01 Cell Signalling lect 1-combined.pdf

CELL SIGNALING OR SIGNAL TRANSDUCTION Prof Dr Seyyedha Abbas Learning Objectives ◼ Differentiate between neurotransmitters, hormones, and cytokines, explaining their distinct roles and sources, and how they function in autocrine, paracrine, endocrine, and synaptic signalin...

CELL SIGNALING OR SIGNAL TRANSDUCTION Prof Dr Seyyedha Abbas Learning Objectives ◼ Differentiate between neurotransmitters, hormones, and cytokines, explaining their distinct roles and sources, and how they function in autocrine, paracrine, endocrine, and synaptic signaling ◼ Describe the mechanisms of receptor-ligand interactions, including the specific types of receptors (ion channel- linked, G-protein-coupled, enzyme-linked, and intracellular), and explain the concepts of receptor affinity and specificity. ◼ Outline the key components and steps of signal transduction pathways, detailing how ligands bind to receptors, and the subsequent roles of second messengers and target proteins, with examples like phosphorylation cascades and G-protein activation ◼ Explain the processes of regulation and termination in signal transduction pathways, discussing mechanisms that maintain cellular homeostasis and the factors that affect the specificity and efficiency of chemical messenger actions ◼ Analyze the impact of altered signal transduction pathways on human health, using examples of diseases such as cancer and diabetes, and evaluate how these pathways can serve as therapeutic targets for drug development. Mya Sthenia is a 37-year-old woman who complains of increasing muscle fatigue in her lower extremities with walking. If she rests for 5 to 10 minutes, her leg strength returns to normal. She also notes that if she talks on the phone, her ability to form words gradually decreases. By evening, her upper eyelids droop to the point that she has to pull her upper lids back in order to see normally. These symptoms are becoming increasingly severe. When Mya is asked to sustain an upward gaze, her upper eyelids eventually drift downward involuntarily. When she is asked to hold both arms straight out in front of her for as long as she is able, both arms begin to drift downward within minutes. Her physician suspects that Mya Sthenia has myasthenia gravis and orders a test to determine whether shehas antibodies in her blood directed against the acetylcholine receptor. Ann O’Rexia, who suffers from anorexia nervosa, has increased her weight to 102 lb from a low of 85 lb. On the advice of her physician, she has been eating more to prevent fatigue during her daily jogging regimen. She runs about 10 miles before breakfast every second day and forces herself to drink a high-energy supplement immediately afterward. Dennis Veere was hospitalized for dehydration resulting from cholera toxin In his intestinal mucosal cells, cholera A toxin indirectly activated the CFTR channel, resulting in secretion of chloride ion and Na ion into the intestinal lumen. Ion secretion was followed by loss of water, resulting in vomiting and watery diarrhea. Dennis is being treated for hypovolemic shock.  When a chemical messenger binds to a cell receptor, the signal it is carrying must be converted into an intracellular response. This conversion is called signal transduction Each cell must contribute in an integrated way as the body grows, differentiates, and adapts to changing conditions. Such integration requires communication that is carried out by Chemical messengers  The eventual goal of such signals is to change actions carried out in target cells by intracellular proteins (metabolic enzymes, gene regulatory proteins, ion channels, or cytoskeletal proteins). Chemical messengers / signaling molecules / ligands  Transmit messages between cells. Secreted from one cell in response to a specific stimulus Travel to a target cell Where they bind to a specific receptor and elicit a response  In the nervous system, these chemical messengers are called neurotransmitters In the endocrine system, they are hormones In the immune system, they are called cytokines Additional chemical messengers include retinoids, eicosanoids, and growth factors.  Depending on the distance between the secreting and target cells, ligands can be classified as 1. Endocrine (travel in the blood to reach target) 2. Paracrine (act on nearby cells) 3. Autocrine (act on the same cell)  General features of ligands Signaling generally follows the sequence: 1. Ligand is secreted from a specific cell in response to a stimulus; 2. ligand diffuses or is transported through blood or other extracellular fluid to the target cell; 3. receptor in the target cell specifically binds the ligand 4. binding of the ligand to the receptor elicits a response; 5. The signal ceases and is terminated. Ligands elicit their response in the target cell without being metabolized by the cell The specificity of the response is dictated by the type of receptor and its location.  Generally, each receptor binds only one specific ligand, and each receptor initiates a characteristic signal transduction pathway that will ultimately activate or inhibit certain processes in the cell. Only target cells, carry receptors for that messenger and are capable of responding to its message.  The means of signal termination is an exceedingly important aspect of cell signaling, and failure to terminate a message contributes to a number of diseases Types of ligandsLigand  Ligands of three major signaling systems in the body 1. THE NERVOUS SYSTEM Secretes two types of messengers: Small molecule neurotransmitters, often called biogenic amines, and neuropeptides. 2. THE ENDOCRINE SYSTEM Endocrine hormones, secreted from specific endocrine glands, transported to target cells through blood.  Polypeptide hormones (e.g., insulin) Catecholamines such as epinephrine (which is also a neurotransmitter), Steroid hormones (derived from cholesterol), and Thyroid hormone (derived from tyrosine) Others; retinoids, derivatives of vitamin A (also called retinol) and vitamin D (derived from cholesterol). 3. THE IMMUNE SYSTEM Cytokines - small proteins regulate a network of responses designed to kill invading microorganisms. Include interleukins, tumor necrosis factors, interferons, and colony-stimulating factors 4. THE EICOSANOIDS include prostaglandins [PG], thromboxanes, and leukotrienes control cellular function in response to injury derived from arachidonic acid, a 20-carbon polyunsaturated fatty acid, usually present in cells as part of the membrane lipid phosphatidylcholine Receptors and Signal Transduction  signals are sensed and processed by cellular signal transduction cascades – comprising of 1. Specific receptors 2. Effector signaling elements 3. Regulatory proteins  Receptor “A protein inside or on the surface of the target cells that binds a specific ligand and initiates the cellular response. When ligand dissociates from receptor, induced response is stopped.” OR proteins containing a binding site specific for a single chemical messenger and another binding site involved in transmitting the message TYPES OF RECEPTORS 1. Intracellur Receptors ◼ Present in nucleus or cytosol ◼ Ligand is small and hydrophobic/lipophilic ◼ E.g. Thyroid hormone, Vit D, steroid hormones  Gene transcription is the process of copying the genetic code from DNA to RNA Most intracellular receptors are gene-specific transcription factors, proteins bind to DNA and regulate the transcription of certain genes  Steroid Hormones (lipophilic) enter directly into cytoplasm - bind to receptor located in the cytoplasm/nucleus In cytoplasm, bind with cytoplasmic receptor, to form hormone-receptor complex - enter nucleus Regulate gene expression directly by binding to DNA hormone response element (HRE)  Hormones Response Element (HRE): A short ( 12 to 20 bp) DNA sequence to which hormone- receptor complex for steroids, retinoid, thyroid hormones and vitamin D bind, altering the gene expression.  CELL SIGNALING OR SIGNAL TRANSDUCTION Lecture # 2 By Dr Seyyedha Abbas  2. Ion channels (Neurotransmitter linked) ◼ Directly control ion flux across the plasm membrane ◼ Two types - Both important in nervous system activity 1. Ligand gated: ◼ Activated when a specific molecule binds ◼ Example: acetylcholine receptor in postsynaptic cell 2. Voltage gated: ◼ Stimulus is electrical, not chemical 3. Cell surface membrane receptors 1. G-Protein – Coupled Receptors 2. Catalytic receptors G-Protein – Coupled Receptors (GPCRs) or 7TM receptors Largest family of cell surface receptors receptors that couple signal transduction to G-proteins Composed of a similar structure i.e. 1. An extra cellular ligand binding region 2. 7 Transmembrane helices (7TM receptor) 3. An intracellular domain that interacts with G-proteins G-protein-linked receptors or seven-transmembrane domain receptors, 7TM receptor  G-Proteins: Bind Guanosine nucleotides (GTP & GDP) Two principal signal transduction pathways involve the G protein-coupled receptors i.e. cAMP and phosphatidylinositol signal pathways Trimeric proteins - 3 subunits – α, β , Ɣ  Types: 1) Gs Stimulates and 2) Gi inhibits Adenylate cyclase. β & γ subunits are identical. α subunits differ.  Catalytic Receptors Catalytic receptors, also known as Enzyme-linked receptors, are a class of receptors that, upon ligand binding, exhibit enzymatic activity cell surface receptor proteins – usually dimeric Transmembrane receptors having an inherent enzyme activity as part of their structure  Contain an extra cellular domain for ligand binding & an intra cellular domain with specific enzyme activity Insulin receptors possess intrinsic tyrosine kinase activity Directly linked to intracellular enzymes ◼ ligand binding and functional domains part of same polypeptide chain ◼ Single transmembrane-spanning domain of 20-25  Receptors involve second messenger molecule Receptors are signal detector – bind Ligands Ligands bind to receptor located on plasma membrane - Second messenger molecules--- intervene between the original message/signal and ultimate effect on the cell - Cellular response  Signaling often requires a rapid response and rapid termination of the message, which may be achieved by a. degradation of the messenger or second messenger b. the automatic G protein clock c. Deactivation of signal transduction kinases by phosphatases, or other means Examples of second messengers 1. cAMP: Glucagon, Calcitonin, CRH, ACTH, FSH, ADH 2. cGMP: Atrial natriuretic factor (ANF), Nitric oxide (NO) 3. Calcium or phosphatidylinositol or both: ADH, Oxytocin, TRH, Gastrin, Angiotensin II 4. Kinase/ phosphatase cascade: Insulin, Growth hormone, Insulin like growth factor (IGF), Prolactin  Types Of Second Messenger pathways 1) Adenylate Cyclase pathway 2) Calcium/ phosphatidylinositol pathway 3) Guanylate cyclase pathway 4) Kinase / phosphatase pathway  1) Adenylate Cyclase pathway Adenylate Cyclase: a membrane bound enzyme that converts ATP to 3/,5/- cyclic adenosine monophosphate (c AMP) Mechanism of action of Adenylate Cyclase pathway Mechanism of action of Adenylate Cyclase pathway ECF which converts ATP to cAMP ◼ A 60-year-old man presents to the emergency department with complaints of muscle weakness and difficulty swallowing. He has a history of myasthenia gravis, an autoimmune disorder affecting neuromuscular transmission. Which of the following ion channels is most likely directly involved in this patient's condition? ◼ A. Voltage-gated sodium channels B. Voltage-gated potassium channels C. Ligand-gated acetylcholine receptors D. Voltage-gated calcium channels E. Ligand-gated GABA receptors ◼ Key: C. Ligand-gated acetylcholine receptors ◼ A patient experiences sudden and severe pain in the chest, along with a sense of impending doom. Upon examination, the patient is found to have a rapid heart rate and elevated blood pressure. Which of the following ion channels is primarily responsible for the initial depolarization of cardiac muscle cells? ◼ A. Ligand-gated acetylcholine receptors B. Voltage-gated sodium channels C. Voltage-gated potassium channels D. Ligand-gated NMDA receptors E. Voltage-gated chloride channels ◼ Key: B. Voltage-gated sodium channels ◼ A 45-year-old woman complains of difficulty sleeping and increased anxiety. She is prescribed a medication that enhances the activity of GABA in the brain. Which of the following ion channels is directly affected by this medication? ◼ A. Voltage-gated sodium channels B. Ligand-gated acetylcholine receptors C. Voltage-gated calcium channels D. Ligand-gated GABA receptors E. Voltage-gated potassium channels ◼ Key: D. Ligand-gated GABA receptors ◼ A 35-year-old male presents with symptoms of peptic ulcer disease, including epigastric pain and nausea. He is prescribed a medication that targets a specific family of receptors to reduce gastric acid secretion. Which family of receptors is most likely targeted by this medication? ◼ A) Tyrosine kinase receptors B) Nuclear receptors C) Ligand-gated ion channels D) G-protein coupled receptors E) Cytokine receptors ◼ Answer: D) G-protein coupled receptors ◼ A new drug is designed to treat heart failure by enhancing the contractility of the heart muscle. It achieves this by stimulating beta-1 adrenergic receptors, which are a type of receptor known for their role in signal transduction involving G-proteins. What is a common feature of these receptors? ◼ A) Intracellular receptor dimerization B) Extracellular ligand-binding region C) Single pass through the cell membrane D) Ligand-gated ion channel E) Direct interaction with transcription factors ◼ Answer: B) Extracellular ligand-binding region ◼ A 45-year-old male presents with symptoms of excessive sweating, palpitations, and hypertension. Laboratory tests reveal elevated levels of catecholamines. Which type of G-protein is most likely involved in the overstimulation of adenylate cyclase, leading to increased production of cAMP in this patient? A) Gq B) Gi C) Gs D) Go E) G12/G13 Key: C) Gs ◼ A patient is treated with a drug that targets G-protein-coupled receptors and reduces intracellular cAMP levels. Which G-protein is most likely activated by this drug? A) Gs B) Gq C) Gi D) Go E) G12/G13 ◼ Key: C) Gi ◼ A 60-year-old woman with a long history of type 2 diabetes is started on insulin therapy. Which of the following best describes the type of receptor that insulin binds to? A) G-protein-coupled receptor with intrinsic GTPase activity B) Catalytic receptor with intrinsic serine/threonine kinase activity C) Catalytic receptor with intrinsic tyrosine kinase activity D) Ion channel-linked receptor with calcium influx activity E) Nuclear receptor with DNA-binding domain Key: C) Catalytic receptor with intrinsic tyrosine kinase activity ◼ A 40-year-old female is found to have a genetic mutation that affects the function of a receptor involved in second messenger signaling. This receptor is located on the plasma membrane and affects intracellular signaling pathways. Which of the following processes is likely disrupted in her cells? A) Direct ion transport across the membrane B) Direct alteration of gene transcription in the nucleus C) Activation of intracellular enzymes through phosphorylation D) Cell-cell adhesion and interaction E) Ligand-independent receptor activation ◼ Key: C) Activation of intracellular enzymes through phosphorylation  CELL SIGNALING OR SIGNAL TRANSDUCTION Lecture # 3 By Prof Dr Seyyedha Abbas which converts ATP to cAMP  cAMP-dependent Adenylate cyclase protein kinase (PKA) ATP cAMP (inactive) cAMP-dependent protein kinase(PKA) (active) ADP ATP Phosphorylated Intracellular Protein Protein effects (active) substrate  Structure OF cAMP-dependent protein kinase Called Protein Kinase A (PKA) R C A Tetramer – Two Regulatory (R) & R C Two Catalytic (C) subunits Complex tetramer is inactive Two molecules of cAMP bind R to two each R subunit disassociates into 2 R subunits R and two catalytically active C units. ◼ Signal amplification -important feature of signal cascades. ◼ One hormone molecule can lead to formation of many cAMP molecules. ◼ Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life- time of the cAMP Termination of Adenylate cyclase pathway 1. Already formed cAMP is cleaved by phosphodiesterase that, itself is activated by phosphorylation - catalyzed by Protein Kinase A. Phosphodiesterase enzymes catalyze: cAMP + H2O → AMP Thus cAMP stimulates its own degradation, leading to rapid turnoff of a cAMP signal 2. Protein Kinase A also activates Phosphatases  ADP ATP Phosphorylated Protein Protein (active) substrate Phosphatase Dephosphorylated Protein (Inactive) Ligand  Calcium/ phosphatidylinositol pathway Phosphatidylinositol 4,5- bisphosphate (PIP2)  2 fatty acids **+ glycerol (C1 & 2) → diacylglycerol Diacylglycerol + PO4 (C3) → Phosphatidic acid (PA) PA+ Inositol (C3) → Phosphatidylinositol (PI) PI + 2 PO4 → phosphatidylinositol 4,5 bisphosphate (PIP2) Phospholipase C (PLC) hydrolyzes PIP2 PIP2 → Diacylglycerol + Inositol Triphosphate **stearic acid (C 1) and arachidonic acid (C 2)  Calcium/ phosphatidylinositol pathway  Calcium/ PI pathway - G protein linked 1. ligand binds with receptor 2. Activates G proteins 3. α subunit gets GTP and detaches from other subunits 4. activates the enzyme Phospholipase C (PLC) 5. PLC activates Hydrolysis of PIP2 – 6. yields diacylglycerol (DAG) and inositol triphosphate (IP3). 7. IP3 Binds to IP3 gated calcium channels on endoplasmic reticulum a. Rapid release of Ca++ ions from ER b. Ca++-calmodulin complex is formed c. In turn binds with enzymes to activate them Calmodulins: Small, calcium binding proteins One molecule binds with 4 molecules of Ca++….. gets activated 8. DAG activates the protein kinase C (PKC) – plays role in cell growth and differentiation ◼ PKC activated by free Ca++ and DAG both ◼ Has regulatory and catalytic domains  4 Ca++-++ calmodulin Cacomplex calmodulin complex Phosphatidyl inositol 1,4,5-triphosphate 3 1 PI3 ER 2 DAG + + Protein Protein Kinase kinase CC Ligand CELL SIGNALING OR SIGNAL TRANSDUCTION Lecture # 4 By Prof Dr Seyyedha Abbas Ligand  3) Guanylate cyclase system Formation of Cyclic guanosine monophosphate (cGMP) in response to various signals like Ligands: Nitric Oxide, peptides Similar to cAMP pathway Enzyme: Guanylate cyclase (forms cGMP from GTP) cGMP Activates cGMP dependent protein kinase OR protein kinase G (PKG) LIGAND 7TM receptor α subunit of G proteins gets GTP Activates Guanylate cyclase GTP cGMP  Guanylate cyclase GTP cGMP PKG (inactive) Dephosphorylated proteins PKG (active) (inactive) Phosphatase Intracellular effects cGMP acts as 2nd messenger in Cardiac atrial tissues Natriuresis Diuresis Smooth muscle relaxation Vasodilation –drugs like nitroglycerine, NO, sodium nitrite, nitroprusside increase intracellular cGMP level 4. Protein Kinases & Phosphatases Protein kinase transfers terminal phosphate of ATP to a hydroxyl group on a protein. Phosphatase dephosphorylates protein by hydrolysis. Tyrosine kinase receptor (catalytic receptor) – activated by INSULIN – a hypoglycaemic hormone Tyrosine kinases are a subclass of protein kinases  Tyrosine kinase dimer – ligand binding leads to dimerization Capable of auto phosphorylation – itself on tyrosine residues In turn phosphorylates other proteins at tyrosine residues Functions as an "on" or "off" switch in many cellular functions Diseases related to signal transduction Cholera toxin 1. When cholera toxin is released from the bacteria in the infected intestine, it binds to the enterocytes 2. triggering endocytosis of the toxin 3. Interferes with the normal action level of G protein. 4. Results in high cAMP levels in infected enterocytes 5. Causes continuous activation of adenylate cyclase. 6. Uncontrolled release of water and sodium into intestinal lumen, leading to diarrhea and dehydration. Various modes of cell signaling Plasma membrane patches (micro domains) – Important for signal transduction 1. Lipid rafts: areas on the cell membrane having predominantly glycosphingolipids, cholesterol & proteins anchored by covalent bonding Lipid rafts have a role in cholesterol transport, endocytosis, G protein signaling and binding of viral pathogens Lipid rafts contain 3 to 5-fold more cholesterol & 50% more sphingolipids (sphingomyelin) as compared with surrounding plasma membrane 2. Caveolae: 50–100 nm plasma membrane invaginations, flask shaped indentations on the areas of lipid rafts Along with other receptor proteins also contain the protein caveolin,– act as scaffolding proteins within caveolar membranes by compartmentalizing and concentrating signaling molecules Involved in endocytosis of cholesterol containing lipoproteins, fusion and budding of viral particles  CELL ADHESION MOLECULES (CAMs) Proteins located on the cell surface - bind with other cells or with the extracellular matrix (ECM) help cells stick to each other and to their surroundings - cell adhesion CAMs are transmembrane receptors - composed of three domains: 1. Extracellular domain - interacts with CAMs 2. Transmembrane domain 3. Intracellular domain - interacts with the cytoskeleton Depending upon the type of CAMs receptor binding is of two types a. Homophilic binding: CAMs of the same kind like Cadherins named for "calcium-dependent adhesion") - transmembrane proteins – play role in cell adhesion within tissues. b. Heterophilic binding: with different kind of CAMs or extracellular matrix (ECM) - like Integrins proteins - function mechanically, by attaching the cell cytoskeleton to the ECM NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF HEALTH SCIENCES Amino Acid Chemistry 2 Dr. Amber Zaidi Assistant Professor MBBS, MPhil, PhD Scholar Department of Biochemistry NUST School of Health Sciences  NSHS Learning Objectives NUST SCHOOL OF HEALTH SCIENCES Describe a Zwitter ion and its significance Describe the functions of amino acids Discuss Amino Acids as Buffers  NSHS Zwitter Ion NUST SCHOOL OF HEALTH SCIENCES A zwitterion is an ion that contains two functional groups. An ion possessing both positive and negative electrical charges. Therefore, zwitterions are mostly electrically neutral (the net formal charge is usually zero).  NSHS Functions of Amino Acids NUST SCHOOL OF HEALTH SCIENCES 1. Enzymes and Hormones 2. Contractile proteins 3. Bone Protein 4. Blood stream proteins  NSHS Isomeric forms of Amino Acid NUST SCHOOL OF HEALTH SCIENCES The amino acids form two stereoisomers that are mirror images of each other. The structures are much like our left and right hands. These mirror images are termed enantiomers  NSHS Acidic and Basic Properties NUST SCHOOL OF HEALTH SCIENCES Amino acids in an aqueous solution contain weakly acidic α- carboxyl groups and weakly basic α-amino groups Each of the acidic and basic amino acids contains an ionizable group in its side chain Thus amino acids can act as Buffers  NSHS A. pH NUST SCHOOL OF HEALTH SCIENCES The concentration of protons ([H+]) in aqueous solution is expressed as pH. pH = log 1/ [H+] or –log [H+] The larger the Ka, the stronger the acid, because most of the HA has dissociated into H+ and A−. Conversely, the smaller the Ka, the less acid has dissociated and, therefore, the weaker the acid.  NSHS Henderson–Hasselbalch equation NUST SCHOOL OF HEALTH SCIENCES This equation demonstrates the quantitative relationship between the pH of the solution and concentration of a weak acid (HA) and its conjugate base (A−)  NSHS Buffer NUST SCHOOL OF HEALTH SCIENCES A buffer is a solution that resists a change in pH following the addition of an acid or base and can be created by mixing a weak acid (HA) with its conjugate base (A−) If an acid is added to a buffer, A− can neutralize it, being converted to HA in the process. If a base is added, HA can likewise neutralize it, being converted to A− in the process.  NSHS Maximum Buffering Capacity NUST SCHOOL OF HEALTH SCIENCES Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid–base pair can still serve as an effective buffer when the pH of a solution is within approximately ±1 pH unit of the pKa. If the amounts of HA and A− are equal, the pH is equal to the pKa.  NSHS Example NUST SCHOOL OF HEALTH SCIENCES A solution containing acetic acid (HA = CH3 – COOH) and acetate (A− = CH3 – COO−) A pKa of 4.8 resists a change in pH from 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3 – COOH) is the predominant species in solution. At pH greater than the pKa, the deprotonated base form (CH3– COO−) is the predominant species. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Dissociation of Alanine NUST SCHOOL OF HEALTH SCIENCES The dissociation constant of the carboxyl group of an amino acid is called K1, rather than Ka, because the molecule contains a second titratable group. The Henderson– Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine: K1 = [H+ ] [II] / [I]  NSHS NUST SCHOOL OF HEALTH SCIENCES where I is the fully protonated form of alanine and II is the isoelectric form of alanine This equation can be rearranged and converted to its logarithmic form to yield pH = pK1 + log [II] / [I] NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Amino group dissociation NUST SCHOOL OF HEALTH SCIENCES The second titratable group of alanine is the amino (−NH3+) group. Because this is a much weaker acid than the –COOH group, it has a much smaller dissociation constant, K2. (Note: Its pKa is, therefore, larger) Release of a H+ from the protonated amino group of form II results in the fully deprotonated form of alanine, form III.  NSHS pKs and sequential dissociation NUST SCHOOL OF HEALTH SCIENCES The sequential dissociation of H+ from the carboxyl and amino groups is summarized in Figure using alanine as an example. Each titratable group has a pKa that is numerically equal to the pH at which exactly one half of the H+ have been removed from that group.  NSHS NUST SCHOOL OF HEALTH SCIENCES The pKa for the most acidic group (−COOH) is pK1, whereas the pKa for the next most acidic group (−NH3 +) is pK2. The pKa of the α-carboxyl group of amino acids is ∼2, whereas the pKa of the α-amino group is ∼9. By applying the Henderson–Hasselbalch equation to each dissociable acid group, it is possible to calculate the complete titration curve of a weak acid. Figure shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the fully deprotonated form (III) NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS a. Buffer pairs NUST SCHOOL OF HEALTH SCIENCES The –COOH/–COO− pair can serve as a buffer in the pH region around pK1, and the –NH3 +/–NH2 pair can buffer in the region around pK2.  NSHS b. When pH=pK NUST SCHOOL OF HEALTH SCIENCES When the pH is equal to pK1 (2.3), equal amounts of forms I and II of alanine exist in solution. When the pH is equal to pK2 (9.1), equal amounts of forms II and III are present in solution.  NSHS c. Isoelectric point pI NUST SCHOOL OF HEALTH SCIENCES At neutral pH, alanine exists predominantly as the dipolar form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, when the sum of the positive charges equals the sum of the negative charges.  NSHS Isoelectric point of Alanine NUST SCHOOL OF HEALTH SCIENCES For Alanine, with only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7) The pI is, thus, midway between pK1 (2.3) and pK2 (9.1). pI corresponds to the pH at which the form II (with a net charge of zero) predominates and at which there are also equal amounts of forms I (net charge of +1) and III (net charge of −1). NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS d. Net charge at neutral pH NUST SCHOOL OF HEALTH SCIENCES At physiologic pH, amino acids have a negatively charged group (−COO−) and a positively charged group(−NH3+), both attached to the α-carbon. Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains. Substances such as amino acids that can act either as an acid or a base are described as Amphoteric.  Buffering the blood, the bicarbonate NSHS NUST SCHOOL OF buffer system HEALTH SCIENCES The pH within our blood is maintained in the slightly alkaline range of 7.35 to 7.45 by the bicarbonate buffer system. Most proteins function optimally at this physiologic pH and their amino acid constituents exist in the chemical form; Exceptions include 1. Some digestive enzymes that function at acidic pH of the stomach between pH 1.5 and 3.5. 2. Lysosomal enzymes also function at an acidic pH range between pH 4.5 and 5.0.  NSHS NUST SCHOOL OF HEALTH SCIENCES Maintaining arterial pH at 7.40 ± 0.5 is important for health; normally the bicarbonate buffer system is able to keep pH within the acceptable range The need for a buffering system can be appreciated by considering that organic acids (e.g., lactic acid) are generated during metabolism and that glucose and fatty acid oxidation generate CO2, the anhydrous form of H2CO3 (carbonic acid).  NSHS Bicarbonate Buffer system NUST SCHOOL OF HEALTH SCIENCES The bicarbonate ion concentration, [HCO3 −], and the carbon dioxide concentration [CO2] influence the pH of the blood. The relatively water-insoluble CO2 is converted by the enzyme carbonic anhydrase to the water-soluble HCO3 − (bicarbonate), which is carried through the blood to the lungs where dissolved CO2 is exhaled. Therefore, lungs regulate the loss and retention of CO2 by altering the breathing rate. Kidneys retain or excrete bicarbonate, H+, ammonia, and other acids/bases that may appear in the blood. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS E. Blood gases and pH NUST SCHOOL OF HEALTH SCIENCES As a consequence of certain disease processes or poisons, blood pH can become abnormal. Acidemia is defined as an arterial pH 7.45. In the bicarbonate buffer system, CO2 is an acid and bicarbonate is a base.  Changes in breathing can impact the acid–base NSHS NUST SCHOOL OF balance HEALTH SCIENCES Acidosis may develop in human body due to 1. Hypoventilation 2. Generation of excess metabolic acids (e.g., lactic acid) 3. Ketoacidosis that can accompany type 1 diabetes mellitus Alkalosis may develop Loss of excess acid through vomiting  NSHS Are compensations 100%? NUST SCHOOL OF HEALTH SCIENCES Neither renal compensation nor compensation by breathing rate changes (respiratory compensation) will bring pH back toward normal physiologic range if excess metabolic acids have been generated. “It should be noted that neither the lungs nor the kidneys can fully compensate or overcompensate for pH imbalances” NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Reference Book NUST SCHOOL OF HEALTH SCIENCES Lippincott Illustrated Reviews 8th edition Chapter 1 Amino Acids and the role of pH NSHS NUST SCHOOL OF HEALTH SCIENCES Mucopolysaccaridoses and Proteoglycans DR ROOMISA ANIS MBBS, MPHILL, PHD SCHOLAR ASSOCIATE PROFESSOR DEPARTMENT OF BIOCHEMISTRY NUSTSCHOOL OF HEALTH SCIENCES Jawad a two year old child is brought to pediatrician by his parents due to concerns about his developmental delays and unusual physical features. They report that Jawad has been having difficulty reaching developmental milestones such as sitting, crawling, and babbling. Additionally, they have noticed that Jawad' abdomen seems larger than usual, and he has been experiencing recurrent ear infections and noisy breathing. Upon examination, the pediatrician observes coarse facial features, including a flattened nasal bridge, protruding tongue, and prominent forehead. Jawad also has hepatomegaly and splenomegaly. There are signs of upper airway obstruction, such as noisy breathing and snoring. Fundoscopic examination reveals corneal clouding. Introduction  Mucopolysaccharidoses are hereditary diseases caused by a deficiency of any one of the lysosomal hydrolases normally involved in the degradation of  Heparan Sulfate,  Dermatan Sulfate, and  Keratin Sulfate  The prevalence of mucopolysaccharidoses is 1:25,000 live births.  Disease becomes apparent only when enzyme activity is less than ~2% of the normal.  Because of inadequate degradation, glycosaminoglycans accumulate in lysosomes of most cells and eventually cause progressive organ damage.  They are progressive disorders characterized by lysosomal accumulation of GAGs in various tissues, causing a range of symptoms, such as skeletal and ECM deformities, and intellectual disability.  All are autosomal-recessive disorders except Hunter syndrome, which has X-linked inheritance.  Affected patients may develop a  Dysmorphicface Lesions of the heart valves,  Hepatomegaly Airway obstruction, Dysfunction of central nervous system.  Splenomegaly  Hernias  Deafness  Cardiomyopathy,  There currently is no cure  Children homozygous for any one of these diseases are apparently normal at birth and then gradually deteriorate.  In severe deficiencies, death occurs in childhood. How are Mucopolysaccharoidoses diagnosed?  Diagnosis relies heavily on measurements of enzyme activities in various cells or body fluids.  Diagnosis is confirmed by measuring the patient’s cellular level of the lysosomal hydrolases.  Incomplete lysosomal degradation of GAGs results in the presence of oligosaccharides in the urine.  These fragments can be used to diagnose the specific mucopolysaccharidoses by identifying the structure present on the non-reducing end of the oligosaccharide, because that residue would have been the substrate for the missing enzyme. What are non reducing ends? Treatment options  Bone marrow and cord blood transplants, in which transplanted macrophages produce the enzymes that degrade GAGs, have been used to treat Hurler and Hunter syndromes, with limited success.  Enzyme replacement therapy is available for both syndromes but does not prevent neurologic damage Austin disease Multiple sulfatase deficiency is a rare lysosomal storage disease in which all sulfatases are nonfunctional because of a defect in the formation of formylglycine, an amino acid derivative required at the active site for enzymatic activity to occur. HURLER SYNDROME (MPS IH) Most Severe Form  Biochemical Defect:  α-L-Iduronidase Deficiency leading to Impaired Degradation of Dermatan Sulfate and Heparan Sulfate  Clinical Features:  Corneal Clouding  Developmental Disability  Dwarfing  Dysmorphic Facial Features  Upper Airway Obstruction  Hearing Loss  Clinical Complications:  Deposition in Coronary Artery → Ischemia → Early Death  Treatment Options:  Bone Marrow or Cord Blood Transplantation: Preferably before Age 18 Months  Enzyme Replacement Therapy:  Available to mitigate symptoms  Milder Forms:  Hurler-Scheie and Scheie Syndromes Early diagnosis crucial for intervention Multidisciplinary care essential for management Prognosis varies depending on severity Hunter Syndrome (MPS II):  Biochemical Defect:  Iduronate sulfatase deficiency leading to degradation of dermatan sulfate and heparan sulfate affected  X-linked Inheritance  Clinical Features:  Wide Range of Severity  No Corneal Clouding  Mild to Severe Physical Deformity  Developmental Disability Treatment Options:  Enzyme Replacement Therapy: Available to alleviate symptoms  No corneal clouding distinguishes it from other MPS types  Focus on managing physical deformity and developmental challenges  Early diagnosis crucial for intervention and management Sanfilippo Syndrome (MPS III):  Rare Genetic Disorder  Four Subtypes (A-D)  Four enzymatic steps necessary for removal of N- sulfated or N-acetylated glucosamine residues from heparan sulfate are blocked  Subtypes and Enzyme Deficiencies:  Type A: Heparan Sulfatase Deficiency  Type B: N-Acetylglucosaminidase Deficiency  Type C: Acetyl CoA:ẞ-Glucosaminide Acetyltransferase Deficiency  Type D: N-Acetylglucosamine 6-Sulfatase Deficiency Clinical Features:  Severe Nervous System Disorders  Developmental Disability  Progressive neurodegeneration  Onset in early childhood  Variable severity among subtypes  Enzyme replacement therapy is under investigation  Supportive care focuses on symptom management and improving quality of life Sly Syndrome (MPS VII)  Rare Genetic Disorder  Biochemical Defect:  Β-glucuronidase deficiency leading to impaired degradation of dermatan sulfate and heparan sulfate Affected  Clinical Features: Short Stature  Hepatosplenomegaly Corneal Clouding  Skeletal Deformity Developmental Disability  Multi-systemic disorder  Variable severity  Corneal clouding distinguishes it from other MPS types  Management focuses on symptom relief and supportive care  Enzyme replacement therapy available for some cases Deficiencies in galactosamine 6- sulfatase and β-galactosidase that degrade keratan sulfate result in Morquio syndrome (MPS IV), A and B, respectively. Deficiencies in arylsulfatase B that degrades dermatan sulfate result in Maroteaux–Lamy syndrome (MPS VI) Introduction to Proteoglycans  Proteoglycans are essential components of the extracellular matrix (ECM) and cell surfaces.  They consist of a core protein with covalently attached linear chains of glycosaminoglycans (GAGs).  The structure resembles a bottle brush, with GAG chains extending from the core protein. Structure of Proteoglycan Monomer  Monomer Structure: Proteoglycan monomers found in cartilage consist of a core protein.  Up to 100 linear chains of GAGs are covalently attached to the core protein.  Each GAG chain can have up to 200 disaccharide units.  Chondroitin sulfate and keratan sulfate are the main types of GAGs in cartilage proteoglycans.  Proteoglycans are grouped into gene families with common structural features (e.g., aggrecan family).  The overall structure of a proteoglycan can be visualized as a bottlebrush, with the core protein as the handle and the GAG chains as the bristles. The GAG chains are much larger than the core protein, and they give the proteoglycan molecule its characteristic extended shape. GAGs-Protein Linkage  GAGs-Protein Linkage: GAGs are attached to the core protein via covalent linkage.  The linkage commonly occurs through a trihexoside (galactose-galactose-xylose) and a serine residue in the protein.  An O-glycosidic bond forms between the xylose and the hydroxyl group of the serine. Aggregate Formation in Proteoglycans  Multiple proteoglycan monomers can associate with one molecule of hyaluronic acid to form proteoglycan aggregates.  The association is not covalent and primarily occurs through ionic interactions between the core protein and hyaluronic acid.  Additional small proteins called link proteins stabilize the association. Importance of Proteoglycans  Proteoglycans play crucial roles in maintaining tissue structure and function.  They contribute to the mechanical properties of cartilage and other connective tissues.  Proteoglycans also regulate cell signaling, cell adhesion, and interactions with growth factors and other ECM components.  When a solution containing GAGs is compressed, the water is squeezed out, and the GAGs are forced to occupy a smaller volume.  When the compression is released, the GAGs spring back to their original, hydrated volume because of the repulsion of their negative charges.  This property contributes to the resilience of cartilage, synovial fluid, and the vitreous  humor of the eye NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF HEALTH SCIENCES GLYCOSAMINOGLYCANS DR ROOMISA ANIS MBBS, MPHILL, PHD SCHOLAR ASSOCIATE PROFESSOR DEPARTMENT OF BIOCHEMISTRY NUSTSCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF Learning Objectives HEALTH SCIENCES Classify of Glycosaminoglycans (GAGS). Outline the structural differences in different types of GAGS. Explain the biochemical roles of different types of GAGs in extracellular matrix (ECM) organization Discuss the clinical importance of some GAGs used in clinical practice  NSHS NUST SCHOOL OF HEALTH SCIENCES A 45-year-old male presents to the clinic complaining of joint pain and stiffness, particularly in his knees and hips. He reports that the symptoms have been progressively worsening over the past year, limiting his ability to perform daily activities such as climbing stairs and walking for extended periods. Additionally, he mentions occasional swelling in the affected joints and difficulty in bending or straightening them fully.  NSHS NUST SCHOOL OF HEALTH SCIENCES Amino sugars Acidic sugars Uronic acids  NSHS NUST SCHOOL OF HEALTH SCIENCES Uronic acids The primary alcohol group of monosaccharides is oxidised to form the corresponding uronic acid. - Glucose is oxidised to form glucuronic acid (GlcUA). - Galactose is oxidised to form galacturonic acid (GalUA). c. L-Ascorbic acid ( Vitamin C) It is formed in some animals (not humans) from glucose. It is considered as a sugar acid.  NSHS NUST SCHOOL OF Amino Sugars HEALTH SCIENCES -These are sugars in which an amino group (NH2) replaces the hydroxyl group on the second carbon e.g. glucosamine (GluN), galactosamine (GlaN) and mannosamine (ManN) -Aminosugars are important constituents of glycosaminoglycans (GAGs) and some types of glycolipids and glycoproteins. Several antibiotics contain amino Sugars which are important for their antibiotic activity  NSHS NUST SCHOOL OF Introduction HEALTH SCIENCES Glycosaminoglycans (GAGs), also known as mucopolysaccharides They are negatively-charged polysaccharide compounds. They are composed of repeating disaccharide units that are present in every mammalian tissue. Their functions within the body are widespread and determined by their molecular structure.  NSHS NUST SCHOOL OF Glycosaminoglycan(GAGs) HEALTH SCIENCES Found in Cartilage Skin Blood vessels Cornea Tendons Ligaments Loose connective tissue  NSHS NUST SCHOOL OF HEALTH SCIENCES Repeating disaccharide units of amino sugar and uronic acid Amino sugars may be acetylated and sulphated as well Presence of carboxylic group and sulphate group gives them a negative charge. Due to negative charge their molecules repel each other Charged groups attract water molecules and produce viscous solutions. They produce gel-like matrix that forms basis of body’s ground substance  NSHS NUST SCHOOL OF GAGS HEALTH SCIENCES Generally associated with a small amount of proteoglycans Consist of up to 95% carbohydrate. Produce the gel-like matrix that forms the basis of the body’s ground substance Hydrated GAGs serve as a flexible support for the ECM, interacting with the structural and adhesive proteins, Also serves as a molecular sieve, influencing movement of materials through the ECM. The viscous, lubricating properties of mucous secretions also result from the presence of GAGs, which led to the original naming of these compounds as mucopolysaccharides.  NSHS Classification Six main classes NUST SCHOOL OF HEALTH SCIENCES Hyaluronic acid Chondroitin Sulphate Dermatan Sulphate Heparan Sulphate Heparin Keratan Sulphate NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF Hyaluronic acid HEALTH SCIENCES Greek Hyalos means “glass”; Hyaluronates can have a glassy or translucent appearance Found in Vitreous humour of eye Synovial fluid Skin Umbilical cord Rheumatic nodule.  NSHS NUST SCHOOL OF Composition HEALTH SCIENCES Sulphate free mucopolysaccharide Contains alternating residues of D-glucuronic acid and N-acetylglucosamine. With up to 50,000 repeats of the basic disaccharide unit. Hyaluronic acid is also an essential component of extracellular matrix of cartilage and tendons It contributes tensile strength and elasticity to tendons NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF HEALTH SCIENCES Some pathogenic organisms secrete Hyaluronidase. Hydrolyze the glycosidic linkages of hyaluronate Tissues become more susceptible to bacterial invasion Spreading factor Similar enzyme in sperm hydrolyzes outer glycosaminoglycan coat around the ovum, allowing sperm penetration.  NSHS NUST SCHOOL OF Chondroiton sulphate HEALTH SCIENCES Found in ground substance of Cartilage Tendons Ligaments Walls of the aorta. Consists of repeating unit of β-Glucuronic acid and N-Acetylgalactosamine sulfate NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Types Depending Upon Position of NUST SCHOOL OF HEALTH SCIENCES Sulphate Group  NSHS NUST SCHOOL OF Functions HEALTH SCIENCES Chondroitin sulfate is a major component of cartilage It contributes to resilience and shock- absorbing properties of cartilage essential for joint health and mobility. Promotes the production of collagen and proteoglycans, which are essential components of healthy cartilage tissue.  NSHS NUST SCHOOL OF HEALTH SCIENCES Contributes to the lubrication of joints by promoting the production of synovial fluid. Contributes to the formation and maintenance of bone matrix.  NSHS NUST SCHOOL OF HEALTH SCIENCES Osteoarthritis affects millions of individuals worldwide. In this disease, joint cartilage is degraded and proteoglycans that normally help provide a cushion for the joint are lost. Without the resilience of the cartilage protecting the joint, there is pain, stiffness, and swelling, with progressive worsening of signs and symptoms. Glucosamine and chondroitin have been reported both to relieve pain and to stop progression of osteoarthritis. These compounds are readily available as over-the-counter dietary supplements in the United States. Based on several well-controlled clinical studies, it appears that glucosamine sulfate (but not glucosamine hydrochloride) and chondroitin sulfate may have a small to moderate effect in relieving symptoms of osteoarthritis  NSHS NUST SCHOOL OF Hip Joint Osteoarthritis HEALTH SCIENCES Avanced End-Stage Hip Hip Joint Joint Normal joint Normal joint Osteoarthritis Osteoarthritis  NSHS NUST SCHOOL OF HEALTH SCIENCES A 55-year-old man, presents to the emergency department with complaints of swelling and pain in his left lower leg. He reports that the symptoms started suddenly yesterday evening and have been progressively worsening since then. He denies any recent trauma to the leg or significant medical history but mentions that he recently took a long-haul flight. Upon examination, his left lower leg is visibly swollen, erythematous, and tender to palpation. His calf circumference is noticeably larger on the left side compared to the right. The ultrasound reveals the presence of a thrombus in the left popliteal vein extending into the proximal veins.  NSHS NUST SCHOOL OF Heparin HEALTH SCIENCES Natural anticoagulant Found in Mast cells of Liver Spleen Lungs Thymus Blood  NSHS NUST SCHOOL OF Composition HEALTH SCIENCES Polymer of repeating disaccharide units of D-Glucosamine (Glc N) and either of the two uronic acids- D-Glucuronic acid 10% L-Iduronic acid 90% Alpha-1→ 4 glycosidic linkage  NSHS NUST SCHOOL OF HEALTH SCIENCES The -NH2 group at C2 and OH group at C6 of D-Glucosamine are sulphated C2 of Uronic acids are sulphated It is strongly acidic.  NSHS NUST SCHOOL OF HEALTH SCIENCES Heparin is a heterogeneous (3000–30,000 kDa), polyanionic oligosaccharide activator of antithrombin III (AT). AT is a slow but quantitatively important inhibitor of thrombin (factor X) and other factors (IX, XI, XII) in the blood-clotting cascade. When heparin binds to AT, it converts AT from a slow inhibitor to a rapid inhibitor of coagulating enzymes. Heparin interacts with a lysine residue in AT and induces a conformational change that promotes covalent binding of AT to the active serine centers of coagulating enzymes, forming a ternary complex and inhibiting pro-coagulant activity. Heparin then dissociates from the complex and can be recycled for anticoagulation.  NSHS NUST SCHOOL OF HEALTH SCIENCES Heparin has an average half-life of 30 min in the circulation, so it is commonly administered by infusion. Heparin does not have fibrinolytic activity; therefore it will not lyse existing clots. In addition to its anticoagulant activity, heparin also releases several enzymes from proteoglycan binding sites on the vascular wall, including lipoprotein lipase, which is often assayed as heparin-releasable plasma lipoprotein lipase activity or post-heparin lipase. Lipoprotein lipase is inducible by insulin, and decreased activity of this enzyme delays plasma clearance of chylomicrons and very low-density lipoprotein (VLDL), contributing to hypertriglyceridemia in diabetes.  NSHS NUST SCHOOL OF Keratan Sulphate HEALTH SCIENCES GAG which does not contain any uronic acid. Formed by repeating units of galactose and N-acetyl glucosamine in beta linkage. Present in cornea, cartilage, bone, and a variety of Horny structures formed of dead cells Horn, hair, hoofs, nails, and claws  NSHS NUST SCHOOL OF Dermatan Sulphate: HEALTH SCIENCES It contains L-iduronic acid and N-acetyl galactosamine in beta -1, 3 linkages. It is found in skin, blood vessels and heart valves.  NSHS NUST SCHOOL OF Synthesis and importance of GAGS HEALTH SCIENCES Synthesized from simple sugars Synthesis requires multienzyme process GAGs are hydrolysed by specific lysosomal hydrolases Deficience of these lysosomal hydrolases result in mucopolysaccharidoses Accumulation of mucopolysaccharides in spleen, liver, joints, heart and coronary arteries. NSHS NUST SCHOOL OF HEALTH SCIENCES NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF HEALTH SCIENCES Extracellular Matrix (ECM) Dr. Amber Zaidi Assistant Professor MBBS, MPhil, PhD Scholar Department of Biochemistry NUST School of Health Sciences  NSHS Learning Objectives NUST SCHOOL OF HEALTH SCIENCES Define Extra-cellular matrix Describe the structure of proteins, carbohydrates, and mineral content of ECM Describe the functions of various molecules of ECM. Describe various forms of Extra-cellular matrix (intercellular space, subcutaneous tissue, cartilage, bone) according to the variations in protein  NSHS Introduction NUST SCHOOL OF HEALTH SCIENCES A large network of proteins and other molecules that surround, support, and give structure to cells and tissues in the body. The extracellular matrix helps cells attach to, and communicate with, nearby cells, and plays an important role in cell growth, cell movement, and other cell functions  NSHS Functions of ECM NUST SCHOOL OF HEALTH SCIENCES 1. ECM adds strength to tendons 2. Involved in filtration in the kidney 3. Role in attachment of skin.  NSHS Components of ECM NUST SCHOOL OF HEALTH SCIENCES Three major categories of extracellular macromolecules make up the ECM: 1. Glycosaminoglycans (GAGs) and proteoglycans, 2. Fibrous proteins, including collagen and elastin, 3. Adhesive proteins, including fibronectin and laminin  NSHS A. Proteoglycans NUST SCHOOL OF HEALTH SCIENCES Proteoglycans are aggregates of GAGs and protein ‘ground substance’. GAGs are also known as mucopolysaccharides and are composed of repeating disaccharide chains where one of the sugars is an N-acetylated amino sugar, either N-acetylglucosamine or N- acetylgalactosamine Examples Chondroitin sulfate, Hyaluronic acid, Keratin sulfate, Dermatan sulfate, Heparin, and Heparan sulfate.  NSHS Properties of GAGs NUST SCHOOL OF HEALTH SCIENCES GAGs repel each other. Slide past each other, producing the slippery consistency GAGs attract positively charged sodium ions, which are found in solution complexed with water molecules Resilience: When compressive forces are exerted on it, the water is forced out and the GAGs occupy a smaller volume. When the force of compression is released, water floods back in, rehydrating the GAGs, much like a dried sponge rapidly soaks up water. This is referred to as resilience. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS B. Fibrous Proteins NUST SCHOOL OF HEALTH SCIENCES Have compact structures resulting from secondary, tertiary, or even quaternary protein structure Examples Collagen and elastin are fibrous proteins in the ECM Minor- Fibronectin, Laminin  NSHS 1. Collagen NUST SCHOOL OF HEALTH SCIENCES Collagen is the most abundant protein in the human body (25% of total body protein mass.) It forms tough protein fibers that are resistant to shearing forces. Occurrence: bone, tendon, and skin. Types 28 distinct types however, over 90% of collagen in the human body is in collagen types I, II, Ill, and IV. Types I, II, and III are fibrillar collagens - linear polymers of fibrils Type IV (and also type VII) is a network-forming collagen that becomes a three-dimensional mesh rather than distinct fibrils.  NSHS Structure of collagen NUST SCHOOL OF HEALTH SCIENCES Collagen molecules are composed of three helical polypeptide a chains of amino acids that wind around one another forming a collagen triple helix Every third amino acid is glycine, because only this amino acid, with the smallest side chain, fits into the crowded central core. Characteristic, repeating sequence of collagen is Gly-X-Y, where X and Y can be any amino acid but most often X is proline and Y is hydroxyproline Because of their restricted rotation and bulk, proline and hydroxyproline confer rigidity to the helix. NSHS NUST SCHOOL OF HEALTH SCIENCES Chain Compositions of Collagen NSHS NUST SCHOOL OF Types HEALTH SCIENCES  NSHS Synthesis of collagen NUST SCHOOL OF HEALTH SCIENCES  NSHS 2. Elastin NUST SCHOOL OF HEALTH SCIENCES Elastin is rich in the amino acids glycine, alanine, proline, and lysine. Similar to collagen, elastin contains hydroxyproline, although only a small amount. No carbohydrate is found within the structure of elastin, and therefore, it is not a glycoprotein. Weak hydrophobic interactions between Valine residues permit the flexibility and extensibility of elastin  NSHS Characteristics of elastin NUST SCHOOL OF HEALTH SCIENCES An interconnected rubbery network that can impart stretchiness to the tissue that contains it. This structure resembles a collection of rubber bands that have been knotted together, with the knots being the desmosine cross-links. Lack an orderly secondary protein structure because elastin can adopt different conformations both when relaxed and when stretched  NSHS Synthesis of Elastin NUST SCHOOL OF HEALTH SCIENCES Cells secrete the elastin precursor, tropeelastin, into the extracellular space. Tropoelastin then interacts with glycoprotein microfibrils including fibrillin, which serve as a scaffolding onto which tropoelastin is deposited. Side chains of some of the lysyl residues within tropoelastin polypeptides are modified to form allysine. In the next step, the side chains of three allysyl residues and the side chain of one unaltered lysyl residue from the same or neighboring tropoelastin polypeptide are joined covalently to form a desmosine cross-link. Four individual polypeptide chains are covalently linked together.  NSHS NUST SCHOOL OF HEALTH SCIENCES Fibronectin- Fibronectin is a glycoprotein present as a structural component of the ECM and also in plasma as a soluble protein. The loss of fibronectin from the surface of many tumor cells may contribute to their release into the circulation, first steps in tumor metastasis Laminin- Laminins are a family of noncollagenous glycoproteins found in basement membranes  NSHS Clinical Significance NUST SCHOOL OF HEALTH SCIENCES Scurvy Dietary deficiency in vitamin C causing aberrant collagen production. Hydroxylation of prolyl and lysyl residues cannot occur, resulting in defective pro-a chains that cannot form a stable triple helix. Blood vessels become fragile, bruising occurs, wound healing is slowed, and gingival hemorrhage and tooth loss occur  NSHS Osteogenesis imperfecta NUST SCHOOL OF HEALTH SCIENCES inherited collagen defects impact individuals throughout their life span "brittle bone disease” because many affected individuals have weak bones that fracture easily Eight forms of Osteogenesis imperfecta are currently known. spinal curvature and hearing loss.  NSHS Ehlers-Danlos syndrome NUST SCHOOL OF HEALTH SCIENCES inherited defects in the structure, production, or processing of fibrillar collagen six primary subtypes Hypermobility followed by the classical forms of EDS are most common (Abnormally flexible and loose joints that extend beyond the normal) range of motion and stretchy but excessively fragile skin and blood vessels are characteristics  NSHS Marfan syndrome NUST SCHOOL OF HEALTH SCIENCES Autosomal dominant trait A mutation occurs in the gene that codes for the fibrillin-1 protein essential for maintenance of elastin fibers. 1. ocular abnormalities ,myopia (near-sightedness) 2. Abnormalities in their aorta. 3. have long limbs and long digits, 4. tall stature, scoliosis (side-to-side or front-to- back spinal curvature) or kyphosis (curvature of the upper spine) 5. abnormal joint mobility, and hyperextensibility of hands, feet, elbows, and knees.  NSHS 4. Adhesive Proteins NUST SCHOOL OF HEALTH SCIENCES Fibronectin and laminin are adhesive glycoproteins secreted by cells into the extracellular space. They contain three different binding domains that link them to cell surfaces and to other components of the ECM, including proteoglycans and collagen Adhesive proteins join ECM components to each other and link cells to the ECM  NSHS 5. Epidermolysis bullosa NUST SCHOOL OF HEALTH SCIENCES Epidermolysis bullosa is a rare heritable disorder characterized by severe blistering of the skin and epithelial tissue. a. simplex: blistering in the epidermis, caused by defects in keratin filaments b. junctional: blistering in the dermal– epidermal junction, caused by defects in laminin c. dystrophic: blistering in the dermis, caused by mutations in the gene encoding type VII collagen.  NSHS Cell Adhesions NUST SCHOOL OF HEALTH SCIENCES Cell-to-ECM and cell-to-cell adhesions are mediated by plasma membrane- anchored proteins called cell adhesion molecules. Collections of adhesion molecules form cell junctions that join cells together within tissues.  A. Adhesion in developing NSHS NUST SCHOOL OF HEALTH SCIENCES tissues Adhesion in developing tissues-Many tissues, including most epithelial tissues, develop from a precursor, the founder cell that divides to produce copies of itself. These newly produced cells remain attached to the ECM and/or to other cells owing to cell adhesion  NSHS B. Cell junctions NUST SCHOOL OF HEALTH SCIENCES Cells in tissues adhere to other cells at specialized regions known as cell junctions, which are classified according to their function 1. Tight or occluding junctions 2. Desmosomes or anchoring junctions (also called maculae adherentes) as well as adherens junctions 3. Hemidesmosomes 4. Gap or communicating junctions NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS C. Cell adhesion molecules NUST SCHOOL OF HEALTH SCIENCES Cell adhesion molecules mediate selective cell-to-cell and cell-to- ECM adhesion. These are all transmembrane proteins that are embedded within the plasma membranes of cells Four families of adhesion molecules function in cell-cell adhesion: a. Cadherins, b. Selectins, c. Immunoglobulin superfamily, d. Integrins  NSHS Adhesion and Diseases NUST SCHOOL OF HEALTH SCIENCES Normal expression and function of adhesion molecules are required to maintain health and to defend against disease. When these normal cell-to-cell and/or cell-to-matrix interactions are interrupted or altered, disease processes can be triggered.  NSHS NUST SCHOOL OF HEALTH SCIENCES Epithelial mesenchymal transition loss of E-cadherin mediated cell-to-cell adhesion occurs during tumor progression and is also required for subsequent tumor spreading or metastasis. Leukocyte adhesion deficiency LAD is an inherited defect in the P2 subunit of lntegrins, which is normally exclusively expressed on leukocytes. Therefore, their leukocytes have an impaired ability to traffic to the sites of infection and recurrent bacterial infections result.  NSHS Pemphigus NUST SCHOOL OF HEALTH SCIENCES blisters develop as a result of failed cell-to- cell adhesion. Pemphigus is an autoimmune condition characterized by the disruption of cadherin-mediated cell adhesions. Antibody binding to desmogleins prevents their function in cell adhesion. Therefore, adjacent epidermal cells are unable to adhere to each other and blisters develop  Increased adhesion molecule NSHS NUST SCHOOL OF HEALTH SCIENCES expression Expression of more than the usual number of adhesion molecules per cell can result in enhanced migration of cells to a region and can lead to inappropriate inflammation. Asthma ICAM-1, a member of the immunoglobulin superfamily that normally facilitates adhesion between endothelial cells and leukocytes after injury or stress, has been implicated in the pathogenesis of asthma Rheumatoid arthritis: In rheumatoid arthritis, synovial inflammation is associated with increased leukocyte adhesion.  NSHS Remodeling of ECM NUST SCHOOL OF HEALTH SCIENCES ECM turnover is mediated by a family of matrix metalloproteinases (MMPs), about 30 zinc endoproteinases with specificity for different components of the matrix.  NSHS Tissue Engineering & ECM NUST SCHOOL OF HEALTH SCIENCES The ultimate goal of tissue engineering is to combine appropriate cells and biomaterials to produce tissue equivalents that mimic normal tissues and organs and can replace damaged or diseased tissues.  NSHS Reference Book NUST SCHOOL OF HEALTH SCIENCES Lippincott Illustrated Reviews Cell and Molecular Biology 3rd edition Chapter 2 “Extracellular Matrix and Cell Adhesion” NSHS NUST SCHOOL OF HEALTH SCIENCES NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF HEALTH SCIENCES Amino Acid Chemistry 1 Dr. Amber Zaidi Assistant Professor MBBS, MPhil, PhD Scholar Department of Biochemistry NUST School of Health Sciences  NSHS Learning Objectives NUST SCHOOL OF HEALTH SCIENCES Define amino acids Classify amino acids on the basis of nutrition, R group and solubility. Explain the formation of peptide bond  NSHS Introduction NUST SCHOOL OF HEALTH SCIENCES Proteins are the most abundant and functionally diverse molecules in living systems Although more than 300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins  NSHS Structure of Amino Acid NUST SCHOOL OF HEALTH SCIENCES Each amino acid has 1. a carboxyl group, 2. a primary amino group and 3. a distinctive side chain or R group At physiologic pH (∼7.4), the carboxyl group of an amino acid is dissociated, forming the negatively charged carboxylate ion (−COO−), and the amino group is protonated (−NH3+) NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Classification of Amino Acids NUST SCHOOL OF HEALTH SCIENCES Amino Acids can be classified as 1. Standard and Non Standard Amino acids 2. Polar and Non polar side chains 3. Acidic and Basic side chains 4. Aliphatic and Aromatic Amino Acids 5. Essential, Non essential and semi essential amino acids 6. Glucogenic, Ketogenic and Glucogenic & Ketogenic Amino acids  NSHS 1. Standard and Non Standard NUST SCHOOL OF HEALTH SCIENCES Standard amino acids are the only amino acids that are encoded by DNA, the genetic material in the cell. They are 20 in number Nonstandard amino acids are produced by chemical modification of standard amino acids.  NSHS 2. Polar and Non polar side chains NUST SCHOOL OF HEALTH SCIENCES Polar Have ionizable groups in side chains (hydrophilic) Examples Glycine, Serine, Threonine, Cystiene Non Polar Have no ionizable group in side chains (hydrophobic) Examples Alanine, Valine, Leucine, Isoleucine NSHS NUST SCHOOL OF HEALTH SCIENCES NSHS NUST SCHOOL OF HEALTH SCIENCES  Comparison Polar side chains Non polar side chains Can loose and gain electron Do not loose or gain electron Can participate in H bonding Do not participate in hydrogen Can participate in ionic bonding bonding Do not participate in ionic bonding NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS 3. Amino acids with acidic side chains NUST SCHOOL OF HEALTH SCIENCES The amino acids aspartic acid and glutamic acid are proton donors. At physiologic pH, the side chains of these amino acids are fully ionized, containing a negatively charged carboxylate group (−COO−). The fully ionized forms are called aspartate and glutamate. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Amino acids with basic side chains NUST SCHOOL OF HEALTH SCIENCES The side chains of the basic amino acids accept protons At physiologic pH, the R groups of lysine and arginine are fully ionized and positively charged. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Histidine is unique NUST SCHOOL OF HEALTH SCIENCES In contrast, the free amino acid histidine is weakly basic and largely uncharged at physiologic pH. However, when histidine is incorporated into a protein, its R group can be either positively charged (protonated) or neutral, depending on the ionic environment provided by the protein. This important property of histidine contributes to the buffering role it plays in the functioning of proteins including hemoglobin Histidine is the only amino acid with a side chain that can ionize within the physiologic pH range  NSHS 4. Aliphatic and Aromatic Amino Acids NUST SCHOOL OF HEALTH SCIENCES Aliphatic Amino Acids An aliphatic amino acid is an amino acid containing an aliphatic side chain functional group Examples : Alanine, isoleucine, leucine, proline, and valine, are all aliphatic amino acids. Aromatic Amino Acids Aromatic amino acids have an aromatic ring present in them Examples :phenylalanine, tryptophan, and tyrosine NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS 5. Essential and Non Essential NUST SCHOOL OF HEALTH SCIENCES a. Essential Amino acids Those amino acids that cannot be synthesized in human body thatswhy must be consumed in diet. b. Non Essential Amino Acids The amino acids that can be synthesized by metabolism of few other amino acids in human body c. Semi Essential Amino acids Amino acids that can be synthesized in human body but in inadequate amount NSHS NUST SCHOOL OF HEALTH SCIENCES 6. Glucogenic , Ketogenic and Gluco NSHS NUST SCHOOL OF HEALTH SCIENCES ketogenic amino acids a. Glucogenic Amino acids Amino acids whose catabolism yields pyruvate or one of the intermediates of the TCA cycle are termed glucogenic. As these intermediates are substrates for gluconeogenesis and can give rise to the net synthesis of glucose in the liver and kidney.  NSHS NUST SCHOOL OF HEALTH SCIENCES b. Ketogenic amino acids Amino acids whose catabolism yields either acetyl CoA (directly, without pyruvate serving as an intermediate) or acetoacetate (or its precursor acetoacetyl CoA) are termed ketogenic. Acetoacetate is one of the ketone bodies, which also include 3- hydroxybutyrate and acetone. Examples Leucine and lysine  NSHS NUST SCHOOL OF HEALTH SCIENCES c. Glucogenic and Ketogenic Amino acids Amino acids that can yield both pyruvate and acetyl CoA are termed as both Glucogenic and Ketogenic NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Abbreviations and symbols for NUST SCHOOL OF HEALTH SCIENCES commonly occurring amino acids Each amino acid has an associated three-letter abbreviation and a one letter symbol.  NSHS One-letter codes NUST SCHOOL OF HEALTH SCIENCES Unique first letter: If only one amino acid begins with a given letter, then that letter is used as its symbol. For example, V = valine. Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most common of these amino acids receives this letter as its symbol. For example, Glycine is more common than glutamate, so G = glycine. NSHS NUST SCHOOL OF HEALTH SCIENCES 3. Similar sounding names: NSHS NUST SCHOOL OF Some one-letter symbols sound like the HEALTH SCIENCES amino acid they represent. For example, F = phenylalanine. 4. Letter close to initial letter: For the remaining amino acids, a one letter symbol is assigned that is close in the alphabet as possible to the initial letter of the amino acid, for example, K = lysine. B is assigned to Asx, signifying either aspartic acid or asparagine Z is assigned to Glx, signifying either glutamic acid or glutamine W is used for tryptophan X is used to represent an unidentified amino acid. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Peptide Bond NUST SCHOOL OF HEALTH SCIENCES In proteins, adjacent amino acids are joined covalently by peptide bonds, which are amide linkages between the α- carboxyl group of one amino acid and the α-amino group of the next amino acid. For example, Valine and Alanine can form the dipeptide valylalanine through the formation of a peptide bond. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS NUST SCHOOL OF HEALTH SCIENCES Peptide bonds are resistant to conditions that denature proteins, such as heat and high concentrations of urea. Prolonged exposure to a strong acid or base at elevated temperatures is required to nonenzymatically break these bonds.  NSHS Properties of peptide bond NUST SCHOOL OF HEALTH SCIENCES A partial double bond, is rigid and shorter than a single bond. This prevents the rotation between C and N Amino acids present within a peptide are called amino acid residues A series of amino acids joined by peptide linkage form a polypeptide chain Various bond formations  NSHS a. Disulfide bond formation NUST SCHOOL OF HEALTH SCIENCES The side chain of cysteine contains a sulfhydryl (thiol) group (−SH), which is an important component within the active site of many enzymes. In proteins, the –SH groups of two cysteines can be oxidized to form a covalent cross-link called a disulfide bond (−S–S–). Two cysteine residues that form a disulfide bond are referred to as cystine. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS b. Phosphorylation and Dephosphorylation NUST SCHOOL OF HEALTH SCIENCES Kinases are enzymes that catalyze phosphorylation reactions. Phosphatases are enzymes that remove the phosphate group. The changes in phosphorylation status of proteins (whether phosphorylated or not), especially of enzymes, alters their activation status; some enzymes are more active when phosphorylated while others are less active. The polar hydroxyl group of serine, threonine, and tyrosine can serve as a site of attachment for phosphate groups. NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS c. Oligosaccharide chain formation NUST SCHOOL OF HEALTH SCIENCES The amide group of few amino acids can serve as a site of attachment for oligosaccharide chains in glycoproteins Asparagine Serine Threonine NSHS NUST SCHOOL OF HEALTH SCIENCES  NSHS Reference Book NUST SCHOOL OF HEALTH SCIENCES Lippincott Illustrated Reviews 8th edition Chapter 1 Amino Acids and the role of pH NSHS NUST SCHOOL OF HEALTH SCIENCES  Vitamin D Dr. Roomisa Anis Associate Professor Department of Biochemistry NSHS Lippincott Illustrated review Biochenistry 8th edition Learning objectives  Recall what are vitamins.  Recall classification of vitamins  Enlist sources of vitamin D  Illustrate the synthesis of vitamin D, noting the effect of light.  Summarise functions of vitamin D What are vitamins? Organic molecules that cannot be synthesized in adequate quantities by humans How vitamins are classified? Introduction  D vitamins are a group of sterols that have a hormone-like function.  Produced by a complex series of enzymatic reactions  Active molecule is 1,25-dihydroxycholecalciferol Active form 1,25-diOH-D3 Calcitriol  Binds to intracellular receptor proteins  D3–receptor complex interacts with DNA in the nucleus of target cells  Stimulates or represses gene transcription  Regulates the plasma levels of calcium and phosphorus  Sources RDA 1 to 70 years: 15 μg/day  Vitamin D occurs naturally in Over 70 years: 20 μg/day  Fatty fish, Liver, and egg yolk.  Milk,unless it is artificially fortified, is not a good source.  Because breast milk is a poor source of vitamin D, supplementation is recommended for breastfed babies. Note: 1 μg vitamin D = 40 international units [IU]. Distribution of vitamin D  Diet  Ergocalciferol (vitamin D2), found in plants  Additional double bond and methyl group in the plant sterol  Cholecalciferol (vitamin D3), found in animal tissues  7-Dehydrocholesterol in skin Metabolism of 7-Dehydrocholesterol in Skin Vitamin D is the  Converted to cholecalciferol in the dermis and “sunshine vitamin”. epidermis of humans exposed to sunlight  Ultraviolet light-mediated, nonenzymatic photolysis reaction. 7-Dehydrocholesterol – Intermediate in cholesterol synthesis  Extent of conversion is related directly to the intensity of the exposure and inversely to the extent of pigmentation in the skin.  An age-related loss of 7-dehydrocholesterol in the epidermis that may be related to the negative calcium balance associated with old age. Absorption Transport of vitamin D  A specific transport protein called the vitamin D–binding protein binds vitamin D3 and its metabolites and moves vitamin D3 from the skin or intestine to the liver. RDA 1- 70 years= 15µg/day Above 70= 20µg/day  Metabolism of vitamin D ? Formation of 1,25-dihydroxycholecalciferol  Site of formation  Liver and kidney  D2 and D3 are not biologically active.  Converted in the body to the active form of the D vitamin by two sequential hydroxylation reactions. First hydroxylation  The first hydroxylation occurs at the 25 position  This hydroxylation is catalyzed by a specific 25-hydroxylase in the liver.  The product of the reaction, 25-hydroxycholecalciferol – predominant form of vitamin D in the plasma  Major storage form of the vitamin.  Transported to the kidney by the vitamin D–binding protein. Second hydroxylation o 25(OH)2-D3 requires hydroxylation at position C1 for full biologic activity.  Accomplished in mitochondria of the renal proximal convoluted tubule.  Three-component mono-oxygenase reaction that requires NADPH, Mg2+, molecular oxygen  Enzyme is 25-hydroxycholecalciferol 1-hydroxylase  Regulation of 25- Hydroxycholecalciferol 1-hydroxylase  25-hydroxycholecalciferol most potent vitamin D metabolite  Formation is tightly regulated by the level of plasma phosphate and calcium ions  25-Hydroxycholecalciferol 1- Hydroxylase activity is increased directly by low plasma phosphate. Or  Indirectly by low plasma calcium  Low calcium triggers the secretion of parathyroid hormone (PTH) from the chief cells of the parathyroid gland.  PTH upregulates the 1-hydroxylase  Thus, hypocalcemia caused by insufficient dietary Ca2+ results in elevated levels of serum 1,25-diOH- D3. Calcitriol inhibits expression of PTH, forming a negative feed back loop It also inhibits 1-hydroxylase Function of vitamin D  The principal function of vitamin D is to maintain the plasma calcium concentration.  Calcitriol achieves this in three ways:  It increases intestinal absorption of calcium  It reduces excretion of calcium (reabsorption in the distal renal tubules)  It mobilizes bone mineral. Three sites of action a. Intestine