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.