobin Containslesser O2thanmyoglobin Agraphplottedwith the amountofO2 boundtothe proteinagainstthepartial pressureofoxygen(pO2 ) Hyperboliccurve Sigmoidcurve Application Stillverysaturatedwith oxygenatthe lowpartialpressureofoxygen(pO2) intissue facilitatingstoragefunction intissue. Effectivetransporterof oxygen,binding withoxygeninthe lungswhere(pO2)is highandreleasingitintissuewhere(pO2) islow .
  43. 43. OxygenbindingaffinityofMyoglobinandHemoglobin Hyperboliccurve of oxygenbinding forMyoglobin Sigmoidcurveof oxygenbinding forHemoglobin At Highpartialpressureofoxygen(pO2)both myoglobin and hemoglobinaresaturatedwithoxygen. Application : Myoglobin stillverysaturatedwith oxygenatthelowpartialpressureofoxygen(pO2)in tissue facilitatingstoragefunctionintissue.Hemoglobinis aeffectivetransporterof oxygen,bindingwith oxygeninthe lungswhere(pO2)ishighandreleasingitintissuewhere(pO2)islow .
  44. 44. Transport of oxygen by hemoglobin PO₂ (mm) of Hg % saturation Inspired air 158 Alveolar air 100 lung 90 97% Capillary bed 40 60% 37% - 40% O ₂ release of oxygen at tissue level
  45. 45. Structural modification of hemoglobin by glycosylation and its application • Glycosylated hemoglobin (Hb A1C): is formed by non –enzymatic reaction of the aldehyde group of Glucose with amino terminal (N –terminal residue) Valine of beta chains of HbA when blood Glucose enters the erythrocytes. • Normal concentration of Glycosylated hemoglobin (Hb A1C): low ( to the extent of 5% of total hemoglobin • Formation of (Hb A1C) is proportional to blood glucose concentration. • Glycosylated hemoglobin (Hb A1C) increases in Diabetes Mellitus : 12% or more of total hemoglobin as serum glucose is high • Since RBC have life span of 120 days , the content of Hb A1C is an indicator of how effectively blood glucose levels have been regulated over the previous 2 or 3 months. • Application of Glycosylated hemoglobin (Hb A1C) estimation : to follow the effectiveness of treatment (management)for Diabetes Mellitus .
  46. 46. Glycosylated hemoglobin (Hb A1C) Glycosylated hemoglobin (Hb A1C): is formed by non- enzymatic reaction of HbA with Glucose when blood Glucose enters the erythrocytes. Application of Glycosylated hemoglobin (Hb A1c) estimation : to follow the effectiveness of treatment for Diabetes Mellitus
  47. 47. Primary structure of Normal protein determines biological functions Unique amino acid sequence specified by genes in a Normal protein Specific amino acid sequence→ confers specific 3 dimensional structure (conformation ) Specific Function arises from conformation Normal physiologically active protein e.g. Normal hemoglobin
  48. 48. Consequences of Altered primary Structure in Abnormal protein Mutation→ altered genetic constitution ( base sequence of DNA) Altered amino acid sequence→ Altered 3 dimensional structure(conformation) Altered or loss of functions arises from Altered conformation of protein Abnormalorphysiologicallyinactiveprotein→diseaseconditione.g.hemoglobinopathy
  49. 49. Structure function relationship of proteins Normal protein • Unique amino acid sequence specified by genes • Specific amino acid sequence→ confers specific 3 dimensional structure ( conformation ) • Specific Function arises from conformation • Normal physiologically active protein e.g. Normal hemoglobin Abnormal protein • Mutation→ altered genetic constitution / base sequence of DNA • Altered amino acid sequence →Altered 3 dimensional structure ( conformation ) • Altered or loss of Function arises from Altered conformation • e.g. abnormal or physiologically inactive protein→ disease condition e.g. hemoglobinopathy
  50. 50. Altered primary Structure of Abnormal hemoglobin Abnormalhemoglobin→ hemoglobinopathy Altered primary Structure leading anemia in early life Sickle cell hemoglobin ( Hb S) no change in amino acid sequence of  chain 6 th amino acid in beta chain of HbA1 (Glutamic acid replaced by Valine in HbS ) HbM (Methemoglobinemia ) Substitution of Tyrosine with Histidine Hb Chesapeake Arginine is replaced by Leucine at 92th amino acid in alpha chain of Hb A Alpha(  ) thalassemia ( normally the rate of synthesis  and  chains identical ) Deficiency or absence of alpha chains → HbA of fetus has tetramer  4 or 4 chains ( enlargement of live and spleen) Beta ( )thalassemia ( more common inherited disease than Alpha thalassemia ) Deficiency or absence of beta chains →hemoglobin found in RBC are HbA2 (  2 2) and HbF (  2 2 )
  51. 51. Deoxygenated HbS • This substitution generates ‘A stick patch’ on the surface of the beta chain of both oxygenated and deoxygenated HbS . • On the surface of deoxygenated normal HbA and deoxygenated HbS , there exists a complement to the sticky patch. • When HbS is deoxygenated ,its sticky patches can bind to the complementary patches on another deoxygenated HbS . • Binding of large number of deoxygenated HbS causes polymerization of deoxygenated HbS forming long fibrous precipitate that mechanically disturbs the red cell→ sickle shaped→ casing lysis and anemia. • This polymerization will not take place when HbS is in oxygenated form as in arterial blood .
  52. 52. Molecular basis of Sickle cell anemia Linus Pauling ( 1954 Noble prize ) reported abnormal electrophoretic mobility & peptide mapping Glutamic acid ( sixth position on beta globin chain ) replaced by Valine (Recessive Mutation ) Hb A & Hb F prevent sickling.
  53. 53. sixth amino acid in beta() chain of HbA1 (Glutamic acid )replaced by Valine in HbS Altered primary Structure leading anemia in early life
  54. 54. Sickle cell disease Binding of large number of deoxygenated HbS causes polymerization of deoxygenated HbS forming long fibrous precipitate that mechanically disturbs the red cell→ sickle shaped→ casing lysis and anemia.
  55. 55. HbM (Methemoglobinemia ) ❖HbM (Methemoglobinemia) : Substitution of Tyrosine with Histidine on hemoglobin result in oxidation of ferrous is to ferric in heme . Hemoglobin in blood is oxidized to methemoglobin. This condition leads to Methemoglobinemia .
  56. 56. HbM (Methemoglobinemia) :Substitution of Tyrosine with Histidine  Substitution of Tyrosine with Histidine oxidation of ferrous is to ferric in heme→
  57. 57. Hb Chesapeake ❖Hb Chesapeake ➢Substitution of Arginine by Leucine at 92th amino acid in alpha chain of Hb A results in increased affinity of hemoglobin for oxygen and does not release as much oxygen to the peripheral tissue as does normal hemoglobin HbA . ➢This leads to tissue hypoxia and polycythemia ( increased number of RBC per unit volume) in ode to meet the oxygen needs.
  58. 58. Quaternary structure of Aspartate trans carbamylase Aspartate trans carbamylase : An allosteric enzyme with 2 subunits : catalytic subunit (C) and the regulatory subunit (R)
  59. 59. Quaternary structure of Lactate dehydrogenase (LDH ) ❖Quaternary structure of Lactate dehydrogenase (LDH ): • Tetramer • two types of polypeptide chains :H and M type • five isoenzymes : 1. LDH 1 →H4 2. LDH2 → H3M 3. LDH3→ H2M2 4. LDH 4→ HM3 5. LDH 5 →M4
  60. 60. Isoenzymes of Lactate dehydrogenase Subunit composition of LDH1 isoenzymes in heart cells favors conversion of Lactate to Pyruvate Subunit composition of LDH5 in muscle cells favors conversion of Pyruvate to Lactate Quaternary structure of Lactate dehydrogenase (LDH ): Tetramer with 2 types of polypeptide chains →H and M type
  61. 61. Structure of Human Insulin In 1953 , Frederick Sanger determined primary structure of Insulin ( a pancreatic protein hormone ) and showed for the first time that a protein has a precisely defined amino acid sequence ( primary structure.)
  62. 62. Insulin and Glucagon(Polypeptide Hormones) Insulin Hormone secreted by  pancreatic cells Polypeptide and a Dimer with 51 amino acids regulates glucose metabolism and Induces hypoglycemia Amino acid sequence varies in different mammalian species Glucagon Hormone secreted by  pancreatic cells Polypeptide and monomer with 29 amino acids regulates glucose metabolism and Induces hyperglycemia Amino acid sequence is same in all mammalian species
  63. 63. Structure of Human Insulin ❖Structure of Human Insulin : described by Sanger(Noble-1955 ) ❖Dipeptide (2 polypeptide chains)of insulin has 51 amino acids A polypeptide chain : 21 amino acids B polypeptide chain : 30 amino acids ❖ Dipeptideofinsulinarerequiredforbiologicalactivityandheldby ➢inter chain disulphide bonds: a. between cysteine residues ( 7th amino acid of A chain and 7th amino acid of B chain ) b. between cysteine residues (20 th amino acid of A chain and 19 th amino acid of B chain) ➢intra chain disulphide bond : between cysteine residues (6 th amino acid of A chain with 11 th amino acid of B chain)
  64. 64. Structure of Human Insulin Carboxy terminal end A chain : Asparagine B chain : Threonine Amino terminal end A chain : Glycine B chain : Phenylalanine A polypeptide chain : 21 amino acids B polypeptide chain : 30 amino acids intra chain disulphide bond : between cysteine residues( 6 th amino acid of A chain with 11 th amino acid of B chain) inter chain disulphide bonds   
  65. 65. Primary Structure of human Insulin S S A chain H2N Gly Cys Cys Cys Cys Asn 1 6 7 11 20 21 S S S S B chain H2N Phe Cys Cys Thr 1 7 19 30 Pig insulin differs from human insulin in only one position ,30th amino acid is alanine instead of Threonine. Insulin from other animals like cattle ,sheep, horse etc. differ from human insulin in having a different sequence of amino acids in the positions 8-9-10 in A chain . This minor altered sequence does not result inappreciable change biological activity.
  66. 66. Amino acid substitution in Primary Structure of human Insulin 8 9 10 Achainof insulin ofspecies Thr Ser Ile Human Ala Ser Val Bovine Thr Ser Ile Pig Ala Gly Val Sheep Thr Gly Ile Horse Aminoacidcompositionat30th amino acidinBchainof insulinofspecies Thr Human Ala Bovine Ala Pig Porcineandhumaninsulinare similar(homologous)exceptC-terminal aminoacidinBchain(Thr→Ala).Itmay produceantibodiesinhumanafter repeated injections.Dealaninatedporcineinsulinwill notproduceanyantibodiesindiabetic patientsevenafterlongtermuse. Aminoacidsequencehasbeenconservedto thegreatextentduringevolution.Human insulinrequiredforreplacementtherapy,is nowsynthesizedbyrecombinantDNA technology.
  67. 67. Site of Insulin Biosynthesis ❖Amino acids form primary structure → definite function ❖Peptide < 10 amino acids ❖Polypeptide > 10 amino acids Preproinsulin (singlepolypeptidechainwith108aminoacids,molwt.11500) ↓ Proinsulin(single polypeptide chain with 86 amino acids, molwt.9000) ↓ Human Insulin ( dipeptide 51 amino acids : polypeptide chain A →21 amino acid and polypeptide chain B →30 amino acids held together b interchain disulfide bridges molwt.5734 ) ➢The gene for insulin synthesis : located on chromosome 11 in beta cells of pancreatic cells. Removal of signal sequence in endoplasmic reticulum Removal of C-peptide in Golgi apparatus
  68. 68. Pre proinsulin ( single polypeptide chain ): 108 amino acids ,mol wt. 11500 Proinsulin ( single polypeptide chain):86 amino acids ,mol wt. 9000 Insulin ( dipeptide chain ):51 amino acids ,mol wt. 5734 Biosynthesis of Insulin from Preproinsulin:1 Removal of signal sequence in endoplasmic reticulum Removal of C- peptide in Golgi apparatus The gene for insulin synthesis : located on chromosome 11 in beta cells of pancreatic cells.
  69. 69. Biosynthesis of Insulin from Preproinsulin:2 In the beta pancreatic cells , insulin and proinsulin combines with Zinc to form complexes. In this form it is stored in the granules of the cytosol which is released in response to various stimuli by exocytosis. Pre proinsulin ( single polypeptide chain ) :108 amino acids ,mol wt. 11500 Proinsulin ( single polypeptide chain):86 amino acids ,mol wt. 9000 Insulin (dipeptide chain):51 amino acids, mol wt. 5734
  70. 70. Structure function relationship of C–peptide and insulin C – peptide has no biological activity . Single polypeptide chain with 86 amino acids  Beta cells of panaceas synthesize insulin as a prohormone – Proinsulin . Biologically active insulin (a dipeptide) is formed by removal of C – peptide ( the central potion ) of proinsulin . C–peptide and insulin are synthesized in equimolar concentration. It useful index for the endogenous production of insulin.
  71. 71. BiochemicalandclinicalaspectsofCollagen Epidermolysis bullosa Scurvy Ehlers-Danlos syndrome
  72. 72. Biochemical aspects of Collagen ❖Biochemical aspects of Collagen: 1. Most abundant fibrous protein(major macromolecules) in human body :70 kg body weight→ 12-14 kg of total protein→5kg of Collagen (1/3 of total protein) 2. Main Component of : connective tissue, skin(70%) ,bone (90%) tendon(85%), cartilage ,teeth and liver(4%) 3. Synthesized by fibroblast in connective tissue and osteoblasts in bone 4. Made up of small fibrils → tropocollagen( fundamental units ) containing 3 polypeptide chains each of them in left-handed helix with 3 amino acid per turn. 5. rich in Glycine and rare amino acids like hydroxyproline, hydroxylysine 6. Cysteine and Tryptophan absent 7. have a triple helical secondary structure and rich in helix destabilizing amino acids (Glycine ,Proline and Hydroxyproline). These amino acids prevent the formation of the usual - helical and - pleated structure. Instead it forms a triple helical secondary structure.
  73. 73. Triple stranded helix Structure of Collagen Made up of small fibrils → Tropocollagen( fundamental units ) containing 3 polypeptide chains each of them in left-handed helix with 3 amino acid per turn.
  74. 74. Triple stranded helix Structure of Collagen • Collagenhas3 polypeptidechainswoundarounditself. Eachpolypeptidechainsubunitis calledalpha-chains. EachofAlpha-chain is twistedintoleft-handedhelix ofthreeresidues perturncomparedwith 3.6forright-handedalpha-helix.Threeoftheseleft–handedhelices arethenwoundtoright-handedsuperhelixtoformastiffrodlike molecule(Triplehelical secondarystructure). • Itisrich in helixdestabilizingaminoacids(Glycine ,ProlineandHydroxyproline).Theseamino acids preventtheformationoftheusual- helicaland-pleatedstructure.Instead,it formsa triplehelicalsecondarystructure. • Every3rdresidueis Glycineandtheonlyaminoacidthatcanfitintothetriplestrandedhelix. • QuarterstaggeredtriplestrandedhelixofCollagenis stabilizedbythestericrepulsionof ringshydroxyproline andhydrogenbondsbetweenthem. • Triplehelicalsecondarystructureimpartsthetensilestrengthofsteeltocollagen(has unusualstrength) ❖Types arrangementofcollagenfibril: a. Parallelbundles:in tendons,cartilage b. Sheets:layered atmanyanglesin skin
  75. 75. Arrangements of collagen fibers in cartilage of bone
  76. 76. Types of Collagen ❖19 different Types of Collagen , composed of 30 distinct polypeptide chains encoded by separate genes. ❖ Numbering for Types of Collagen: Roman numerals I, II, III….XIX ❖Structure of collagen types : in principle , all types of collagen are triple helical structures . The triple helix may occur throughout the molecule or only a part of the molecule. ❖Each one suited to performed specialized function in tissue ❖e.g. Collagen Type I →skin, Collagen Type II → bone
  77. 77. Most abundant types of collagen found in human tissue and their distribution Type of collagen Distribution Composition of triple helix I Skin , bone, tendon , cornea 2 -1(I), -2(I) II Articular cartilage, intervertebral disc, vitreous body 3-1(II) III Fetal skin ,cardiovascular system, reticular fibers 3-1(III) IV Basement membrane 2 -1(IV), -2(IV) V Placenta, Skin 2 -1(V), -2(V)
  78. 78. Structure of collagen Type 1 ❖ Structure of collagen Type 1: 1. Triple stranded helical structure present throughout the collagen molecule 2. Shape : rod-like molecule → 1.4 nm diameter and 300 nm length 3. Number of Amino acid residues : 1000 per for each polypeptide chain (3000 /molecule) 4. Amino acid contribution : 1/3 rd of amino acids are Glycine (every third amino acid in collagen is Glycine. 5. Repetitive amino acid sequence : (Gly – X –Y )n ,where X and Y represent other amino acids 6. Proline and hydroxyproline : 100 per for each polypeptide chain 7. Function of Proline and hydroxyproline : confer rigidity to the collagen molecule 8. Collagen Fibril formation : Triple helical molecule of collagen assemble to form elongated fibrils . It occurs by a quarter staggered alignment i.e. each triple helix is displaced longitudinally from its neighbor collagen molecule by about one-quarter of its length 9. Collagen Fiber formation : Collagen Fibrils assemble to form rod like fibers . 10. Strength of Collagen Fiber : contributed by covalent cross linking of formed between Lysine and hydroxylysine and also between Proline and hydroxyproline.
  79. 79. Collagen molecules in Collagen fibers Triple helical molecule of collagen assemble to form elongated fibrils . Triple stranded helical structure present throughout the collagen molecule Collagen Fibrils assemble to form rod like fibers . Repetitive amino acid sequence (Gly – X –Y )n Proline and hydroxyproline confer rigidity to the collagen molecule
  80. 80. Arrangement of Tropocollagen molecules in collagen fibril Heads of Tropocollagen molecules 64 nm Cross striations Sections of Tropocollagen moleculeCollagen Fibril formation : Triple helical molecule of collagen assemble to form elongated fibrils . It occurs by a quarter staggered alignment i.e. each triple helix is displaced longitudinally from its neighbor collagen molecule by about one-quarter of its length.
  81. 81. Tropocollagen molecule ❖Tropocollagen : Subunits of Collagen • Shape : rod shaped • Length : 300nm • Thickness : 1.4 nm • Molecular weight : 300,000 • Constituent polypeptides: three helically interwind polypeptides of equal length (each with 1000 amino acid residues) • Primary structure of collagen : all 3 or two out of three chains have identical in amino acid sequence. Rich in Glycine (35%)and Alanine(11%) , Gly-Pro-X or Gly-Hpr-X or • Repetitive amino acid sequence : (Gly – X –Y )n ,where X and Y represent other amino acids • Secondary structure of collagen : Each of three polypeptide chains of tropocollagen is itself -helix. Proline and hydroxyproline form bends in polypeptide chains that they are not compatible with -helix structure.
  82. 82. Collagen fibrils ❖Collagen fibril : • Triple helical molecules are associated into Collagen fibrils. • It consists of recurring polypeptide subunits called tropocollagen, arranged head to tail in parallel bundles . The heads of the tropocollagen molecules are staggered along the length of fibers ,accounting for the characteristic 64 nm spacing of the cross striations in most collagens . • A section of tropocollagen molecule shows the backbone of triple helix . Each of three polypeptide chains of tropocollagen is itself -helix whose pitch and spacing is determined by the rigid R group of the numerous Proline and hydroxyproline residue . • The gap between the end of one triple helix and the beginning of the next where there is the deposition of hydroxyapatite crystals in bone formation.
  83. 83. Constituent amino acids of triple stranded helix Structure of Collagen • ScvConstituent amino acids of Collagen % of total amino acids Glycine 33 Proline and hydroxy proline 21 Lysine and hydroxy Lysine 3 Alanine 11 Arginine 5 Cysteine and Tryptophan absent Scurvy:vitaminCdeficiency→failure ofhydroxylationofProlineandLysineleadstoreducedhydrogen bonding→weaknessofcollagen→Brittlebonedisease:mutation→replacementofcentralGlycine
  84. 84. Triple helical secondary structure of Collagen
  85. 85. Forces stabilizing Triple helical secondary structure of Collagen ❖Forces stabilizing Triple helical secondary structure of Collagen: 1. Hydrogen bonds : three left-handed helices are bound together by interchain hydrogen bonds. 2. Lysinonorleucine bond: covalent cross links both within and between triple helical units further stabilize Collagen fibers. 3. Electrostatic interactions 4. Hydrophobic interactions
  86. 86. Covalent cross-links in Collagen fibers • Strength of Collagen Fiber : contributed by covalent cross linking formed between Lysine and hydroxylysine and also between Proline and hydroxyproline. • Covalent cross links are formed both within and between triple helical units further stabilize Collagen fibers. • The degree of covalent cross-linking in Collagen molecule increases with age . • In Elder individuals : skin, blood vessels (Collagen containing tissue) become less elastic and more stiff → health complications
  87. 87. Skin :Collagen containing tissue In Elder individuals , skin, blood vessels (Collagen containing tissue) become less elastic and more stiff → health complications
  88. 88. Collagen and calcific aortic valve stenosis(CAVS)
  89. 89. Biosynthesis of collagen ❖Biosynthesis of collagen: collagen is an extracellular protein but synthesized as an intracellular precursor molecule before becoming a mature collagen fibril. • Site : fibroblast ,osteoblasts in bones , chondroblasts in cartilage, odontoblasts in teeth • Cellular location : ribosomes in endoplasmic reticulum (ER) • Precursor : preprocollagen (a single polypeptide chain) with leader peptide at amino terminal 20000 MW and carboxy terminal 35000MW.Both are not present in mature collagen. • Function of preprocollagen: contains a signal peptide which directs the protein to each endoplasmic reticulum (ER) • Synthesis of procollagen : from preprocollagen in (ER) after cleavage of a signal peptide • Post transcriptional modification of procollagen : hydroxylation, glycosylation and disulfide formation . Followed by its secretion in extracellular medium by the way of Golgi complex . • Synthesis of collagen in extracellular medium : from preprocollagen after action of aminopeptidase and carboxypeptidase to remove terminal amino acids. This followed by a spontaneous assembly of polypeptide chains to form triple helical structure (with 1000 amino acids each) of collagen .
  90. 90. Types of cross links in collagen Lysine Lysil oxidase Allolysine Allysine Lysine H2O H2O Aldol condensation Schiff base Reduction Lysinonoleucine
  91. 91. Synthesis of Collagen
  92. 92. Structural modification of Collagen during its Synthesis Procollagen Tropocollagen Collagen Glycosylationloss of peptide potion from N-terminal and C-terminal Each of the 3 chains is in a left handed helix with 3 amino acids per turn. 3 Chains are further twisted in right handed way to give cable like structure. Hydroxylation of Proline and Lysine by Lysyl hydroxylase and Proline hydroxylase in presence of vitamin C→ Cross linking of hydroxy proline and hydroxy lysine Since vitamin C is required for collagen synthesis ,a connective tissue , there is a delay in wound healing process in vitamin C deficiency.
  93. 93. Intracellular and extracellular alterations of Collagen during post-translational processing Intracellular alterations of Collagen Extracellular alterations of Collagen Hydroxylation of Proline and some Lysine residues Formation of intra and interchain crosslinks Glycosylation of some of the hydroxylysine residue Oxidative deamination of epsilon amino groups of Lysine and hydroxylysine residues Formation of intrachain and interchain disulphide bonds ,mainly in the carboxy and amino terminal ends Cleavage of 25-35 kD portions at both carboxy and amino terminal ends Formation of triple helix Formation of quarter staggered alignment
  94. 94. Functions of Collagen ❖Functions of Collagen : triple helical molecules are associated into fibrils. There is gap between the end of one triple helix and the beginning of the next where there is deposition of hydroxyapatite crystals in bone formation. 1. Gives tensile strength, support and shape to tissue . To break a collagen fiber of 1 mm in diameter, a load of 10-40 kg is needed. In disease status tensile strength is reduced. 2. Contributes to proper alignment of cells ,which in turn help in cell proliferation and their differentiation to different tissue and organs . 3. Collagen which is exposed in blood vessels contributes to thrombus formation. ❖Collagen can be converted to a. gelatin by boiling by splitting off some amino acids .Gelatin is highly soluble and easily digestible. It forms gel on cooling and is provided as diet for convalescents and invalids. But it lacks essential amino acid Tryptophan. b. a tough hard substance on treatment with tannic acid (tannic process)
  95. 95. Genetic aspects of Collagen Synthesis ❖Genetic aspects of Collagen Synthesis : 1. Complex process 2. Involves at least 30 genes in human 3. about 8 post –transcriptional modifications 4. Inherited diseases due to gene mutations linked with collagen synthesis: a. Ehlers-Danlos syndrome b. Alport syndrome c. Osteogenesis imperfecta d. Epidermolysis bullosa
  96. 96. Abnormalities associated with collagen synthesis Disease Abnormalities associated with collagen synthesis Ehlers-Danlos syndrome Inherited disorders characterized by hyperextensibility of skin and abnormal tissue fragility , hypermobile and lax joints Alport syndrome Defect in formation of type IV collagen fibers found in the basement membrane of renal glomeruli→ hematuria and renal disease Osteogenesis imperfecta Characterized by abnormal bone fragility due to deceased synthesis of collagen Epidermolysis bullosa due to alteration in in the structure of type VII collagen fibers→ skin breaks and blister formation even with minor trauma Scurvy Deficiency of vitamin C→ defective post translational modification of collagen→ bleeding gums ,poor wound healing, subcutaneous hemorrhage Lathyrism (disease of bone deformities ) CausedbyconsumptionofLathyrussativa(kesaridal)containingtoxiccompound BetaOxalylAminoAlanine(BOAA).BOAAinhibitsenzymeLysyloxidaseand interfereswiththecrosslinkingoflysineaminoacidresiduesincollagen.
  97. 97. Types of Ehlers-Danlos syndrome ❖Types of Ehlers Danlos syndrome : • Ehlers-Danlos syndrome type V: inherited deficiency of Lysyl oxidase(copper requiring enzymes)→prevents cross-linking of collagen→ arterio-vascular and skeletal changes. • Ehlers-Danlos syndrome type VI: inherited deficiency of Lysyl hydroxylase →abnormalities of the eye ,severe scoliosis (abnormal vertebral curvature) and hyperextensibility of skin and joints. • Ehlers-Danlos syndrome type VII: non-serving of procollagen as a substrate for the procollagen amino protease →hip dislocation , increased skin elasticity and short stature.
  98. 98. Ehlers-Danlos syndrome : Clinical manifestations Hyperextensibility of skin and joints severescoliosis (abnormalvertebralcurvature) Ehlers-Danlos syndrome
  99. 99. Alport syndrome Alport syndrome :Defectinformationof typeIVcollagenfibersfoundinthebasementmembraneof renalglomeruli→ hematuriaandrenaldisease
  100. 100. Alport syndrome :Clinical manifestations Visual abnormality Deafness Glomerular Nephritis
  101. 101. Epidermolysis bullosa Epidermolysisbullosa:duetoalterationininthestructureoftypeVIIcollagenfibers→skinbreaksandblister formationevenwithminortrauma Epidermolysis bullosa
  102. 102. Osteogenesis imperfecta : Clinical manifestations Osteogenesisimperfecta:Characterizedby abnormalbonefragilityduetodeceasedsynthesisofcollagen
  103. 103. Marfan's syndrome ❖An autosomal dominant trait. ❖Molecular basis : defect in the gene coding for fibrillin -1 located on chromosome 15 → deficient of deposition of fibrillin -1 and elastin which are components of microfibrils or defect in the gene coding for fibrillin -2 located on chromosome 5 → deficient of deposition of fibrillin -2→congenital contractual Arachnodactyly. ❖Clinical manifestations of Marfan's syndrome : a. Arachnodactyly (long digits) b. Ectopia lentis(dislocation of lenses) c. Hyperextension of joints d. Aortic aneurism
  104. 104. Molecular basis of Marfan's syndrome Molecular basis : defect in the gene coding for fibrillin -1 located on chromosome 15 → deficient of deposition of fibrillin -1 and elastin which are components of microfibrils or defect in the gene coding for fibrillin -2 located on chromosome 5 → deficient of deposition of fibrillin -2→congenital contractual Arachnodactyly. An autosomal dominant trait
  105. 105. Clinical manifestations of Marfan's syndrome
  106. 106. RoleofLysyloxidasecrosslinkingoflysineaminoacidresiduesincollagen
  107. 107. Hyperhomocysteinemia Accumulation of Homocysteine Reaction of Homocysteine with Lysyl aldehyde formed by Lysyl oxidase Prevention of cross- linking of Lysine residues in connective tissue Skeletal deformities ,vascular and ocular defects
  108. 108. Hyperhomocysteinemia ❖Normal Homocysteine levels (blood): 5-15 micromoles/L ❖Hyperhomocysteinemia : homocysteine levels (blood) increased 50-100 times→ increased risk of coronary artery diseases ,urinary excretion of homocysteine increases( >300 mg/24 hr.). ❖Causes of Hyperhomocysteinemia: a. Vitamin B6 and/or B12 deficiency b. Hypothyroidism c. Tobacco smokers d. Alcoholics →chronic pancreatitis e. Congenital diseases f. Pre –eclampsia of Pregnancy g. Elderly persons
  109. 109. Congenital Hyperhomocysteinemia ❖Congenital Hyperhomocysteinemia : due Cystathionine beta-synthase deficiency ❖Clinical Signs and symptoms of Hyperhomocysteinemia : a. Mental retardation b. Charley Chaplin gait c. Skeletal deformities d. ocular defects: glaucoma myopia , Ectopia lentis(dislocation/ subluxation of lenses) e. vascular defects: intravascular thrombosis ❖Molecular basis /changes : increased Homocysteine→ activation of Hageman’s factor→ increased platelet adhesiveness → intravascular thrombosis→ life threatening ❖Biochemical changes : increased serum Homocysteine and Methionine levels, increased urinary excretion of Homocysteine( > 300mg/24 hr.), reduced plasma cysteine levels
  110. 110. Clinical manifestations of Congenital Hyperhomocysteinemia
  111. 111. Management of Hyperhomocysteinemia ❖Management of Hyperhomocysteinemia : a. Dietary supplementation of Vitamin B6( 500mg /per day) and/or B12 b. Diet Low in Methionine and rich in Cysteine supplemented ❖Cyanide – nitroprusside Biochemical test for diagnosis (in urine): positive ➢Other diseases associated with Hyperhomocysteinemia: neurological disorders
  112. 112. Protein structure-function relationship of Menke’s kinky hair syndrome ❖Menke’s kinky hair syndrome : • An x-linked defect (affects only male child). • Molecular basis :absence of an intracellular copper binding ATPase protein(mutation in ATP7 A gene)→dietary copper absorbed from GI tract; but cannot be transported to the blood . • Copper that has entered into intestinal cells is not able to get out of the cell and so it gets accumulated there . Therefore , Copper(a constituent of Lysyl oxidase)is not available for metabolism ,resulting in defective cross linking in collagen molecule of connective tissue. • Defective Vascular( weakening of walls of major blood vessels including aorta →aneurysm→ fatal rupture of aorta→ cardiac failure) and connective tissues. • Child dies in infancy. ➢Copper binding ATPase protein present in intestinal cells are different from that present in liver and extrahepatic tissues . Therefore , Clinical manifestations of Wilson’s disease and Menke’s disease are different .
  113. 113. Menke’s disease Absence of an intracellular copper binding ATP ase protein( mutation in ATP7 A gene) Accumulation of Copper in intestinal cells (Copper that has entered into is not able to get out of the cell and so it accumulated gets there) Unavailability of Copper for metabolism and function of Lysyl oxidase Defective cross-linking in collagen molecule of connective tissue Defective formation of Vascular and connective tissues Death of the Child in infancy
  114. 114. Menke’s kinky hair syndrome
  115. 115. Scurvy Scurvy: Deficiency of vitamin C→ defective post translational modification( hydroxylation )of collagen → fragility of blood vessels → bleeding gums ,poor wound healing, subcutaneous hemorrhage Scurvy
  116. 116. Lathyrism Lathyrism :CausedbyconsumptionofLathyrussativa(kesaridal)containingtoxiccompoundBetaOxalyl AminoAlanine(BOAA).BOAAinhibitsenzymeLysyloxidaseandinterfereswiththecrosslinkingoflysine aminoacidresiduesincollagen. Lathyrussativa
  117. 117. Lathyrism
  118. 118. Degradation of collagen by Collagenase Collagen peptides Amino acids Collagenase ❖ Clinical applications of degradation of Collagen by collagenase: • Reabsorption of bone and cartilage • Osteoporosis • Postpartum involution of uterus • Rickets • Paget’s disease • Osteoarthritis • Rheumatoid arthritis • Scurvy • Gas gangrene • Tumor metastasis ➢ Adult human tissue do not have significant amount of collagenase activity. ➢ Tissue collagenase is active in animals whose tissue undergo a degree of remolding e.g. tadpoles Peptidase ❖ Gas gangrene: • Collagenase produced by Clostridium histolyticum splits each polypeptide chain at the site indicated ( X-Gly-Pro-Y) • Connective tissue barriers destroyed by bacterium →invasiveness ✓ Collagen: a protein resistant to action of by ordinary proteolytic enzymes.
  119. 119. Clinical applications of degradation of Collagen structure by Collagenase Gas gangrene : degradation of collagen structure by collagenase by bacterium Clostridium Histolyticum Osteoarthritis and Rheumatoid Arthritis
  120. 120. Connective tissue proteins Connective tissue proteins Foundinlarge quantities Functionsof proteins Abnormities associatedwithprotein Elastin Lungs,elasticligaments, arterialbloodvessels Extensibilityand elasticityoftissue Williams'ssyndrome:impairmentofelastinsynthesis duetogenemutation→defectiveconnectivetissue andcentralnervoussystem,pulmonaryemphysema Fibrillin Myofibrilsfoundinvarious tissue extensibilityofmuscles Marfansyndrome:impairmentoffibrillin synthesisduetogenemutation→hyperextensibilityof jointsandskeletalsystem→longdigitsandtallness, Cardiovascularcomplications(e.g.AbrahamLincoln) Fibronectin Connectivetissue InvolvedinInteraction ofcellswithextracellular matrix,celladhesion ,cellmigration Tumorcellmetastasis: impairmentoffibronectin synthesisduetogenemutation→lackofcelladhesion amongtumorcells→cellmigration→metastasis Laminin Basallaminaofglomerular membraneofrenalcells, Extracellularprotein Involvedinneuronal growthandnerve degeneration Alzheimer'sdisease→excessivefibronectinsynthesis duetogenemutation→highconcentrationofLaminin
  121. 121. Elastin ❖Elastin: 1. A Connective tissue protein imparting high tensile strength 2. Occurrence : the major component in yellow elastic fibers of connective tissue →lungs, elastic ligament , arterial blood vessels( especially large vessels like aorta ,tendons 3. Formed in large amount in uterus during pregnancy 4. Are hydrolyzed by pancreatic elastase enzyme
  122. 122. Amino acid composition of Elastin ❖Tropoelastin(the basis subunit of elastin fibrils): • contains about 800 amino acid residues • Rich in non-polar amino acids such as Alanine, Leucine, Valine , Isoleucine and Proline . • Contain high amounts of Glycine, Proline(like collagen) • One-third the residues are Glycine but No repeat sequence of (Gly-X-Y)n (unlike collagen) • Less hydroxyproline • Do not contain Cysteine, Methionine , Histidine , 5-hydroxylysine, glycosylated hydroxylysine. • No triple helix
  123. 123. Biosynthesis of Elastin Biosynthesis : is synthesized as Tropoelastin Tropoelastin Post-translational modifications formation of hydroxyproline Elastin ➢Collagen has aldol cross links, while elastin has Desmosine cross links. ➢When elastin matures , Desmosine cross links are formed from lysine residues. ➢Once elastin matures , elastin is very stable ,turn overrate is very low due to different crosslinks .
  124. 124. Desmosine: Cross links of Elastin ❖Cross links of elastin: • More complex than those in collagen. • the major cross links in elastin are Desmosine. • are formed from 4 Lysine residues. Some Lysine residue of Tropoelastin get oxidized by lysine oxidase( copper containing enzyme) to aldehyde derivative of lysine called Allysine. 3Allysine + unmodified Lysine →lysinonoleucine cross links of Desmosine (by condensation) • permit the elastin to stretch in two dimensions and subsequently recoil during the performance of its physiologic functions. • are destroyed by elastase . ✓Deficiency of alpha-trypsin (an inhibitor elastase)can result in Emphysema.
  125. 125. Cross links of elastin
  126. 126. Emphysema: a clinical condition related to loss of Elastin function Deficiency of alpha-trypsin (an inhibitor elastase)can result in emphysema.
  127. 127. Comparison of primary structure of Collagen and Elastin Collagen Elastin Many different genetic type One genetic type It has no capacity to stretch It has capacity to stretch and subsequently to recoil Primarystructurehasrepeating(Gly-X-Y)sequences Primarystructurehasnorepeating(Gly-X-Y)sequences Formation of triple helix secondary structure No triple helix secondary structure Presence of Hydroxylysine absence of Hydroxylysine Presence of Glycosylated hydroxylysine absence of Glycosylated hydroxylysine Formation of intramolecular aldol cross links Formation of intramolecular Desmosine cross links
  128. 128. Abnormalities associated with elastin biosynthesis Disease Molecularbasis ClinicalManifestation William-Beurensyndrome Deletionofgeneforelastinonchromosome7 Severedevelopmentalabnormalitiesin connectivetissuealloverthebody. Defectiveconnectivetissueandcentral nervoussystem,pulmonary emphysema Pseudoxanthomaelasticum Inheriteddefectinformationofelastin Inheriteddisorderscharacterizedby hyperextensibilityofskinandabnormal tissuefragility,hypermobileandlax joints(similartoEhlers-Danlos syndrome) Copperdeficiency Blockstheformationofaldehydeswhichare essentialforcross-linking.Somelysineresidues areoxidizedbycoppercontainingLysyloxidase andresultingaldehydederivativewhichcondense withunmodifiedlysinetoformLysinonorleucine. Reducedcrosslinkageofcollagen
  129. 129. Human fibrinogen ❖Human fibrinogen ( factor I): 1. Soluble glycoprotein 2. 2-3 % plasma protein(plasma fibrinogen concentration: 0.3g/dl) 3. Consist of 6 polypeptide chains → two A  ,two B , two  4. Structural formula= (A )2 (B )2  2
  130. 130. Structure –function relationship of fibrinogen Fibrinogen Fibrin monomer formation Fibrin monomers stick together to form hard clot formation Stabilization of clot formation by cross linking between Glutamine and Lysine Red cells get entangled in fibrin clot → red color of clot Proteolytic cleavage catalyzed Thrombin (IIa)→ Release of fibrinopeptides A and B Prothrombin→ Thrombin(IIa)
  131. 131. Fibrinogen Schematic diagram of fibrin clot formation from fibrinogen Fibrin monomer Proteolytic cleavagecatalyzed Thrombin(IIa) → Releaseof fibrinopeptidesAandB Fibrin clot Prothrombin→ Thrombin(IIa) Stabilizationofclotformation bycrosslinkingbetween GlutamineandLysine Redcellsgetentangledinfibrinclot→redcolorofclot
  132. 132. Biochemistry of Albumin ❖Biochemistry of Albumin: • Plasma concentration : 3.5 - 5.5 gm/dl(60% of plasma proteins ) • Molecular weight : 69000 • Structure : a single polypeptide with 585 amino acids with 17 disulfide bridges • Site of synthesis : liver ( 12gm/day→25% of total hepatic protein) • Half life : 20 days • Application of Measurement of Plasma concentration→ Liver function test
  133. 133. Functions of Albumin (Globular proteins) ❖ Functions of Albumin : 1.Nutritive(serum albumin ,ovalbumin , Lactalbumin): serves as source of amino acids for protein synthesis particularly in nutritional deprivation of amino acids. 2. Transport :binds and transports plasma free fatty acids ,bilirubin, steroid hormones ,Calcium and Copper in circulation 3. Buffering function : among the plasma proteins, albumin has maximum buffering capacity(lower than bicarbonate buffer system). 4.Osmotic function : due to high concentration and low molecular weight .It plays predominant role in maintaining blood/plasma volume and body fluid distribution. It contributes to 75-80% of the total plasma osmotic pressure(25 mm Hg) . Hypoalbuminemia:Lowplasmaalbumin<2g/dl(e.g.kwashiorkor, nephroticsyndrome,cirrhosis )→edema TherapeuticuseAlbumin:treatmentof burns,hemorrhageandkwashiorkor
  134. 134. Protein misfolding and diseases
  135. 135. Four orders of protein structure
  136. 136. Protein misfolding • The process of Protein folding is complex. ❖Causes of Protein misfolding : 1. Spontaneous 2. Gene mutations ❖Consequences of Protein misfolding : misfolded protein usually get degraded . However ,as the individual age progresses , misfolded protein get accumulated and cause number of diseases. ❖Group of diseases due to Protein misfolding : a. Prion diseases b. Amyloidosis
  137. 137. Prion diseases due to Protein misfolding ❖Prion: represents proteinous infectious agents. ❖Prions protein(PrP): 1. the altered forms of normal proteins. 2. No difference in the primary structure (amino acid sequence) and post- translational modifications observed. 3. Certain changes in three –dimensional structure. 4. Major alterations is the replacement of alpha-helices by beta-sheets in PrP which confers resistance to proteolytic digestion of Prions protein. 5. highly infectious agents and can act as template to convert non-infectious proteins with alpha-helices to infectious form . 6. The process continues in exponential manner to accumulate a large number of prion proteins in tissue.
  138. 138. Prion: represents proteinous infectious agents Apolipoprotein E 2 (APO-E2) : responsible for production of chaperons of Tau protein→ the risk for Alzheimer's disease.
  139. 139. A model for the formation of infectious prions Alpha-helix of a protein(non-infectious) Infectious prion (with beta –sheets} Two molecules of Infectious prions (with beta-sheets) These two molecules of Infectious prions separate and convent another two non- infectious proteins to Infectious prions with beta –sheets. Exponential increase in Infectious prions interaction
  140. 140. A model for the formation of infectious prions
  141. 141. OXPHOS(Oxidative phosphorylation)Diseases • Defects in mitochondrial genome will lead to myopathies. Leber’s Hereditary Neuropathy (LHON)is caused by a single base mutation which alters one Arginine to Histidine in NADH Coenzyme Q reductase. OXPHOS(Oxidative phosphorylation)Disease Clinical features Leber’s Hereditary Neuropathy (LHON) Complex I defect, blindness ,cardiac conduction defects Leigh’s syndrome Complex I defect , NDUFS gene defect, movement disorders Myoclonic epilepsy ragged red fiber disease (MERRF) Myoclonic epilepsy, myopathy , dementia Mitochondrial encephalopathy lactic acidosis stroke like episodes ( MELAS) Complex I defect , lactic acidosis, stroke , myopathy, seizures, dementia
  142. 142. Amyloidosis ❖Amyloidosis: refers to the altered proteins with beta sheets that accumulate in the body particularly in the nervous system. ❖Amyloids : 1. Extracellular Proteins found in tissue and body fluids resembling starch. 2. pathological deposit formed by protein misfolding or due to gene mutations associated with a group of disorders collectively called amyloidosis. 3. pathological deposit exert pressure on the vital organs and eventually cause their death. 4. at least 15 different proteins found in a amyloidosis 5. are not infectious agents as prion proteins. 6. accumulate as the age advances (aging). 7. implicated in degenerative diseases ( e.g. Alzheimer'sdisease) and multiple myeloma. 8. Secondary amyloidosis associated with inflammatory or infectious diseases. 9. Familial amyloidosis: inherited genetic mutations 10. Diagnosis / Detection of amyloidosis: Amyloids + Congo red + polarized light→ Apple green fluorescence
  143. 143. Pathogenesis of Amyloidosis
  144. 144. Protein misfolding in Amyloidosis
  145. 145. Diseases associated with Amyloidosis due to Protein misfolding Diseases Abnormal misfolded Protein Alzheimer’s disease Beta amyloid Cystic fibrosis CFTR Parkinson’s disease Alpha synuclein Huntington’s disease Huntingtin Creutzfeldt Jakob disease Prion
  146. 146. ❖Chaperones : • Three dimensional conformation of proteins important for biological functions. • some proteins can generate the functionally active conformation spontaneously e.g. ribonuclease. • Majority of attain correct conformation ,through assistance of certain proteins called Chaperones. Role of Chaperones in protein folding
  147. 147. Functions of Chaperones ❖Functions of Chaperones : 1. are heat shock proteins. 2. facilitate and favor interactions on the polypeptide surfaces to finally give the specific conformation of a protein. 3. can reversibly bind to hydrophobic regions of unfolded proteins and folding intermediates. 4. can stabilize intermediates and prevent the formation of incorrect intermediates. 5. prevent undesirable interactions with other proteins. 6. these activities of chaperones help the protein to attain compact and biologically active conformation.
  148. 148. Functions of Chaperones Chaperones facilitate and favor interactions on the polypeptide surfaces to finally give the specific conformation of a protein. Chaperones can reversibly bind to hydrophobic regions of unfolded proteins and folding intermediates.
  149. 149. Types of chaperones ❖Types of chaperones : 1. heat shock protein (Hsp)system : consist of 70 kDa heat shock protein (Hsp 70) and 40 kDa heat shock protein (Hsp 40) . These proteins can bind individually to the substrate protein and help in formation of protein folding. 2. Chaperonin system : a large oligomeric assembly which forms a structure into which the folded proteins are inserted. It mainly consist of Hsp 60 and Hsp 10 i.e. 60 kDa and 10 kDa Hsp. They are required at later part of protein folding process and work in association with Hsp 70 system.
  150. 150. Chaperonin system : a Type of Chaperone It is a large oligomeric assembly which forms a structure into which the folded proteins are inserted It is required at later part of protein folding process and work in association with Hsp 70 system.
  151. 151. Heat shock protein (Hsp)system: a Type of Chaperone Heat shock protein (Hsp)system : consist of 70 kDa heat shock protein (Hsp 70) and 40 kDa heat shock protein (Hsp 40) . These proteins can bind individually to the substrate protein and help in formation of protein folding.
  152. 152. Protein misfolding and diseases ❖Protein misfolding and diseases : The failure of a protein to fold properly and generally leads to its rapid degradation. Prions (proteinous degraded infectious agents) are aggregates of misfolded proteins or their partially degraded products . Prions exhibit the characteristics of viral and microbial pathogens. ❖Protein misfolding and Cystic fibrosis (CF): 1. A common autosomal recessive disease 2. with mutations that result in abnormal protein cystic fibrosis transmembrane conductance regulator (CFTR) 3. Mutated CFTR cannot fold properly ,not being able to get glycosylated and transported . Therefore ,CTFR gets degraded. ❖Protein misfolding and neurological diseases : Prion are implicated in Alzheimer’s disease , mad cow disease , Huntington’s disease, Creutzfeldt – Jacob disease
  153. 153. Cystic fibrosis (CF) and Alzheimer'sdiseasearedueProtein misfolding The failure of a protein to fold properly and generally leads to its rapid degradation.
  154. 154. Alzheimer’s disease and chaperones PrionsareimplicatedinAlzheimer’sdisease.Alzheimer'sdiseaseis duecellular accumulationofaggregatesofmisfoldedproteins(withbetasheets)ortheir partially degradedproductsinthebodyparticularlyinthenervoussystem.
  155. 155. Alzheimer'sdisease ❖Alzheimer'sdisease was reportedbyAloysiusAlzheimerin1906. ❖Alzheimer'sdisease: 1. aneurodegenerativedisease→seriouspsychologicalproblem. 2. affects5-10%ofthepeopleabove60yearsofage. 3. asthediseaseprogresses,thepatientmayentervegetativestate(affectentirefamily). 4. maydieafter10yearsafterthefirstonsetofthediseasesymptoms/manifestation. ❖Molecularbasis:ApolipoproteinEpromotestheconformationchangeofalphaamyloid tobetaamyloid.Followedbyselfaggregationofbetaamyloidsinthenervoussystem.
  156. 156. ClinicalmanifestationsofAlzheimer'sdisease ❖ClinicalmanifestationsofAlzheimer'sdisease: a. Memoryloss/dementia b. Confusion c. Hallucinations d. Personalitychangeswithabnormalbehavior→patients entervegetativestatewithno comprehensiveoftheoutsideworld. e. Requireroundtheclockcareandprotection f. Shakespeare’sKingLear(whoislosinghismemoryandbecomingdisoriented)isawell –knownexample.
  157. 157. Genes associated with Alzheimer's disease Genes associated with Alzheimer's disease Located onChromosome number Amyloidprecursorprotein(APP) 21 Presenilin-1 14 Presenilin-2 1 AD-3 14 AD-4 1 APO E4(Apolipoprotein E4) 19 Gene S 182 14
  158. 158. PathologicalhallmarksofAlzheimer'sdisease ❖PathologicalhallmarksofAlzheimer'sdiseaseinclude: • Cerebralamyloiddeposition-Amyloid precursor protein (APP) • NeurofibriltanglesinCNS(Tauproteins) • Senileneuroticplaques ➢Inflammationwithinthebrainplaysa roleindevelopmentAlzheimer'sdiseaseandlong termuseofanti-inflammatorydrugwasfoundtoreducetheincidenceofdisease. ➢Aluminumtoxicity:Alpha-helicesofAPPundergoconformationchangetobeta-pleated sheetsinpresenceofAluminum.ThisAPPproteinwithabnormalconformationbeing insolublegetsdepositedinCNSleadingtoDementia.
  159. 159. Alpha()secretase( proteolytic enzyme) ❖Alpha ()secretase: 1. Expressed and present on cell surfaces(trans-membrane region). 2. A proteolytic enzyme. 3. Are members of ADAM(a Disintegrins and metalloprotease domains). ➢Secretase complex is a prime target for pharmacological interventions of AD. Amyloid precursor protein(APP- a transmembrane protein ) Alpha ()secretase Beta ()secretase Gamma( )secretase Soluble Alpha protein (sAPP)into the extracellular environment insolubleproteins withAlpha()andbeta ()regions–toxic conformation→getdeposited
  160. 160. Structure-functionrelationshipofAmyloid precursor protein(APP) in Alzheimer's disease Criteria Normal Amyloid precursor protein( APP) Amyloid precursor protein (APP) in Alzheimer's disease Genetic aspect Itiscodedbyagenelocatedonalongarmof chromosome21.Normalconstituentofserum andalsofoundintransmembraneregion. Mutation of a gene coding for APP→ substitution of Valine by Isoleucine Solubility Soluble Insoluble(cannot be degraded by Cathepsins and are deposited in neurons)→neurotic plaque Substrate for Alpha ()secretase( proteolytic enzyme) Beta ()secretase( proteolytic enzyme) Product of secretase catalysis Soluble protein(sAPP) Insoluble beta- protein with 40-42 amino acids gets deposited → neurotoxic effect →dementia(Alzheimer's disease)
  161. 161. Selfaggregationofbeta-amyloidsinthenervoussysteminAlzheimer'sdisease Action of beta and gamma secretase on the Amyloid precursor protein(APP) can result in toxic conformation , called alpha-beta peptide (40-42amino acids )with neurotoxic activity→ dementia of Alzheimer'sdisease.
  162. 162. Structure-functionrelationshipofBeta-Amyloid precursor protein(APP) in Down syndrome Trisomyofchromosome21/mutationofagenecodingfor Amyloidprecursor protein(APP) Substitution of Valine by Isoleucine Increased rate of biosynthesis of Beta-APP Deposition of insoluble Amyloid plaques Dementia
  163. 163. sAPP and Alzheimer's disease ❖ sAPP : soluble form of Amyloid precursor protein results from its proteolytic cleavage by -Secretase. It is released from neurons in response to Electrical activity. ❖Functions of sAPP in Modulation of : 1. Synaptic plasticity 2. Synaptogenesis 3. Neuronal excitability 4. Neurite outgrowth 5. Neuronal Cell survival sAPP C-Gmp (guanosine 3’,5’ –cyclic monophosphate ) Modulation of activities of K-channels Signaling pathway Beta APP sAPP : a soluble neuroprotective protein APP with C-terminal end of truncated (s-APP ) +amyloid beta- peptide(A –beta) with neurotoxic activity -Secretase - secretase Alzheimer's disease: is associated with mutation in secretase leading to decreased levels of sAPP protein and or elevated levels of A-beta( APP )protein→ Dementia - secretase amyloid beta peptide with neurotoxic activity
  164. 164. Regulation of Beta-Amyloid precursor protein(APP) synthesis by 25- hydroxy cholesterol ➢27- hydroxy cholesterol(27OHC)→ regulate a number of key enzymes within the brain→ regulate synthesis of Beta- Amyloid precursor protein(APP)→ prevent dementia/ Alzheimer's disease . ➢27- hydroxy cholesterol→ suppresses expression of gene responsible for synthesis of Arc( a cytoskeletal-associated protein responsible for memory consolidation). ➢Alzheimer's disease is associated with low levels of Arc.
  165. 165. Toxic effects of Beta-Amyloid precursor protein(APP) on neurofibrils ❖Toxic effects of Beta -Amyloid precursor protein(APP) on neurofibrils: 1. Beta -Amyloid precursor protein(APP) cause oxidative injury and changes 2. Changes in intracellular calcium homeostasis 3. Cytoskeletal recognition ➢The gene coding beta-APP is located on chromosome 21 .Therefore , in Trisomy 21(Down syndrome) the rate of production of Beta-Amyloid precursor protein(APP) is increased leading to early onset of Alzheimer’s in patients with Down syndrome.
  166. 166. Tauproteins ❖Tauproteins: • Microtubuleassociatedproteinsthatareabundantinneuronsinthecentralnervous system.Itenhancespolymerizationoftubulin. • PhosphorylationofTauisregulatedbyahostofkinasese.g.PKN(Serine/Threonine kinases) ActivationofPKN(Serine/Threoninekinases) PhosphorylationofTau Disruptionofmicrotubuleorganization Pathogenesis of Alzheimer's disease and other Tauopathies Self –assembly of tangles of paired helical filaments and straight filaments HyperphosphorylationofTau(Tauinclusions) Enhanced activity of protein kinases and diminished activity of phosphatases
  167. 167. Structure-functionrelationshipofTauproteinsinAlzheimer's disease Criteria Normal Tau proteins Tau proteins in Alzheimer's disease Solubility Soluble and catabolized easily Insoluble(cannot be degraded by Cathepsins and are deposited around neurons) Function Required for Stabilizing axonal microtubules and facilitating the communication channels in nerve fibers. Loss stability of microtubules ,hyper polymerization of tubulins in neurons →deceased synthesis of Acetyl CoA→ dementia . Diabetes mellitus ,hypertension are risk factors for Alzheimer's disease.
  168. 168. BiochemicalchangesduetoTauproteinsinAlzheimer's disease Tauopathies:Biochemical changes duetoTau proteins in Alzheimer's disease : 1. Threonine /Serine kinases catalyze phosphorylation of Tau proteins → self-assembly of paired helical filaments of Tau proteins → tangles of Tau→ disruption of microtubules in neurons of CNS → Dementia 2.Changes in intracellular Calcium homeostasis .
  169. 169. Dementia :due to Mutation of a gene coding for Transmembrane protein Presenilin-1 and Presenilin-2 Mutation of a gene coding for Transmembrane proteins Presenilin-1 and Presenilin-2 Excessive production of Beta- Amyloid precursor protein(APP) Deposition of insoluble Amyloid plaques in neurofibrils Dementia
  170. 170. Apolipoprotein E4 associated with Familial Alzheimer's disease ❖Apolipoprotein E : Arginine rich protein. It is present in chylomicrons, LDL, and VLDL . It is a ligand for hepatic uptake. Normal Blood levels Apo E 2 mg/dL. Gene for Apolipoprotein E is polymeric and located on chromosome 19 . It has has 3 alleles and therefore 6 possible combinations . 1. ABC 2. ACB 3. BAC 4. BCA 5. CAB 6. CBA ❖Liver and also Astrocytes make Apo-E. It is involved in cellular transport of lipids in CNS. Apo-E is associated with lipoprotein glomerulopathy. Apo-E has four isoforms viz. ApoE- I, II, III, IV. ❖Two of these isoforms i.e. Apolipoprotein E- II and Apolipoprotein E- IV, increase the risk for Alzheimer's disease by eight folds. ❖Apo E- IV is implicated in the development of senile dementia and Familial Alzheimer's disease (AD). ❖Familial Alzheimer's disease: 30% cases of (AD) with genetic background are associated with Apolipoprotein E- IV . ➢Apolipoprotein E- II (Apo E -II) : responsible for production of chaperons of Tau protein→ the risk for Alzheimer's disease.
  171. 171. CerebralamyloiddepositionbyApolipoproteinE4inAlzheimer'sdisease Alzheimer'sdisease:ApolipoproteinE4promotestheconformationchangeofalphaamyloidtobetaamyloid
  172. 172. Zinc deficiency and Alzheimer's disease ❖Zinc binds to beta amyloid to form plaque →dementia → Alzheimer's disease
  173. 173. Glutamate transporters in Alzheimer's disease • L –Glutamate : an excitatory neurotransmitter in mammalian CNS. • EAAT : high affinity glutamate transporters (5 types viz EAAT 1-5) • Neural EAATs : play specialized roles at neural synapses. • The transporters EAAT1 and EAAT2 present in glial neural cells and are responsible for majority of Glutamate uptake. • Dysfunction of EAAT is implicated in pathology of neurogenerative conditions including Alzheimer's disease( other conditions : Huntington’s disease ,epilepsy , ischemic stroke, amyotrophic lateral sclerosis). • Trafficking ,splicing, and post-transcriptional modifications of EAAT are exploited in treatments of these conditions.
  174. 174. Clinical aspects of N-acetyl –D-aspartate receptors and Transcription factor NFKB ❖Genetic mutations or age-related changes in N-acetyl –D-aspartate receptors and Transcription factor NFKB may promote neuronal degeneration indicated in Alzheimer's disease by increasing production of A-beta and or decreasing levels of neuroprotective s-APP-alpha .
  175. 175. Role of Glutathione in prevention of Alzheimer's disease Alzheimer's disease: is due cellular accumulation of aggregates of misfolded proteins or their partially degraded products . The term Prion ( proteinous infectious agents ) is coined to collectively represent them. Reduced Glutathione prevents amyloid formation by activating enzyme Glyoxalase and thus restores memory.
  176. 176. Diagnostic tests for Alzheimer's disease ❖Diagnostic tests for Alzheimer's disease 1. Elevated levels of Tau proteins in CSF 2. Elevated levels of soluble -Amyloid precursor protein Beta in CSF (sAPP) 3. Decreased levels of (s-APP) 4. Serum DHEA ➢APP-beta mutations are linked to some inherited forms of Alzheimer's disease. ➢ s-APP is approximately 100-fold more potent than (s-APP) in protecting hippocampal neurons against excitotoxicity.
  177. 177. Management of Alzheimer's disease 1. N –methyl aspartate an antagonist → slows the progression of disease 2. Omega-3-fatty acids → prevention disease 3. Long term anti-inflammatory drugs→ reduce incidence of disease 4. Beta() and Gamma ()secretase inhibitors ➢ Limitation of Gamma ()secretase inhibitor therapy : Gamma ()secretase is required by variety of physiological substrates therefore ,there is a need to develop substrate specific compounds.
  178. 178. Alpha-synuclein and Parkinson’s disease ❖Parkinson’s disease: • A degenerative disease –affecting muscular coordination. • Two genes are associated with this disease 1. Gene for Alpha-synuclein is mutated in Parkinson’s disease. Alpha- synuclein is found in Lewy bodies(=inclusion bodies) found in many regions of the brain in this disease . Lewy bodies are responsible for neuronal degeneration of Parkinson’s and Alzheimer’s disease. 2. Gene Codes for protein- Parkin which is associated with juvenile form of Parkinson’s disease.
  179. 179. Cystic Fibrosis ❖Cystic Fibrosis (CF) is a multisystem disease that presents • in neonates ,with failure to pass the first feces containing bile, intestinal debris and mucus ( meconium ileus ) • in early childhood with respiratory infections • in the adults ❖Inheritance of Cystic Fibrosis (CF) : autosomal recessive disorder
  180. 180. Pathogenesis of Cystic Fibrosis ❖Cystic Fibrosis arises due to mutations in gene located on chromosome 7 encoding the Cystic Fibrosis transmembrane regulator (CFTR ) protein that regulates transmembrane chloride transport . ❖ The most common mutation is F 508 mutation which refers to deletion of three base pairs , resulting in the absence of Phenylalanine at position 508 in amino acid sequence of CFTR .
  181. 181. Structure-functionrelationshipofCystic Fibrosis transmembrane regulator (CFTR ) ❑Absence of Cystic Fibrosis transmembrane regulator (CFTR ) /chloride channel leads to following Consequences : • Exocrine pancreatic insufficiency with impaired secretion of sodium, bicarbonates and water resulting in increased viscosity (mucoviscoidosis) ,obstruction of pancreatic duct , pancreatic fibrosis and obstruction of pancreatic tissue. • Chronic airways infection that affects mucus secretion in the bronchi with recurrent respiratory infections ,bronchiectasis and chronic lung disease • Malabsorption ,cirrhosis of liver and cholelithiasis due to defective secretion of chloride and water • abnormal sweat gland function due to excessive excretion of sodium and chloride in sweat • Abnormal urogenital functions
  182. 182. Diagnosis of Cystic Fibrosis ❖Diagnosis of Cystic Fibrosis is based on: 1. Clinical symptoms 2. Measurement of pilocarpine induced sweat electrolyte concentration: 3. Na ⁺ and Cl⁻ in sweat ( 70 mmols /L or mequ /L ) 4. Neonatal screening test : increased plasma immunoreactive trypsin 5. Prenatal screening test : for F 508 mutation
  183. 183. Diagnosis of Cystic Fibrosis
  184. 184. Sweat test for Diagnosis of Cystic Fibrosis

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