6 Signal Transduction

Define signal transduction

• Signal transduction is the process of transmitting information from the extracellular environment into the cell.

• Extracellular signals—also known as primary messengers or mediators—bind to specific cell surface receptors.

• The binding event triggers a signaling cascade that ultimately leads to a biological response.

what are the methods of cellular signal transmission?

• Endocrine signalling

• Paracrine signalling

• Autocrine signalling

• Gap junctions

describe the mechanism, function and significance of autocrine signalling

• Mechanism: A cell releases signalling molecules that bind to receptors on its own surface.

• Function: Facilitates self-regulation and can reinforce a particular cellular response within the same cell.

• Significance: Common in processes where feedback loops are needed for maintaining cell function or survival.

describe the mechanism, function and significance of endocrine signalling

• Definition: Involves the release of hormones by specialized endocrine cells into the bloodstream, allowing the signal to travel long distances to reach target cells throughout the body.

• Examples:

  • Insulin secreted by the pancreas affects glucose uptake in muscle and fat cells.

  • Thyroid hormones influence metabolism in virtually all tissues.

• Characteristics:

  • Slow but long-lasting effects.

  • Very specific due to receptor binding on target cells.

  • Regulated through feedback loops (e.g., hypothalamic-pituitary axis).

describe the mechanism, function and significance of paracrine signalling

• Definition: A signalling molecule is released by a cell and acts on neighbouring cells in the local environment.

• Examples:

  • Growth factors like fibroblast growth factor (FGF) during wound healing.

  • Nitric oxide (NO) acting on nearby smooth muscle cells to cause vasodilation.

• Characteristics:

  • Local and fast-acting.

  • Important in processes like inflammation, tissue repair, and development.

  • Signal is often rapidly degraded or taken up to limit the range.

describe the mechanism, function and significance of gap junctions

• Definition: Specialized intercellular connections that allow direct communication between neighbouring cells.

• Function: Enable the passage of ions, metabolites, and small signalling molecules.

• Role in Tissue Coordination: Essential for synchronizing activities in tissues such as cardiac and smooth muscle.

Describe the Steps of Signal Transduction

• Synthesis: The extracellular signalling molecule is synthesized by the signalling cell.

• Release: The signalling molecule is secreted into the extracellular space.

• Transport: The signal is transported through the extracellular medium to the target cell.

• Reception: A specific receptor on the target cell binds the signalling molecule.

• Response: Binding initiates changes in metabolism, function, or development.

• Termination: The signal is removed or degraded, ending the cellular response and resetting the system.

Describe the Structural Organization of a Signalling Pathway

• Extracellular Ligands: The signals that initiate the pathway.

• Receptors: Proteins that recognize and bind the extracellular signal.

• Intracellular Mediators: Include enzymes and second messengers that propagate the signal.

• Second Messengers: Small molecules that amplify and distribute the signal within the cell.

• Enzymes: Such as kinases and phosphatases that modify other proteins.

• Adaptor Proteins: Facilitate the assembly of signalling complexes.

• Effectors: The final targets that execute the cellular response.

what are the 2 main types of ligands/extracellular signals?

1. Water-soluble ligands.

• They cannot diffuse through the cell membrane (because it is hydrophobic).

• Therefore, they bind to a ligand-binding site exposed on the extracellular region of the membrane receptor.

• Examples of such ligands include amines, amino acids, peptides, and proteins.

2. Lipid-soluble ligands.

• They can freely cross the cell membrane and bind to cytosolic receptors.

• The ligand-receptor complexes then diffuse through the nuclear membrane and accumulate in the nucleus, where they modulate DNA transcription.

• In this sense, these receptors are called nuclear receptors.

• Examples of such ligands include steroid hormones (progesterone, estrogens, testosterone) and non-steroid hormones (thyroxine and triiodothyronine), vitamin D3, and retinoic acid.

what are the 2 main types of receptors?

• Membrane (cell-surface) Receptors for water-soluble extracellular signalling molecules that cannot pass through the cell membrane

• Intracellular Receptors for lipid-soluble extracellular signals that can freely cross the cell membrane. They are divided into type 1 (mainly in the cytoplasm) and type 2 (mainly in the nucleus)

define Latent Gene Regulatory Proteins

“Latent” gene regulatory proteins are transcription factors or co-regulators that are sequestered in an inactive state—often via proteolytic turnover or cytoplasmic retention—until a signalling event stabilizes and/or activates them, allowing nuclear translocation and target‐gene activation

define Scaffold Proteins

Scaffold proteins simultaneously bind multiple members of a signalling cascade, tethering them into complexes that enhance pathway specificity, speed, and localization

define Relay Proteins

Relay proteins simply pass the activated state from one component to the next (e.g., kinase to kinase), forming the core “linear” chain of a signalling pathway

define Bifurcation Proteins

Bifurcation proteins branch a single upstream signal into two or more distinct downstream pathways, enabling a single cue to generate multiple cellular outcomes

define Adaptor Proteins

Adaptor proteins possess interaction domains (e.g., SH2, SH3) and function as molecular bridges, linking specific signalling proteins without enzymatic activity of their own

define Amplifier Proteins

Amplifier proteins escalate signal strength by activating multiple downstream targets or by generating large numbers of small-molecule mediators (e.g., kinases, ion channels, cyclases)

define Transducer Proteins

Transducer proteins convert one form of signal into another—such as converting ligand-induced conformational change into kinase activation—thereby propagating the message in a new chemical form

define Integrator Proteins

Integrator proteins collect and integrate inputs from multiple, independent signalling pathways, synthesizing them into a unified downstream response

define Anchoring Proteins

Anchoring proteins (e.g., A-kinase anchoring proteins, AKAPs) tether key enzymes and effectors to specific subcellular locales, creating spatially constrained signalling “microdomains”

define Modulator Proteins

Modulator proteins regulate the activity or sensitivity of core signalling components—often via feedback loops or post-translational modifications—to fine-tune pathway output

define and describe the types of Molecular Intracellular Switches

• Many intracellular signalling proteins act as switches, toggling between active and inactive states.

• Protein Kinases/Phosphatases: Regulate activity through phosphorylation.

  • Protein Kinases: Enzymes that add phosphate groups to proteins (phosphorylation), often leading to activation.

  • Protein Phosphatases: Enzymes that remove phosphate groups (dephosphorylation), turning off signals.

  • Phosphorylation cascades can rapidly amplify the signal, leading to a significant cellular response. Sequential activation of protein kinases, where one kinase activates the next. A small initial signal is magnified, allowing for a robust and rapid cellular response. Critical for both signal amplification and eventual signal termination.

• GTP-Binding Proteins: Function by binding and hydrolysing GTP. They possess GTPase activity, which allows them to hydrolyze GTP to GDP, thereby turning themselves off. These proteins toggle between:

  • Active State: Bound to GTP.

  • Inactive State: Bound to GDP.

  • Large Heterotrimeric G-Proteins: Typically associated with GPCRs, mediating signals from the cell surface.

  • Small Monomeric GTPases: Involved not only in signaling but also in processes like vesicular trafficking.

• The dynamic switching is essential for both activating and deactivating the signalling cascade.

describe the role, function and interaction domains of adaptor proteins

• Role: Serve as molecular docking platforms rather than having catalytic activity.

• Function:

  • Complex Assembly: Help bring together different signalling molecules.

  • Mediating Crosstalk: Enable interactions between different signalling pathways.

• Key Domains:

  • SH2 Domains: Bind to phosphotyrosine residues.

  • SH3 Domains: Bind to proline-rich sequences.

describe the mechanisms and importance of signals to turn off the transduction pathway

• Importance of Deactivation: For the cell to be ready for new signals, the active signalling molecules must be inactivated.

• Mechanisms:

  • Dephosphorylation: Removal of phosphate groups by phosphatases.

  • GTP Hydrolysis: Conversion of GTP to GDP in GTP-binding proteins.

• Resetting the pathway to ensure controlled, timely responses and prevent overactivation.

What are the Characteristic Features of Signal Pathways?

• Integration: Combining information from various signals.

  • The coordination and interaction of multiple signaling pathways.

  • Allows cells to combine different types of information to finely tune the cellular response.

  • Ensures that the cell’s response is balanced and context-specific.

• Convergence: Different receptors can trigger a common downstream response.

  • Signals from different independent receptors can merge to activate a common effector.

  • This redundancy increases the robustness of the signaling system, ensuring that essential responses are activated even if one pathway is compromised.

• Divergence: A single signal may activate multiple pathways.

  • A single extracellular ligand can activate multiple downstream effectors.

  • Leads to a variety of cellular responses from one signaling event.

  • Demonstrates how complex and versatile cellular responses can be, even from a single signal.

• Crosstalk: Overlapping components allow for coordinated regulation across pathways.

  • Occurs when the same signaling molecules participate in more than one signaling pathway.

  • Allows for communication and coordination between different pathways.

  • Enhances the cell’s ability to respond to a dynamic and changing environment.

describe the role of protein acylation

• Fatty Acid Acylation Activates Signalling Molecules Upon Translocation to the Cell Membrane

• Post-Translational Modification: Fatty acid acylation attaches fatty acid chains to proteins.

• Functions:

  • Membrane Anchoring: Helps anchor signaling proteins to the cell membrane.

  • Stabilization: Enhances protein-protein interactions.

  • Regulation: Modulates enzyme activities, particularly within mitochondria.

• Mutations that prevent acylation can impair or reduce the biological function of these proteins.

• Specific Example: Ras protein requires lipid modifications.

  • Farnesylation: A type of prenylation that adds a farnesyl group.

  • Acylation: Further modification that ensures proper membrane attachment.

  • These modifications are crucial for anchoring Ras to the Cell Membrane, where it can effectively participate in signal transduction.

what are the types of plasma membrane receptors?

• lon channel-coupled

• G-protein-coupled (guanine nucleotide-binding proteins)

• Receptors with enzymatic activity:

  • Receptors with kinase activity: - with tyrosine kinase - with serine/threonine kinase - dual - specific

  • Receptors with protein phosphatase activity

  • Receptors with guanylate cyclase activity

• Receptors associated with the functions of other tyrosine kinases

• Death-receptors associated with apoptosis

• Integrins (receptors regulating cell adhesion)

give the definition, function, significance and example of ion channel-coupled receptors

• Receptors that form part of or are directly linked to ion channels.

• Function: Change the permeability of the cell membrane by opening or closing ion channels in response to ligand binding.

• Significance: Key in rapid signal transmission, particularly in neuronal communication

• E.g. Acetylcholine Receptors

  • Structure: Comprised of several subunits (α₂, β, γ, δ) that assemble to form a functional ion channel.

  • Post-Translational Modification: The β, γ, and δ subunits can be phosphorylated by protein tyrosine kinases.

  • Classes:

    • Muscarinic receptors: Respond to muscarine.

    • Nicotinic receptors: Respond to nicotine.

  • Mechanism: Ligand binding triggers Na⁺ influx and K⁺ efflux, leading to depolarization and initiation of an action potential.

give the definition, function, structure and significance of G-Protein-Coupled Receptors (GPCRs)

• A large and diverse group of receptors that activate intracellular G-proteins.

• Function: Transduce extracellular signals into intracellular responses via second messengers.

• Importance: Involved in many physiological processes, from sensory perception to hormonal responses.

• Characterized by seven transmembrane alpha-helices.

• Possess extracellular ligand-binding domains and intracellular domains that interact with G-proteins.

• Structural Variations: Different classes have unique structural motifs that determine ligand specificity and signalling mechanisms.

GPCR Classification

• Family A (Class I):

  • Largest group; contains conserved regions.

  • Notable feature: Palmitoylated cysteine at the C-terminus.

  • Examples: Rhodopsin, adrenergic, histaminergic, dopaminergic, muscarinic, and tachykinin receptors.

• Family B (Class II):

  • Features a long N-terminal domain with six conserved cysteines forming disulfide bridges.

  • Examples: Glucagon, calcitonin, secretin, and PTH receptors.

  • Signalling: Often activates adenylate cyclase via Gs-protein.

• Family C (Class III):

  • Binds neurotransmitters like glutamate or GABA.

  • Roles include the regulation of Ca²⁺ metabolism and taste.

  • Includes metabotropic glutamate receptors (mGluR) with allosteric sites that are potential drug targets for disorders such as Parkinson’s and schizophrenia.

General Overview of a G-Protein Signalling Pathway

• Key Components:

  1. Membrane Receptor: Activated by an extracellular ligand.

  2. G-Protein: Activated by the binding of GTP.

  3. Effector Protein: Receives signals from the G-protein (e.g., adenylate cyclase).

  4. Second Messenger: Small molecules (e.g., cAMP) produced upon effector activation.

• Upstream: Refers to the receptor.

• Downstream: Refers to the effector and subsequent signaling events.

describe the composition and role of Heterotrimeric G-Proteins

• Composition:

  • Consist of three subunits: α, β, and γ.

• Roles of Subunits:

  • α-Subunit: Determines effector specificity; binds GTP.

  • β- and γ-Subunits: Involved in regulation of enzymes (e.g., phospholipase A₂, phospholipase C-β) and ion channels.

• Signaling Diversity:

  • Different combinations of subunits allow for a variety of signaling outcomes.

• Main G-Protein Families:

  • Gsα - activates adenylate cyclase and Ca2+ channels in all tissues. Increase cAMP. It is a substrate of cholera toxin.

  • Giα - activates PLC and PLA2 and K+ channels and inhibits cAMP, adenylate cyclase and Ca2+ channels

  • Gqα - activates PLC, found in all tissues. Increases DAG. IP3.

  • G12α - found in all tissues, regulates Nat/H+ antiport, electrical Ca2+ dependent channels, eicosanoid cell signals, and activates Rho proteins.

  • Other G-proteins: G11, G14, G15, G16 (G15 and G16 are in hematopoietic tissues).

describe the 3 main pathways of G-Protein signalling

• The mammalian genome encodes ~20 different α-subunits, 6 β-subunits, and 12 γ-subunits.

• Activation of Adenylate Cyclase (AC) ↑ cAMP

  • Hormones: ß1-Adrenergic agonists (adrenaline, noradrenaline), calcitonin, PTH, ADH, TSH, FSH, ACTH, LH, glucagon.

• Activation of Phospholipase C (PLC) ↑ IP3 & DAG

  • Hormones: α1-Adrenergic agonists (adrenaline, noradrenaline), oxytocin, hypothalamic hormones, eicosanoids.

• Inhibition of Adenylate Cyclase & Activation of Phosphodiesterase ↓ cAMP

  • Hormones: α2-Adrenergic agonists (adrenaline, noradrenaline).

Blockade of G-Protein Signalling

• by Bacterial Toxins

• Cholera Toxin:

  • Mechanism: ADP-ribosylates Gsα, preventing GTP hydrolysis.

  • Outcome: Persistent activation of Gs leads to increased cAMP and excessive secretion of Cl⁻ ions, resulting in severe diarrhea.

• Pertussis Toxin:

  • Mechanism: ADP-ribosylates Giα, inhibiting its normal receptor interactions.

  • Outcome: Inhibits adenylate cyclase while activating other pathways (PLA₂, PLC), leading to disruption of normal hormone signaling.

What are the Receptors with Enzymatic Activity?

• Not all receptors rely solely on secondary messengers.

• Some have intrinsic enzymatic functions or are directly associated with enzymes.

• Main Groups:

  1. Receptors with protein kinase activity.

  2. Receptors with protein phosphatase activity.

  3. Receptors with guanylate cyclase activity.

• According to their localization, two groups of protein kinases are distinguished:

  • Receptor kinases in the membrane.

  • Free protein kinases in the cytosol.

• Depending on the amino acid they phosphorylate, protein kinases are divided into three groups:

  • protein tyrosine kinases

  • protein serine/threonine kinases

  • dual-specificity kinases

What are the Ligands for Receptor Tyrosine Kinases (RTKs)?

• Nerve Growth Factor (NGF)

• Platelet-Derived Growth Factor (PDGF)

• Fibroblast Growth Factor (FGF)

• Epidermal Growth Factor (EGF)

• Insulin and Insulin-like Growth Factor-1 (IGF-1)

• Ephrins (Eph)

• Vascular Endothelial Growth Factor (VEGF)

• Hepatocyte Growth Factor (HGF)

• Stem Cell Factor (SCF, binds to c-Kit receptor)

Key Steps in RTK-Mediated Signalling Pathways

• Activation Mechanism: Ligand binding induces dimerization and autophosphorylation (cross-phosphorylation) of tyrosine residues.

• Signal Propagation: Phosphorylated tyrosine residues serve as docking sites for downstream signalling proteins.

  • Outcome: Initiates a cascade of phosphorylation events that ultimately affect gene expression and cellular behaviour.

describe the MAPK (Mitogen-Activated Protein Kinase) Pathway

• Most ligands are cytokines, growth factors, hormones, and

  neurotransmitters, selectively activating these cascades.

• MAPK pathways operate through sequential phosphorylation, leading to transcription factor activation and gene expression regulation.

• Mitogen-Activated Protein Kinases (MAPKs) are a family of Serine/Threonine kinases involved in key cellular processes: Cell proliferation, Cell differentiation, Cell migration, Cell death (apoptosis)

describe the types of Ras and its link to cancer

• Ras Isoforms:

  • H-Ras, K-Ras, and N-Ras.

• Common mutations in Ras proto-oncogenes (found in ~25% of human tumors) often lead to loss of GTPase activity.

• Mutated Ras remains permanently active, promoting uncontrolled cell proliferation.

• K-Ras mutations, for example, are prevalent in colorectal carcinomas.

• Dysregulated Ras signaling is a key driver in oncogenesis.

give an example of Receptors with Protein Phosphatase Activity

• Key Example – CD45:

  • First characterized transmembrane protein tyrosine phosphatase.

  • Critical in T-cell activation by regulating the activity of the Src-family kinase Lck.

• Mechanism:

  • CD45 dephosphorylates the inhibitory tyrosine residue (Y505) on Lck, enabling its activation.

  • It can also dephosphorylate an activating residue (Y394) to maintain basal kinase activity.

• Biological Role: Ensures proper regulation of T-cell receptor signaling and immune response.

give the function and example of Receptors with Guanylate Cyclase Activity

• Function: Convert GTP to cyclic GMP (cGMP), a second messenger.

• Examples: Receptors for natriuretic peptides (ANP), including ANPR-A, ANPR-B, and ANPR-C.

• Physiological Relevance: Regulates cardiovascular functions such as vasodilation and blood pressure.

explain the difference between Primary vs. Secondary Messengers

• Primary messenger: The extracellular signal (e.g., hormones, neurotransmitters).

• Secondary mediators: Molecules that carry the signal inside the cell.

give the types and examples of secondary mediators

• Cyclic Nucleotides:

  • cAMP: Activates protein kinase A (PKA).

  • cGMP: Activates protein kinase G (PKG).

• Lipid Mediators:

  • Diacylglycerol (DAG): Activates protein kinase C (PKC).

  • Inositol-3-phosphate (IP3): Increases intracellular Ca²⁺ levels.

  • Additional lipids: Ceramide and sphingosine-1-phosphate.

• Calcium and Calmodulin:

  • Elevations in Ca²⁺, along with calmodulin, activate Ca²⁺/calmodulin-dependent protein kinases (types I, II, and III).

• Hormone-Receptor Complexes:

  • Example: Steroid hormones that directly affect gene expression.

describe the role, mechanism of action and receptor regulation of cAMP as a Key Second Messenger

• Fundamental Role:

  • First identified second messenger, critical for transmitting extracellular signals.

  • Involved in compartmentalizing signals within the cell.

• Mechanism of Action:

  • cAMP is synthesized from ATP and plays a pivotal role in regulating various cellular responses.

  • Selectively activates isoforms of protein kinase A (PKA) to modulate metabolism, gene transcription, and other processes.

• Receptor Regulation:

  • Multiple G protein–coupled receptors (GPCRs) such as adrenergic, CRH, and glucagon receptors influence cAMP levels.

describe the production of Adenylyl Cyclase (AC) and cAMP

• Enzymatic Conversion:

  • ATP is converted to cAMP by Class III adenylyl cyclases.

• Isoforms of AC:

  1. Transmembrane Adenylyl Cyclases (tmACs):

     • Humans have 9 types, regulated by GPCRs.

     • Play a critical role in responding to extracellular signals.

  2. Soluble Adenylyl Cyclase (sAC):

     • Not regulated by G-proteins.

     • Directly activated by intracellular Ca²⁺ and bicarbonate (HCO₃⁻), functioning as a metabolic sensor.

describe GPCR Signalling & Adenylyl Cyclase Regulation

• Signal Initiation:

  • Extracellular ligands (e.g., neurotransmitters, hormones, adrenaline) bind to GPCRs.

• G-Protein Regulation:

  • Gs Protein (Stimulatory): Activates adenylyl cyclase to increase cAMP production.

  • Gi Protein (Inhibitory): Inhibits adenylyl cyclase, reducing cAMP levels.

• Balances cellular responses by modulating the levels of the second messenger cAMP.

describe the mechanism of G-Protein Activation

• Inactive State

  • G-proteins are heterotrimeric, consisting of α, β, and γ subunits, and are bound to GDP in the inactive state.

  • Membrane Anchoring Achieved through post-translational modifications: prenylation of βγ and myristoylation of the α-subunit.

• Activation Process:

  • Ligand Binding: Causes a conformational change in the receptor.

  • GDP/GTP Exchange: GDP is replaced by GTP on the α-subunit.

  • Subunit Dissociation: The GTP-bound α-subunit dissociates from the βγ dimer and interacts with effector proteins (e.g., adenylyl cyclase).

describe Adrenaline Activation of the Adenylyl Cyclase System

• Inactive State:

  • G-protein α-subunit is bound to GDP.

• Activation:

  • Adrenaline binds to its receptor.

  • Triggers GDP-to-GTP exchange; the α-subunit activates adenylyl cyclase.

• cAMP & PKA Activation:

  • Newly formed cAMP binds to the regulatory subunits of the PKA tetramer.

  • Catalytic subunits are released and phosphorylate various substrates, including proteins that translocate to the nucleus to affect gene expression

• Initiates metabolic and transcriptional responses, such as glycogenolysis.

describe cAMP & Transcription Regulation

• Role of CREB:

  • cAMP influences gene transcription primarily via the cAMP response element-binding protein (CREB).

• Mechanism:

  • PKA phosphorylates CREB on a serine residue, enhancing its transcriptional activity.

    • Activated CREB binds to the cAMP response element (CRE) in DNA, leading to the transcription of genes involved in catabolic pathways (e.g., glycolysis, glycogenolysis, and lipolysis).

describe Adrenaline Activation of Glycogenolysis

• Pathway: Adrenaline triggers a cAMP-dependent signaling cascade.

• Mechanism:

  • Binding of adrenaline to its receptor elevates cAMP levels.

  • Activation of PKA leads to phosphorylation of key enzymes that catalyse the breakdown of glycogen.

• Physiological Effect: Provides rapid energy mobilization during stress or increased energy demand.

list the enzymes regulated by phosphorylation by PKA and their pathway

• Glycogen Synthase - glycogen synthesis

• Phosphorylase Kinase - glycogen breakdown

• Pyruvate Kinase - Glycolysis

• Pyruvate Dehydrogenase - Pyruvate to acetyl-CoA

• Hormone-sensitive Lipase - Triacylglyeride breakdown

• Tyrosine Hydroxylase - Synthesis of DOPA, dopamine, norepinephrine

• Histone H1 - Nucleosome formation with DNA

• Histone H2B - Nucleosome formation with DNA

• Protein phosphatase 1 Inhibitor 1 - Regulation of protein dephosphorylation

• CREB - cAMP regulation of gene expression

• PKA cosensus sequence - XR(R/K)X(S/T)B (B = hydrophobic amino acid)

describe the formation, isoforms and mechanism of Nitric Oxide (NO) signalling

• NO is a short-lived gas & free radical, involved in various physiological & pathological processes

• Synthesized from L-arginine via NO synthase (NOS), producing L-citrulline as a by product

• NOS Isoforms:

  • nNOS (Type I, neuronal) & eNOS (Type III, endothelial)

    • Constitutively expressed (latent enzymes)

    • Require high Ca2+ levels for activation

  • iNOS (Type II, inducible)

    • Ca2+-independent due to strong binding to Ca2+/calmodulin

• NO Mechanism:

  • Diffuses into neighboring cells → activates soluble guanylate cyclase (sGC).

  • Increases cGMP levels, regulating enzymes & ion channels.

  • L-arginine → L-citrulline + NO

describe the Regulation of eNOS Activity

• eNOS Function: Endothelial nitric oxide synthase (eNOS) produces NO, a critical regulator of vascular tone.

• Stimuli: Shear stress, acetylcholine, VEGF, bradykinin, estrogen, S-1P, H2O2, and angiotensin-II stimulate eNOS activity.

• Regulatory Mechanisms:

  • Localization: eNOS is targeted to caveolae in the endothelial cell membrane via myristoylation and palmitoylation.

  • Inhibition: Caveolin-1 binds to and inhibits eNOS.

  • Activation:

    • Calmodulin (CaM) displaces Caveolin-1 when Ca²⁺ levels rise.

    • Heat shock protein 90 (Hsp90) and Akt-mediated phosphorylation further enhance eNOS activity.

Cellular Signaling Cascades Regulating eNOS Activity

1. Shear Stress:

   • Activates G-proteins leading to PI3K, PDK, and the cAMP pathway, which phosphorylate and activate eNOS.

2. Growth Factors & Hormones:

   • VEGF, estrogen, S1P, and bradykinin trigger PI3K/Akt and PLC-γ pathways, increasing intracellular Ca²⁺ and DAG.

3. Metabolic Stress:

   • ATP breakdown activates cAMP-dependent PKA, which phosphorylates eNOS.

4. Protein Interactions:

   • Proteins like Dynamin-2 and Porin interact with eNOS to promote its Ca²⁺-dependent activation.

5. Substrate Availability:

   • Efficient arginine uptake is necessary as arginine directly interacts with eNOS in caveolae.

describe the Biological Role of eNOS

• NO Production: eNOS synthesizes nitric oxide (NO) in endothelial cells.

• Physiological Effects of NO:

  • Acts as a potent vasodilator, helping lower blood pressure.

  • Inhibits platelet aggregation, leukocyte adhesion, and vascular smooth muscle proliferation.

  • Plays a role in preventing angiogenesis.

• Dysregulation of eNOS activity is linked to vascular diseases such as atherosclerosis and hypertension.

• Acetylcholine-mediated eNOS activation leads to NO release, which diffuses into smooth muscle cells to induce relaxation and increase blood flow.

• Nitroglycerin induces vasodilation

describe the role of NO in the Relaxation of Smooth Muscle Cells

• NO diffuses from endothelial cells into adjacent smooth muscle cells.

• It activates soluble guanylate cyclase, converting GTP into cGMP.

• Elevated cGMP levels induce smooth muscle relaxation and vasodilation.

• This mechanism is the basis for the action of nitroglycerin, used to treat angina by dilating blood vessels.

• Regulation: Phosphodiesterases (PDEs) degrade cGMP to GMP, ensuring that the signal is transient.

• Sildenafil (Viagra) inhibits PDE5, preventing cGMP breakdown and prolonging its vasodilatory effects.

Describe the Extracellular Fate of Nitric Oxide

• NO reacts non-enzymatically with O₂ and H₂O in the extracellular milieu.

• Products: Nitrate (NO₃⁻) and nitrite (NO₂⁻).

• Physiological significance: These stable anions serve as reservoirs for NO bioactivity and can be recycled back to NO under hypoxic conditions

NO-Mediated Toxicity & Peroxynitrite Formation

• NO + O₂⁻ → Peroxynitrite (ONOO⁻)

• Peroxynitrite effects:

  • DNA damage: Causes strand breaks and base modifications leading to fragmentation.

  • Lipid peroxidation: Initiates free-radical chain reactions in membranes, compromising integrity and fluidity.

• Overproduction of ONOO⁻ is implicated in inflammatory diseases, neurodegeneration, and ischemia–reperfusion injury.

Mitochondrial Impact of Peroxynitrite

• Targets within the Electron Transport Chain (ETC):

  • Complexes I–IV are susceptible to nitration and oxidation, impairing electron flow and ATP synthesis.

• Effect on MnSOD (Mitochondrial Superoxide Dismutase):

  • ONOO⁻ inactivates MnSOD, diminishing mitochondrial antioxidant defense.

• Enhanced ROS production: Accumulation of O₂⁻ and H₂O₂ exacerbates oxidative stress, triggering mitochondrial permeability transition and apoptosis.

Regulation of Protein Activity by S-Nitrosylation

• NO covalently modifies thiol (–SH) groups on cysteine residues to form S-nitrosothiols (RSNOs).

• Alters protein conformation, activity, localization or interactions—akin to phosphorylation.

• S-nitrosylation of ion channels, receptors, and enzymes modulates cardiovascular tone, neurotransmission, and immune responses. ​

Stability & Reversibility of S-Nitrosothiols

• Chemical Lability: RSNO bonds are unstable — sensitive to light and oxidative environments, leading to spontaneous NO release.

• Enzymatic Denitrosylation:

  • Thioredoxin/Thioredoxin Reductase (Trx/TR) System: Catalyzes removal of the –NO group, restoring the free thiol and terminating the nitrosylation signal.

  • Provides dynamic control over S-nitrosylation-dependent signaling pathways.

describe the the role of iNOS and NO in Innate Immunity

• Inducible Nitric Oxide Synthase (iNOS): Expressed in macrophages in response to infection or inflammatory signals.

• Role in Immunity:

  • High levels of NO are produced to combat pathogens and tumor cells.

  • NO, in conjunction with superoxide, acts as a potent antimicrobial and cytotoxic agent.

• Microbial components such as bacterial and fungal cell wall elements stimulate iNOS expression.

Activation of iNOS Expression

1. Bacterial & Fungal Cell Wall Components

• LPS (lipopolysaccharides) from Gram-negative bacteria bind to LBP (LPS-binding protein).

  • SOCS1 (Suppressor of Cytokine Signaling 1) inhibits the signaling cascade triggered by LPS.

  • This serves as a regulatory mechanism to prevent overactivation of the immune response and limit excessive NO production.

• CD14 (LPS receptor) on macrophages/neutrophils interacts with TLR4 (Toll-like receptor 4) and MD2.

• This triggers intracellular signaling cascades via adaptor proteins.

• TRAF6 & p38 are activated, leading to IKK (IκB kinase) phosphorylation.

• NF-KB is released, translocates to the nucleus, and induces iNOS transcription.

  • Normally, NF-κB is trapped in the cytoplasm because it is bound to a protein called IκB (inhibitor of NF-κB).

  • IKK phosphorylates (adds phosphate groups to) IκB.

  • Phosphorylated IκB is marked for destruction by the proteasome.

  • Once IκB is destroyed, NF-κB is free to move into the nucleus.

  • In the nucleus, NF-κB turns on genes related to inflammation, immune response, and cell defense.

2. Cytokines from Infected Cells

• Interferons (IFNs) exhibit antiviral, immunostimulatory, antiproliferative, and antitumor effects.

• IFN-α - secreted by leukocytes.

• IFN-ß - secreted by fibroblasts.

• IFN-γ - produced by lymphocytes, activates JAK-STAT signaling.

• Leads to IRF1 (Interferon Response Factor-1) synthesis, stimulating iNOS mRNA transcription.

give the Calcium Concentration Ranges

• Extracellular Ca²⁺: Approximately 5 mmol/L.

• Free intracellular Ca²⁺: Ranges from 0.1 to 10 μmol/L.

• Organelle-bound Ca²⁺ (e.g. mitochondria and ER): Ranges from 1 to 20 μmol/L

describe the mechanisms for Maintaining Low Cytosolic Ca²⁺

• Ca²⁺-ATPase: Enzymes such as SERCA (in the ER) and PMCA (at the plasma membrane) pump Ca²⁺ out of the cytosol using ATP hydrolysis.

• Ca²⁺-Binding Proteins:

  • Calbindin in the cytosol.

  • Calreticulin in the ER

  • both act as buffers

• Na⁺/Ca²⁺ Exchanger (NCX):

  • Particularly active in excitable cells (e.g., muscle and neurons).

  • Uses the Na⁺ electrochemical gradient to export Ca²⁺ when intracellular levels rise significantly.

Ca²⁺-Mediated Signalling Pathway in Excitable Cells

• in Neurons, Muscle, Pancreatic Endocrine Cells

• Depolarization: Opens voltage-dependent Ca²⁺ channels (VDCC).

• Triggers neurotransmitter and hormone secretion.

• Induces muscle contraction.

• Example: In pancreatic β-cells, the closure of K⁺-ATP channels causes membrane depolarization that opens VDCC.

Ca²⁺-Mediated Signalling Pathway in Non-Excitable Cells

• Receptor-Ligand Binding: Activates Ca²⁺ entry through voltage-independent Ca²⁺ channels (VICC).

• IP₃ Pathway:

  • Activation of receptors generates inositol trisphosphate (IP₃).

  • IP₃ binds to ER receptors, releasing Ca²⁺ into the cytosol.

Short-Term Cellular Responses of Ca²⁺ as an Intracellular Second Messenger

• Excitation-Contraction Coupling: Critical for skeletal and cardiac muscle function.

• Stimulus-Secretion Coupling: Essential for endocrine and neuronal cells.

• Mechanisms Involved:

  • Post-translational modifications (for example, phosphorylation of ion channel subunits).

  • Activation of Ca²⁺-dependent kinases such as CaM-kinase II and protein kinase C (PKC).

  • Stimulation of Ca²⁺-sensitive adenylyl cyclases that lead to cAMP signalling.

Long-Term Cellular Responses of Ca²⁺ as an Intracellular Second Messenger

• Gene Expression: Influences cell proliferation, differentiation, apoptosis, and neuronal adaptation.

• Regulation:

  • Involves Ca²⁺-regulated proteins such as hormones, neuropeptides, ion channels, and oncogenic proteins.

  • Ca²⁺-responsive gene elements modulate transcription.

• Low cytosolic/ER Ca²⁺ can inhibit cell proliferation.

Calmodulin (CaM) and Its Cellular Functions

• CaM is a Ca²⁺-dependent protein predominantly located in the cytosol.

• Structurally and functionally analogous to troponin C found in muscle tissue.

• Frequently acts as a regulatory subunit within various protein complexes.

• Modulates the activity of several kinases, enzymes involved in cyclic nucleotide metabolism, and proteins responsible for ion transport.

• Regulated Cellular Processes:

  • Regulation of the actin-myosin complex critical for smooth muscle contraction.

  • Control over microfilament-mediated activities, including:

    • Cell motility.

    • Mitosis.

    • Granule exocytosis.

    • Endocytosis.

• Additional Ca²⁺-Binding Proteins Mentioned:

  • Annexins, sorcin, light myosin chains, beta-actin, calcineurin, and calpain are listed as other proteins that bind calcium.

List the Enzymes Regulated by Ca²⁺/Calmodulin

• Cyclases:

  • Adenylate cyclase and guanylate cyclase, which synthesize cyclic nucleotides.

• Protein Kinases:

  • Ca²⁺-dependent protein kinase, myosin kinase, cAMP-dependent protein kinase.

• ATPases and Phospholipases:

  • Ca²⁺/Mg²⁺-ATPase, phospholipase A2.

• Other Kinases & Phosphorylases:

  • Ca²⁺/phospholipid-dependent protein kinase, phosphorylase kinase.

• Phosphatases and Other Enzymes:

  • Phosphoprotein phosphatase 2B.

  • Phosphodiesterase (breaks down cyclic nucleotides).

  • Metabolic enzymes such as pyruvate carboxylase, glycerol-3-phosphate dehydrogenase, pyruvate dehydrogenase, glycogen synthase, and pyruvate kinase.

Key Pathways Producing Lipid Mediators

• Phospholipase A2 (PLA2) Route:

  • Involves the release of arachidonic acid (AA) from membrane phospholipids.

• Phosphatidylinositol Route:

  • Involves the generation of inositol phosphates (through the phospholipase C pathway) and subsequent signals.

• Sphingomyelin System:

  • Leads to the production of signalling molecules such as ceramide and sphingosine-1-phosphate (S1P).

Arachidonic Acid Pathway & Lipid Mediators

• Arachidonic Acid (AA): A polyunsaturated fatty acid released from membrane phospholipids through PLA2 activity.

• Cyclooxygenase (COX) Pathway:

  • Isoforms:

    • COX-1: Constitutively expressed.

    • COX-2: Inducible under inflammatory conditions.

  • Converts AA into prostaglandins, prostacyclins (PGI₂), and thromboxanes (TXA).

• Lipoxygenase (LOX) Pathway:

  • AA is converted to 5-HPETE.

  • 5-HPETE is further metabolized to Leukotriene A₄ (LTA₄).

  • Leukotriene Derivatives:

    • LTB₄: Promotes inflammation by inducing chemotaxis and degranulation of polymorphonuclear leukocytes.

    • LTC₄, LTD₄, LTE₄: Involved in vasoconstriction and bronchospasm, playing roles in asthma and anaphylaxis.

• Lipoxins, which are also derived from AA, act as anti-inflammatory mediators

Phosphatidylinositol Pathway of Phospholipase C

• Involves cleavage of membrane phospholipids to generate diacylglycerol (DAG) and inositol-3-phosphate.

• Plays a key role in cellular signalling via second messengers.

Phosphatidylinositol-3 Kinase (PI3K) Pathway

• PI3K is a Heterodimer - Comprised of 2 subunits

  • a regulatory subunit (p85, 85 kDa, contains SH2 domains, interacts with tyrosine-phosphorylated receptors or PTKs)

  • a catalytic subunit (p110, 110 kDa, enzymatic function, phosphorylates substrates.s

• Phosphorylates phosphatidylinositols at the 3′ position on the inositol ring.

• Activation: Triggered by receptor tyrosine kinases (RTKs) such as those for PDGF, EGF, Insulin, IGF-1, HGF, and NGF.

• Downstream Signaling Events:

  • PI3K generates PIP3 (Phosphatidylinositol 3,4,5-triphosphate)

  • PIP3 activates PDK1, which binds to the PH domain of PKB/Akt

  • Cellular Outcomes: Regulation of adhesion, chemotaxis, apoptosis, secretion, glucose transporter translocation, platelet activation, and reorganization of the cytoskeleton.

• Regulation by PTEN:

  • PTEN (Phosphatase and Tensin Homolog) dephosphorylates PIP3 back to PIP2.

  • This activity serves as a negative regulatory mechanism, reducing Akt activation and modulating cell survival and proliferation.

  • Ligand binding to membrane receptors activates PI3K

  • PI3K phosphorylates PIP2 (PI(4,5)P) → forms PIP3 (PI(3,4,5)P)

  • PIP3 recruits PDK1 to the plasma membrane

  • PDK1 phosphorylates and activates AK1 (PKB), regulating various cellular processes

  • PTEN (Lipid Phosphatase) dephosphorylates PIP3 → increases PIP2 levels

  • This reduces AKT activity, modulating cell signaling.

mTOR (Mammalian Target of Rapamycin)

• mTOR: A serine/threonine kinase that functions as a central regulator in the PI3K/Akt pathway.

• Roles: Controls cell growth, proliferation, metabolism, survival, and protein synthesis.

• Dysregulation: Overactivity of mTOR can lead to tumorigenesis by promoting anabolic processes while inhibiting autophagy.

• mTOR Inhibitors: Rapamycin (Sirolimus) and its analogs are employed both as immunosuppressants (to prevent transplant rejection) and as targeted cancer therapies.

• mTOR Complexes:

  • mTORC1:

    • Rapamycin-sensitive

    • Sensor for nutrients and energy status.

    • Activated by PI3K/Akt in response to insulin, IGF-1, IGF-2, PDGF, EGF, HGF.

    • Requires amino acids, ATP, oxygen; inhibited by low ATP (AMPK activation) & hypoxia.

    • Regulates protein and lipid metabolism and suppresses autophagy.

    • Target in cancer therapy (Rapamycin analogs).

  • mTORC2:

    • Regulation of the cytoskeleton and cell survival (e.g. via Akt Ser473 phosphorylation)

    • Does not respond to nutrients

Ceramide in Apoptosis Regulation

• Role as a Secondary Messenger: Ceramide is crucial in the initiation phase of apoptosis.

• Extrinsic Apoptotic Pathway:

  • Mediated by tumor necrosis factor (TNF) receptors and the formation of a death domain (DD) signalling complex that activates transcription via the JNK pathway

• Intrinsic Apoptotic Pathway:

  • Alters mitochondrial function in response to stress signals (UV radiation, oxidative stress, ionizing radiation, heat shock)

• Some tissues may undergo apoptosis with minimal ceramide contribution.

• In disorders such as Niemann–Pick disease (associated with a sphingomyelinase defect), a deficiency in ceramide leads to diminished apoptosis.

Sphingosine-1-Phosphate (S1P) Signalling

• Synthesis and Activation:

  • Formation of S1P: Sphingosine is phosphorylated by sphingosine kinase 1 (SphK1).

  • Stimulatory Factors for SphK1: Growth factors, chemokines, oxidative LDL, ATP, acetylcholine, vitamin D3, and several cytokines (such as PDGF, VEGF, TNF-α, EGF, and basic fibroblast growth factor).

• Autocrine/Paracrine Action: S1P is secreted and acts via its G-protein-coupled receptors (S1PRs) on the cell surface.

• Receptor Cross-Talk: Activation of the PDGF receptor can lead to subsequent activation of FAK, Src, and Rac; these signals promote cell migration.

• Downstream Pathways:

  • Extracellular S1P: Activates S1PR1, which in turn triggers the JAK2/STAT3 pathway; this pathway regulates the expression of IL-6 and S1PR1.

  • Intracellular S1P: Can activate NFκB, which feeds back to regulate SphK1 expression.

• The sustained activation through the S1P-STAT3-NFκB loop may drive cell transformation and tumor progression.

give the definition and function of a hormone

• Any substance in the body that transmits a signal and leads to a change at the cellular level (a change in cellular behaviour)

• Hormones are biochemical messengers produced by endocrine glands or cells and released into the bloodstream.

• They trigger specific changes in target cells that possess the appropriate receptors.

• Classification by solubility:

  • Hydrophobic - Cross membranes easily

  • Hydrophilic - Require membrane receptors

Classification of hormones and signalling molecules based on their chemical structure

• Peptide and Protein Hormones:

  • Examples: Insulin, Glucagon, Growth Hormone (GH), Adrenocorticotropic Hormone (ACTH).

• Steroid Hormones:

  • Examples: Cortisol, Aldosterone, Estrogens (e.g., Estradiol), Androgens (e.g., Testosterone).

• Amino Acid-Derived Hormones:

  • Examples: Thyroid Hormones (T3, T4), Catecholamines (e.g., Epinephrine, Norepinephrine, Dopamine), Melatonin.

• Lipid-Derived Hormones (Eicosanoids):

  • Examples: Prostaglandins, Leukotrienes, Thromboxanes.

• Glycoprotein Hormones:

  • Examples: Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), Thyroid-Stimulating Hormone (TSH).

• Gaseous Signaling Molecules:

  • Examples: Nitric Oxide (NO), Carbon Monoxide (CO), Hydrogen Sulfide (H₂S).

Describe the main groups of hormones by Action and Properties

• Group I Hormones:

  • Types: Steroids, iodothyronines (e.g., thyroid hormones), calcitriol, retinoids.

  • Solubility: Lipophilic.

  • Transport: Often require binding to plasma transport proteins.

  • Plasma Half-Life: Generally long (hours up to days).

  • Receptor Localization: Act on intracellular receptors.

  • Secondary Mediators: Typically do not require classic secondary messengers.

• Group II Hormones:

  • Types: Polypeptides, proteins, glycoproteins, catecholamines.

  • Solubility: Hydrophilic.

  • Transport: Circulate freely in plasma without specific binding proteins.

  • Plasma Half-Life: Short (minutes).

  • Receptor Localization: Act via receptors on the plasma membrane.

  • Secondary Mediators: Often signal through systems such as cAMP, cGMP, Ca²⁺, lipid mediators, or kinase cascades.

Pituitary Gland & Hypothalamic Control

• Pituitary Anatomy:

  • Composed of two lobes:

    1. Adenohypophysis (Anterior Lobe).

    2. Neurohypophysis (Posterior Lobe).

• Hypothalamic Influence:

  • The hypothalamus synthesizes releasing hormones that are secreted into the portal circulation of the anterior pituitary

  • These releasing hormones activate intracellular signalling pathways (involving Ca²⁺, IP₃, and PKC) leading to exocytosis of pituitary hormones

• The released tropic hormones regulate growth and activity in target endocrine glands, forming the basis of the hypothalamic–pituitary axis.

Molecular Characteristics of Trophic - Anterior Pituitary Hormones

• The anterior pituitary secretes tropic hormones which regulate growth and secretion in other endocrine glands.

• Glycoprotein Hormones (TSH, FSH, LH):

  • Composed of two peptide chains:

    • β-chain: Specific and Biologically active

    • α-chain: Identical in all three hormones.

• Growth Hormone & Prolactin:

  • Structurally similar; both are gene duplicates (~200 amino acids) with similar sequences.

• POMC-Derived Hormones:

  • Precursor molecule is processed via specific proteolysis to yield multiple biologically active hormones such as ACTH, β-endorphin, β-lipotropin, α-MSH, and β-MSH.

Hypothalamic-Pituitary-Thyroid Axis

• Hypothalamus: Produces thyrotropin-releasing hormone (TRH).

• TRH is transported via portal circulation to the pituitary

• Pituitary: In response to TRH, the adenohypophysis secretes thyroid-stimulating hormone (TSH)

• Thyroid Gland: TSH stimulates the thyroid to produce and release thyroid hormones (T4 and T3).

• The hypothalamic TRH prompts intracellular signalling in the pituitary, preparing the gland to release TSH, which then influences thyroid function.

• TRH

  • Structure: Modified tripeptide, synthesized as a prohormone.

  • Stimulates TSH subunit biosynthesis & glycosylation.

  • Receptor downregulation via negative feedback from TSH & thyroid hormones.

Intracellular Signalling Pathways of TRH

• TRH receptor (TRH-R) activation → Stimulates phospholipase C (PLC).

• InsP3 production → Triggers Ca2+ release from intracellular stores → TSH exocytosis

• Key signalling cascades:

  • Ca2+/Calmodulin-dependent kinase (Ca2+/CamKin)

  • Protein kinase C (PKC)

  • MAPK pathway

• Transcription factors activated: CREB, AP-1, Elk-1.

TSH Receptor and Signal Transduction

• Structure of the TSH Receptor (TSHR): Glycoprotein receptor with three domains:

  • Extracellular domain: Binds thyroid-stimulating hormone (TSH).

  • Transmembrane domain: Spans the plasma membrane (7-transmembrane G protein-coupled receptor—GPCR).

  • Intracellular (cytoplasmic) domain: Interacts with G proteins to initiate intracellular signaling cascades.

• TSH binds to the extracellular domain of TSHR → triggers a conformational change → activates G proteins (mainly Gs and Gq subtypes).

• Intracellular Signaling Pathways Activated:

  • cAMP pathway (via Gs protein): Activates adenylyl cyclase → ↑ cAMP → activates PKA → Stimulates transcription of genes involved in thyroid hormone synthesis and thyrocyte proliferation

  • Phospholipase C pathway (via Gq protein): Activates PLC → ↑ IP₃ (releases Ca²⁺ from ER) and DAG (activates PKC → modulates protein phosphorylation and secretion)

• Key Cellular Effects of TSH:

  • Stimulates:

    • Iodide uptake via sodium-iodide symporter (NIS).

    • Thyroglobulin (Tg) synthesis and iodination.

    • Formation of iodothyronines (T3, T4) within the colloid.

    • Proteolysis of thyroglobulin → releases T3 and T4 into circulation.

    • Peripheral deiodination of T4 to active T3 (modulation of local thyroid hormone levels).

  • Promotes:

    • Thyroid follicular cell growth and division (trophic effect).

• Negative Feedback Regulation of T3/T4:

  • Suppress TSH gene expression and secretion at the pituitary level.

  • Act through thyroid hormone receptors (TRs) in the pituitary to:

    • Inhibit TSH β-subunit transcription.

    • Reduce glycosylation and processing of TSH.

  • This feedback ensures homeostatic control of thyroid hormone levels

Thyroid Hormones (T4 & T3) Production & Regulation

• Synthesis:

  • Thyroglobulin Iodination:

    • Thyroid hormones are produced by iodinating tyrosine residues within thyroglobulin (Tg), which is then stored in the colloid and hydrolyzed to release T4 and T3.

  • T4 (thyroxine) is produced in larger amounts compared to T3 (triiodothyronine)

• Converstion by Deiodination:

  • T4 is converted to the more active T3 through deiodination processes.

  • Depending on the location (thyroid itself, target tissues, or peripheral tissues), different deiodination pathways modify T4 into active T3 (by 5’ deiodination) or the inactive reverse T3 (rT3, by 5-deiodination).

• Secretion:

  • T4 and T3 are released via Tg hydrolysis (colloid droplets)

• Iodide Recycling:

  • Iodide is recovered for reuse after hormone release.

• Daily Tg turnover

  • 100 micrograms released unchanged

  • only 25 micrograms hydrolysed to meet T4 demand

Thyroid Hormone Metabolism: Deiodinases

• Type I Deiodinase (D1):

  • Catalyzes both 5’ (activating) and 5-deiodination (inactivating) reactions.

  • Found in liver, kidney, thyroid, pituitary, and CNS.

  • Exhibits a high Km for T4, producing both T3 and rT3; activity is increased in hyperthyroidism.

• Type II Deiodinase (D2):

  • Catalyzes 5’-deiodination exclusively to produce active T3.

  • Present in the brain, brown adipose tissue, placenta, and pituitary.

  • Has a lower Km than D1, helping to maintain intracellular T3 levels; its activity can increase in hypothyroidism.

• Type III Deiodinase (D3):

  • Catalyzes 5-deiodination, effectively inactivating T4 to rT3.

  • Mainly found in the brain and placenta to prevent excess T3 accumulation in sensitive tissues.

Metabolism/Degradation of T3 and T4

• T4 metabolism:

  • Sulfation, decarboxylation, deamination, glucuronidation.

  • Liver: T4 undergoes deamination & decarboxylation → forms TETRAC (tetraiodothyroacetate).

• T3 metabolism:

  • Sulfation (T3S).

  • Conversion to TRIAC (triiodothyroacetate).

• Half-life in serum:

  • T4 → 7 days.

  • T3 → 1 day.

Effects of Thyroid Hormones (TH) on Metabolism

• Impact on Lipid Metabolism:

  • Enhances fat mobilization, resulting in higher free fatty acid concentrations in the blood.

  • Stimulates fatty acid oxidation in various tissues.

• Impact on Carbohydrate Metabolism:

  • Increases glucose uptake in an insulin-dependent manner.

  • Promotes both gluconeogenesis and glycogenolysis to elevate blood glucose levels.

• Metabolic Rate Stimulation (except brain. spleen, testes):

  • Increases mitochondrial number and size as well as respiratory chain enzyme synthesis.

  • Upregulates Na⁺/K⁺ ATPase activity and affects membrane permeability, thereby influencing the metabolic rate.

Thyroid Hormones & Metabolic Regulation (BMR & REE)

• 15-40% of resting energy supports Na+/K+ ATPase activity.

• Basal Metabolic Rate (BMR) & Resting Energy Expenditure (REE):

  • TH play a critical role as regulators of BMR—excess TH can elevate BMR by up to 100%, while TH deficiency may drop it by around 50%.

  • They enhance overall metabolic activity (increased oxygen consumption, ATP hydrolysis, and heat production).

  • TH can alter mitochondrial functions, including uncoupling oxidative phosphorylation and modifying mitochondrial hydrogen shuttles, thereby affecting the energy usage of major organs.

  • REE (energy for major organs) is highly TH-sensitive. (BMR & REE differ by ~10% and are often used interchangeably.)

• Direct correlation between TH levels, energy expenditure, and body weight.

• Hyperthyroidism (Hypermetabolic state) ↑ REE, weight loss, ↓ cholesterol, ↑ lipolysis & gluconeogenesis.

• Hypothyroidism (Hypometabolic state) ↓ REE, weight gain, ↑ cholesterol, ↓ lipolysis & gluconeogenesis.

• TH regulate metabolism via brain, white & brown adipose tissue, skeletal muscle, liver, pancreas.

Thyroid Disorders

• Prevalence & Demographics:

  • Thyroid disorders affect approximately 3% of the population and are notably 9 times more common in women.

  • They can occur at any age.

• Autoimmune Thyroid Diseases (AITD):

  • Over 95% of thyroid dysfunctions are autoimmune in nature, where antibodies either inhibit or stimulate thyroid hormone production.

    • Blocking Antibodies: Lead to reduced T4 production with a compensatory increase in TSH (hypothyroidism).

    • Stimulating Antibodies: Mimic TSH action, leading to thyroid overactivity (hyperthyroidism, e.g., thyrotoxicosis). Increased T4 production with a decrease in TSH

• Central Hypothyroidism (hypothalamic/pituitary dysfunction):

  • Caused by TSH suppression due to pituitary tumors

  • TSH-secreting pituitary tumors exist but are rare

Clinical Cases of Hypothyroidism and Hyperthyroidism

• Hypothyroidism - Hashimoto’s Thyroiditis

  • elevated TSH suggest primary thyroid dysfunction

  • thyroxine replacement therapy treatment

• Hyperthyroidism - Graves’ Disease

  • TSH supression due to high circulating TH

  • antibodies bind TSH receptor, mimic TSH causing excess TH production

  • antithyroid drug carbimazole treatment

Consequences of iodine deficiency in pregnant women

• Thyroid hormones play an essential role in neuronal migration, myelination, synaptic transmission during the early embryonic development of the nervous system.

• The thyroid gland of the fetus does not produce thyroid hormones until approximately the 20th week of pregnancy.

• Until then, it is dependent on maternal T4, which passes through the placenta into the fetus in very small amounts.

• mental retardation of the fetus;

• brain damage to both the fetus and young children in the first few years of life;

• high infant mortality rate;

• miscarriages and premature birth;

• fetal growth retardation;

• neonatal hypothyroidism;

Hormonal Control of Steroid Hormone Synthesis

• Cholesterol is the precursor of all steroid hormones.

• Pituitary hormones regulate steroid hormone synthesis:

  • LH (Luteinizing Hormone) → progesterone, testosterone

  • ACTH (Adrenocorticotropic Hormone) → cortisol

  • FSH (Follicle-Stimulating Hormone) → estradiol

  • Angiotensin II/III → aldosterone

• The specific hormone synthesized depends on receptor expression in target cells.

Describe the Key Features and Functions of Steroid Hormones

• Lipophilic (freely cross the membranes), derived from cholesterol (common precursor)

• Produced upon stimulation and need, not stored or synthesised in advance

• Synthesis enzymes in the mitochondrial + smooth ER

• Require carrier proteins in blood due to water-insolubility:

  • CBG (corticosteroid-binding globulin) for cortisol

  • SHBG (sex hormone-binding globulin) for testosterone and estradiol

• Metabolism in peripheral tissues modifies and modulates hormonal activity (esp. cortisol, not aldosterone)

• Functional categories:

  • Glucocorticoids – carbohydrate metabolism e.g. Cortisol

  • Mineralocorticoids – mineral and electrolyte balance e.g. Aldosterone

  • Gonadocorticoids / Sex hormones – reproductive roles

• Other Functions:

  • Inflammation control

  • Stress response

  • Mood, Cognition

  • Bone health and metabolism

describe Cholesterol Transport for Steroid Hormone Synthesis

Cholesterol Transport:

• Cholesterol (from acetyl CoA via de novo synthesis or lipoproteins in blood)

• Transported to mitochondria by StAR

• Delivered to P450scc (inner membrane)

• Converted to pregnenolone (first steroid intermediate)

Importance:

• This is the initial and rate-limiting step in all steroid hormone biosynthesis.

• Pregnenolone can then enter pathways to form:

  • Cortisol (glucocorticoid)

  • Aldosterone (mineralocorticoid)

  • Testosterone/estradiol (gonadocorticoids)

Key Proteins:

1. StAR (Steroidogenic Acute Regulatory Protein):

   • Location: Outer mitochondrial membrane

   • Function: Transfers cholesterol from the outer to inner mitochondrial membrane

   • Domain: Has a START domain (StAR-related lipid transfer domain)

   • Importance: Rate-limiting step in steroid hormone synthesis

   • Regulation: Stimulated by ACTH (via cAMP → PKA)

2. PBR (Peripheral Benzodiazepine Receptor) – now renamed TSPO (Translocator Protein):

   • Binds cholesterol and may assist in its import into mitochondria

   • Works with StAR in transporting cholesterol to the inner membrane

3. SBP (StAR-binding protein):

   • Less well characterized, possibly involved in stabilizing or transporting cholesterol

4. START family proteins:

   • A family of proteins related to lipid transfer (StAR is one of them)

Conversion of Cholesterol → Pregnenolone

• Enzyme: P450scc (Cytochrome P450 Side Chain Cleavage enzyme)

  • Also called Desmolase or SCC enzyme

  • Encoded by: CYP11A1 gene

  • Location: Inner mitochondrial membrane

  • Reaction:

    • Cleaves the C20–C22 bond in cholesterol

    • Produces pregnenolone, the common precursor of all steroid hormones

• Cofactors Required:

  • NADPH – provides electrons

  • Adrenodoxin and adrenodoxin reductase – electron carriers

  • Cytochrome P450 – hydroxylation before cleavage

Aldosterone - Function & Mechanism

• Target organs: kidneys, salivary glands, colon

• Targets ENaC (Epithelial Sodium Channel) → increases Na⁺ reabsorption, water retention, promotes K⁺ excretion

• Regulates gene expression of ENaC → genomic effects, enhances mRNA production

• Local aldosterone production also exists in some tissues e.g. cardiomyocytes, blood vessels, brain

Cortisol Peripheral Metabolism

• 11β-HSD Type 1

  • Location: Liver, adipose tissue, brain, skin

  • Function: Converts inactive cortisone → active cortisol (keto group at C11 to OH-group)

  • Net effect: Amplifies cortisol action in these tissues

• 11β-HSD Type 2

  • Location: Kidney, colon, salivary glands (aldosterone target tissues)

  • Function: Converts active cortisol → inactive cortisone (OH-group at C11 to a keto-group)

  • Purpose: Protects mineralocorticoid receptors (MR) from being activated by cortisol (which is abundant and has affinity for MR)

  • This allows aldosterone (which is more specific) to act effectively.

• Without 11β-HSD type 2, cortisol would overstimulate mineralocorticoid receptors, leading to hypertension and hypokalemia — this happens in apparent mineralocorticoid excess (AME), a rare genetic disorder.

CRH (Corticotropin-Releasing Hormone) Role in Cortisol Release

• Structure: 41 amino acid peptide

• Source: Secreted by the paraventricular nucleus (PVN) of the hypothalamus

• Function:

  • Stimulates the anterior pituitary to release ACTH

  • ACTH → stimulates adrenal cortex to produce cortisol

• Signalling pathway: Activates cAMP second messenger system via Gs-coupled receptor

• Example: During stress, CRH release increases → more ACTH → more cortisol → mobilizes glucose and suppresses immune responses.

• Negative Feedback by Cortisol

ADH (Antidiuretic Hormone) Role in Cortisol Release

• Vasopressin

• Structure: 9 amino acid peptide

• Source: Synthesized in supraoptic and paraventricular nuclei of the hypothalamus; stored and released from the posterior pituitary

• Main roles:

  • Regulates water balance via kidneys

  • In HPA axis, acts synergistically with CRH to enhance ACTH release

• Mechanism: Modulates intracellular calcium channels (via V1b receptors in the pituitary)

• Example: In hypovolemia (low blood volume), ADH increases → helps conserve water and supports ACTH release → ↑ cortisol to maintain blood pressure and glucose.

• Negative Feedback by Cortisol