Lecture 3

Proteins and their Roles in Physiology

• Proteins are the working parts of physiology. They include:

  • Enzymes

  • Transporters

  • Receptors

  • Structural components

  • Motor proteins

  • Hormones

• Their proper function underpins almost all cellular processes and organismal physiology.

Protein-Facilitated Movement of Ions and Molecules

• Most substances move into or out of cells through protein channels.

• Movement depends on:

  • Substance gradient

  • Electrochemical gradient

  • Channel structure

• Key terms from Fig. 4.17: inheritance, expression levels, location.

General Physiological Processes (Overview from Fig. 1.8)

• Mouth, Gut, Skin; ICF vs ECF; Na⁺, H⁺ dynamics

• Transporting fluid system:

  • “blood” as the circulating fluid

• Exchange surfaces (e.g., gills)

• External environment interaction

• Core physiological processes: respiration, digestion, circulation, excretion

Transport Across Membranes: Types of Transport

• Sodium transport can be classified as:

  • Passive transport

  • Secondary active transport

  • Primary active transport

• Primary Active Transport (Na⁺/K⁺-ATPase) – Fig. 4.10 details

  • Stoichiometry (per ATP hydrolyzed):
    $3\,\mathrm{Na^+<em>{out}} \quad\text{and}\quad 2\,\mathrm{K^+</em>{in}}$

  • Mechanism (cycle):

  1. Binding of Na⁺ ions (outside) to the pump in E1 conformation

  2. ATP binds and is hydrolyzed; phosphorylation of the pump

  3. Eversion to E2 conformation; Na⁺ is released to the outside

  4. 2 K⁺ ions bind from outside

  5. Dephosphorylation triggers return to E1 conformation

  6. 2 K⁺ are released inside

  • Energy source: ATP hydrolysis with affinity changes driving conformational shifts

  • Overall effect: maintains Na⁺ and K⁺ gradients essential for membrane potential and cell volume regulation

• Other transport types (Fig. 4.13) – Endocytosis, Exocytosis, Transcytosis

  • Endocytosis can be receptor-mediated

  • Requires GTPases (e.g., dynamin in vesicle scission)

  • Transcytosis combines endocytosis and exocytosis across a cell

  • Exocytosis releases material to the extracellular space; essential for secretion

  • Receptor-mediated endocytosis as a specific, targeted form

  • Examples are not exhaustively listed in the transcript; the mechanism is highlighted

Protein Channels and Types of Channel Gating

• Cells control movement via channels; three major types:

  • Leak channels: not gated, but can be modified; help establish resting membrane permeability

  • Voltage-gated channels: gating controlled by changes in membrane potential; opening/closing in response to voltage changes

  • Ligand-gated channels: gating controlled by binding of a ligand (neurotransmitter, hormone, etc.)

• Leak channels provide a baseline conductance that shapes the resting membrane potential

Voltage-Gated Channels and Action Potentials

• Voltage-gated Na⁺ channels and K⁺ channels coordinate action potentials

• Phases illustrated in Fig. 9.5:

  • Resting membrane potential (typical value around $-70\ \mathrm{mV}$)

  • Threshold for excitation (roughly $-55\ \mathrm{mV}$)

  • Rapid activation of Na⁺ channels leading to depolarization (membrane potential rises toward $+40\ \mathrm{mV}$)

  • Inactivation of Na⁺ channels

  • Opening of K⁺ channels (slower) leading to repolarization and return toward resting potential

  • Hyperpolarization briefly below resting potential due to continued K⁺ efflux

  • Refractory period following an action potential during which another action potential cannot fire immediately

• Key dynamic sequence (summarized):

  • Activation of Na⁺ channels increases positivity inside; Na⁺ influx drives up the membrane potential

  • Subsequently, delayed K⁺ channel opening causes efflux of K⁺, driving repolarization

  • Na⁺/K⁺ ATPase pumps restore ion gradients after the spike

Action Potential Propagation

• Action potentials propagate along neurons as waves of depolarization

• Involves extracellular voltage changes and cytoplasmic currents

• Depolarizing, resting, and repolarizing phases propagate along the axon to transmit signals

Receptors and Signaling: Intracellular vs Extracellular Receptors

• Intracellular receptors:

  • Bind lipophilic ligands (e.g., steroids, vitamin A, retinoids, thyroid hormones)

  • Hormone response elements (HREs) on DNA are binding targets; receptor binding influences transcription

• Extracellular receptors:

  • Bind hydrophilic ligands outside the cell (peptide hormones, neurotransmitters)

  • Relay signals into the interior of the cell via various mechanisms

  • Receptor types include:

  • Enzyme-linked receptors (kinases and integrins)

  • Ion Channel-linked receptors

  • G-protein coupled receptors (GPCRs)

• Intracellular Lipophilic Ligands (examples):

  • Estrogen receptor

  • Androgen receptor

  • Glucocorticoid receptor

  • Thyroid hormone receptor

  • Retinoid X receptor (RXR)

Thyroid Hormone Receptor (TR) and RXR: Mechanistic Detail

• TR forms a heterodimer with RXR at thyroid hormone response elements (TREs) on DNA

• Without thyroid hormone (TH):

  • TR-RXR recruits a corepressor complex

  • Corepressor recruits histone deacetylases (HDACs)

  • HDACs remove acetyl groups from histone tails, increasing histone-DNA attraction and condensing chromatin

  • Gene transcription is repressed (referred to as the “without TH” state; sometimes labeled TR3 when unbound)

• With thyroid hormone:

  • TH binds TR, causing a conformational change

  • Corepressor dissociates from TR-DNA complex

  • Coactivators are recruited and contain histone acetyltransferase (HAT)

  • Histones become acetylated, reducing positive charge and loosening DNA-histone interactions

  • Chromatin decondenses and transcription is enabled (the TR3 state with TH)

• This sequence illustrates epigenetic regulation at the level of chromatin accessibility in response to hormone signals

Estrogen and Gene Expression Time Course (Intracellular Effects)

• Intracellular hormones like estrogen can modulate egg-related gene expression

• Distinct time course of mRNA activation observed after estrogen injection

• Highlights that transcriptional responses to hormones can be rapid or delayed depending on the target genes and chromatin context

Extracellular Receptors and Signaling Architecture

• Receptor architecture features:

  • NH₂ (extracellular) ligand-binding domain

  • COOH (intracellular) phosphorylation sites

  • Extracellular space and plasma membrane components

  • Cytosolic G-protein-binding sites

• These receptors activate intracellular signaling cascades that regulate cellular responses

Intracellular Second Messengers

• Receptors activate intracellular mediators (second messengers):

  • Cyclic AMP (cAMP)

  • Calcium ions (Ca²⁺)

  • Inositol triphosphate (IP₃)

  • Diacylglycerol (DAG)

• Role: amplify and propagate signals within the cell to elicit appropriate responses

G-Protein-Coupled Receptors (GPCRs): Core Signaling Pathways

• GPCRs activate multiple effector pathways via G-proteins (Gs, Gi, Gq)

• Common effector pathways include:

  • Gs: adenylyl cyclase → cAMP → protein kinase A (PKA; stimulatory)

  • Gq: phospholipase C (PLC) → DAG and IP₃; DAG activates protein kinase C (PKC) and IP₃ raises intracellular Ca²⁺

  • Gi: inhibits adenylyl cyclase → ↓ cAMP → ↓ PKA (inhibitory)

• This framework explains how extracellular ligands can produce diverse, context-dependent intracellular responses

Extracellular Receptors: Context-Dependent Responses

• Receptors show context-dependent responses depending on cell type, receptor subtype, and downstream signaling components

• Examples of stimulatory versus inhibitory signals:

  • Stimulatory: epinephrine (β-adrenergic pathways often increase cAMP)

  • Inhibitory: insulin (pathways that may dampen certain cascades or activate distinct signaling modules)

• Receptor architecture includes:

  • Extracellular ligand-binding domain

  • G-protein-binding sites

  • Phosphorylation sites on the cytoplasmic side

Epigenetics: Overview and Core Concepts

• Epigenetics refers to On/OFF switches of gene transcription that regulate access to DNA without changing the underlying DNA sequence

• Epigenetic processes are influenced by environmental factors and can modulate gene expression

• Some epigenetic marks can be transmitted across generations (transgenerational epigenetic inheritance) via modifications to germ cell DNA

• Some marks persist across generations, though the extent and mechanisms vary

• Epigenetics bridges gene regulation, development, aging, and disease

Major Epigenetic Mechanisms

• Histone acetylation

• Histone methylation

• DNA methylation

• These processes interact to regulate chromatin structure and gene expression

Histone Acetylation

• Occurs on histone tails, often on the N-terminal regions rich in lysine (Lys) and arginine (Arg)

• Acetylation is added to lysine residues and reduces the positive charge of histones

• Consequences:

  • Decreases histone-DNA affinity

  • Leads to chromatin decondensation and gene activation

• Enzymes involved:

  • Histone Acetyl Transferase (HAT): adds acetyl groups, promoting gene expression (the “on” switch)

  • Histone Deacetylase (HDAC): removes acetyl groups, repressing gene expression (the “off” switch)

• Epigenetic implication: acetylation state can dynamically regulate transcription in response to cellular signals

Histone Methylation

• Also occurs on histone tails (primarily at lysine residues)

• Methylation can prevent acetylation and influence chromatin state

• Enzymes:

  • Polycomb Repressive Complex II (PRC2): adds methyl marks, generally associated with transcriptional silencing (the “off” switch)

  • “Jumonji” family demethylases (e.g., JMJD3): remove methyl marks, reactivating transcription (the “on” switch in certain contexts)

• Example given: bone remodeling involves a balance of osteoblast creation and apoptosis, influenced by histone methylation status

DNA Methylation

• Occurs on cytosine residues in DNA, especially at CpG dinucleotides

• CpG islands: stretches of DNA rich in cytosine and guanine (CpG) and often found near gene start sites

• Enzymes:

  • Methyltransferases (DNMTs): add methyl groups to cytosine, usually hindering gene expression (the “off” switch)

  • Demethylases (e.g., TET family, other demethylases like AlkB-related enzymes): remove methyl groups, often enabling gene expression (the “on” switch)

• Sequence example: ATCTCGCCG illustrating CpG context

Epigenetics: Methylation Across Evolution

• Methylation mechanisms across life forms:

  • Bacteria: 6mA (N6-methyladenine) and other methylation types

  • Eukaryotes: 5mC (5-methylcytosine) dominating in vertebrates

  • Enzymes involved vary by lineage (examples include DNMTs in many eukaryotes; Dam-like methyltransferases in bacteria; Tet and AlkB family in demethylation processes)

• The figure (Lyko, 2018) highlights conservation and loss of methylation systems across taxa, with some lineages showing loss of certain methyltransferases or demethylases

DNA Methylation and Cancer: Clinical Relevance (Michalak et al., 2019)

• Chromatin methylation regulates gene expression, DNA repair, and DNA replication

• Dynamic methylation changes are essential for cell fate determination and development

• Mutations or dysregulation of factors that regulate DNA, RNA, and histone methylation are linked to aging, development disorders, and cancer

• Epigenetic therapies aim to reset methylation imbalances and are being explored clinically

• Methylation status can serve as diagnostic or prognostic biomarkers and may guide combination epigenetic therapies to enhance immune surveillance or improve responses to chemotherapies