Cellular Dynamics: Adhesion, Signaling, and Cancer Biology

Posted on Sep 3, 2025 in Biotechnology

Cytoskeleton Structure and Function

Actin Microfilaments

Actin is the most abundant intracellular protein in eukaryotic cells. While some organisms may have only one gene, humans possess six actin genes that encode different isoforms. Four α-actin isoforms are present in various muscle cells, while β and γ actin isoforms are found in non-muscle cells. These isoforms differ by only 4-5 amino acids.

Actin exists as a monomer, G-actin (globular form), and a polymer, F-actin (filamentous form).

Functions of Actin

• Local restructuring of the cell, mediated by actin.
• Actin polymerization/depolymerization is crucial for mobile structures that extend and/or contract.
• Phagocytosis: An actin-mediated process to surround large particles (e.g., bacteria, dead cells). Apoptotic cells are also removed by phagocytosis.
• Cell movement: Cells extend and contract their actin cytoskeleton to move or change shape. Pseudopodia are extensions of the cytoplasm used by amoebas to “walk” over surfaces.

Loss of filamin results in no movement (e.g., melanoma with filamin mutation). Restoration of filamin allows normal lamellipodia and high mobility.

Drugs Affecting the Actin Cytoskeleton

• Cytochalasin D: A toxin from fungi that depolymerizes microfilaments. It binds to the + end of filaments, blocking further addition of G-actin subunits. If added to live cells, the actin cytoskeleton disappears, inhibiting locomotion and cytokinesis.
• Phalloidin: A toxin from Amanita phalloides (death cap fungus). It binds subunits in F-actin and locks them together, preventing depolymerization.

Microtubules: Characteristics and Functions

Microtubules are hollow tubes found in every eukaryotic cell, with a diameter of 25nm. They are made of tubulin proteins (α and β), which form heterodimers that join to create a microtubule.

Microtubule Functions

• Form motile components in cilia and flagella.
• Facilitate chromosome separation in mitosis (mitotic spindles).
• Provide a scaffold for cell shape, integrity, and organelle position.

Microtubule assembly involves growth and shrinkage from the + end, while the minus end is stabilized by the centrosome. Microtubule-associated proteins (MAPs) cross-link microtubules to form bundles, increasing stability.

Drugs Disrupting Microtubule Dynamics

These drugs are used to treat cancer by targeting rapidly dividing cells.

• Taxol (paclitaxel): Binds to microtubules and stabilizes them, preventing depolymerization. It arrests cells in mitosis and disrupts the movement of organelles.

Intermediate Filaments: Structure, Function, and Disease

Intermediate filaments are found throughout the cell, especially in the cytoplasm, extending to the plasma membrane. They are also present in the nucleus (lamins). These filaments are found in most animal cells, particularly those undergoing mechanical stress. They may connect microtubules, showing similar spatial patterns.

Structure of Intermediate Filaments

Intermediate filaments have a central α-helical core flanked by globular N and C terminal domains. The core has four long helices with three non-helical spacers, forming coil-coil dimers (homo or hetero).

Functions of Intermediate Filaments

• Provide mechanical support for membranes.
• Form an internal framework to support cell shape and resilience.
• Are anchored at specialized cell junctions (e.g., keratin maintains skin integrity).

Disease Related to Intermediate Filaments

• Epidermolysis Bullosa Simplex (EBS): Caused by mutant keratin proteins, leading to blistering skin. Without keratin bundles, skin cells are fragile and easily damaged.

Comparison of Cytoskeletal Filaments

• Microtubules (MT): Easily deformed but cannot be stretched without rupture.
• Actin Microfilaments (MF): More rigid, some stretch but rupture easily.
• Intermediate Filaments (IF): Flexible and withstand large mechanical stresses, best for maintaining cell integrity.

Cell Junctions and Adhesion

Main Types of Cell Junctions

Cell junctions are structures that connect cells together, anchoring, occluding, channel-forming, or relaying signals. Interactions of the cytoskeleton with junctions are fine-tuned by IgCAM and cadherin.

How Cells Form Tissues

Cells anchor to other cells and/or surrounding material. Tissues consist of cells plus a network of molecules in the intracellular space, filled with a mixture of proteins and sugars secreted by cells.

Types of Junctions

• Anchoring Junctions:
  • Adherens Junction: Composed of intracellular actin filaments and transmembrane cadherins. Cadherins are proteins that mediate adhesion in a calcium-dependent manner. They bind to other cadherins on neighboring cells, anchoring them together.
  • Desmosome: Composed of intracellular intermediate filaments and non-classical cadherins (e.g., desmoglein, desmocollin). Provides mechanical strength to tissues.
• Occluding Junctions:
  • Tight Junctions: Allow epithelial layers to function as barriers. Composed of proteins called claudins and occludins that tightly seal adjacent plasma membranes. Different levels of permeability can be regulated by factors such as nutrients and intestinal bacteria. Increased permeability can lead to conditions like “leaky gut” syndrome.
• Channel-Forming Junctions:
  • Gap Junctions: Made of connexins (invertebrates) and innexins (in Drosophila and C. elegans). They allow passage of ions and small metabolites (up to 1000 Daltons). Formed by six transmembrane proteins creating a hemi-channel (connexon) in each membrane. Two hemi-channels meet to form a gap of 2-4nm between cells.

Transient Cell-Cell Interactions: Immune Cells

Immune cells are migratory and can roam through blood and tissues. They utilize two main adhesion systems:

• Selectins: Bind to sugars on target cells. Expressed by endothelial cells and immune cells. Types include L-selectin (leukocytes), P-selectin (platelets), and E-selectin (endothelial cells).
• Ig-CAMs: Have antibody-like protein domains. Over 65 different CAMs exist in various tissues. They bind with integrins or other CAMs, providing weaker interactions than cadherins.

Selectin-Mediated Leukocyte Rolling

Immune cells use selectins to roll along blood vessels. Attachment to actin filaments helps cells push through blood vessels and tissues.

Signaling Junctions: Synapses

Synapses involve long-term interactions between neurons. They require cells to adhere to each other using cadherin and Ig-like CAMs. Actin filaments play a role in the formation and function of synapses.

Cell Adhesion and Extracellular Matrix (ECM)

Extracellular Matrix (ECM)

The ECM is the non-cellular component of a tissue, including basal lamina, fibers, and proteins. It is produced by the cells within it and determines the physical properties of tissues (e.g., bone vs. muscle vs. fat).

Connective Tissue

Connective tissue is composed of individual cells and the ECM they are embedded in. The ECM includes proteins, sugars, and fibers (collagen, elastin). Cells in the ECM do not contact other cells and are not organized into organs.

Types of Connective Tissue Cells

• Fibroblasts: Least specialized, most common.
• Macrophages: Immune cells.
• Mast cells: Immune cells.
• Plasma cells: Produce antibodies.
• Chondrocytes: Cartilage cells.
• Osteoblasts: Secrete bone matrix protein.
• Osteocytes: Bone cells, most specialized.
• Adipocytes: Fat cells.
• Myoepithelial cells: Contractile cells.

The ECM is continually renewed by the cells within it.

Cell-Matrix Adhesions

• Focal Adhesions: Intracellular actin filaments + transmembrane integrins anchor cells to the basal lamina.
• Hemi-desmosomes: Intracellular intermediate filaments + transmembrane integrins anchor cells to the basal lamina.

Components of the Basal Lamina and ECM

• Basal Lamina: The layer between the cell and the amorphous connective tissue.
• Lamin: Adhesive protein for stickiness.
• Collagen: Fibrous protein for strength.
• Integrin: Connects cells to the basal lamina.
• Fibronectin: Major adhesive protein in ECM.

Glycosaminoglycans (GAGs)

GAGs are long, unbranched chains of sugar molecules (polysaccharides) composed of paired sugar residues (disaccharides) like N-acetylglucosamine or N-acetylgalactosamine. They are hydrophilic, attracting water and forming a gel that withstands pressure (e.g., in knee joints).

Hyaluronic Acid: An Extreme GAG

Hyaluronic acid is not made in the ER/Golgi; it is produced on the outer surface of the plasma membrane. It creates a cell-free space for cell migration and is produced in large quantities during wound repair, inflammation, and cell migration. It is a major component of synovial fluid in joints for lubrication and compression resistance. It also drives cancer cell metastasis by helping cancer cells stick to new environments.

Collagen: Structure and Importance

Collagen is a fibrous protein secreted by connective tissue cells. It is a major component of the ECM, making up 25-30% of protein in the body. There are 25 different collagens, each with a different gene.

Collagen Structure

Collagen has a triplet of amino acids (Gly-Pro-X, where X can be any amino acid), which is converted to hydroxyproline. This forms a triple helix structure, with hydrogen bonds between hydroxyproline residues. Collagen molecules form fibrils and fibers, making connective tissue strong. Collagen is continually broken down and renewed.

Scurvy: A Collagen Deficiency

Scurvy is a collagen deficiency due to low vitamin C, as vitamin C is required for the hydroxylation of proline. Loss of hydroxyproline leads to unstable collagen.

Elastin: Flexibility and Disease

Elastin provides flexibility to connective tissue. It contains lots of proline and glycine, similar to collagen but with less hydroxylation or glycosylation. After secretion, elastin assembles into a network of fibers; cross-linking between fibers is essential for elasticity.

Marfan Syndrome

Marfan syndrome is a defect in cross-linking elastin fibers, leading to non-stretchy connective tissue.

Connective Tissue and Blood Vessel Health

Blood Vessel Damage

Blood vessels can be prone to damage, which can lead to various health issues.

Ehlers-Danlos Syndromes (EDS)

EDS are a group of disorders affecting connective tissue, usually caused by mutations in collagen genes. Specific symptoms depend on which collagen gene is affected, but common symptoms include unstable joints, fragile skin, and blood vessel problems.

The Science of Stretch: Connective Tissue and Acupuncture

Connective Tissue: A Body-Wide Network

Connective tissue is crucial for connecting various parts of the body: it joins the thigh to the calf, the hand to the arm, and the breastbone to the clavicle. It allows muscles to glide past one another and supports organs. It acts as a net and adhesive, holding cells in place and relaying messages.

Acupuncture Research Insights

Acupuncture Study #1: Tissue Stretch and Fibroblast Morphology

Conclusions indicated that pulling collagen and elastic fibers during needle manipulation affects local cells. The study used ex vivo and in vivo models to investigate tissue stretch on mouse subcutaneous tissue fibroblast morphology. Findings showed that tissue stretch ex vivo (average 25% elongation from 10 min to 2 h) significantly increased fibroblast cell body perimeter and cross-sectional area (ANOVA P<0.01). At 2 hours, fibroblast cross-sectional areas were 201% greater in stretched than unstretched tissue. This implies that dynamic responses of fibroblasts to tissue length changes are important for understanding movement, posture, and therapies like physical therapy, massage, and acupuncture.

Acupuncture Study #2: Cytoskeletal Involvement

In in vivo stretching, mice were bent for 30 minutes, and connective tissue was fixed/removed. Cells were stained with phalloidin to measure cell size. Phalloidin prevents the actin cytoskeleton from remodeling. In ex vivo stretching, actin microfilaments were visualized with phalloidin, showing that cells enlarged and actin microfilaments were redistributed.

Acupuncture Study #3: Distance of Response and Signaling

This study investigated the interaction between the needle, extracellular matrix (ECM) fibrous proteins, and fibroblasts, with and without needle rotation. Twisting a needle in the ECM pulls on the actin cytoskeleton of nearby cells.

Signaling Molecules in Acupuncture

Adenosine, AMP, ADP, and ATP are released during acupuncture. Adenosine receptors are required for acupuncture to reduce pain in mice; mice with mutant adenosine receptors show different responses. Adenosine is also released during acupuncture in humans, causing Ca²⁺-mediated signaling.

Other Effects of Stretch

Stretch-induced apoptosis occurs when adaptive reorientation of annulus fibrosus cells is restricted.

Cellular Signaling Mechanisms

How Signals are Relayed

Signaling begins when a ligand protein binds to a receptor, initiating a series of events known as signal transduction. Second messengers are molecules that relay signals inside the cell, and enzymes facilitate the signaling process.

Types of Receptors

• Cell Surface Receptors: For hydrophilic ligands that cannot cross the plasma membrane.
• Intracellular Receptors: For hydrophobic ligands that can cross the membrane.

Key Second Messenger Molecules

• cAMP: Cyclic adenosine monophosphate.
• DAG: Diacylglycerol.
• IP3: Inositol trisphosphate.

These molecules are generated in response to receptor activation.

Enzyme-Linked Receptors and Pathways

Enzyme-linked receptors signal to alter cell activity through various pathways. For example, phospholipase C activates protein kinase C (PKC). The RAS-MAPK Pathway links to transcription factors and gene expression, involving a series of phosphorylation events.

Signal Transduction Process Overview

1. Recognition: Ligand binds to receptor.
2. Transfer and Transmission: Via signaling molecules (second messengers).
3. Cellular Response: Leads to a specific cellular action.
4. Termination of Response: Can involve enzyme activity, such as kinases adding a phosphate group to a substrate protein, and phosphatases removing a phosphate group.

Multiple Enzymes in Signal Transduction

• Kinase: Adds a phosphate group.
• Phosphatase: Removes a phosphate group.
• GTPase: Hydrolyzes GTP (active with GTP).
• Guanine Nucleotide Exchange Factor (GEF): Swaps GDP for GTP, activating GTPase.
• GTPase Activating Protein (GAP): Speeds up hydrolysis of GTP, ending GTPase activity.
• Adenylate Cyclase: Generates cyclic AMP.

Three Classes of Receptors

• Ion Channel-Coupled Receptors: E.g., acetylcholine receptor at the nerve-muscle junction.
• G-Protein Coupled Receptors (GPCRs): E.g., adrenaline, serotonin, histamine, dopamine.
• Enzyme-Coupled Receptors: Usually a kinase (e.g., Receptor Tyrosine Kinase, RTK).

Receptor Tyrosine Kinase (RTK) Signaling

Ligands for signaling receptors are found in the extracellular matrix (ECM); they bind to proteins and proteoglycans, stored for later use. When a ligand binds to an RTK, it brings two receptors together, leading to phosphorylation, which activates Ca²⁺, phospholipase C, RAS, and MAP kinase.

RTK Pathway 1: Ca²⁺ Release

Phospholipase C (PLC) cleaves phospholipid PIP2, generating second messengers:

• DAG: Activates PKC.
• IP3: Opens Ca²⁺ channels in the ER.

Result: Increased intracellular Ca²⁺ and PKC activation.

RTK Pathway 2: RAS-MAPK Activation

An adaptor protein (GRB2) binds to phosphorylated RTK. It recruits GEF (SOS) for RAS; GEF swaps GDP for GTP, activating RAS. Active RAS initiates a phosphorylation cascade.

Signaling and Transcription

Environmental signals activate specific transcription factors; for example, the presence of glucose activates genes for glucose breakdown.

Process of Initiating Transcription

Ligand binds to receptor → activated PLC → generated DAG, IP3 → activates PKC → alters gene expression. Transcription factors recognize specific DNA sequences, bind, and open chromatin by acetylating histones.

RTK Pathway 2: MAPK Activation Cascade

Involves a cascade of kinases: MAP3K (e.g., RAF) → MAP2K (e.g., MEK) → MAPK (e.g., ERK). Result: Changes in gene expression and cell function.

G-Protein Coupled Receptors (GPCRs)

GPCR Structure and Activation

Ligand binding to a GPCR activates a trimeric G-protein. GPCRs have seven transmembrane domains, one site for G-protein binding, and one site for ligand binding.

Types of G-Proteins

• Trimeric G-proteins: Activated by GPCRs. Types include:
  • Inhibitory (Gi): Inhibits signaling.
  • Stimulatory (Gs): Stimulates signaling.
  • Gq: Activates phospholipase C (PLC).
• Monomeric G-proteins: Such as RAS.

GPCR Signaling Pathways

Pathway 1: PLC to PKC

Activated by GPCR-ligand binding, generating second messengers from PIP2.

Pathway 2: Adenylate Cyclase to PKA

Ligand binding activates adenylate cyclase, which converts ATP to cAMP. cAMP binds to PKA, activating it. Activated PKA then goes to the nucleus and activates CREB (cAMP response element-binding protein), leading to gene expression changes.

Effects of Ligand Binding and Drug Targeting

Ligand binding can alter cell function by stimulating or inhibiting specific GPCRs. For example, adrenaline (epinephrine) binds to various adrenergic receptors (α1, α2, β1, β2) and has different effects depending on the tissue.

Targeting GPCRs with Drugs

• Beta-blockers: Block β2 adrenergic receptors, used for hypertension (e.g., Propranolol).
• Beta-agonists: Activate β2 adrenergic receptors, used for asthma treatment (e.g., Isoprenaline).

Caffeine and GPCRs

Caffeine binds to adenosine receptors (A1 and A2A), preventing adenosine from binding. This leads to blood vessels not dilating and neurons not unwinding, preventing sleepiness. The “caffeine nap” concept suggests that after sleeping, adenosine receptors clear faster, allowing caffeine to bind without adenosine.

Endocannabinoid Receptors and Biased Signaling

Endocannabinoid Receptors

• CB1: Primarily in neurons; regulates brain functions.
• CB2: Primarily in immune cells; regulates pain perception.

Both receptors interact with Gαi/o proteins, affecting signaling pathways.

Biased Signaling

Some ligands activate Gα proteins, while others activate the β-arrestin pathway. This can lead to different cellular responses based on the ligand-receptor interaction.

Ligands for Cannabinoid Receptors

• Endocannabinoids: Naturally synthesized by the body.
• Phytocannabinoids: Naturally synthesized by plants.

Cannabinoid Receptor Interactions

• THC: Primarily binds to CB1 receptors (CB1 > CB2 in binding affinity).
• CBD: Antagonizes (blocks) CB1 and CB2 receptors.

Cell Membrane: Structure and Dynamics

Cell Membrane Functions

Organelles are defined by membranes (e.g., nucleus, ER, Golgi, mitochondria, lysosomes). Their primary function is to separate specialized environments. The plasma membrane, composed of a lipid bilayer with embedded proteins, encloses the cell, separating the cytosol from the extracellular environment.

Lipid Bilayer Structure

The lipid bilayer is a thin (~50Å) film of lipids and proteins, impermeable to most water-soluble molecules (hormones, drugs). It is a dynamic, fluid structure where molecules move within the membrane.

Amphiphilic Molecules

These molecules expose hydrophilic heads to water, while their hydrophobic tails can form spherical micelles or bilayers. The membrane also possesses a self-sealing property, where small tears lead to spontaneous rearrangement to eliminate free edges.

Phospholipids: Essential for Membranes

Phospholipids are essential for functional membranes. All molecules are amphiphilic, with a polar head (containing a PO₄ group) and two fatty acid tails (one unsaturated and one saturated).

Membrane Fluidity

Fluidity depends on composition and temperature. A phase transition occurs with changes in fluidity, influenced by fatty acid composition, sterol content, and temperature.

Components of the Lipid Bilayer

• Glycolipids: Sugar-containing lipids found on the surface of eukaryotic plasma membranes, made in the Golgi and important for cell recognition.
• Cholesterol: Present in animal plasma membranes (up to one molecule per phospholipid). It adjusts fatty acid composition to maintain fluidity, preventing freezing and excessive fluidity.

Eukaryotic cell membranes can contain up to 100,000 different lipid species. Lipid species and proteins are not randomly distributed; they can form nanoclusters or larger assemblies. Lipid rafts are specialized domains where specific proteins and lipids are concentrated.

Lipid Movement and Asymmetry

Lipid molecules move laterally within a monolayer rapidly (~10⁷ times per second), allowing for rapid lateral diffusion (the “flip-flop”).

Phospholipid Translocators

• Flippase: Moves outer phospholipids to the inner leaflet, using ATP to catalyze the flip-flop process.
• Floppase: Moves inner phospholipids to the outer leaflet, also using ATP for its function.
• Scramblase: Scrambles phospholipids nonspecifically between inner and outer leaflets, and does not require ATP; the exchange is energetically neutral. It is activated by Ca²⁺ ions.

These proteins are crucial for maintaining lipid asymmetry in the membrane, which is important for various cellular functions, including the budding of transport vesicles. Lipid asymmetry is vital for converting extracellular signals into intracellular responses.

Membrane Curvature and Vesicle Trafficking

The net translocation of lipids contributes to membrane curvature, aiding in vesicle formation during post-Golgi trafficking.

Phospholipases

These enzymes cleave specific phospholipids in the membrane, activated by extracellular signals, generating fragments that act as intracellular mediators.

Implications of Dysfunction

Genetic mutations in flippases, floppases, and scramblases can lead to severe diseases like Angelman syndrome. During cell death, when ATP is depleted, only scramblase remains active, signaling for cell removal or phagocytosis.

Endoplasmic Reticulum and Toxins

Cholera Toxin: Mechanism of Action

The ganglioside GM1 acts as a receptor for cholera toxin on intestinal epithelial cells. Entry of cholera toxin into the cell causes increased intracellular levels of cAMP, leading to massive secretion of ions and water into the intestine, resulting in profuse diarrhea.

Cholera toxin binds to the plasma membrane via its B subunit, is internalized, and delivered to endosomes. The A subunit is released in the ER, refolds, and activates adenylate cyclase, increasing cAMP levels.

Toxins and Membrane Trafficking

Many pathogens produce pore-forming toxins (PFTs) that damage cell membranes, leading to leakage and lysis. PFTs are rich in β-sheets and form β-barrel structures that create channels in the lipid bilayer. Pore sizes can range from 15–30 Å to 250-300 Å.

Structure and Function of the ER

All eukaryotic cells have an ER, which is crucial for lipid and protein biosynthesis. The ER consists of branching tubules and flattened sheets, forming a continuous internal space called the ER lumen. The transitional ER transports material to the Golgi apparatus. The ER and Golgi lumens are topologically equivalent to the cell’s exterior.

Sorting Signals

Sorting signals are specific amino acid sequences recognized by sorting receptors. These receptors are reused after targeting proteins, and most sorting receptors recognize classes of proteins rather than individual species.

Golgi Apparatus

The Golgi apparatus receives lipids and proteins from the ER and modifies them (e.g., glycosylation) before dispatching them to various destinations.

Rough ER

The Rough ER synthesizes proteins for the cell surface, lysosomes, Golgi, or secretion. It appears rough due to ribosomes on its surface, produces transmembrane proteins, and is involved in protein folding and post-translational modifications.

Smooth ER

The Smooth ER lacks ribosomes and is involved in detoxifying lipid-soluble molecules and synthesizing lipids and cholesterol. It contains enzymes like cytochrome p450 for metabolizing drugs and harmful compounds. In muscle cells, the smooth ER (sarcoplasmic reticulum) regulates calcium (Ca²⁺) release for muscle contraction.

Transmembrane Proteins

Transmembrane proteins extend through the lipid bilayer, with hydrophobic regions passing through the membrane.

Protein Synthesis in the ER

Entry of Proteins into the ER

Proteins enter the rough ER through a process involving a signal sequence. Secreted proteins are released outside the cell (e.g., hormones, signaling proteins, pancreatic enzymes, blood proteins, coagulation factors, and antibodies). Transmembrane proteins may function in the ER or be destined for the plasma membrane or other organelles.

All proteins entering the ER have an ER signal sequence, which is cleaved off by a signal peptidase in the ER membrane before the polypeptide chain is complete.

Signal-Recognition Particle (SRP)

The Signal-Recognition Particle (SRP) cycles between the ER membrane and the cytosol, binding to the nascent signal peptide. It binds to hydrophobic segments of the unfolded polypeptide chain, inhibiting further translation by the free ribosome in the cytosol.

Ribosomes and Translation

Ribosomes read mRNA sequences to determine the amino acid sequence of proteins. Amino acids are carried to the ribosome by tRNA, which binds to the mRNA via an anticodon sequence. After protein synthesis, the two subunits separate and can be reused. Both types of ribosomes are structurally and functionally identical and come from the same pool; they differ only in the proteins they are synthesizing at any given time.

Co-translational Translocation

Proteins can be made in the cytoplasm, but they require proper folding and compartmentalization. The Sec61 complex is a water-filled channel through which the polypeptide chain passes.

Protein Structure and Folding

Protein Purification and Oligomerization

Protein purification is the process of isolating a specific protein from a complex mixture. Protein oligomerization refers to oligomeric proteins consisting of more than one subunit, possessing a quaternary structure. These can be homo-oligomers (same protein) or hetero-oligomers (different proteins).

Chaperones and Protein Folding

Chaperones assist in the folding and assembly of proteins. They prevent newly synthesized polypeptides and assembled subunits from aggregating into non-functional structures. Proteins can misfold into harmful aggregates, which can kill cells. In the ER, as peptides emerge from the pore, folding and post-translational modifications begin co-translationally.

Protein Folding and Chaperones in the ER

BiP and Unidirectional Translocation

Binding protein (BiP) binds the polypeptide chain as it emerges from the pore into the ER lumen. ATP-dependent cycles of BiP binding and release drive unidirectional translocation.

Cysteine Bonding

Cysteine needs to be disulfide bonded for proper folding. This provides rigidity and acts as a “pinch point” during folding to stabilize proteins.

ER Chaperones

• Calnexin: Interacts with N-glycans near the ER membrane.
• Calreticulin: Binds mainly to glycoproteins in the ER lumen.

These are scaffold chaperones that recognize N-linked sugars with a single glucose (monoglucosylated). Unfolded proteins undergo cycles of glucose trimming and addition until fully folded. Misfolded proteins are toxic to cells.

Glycosylation: N-Glycans and Functions

N-Glycans in Protein Folding

N-glycans are key in folding polypeptides in the ER and maintaining protein solubility and conformation. A lack of glycosylation leads to incorrect folding, aggregation, and degradation. N-glycans force amino acids near them into a hydrophilic environment, keeping loops on the surface. Glycans also provide a shield from proteases, antibody binding, and unwanted interactions.

Functions of Glycosylation

• Specific recognition by lectins (glycan-binding proteins).
• Some glycan receptors mediate clearance, turnover, and intracellular trafficking of soluble blood-plasma glycoproteins.
• Pathogens may mimic host glycans to evade immune response.

A-B-O Blood Type Antigens

A-B-O blood type antigens are oligosaccharides on glycoproteins and glycolipids found on erythrocytes and other cells.

• A antigen: N-acetylgalactosamine attached to outer galactose.
• B antigen: Extra galactose residue attached to outer galactose.

Antibodies against A or B alleles form after birth. The physiological functions of ABO blood group antigens are unknown but are associated with various disease risks.

Glycosylation and Cell Adhesion Molecules

N-Linked Glycosylation Process

Free sugars from the cytoplasm attach to a glycolipid called lipid-linked oligosaccharide (LLO). The lipid part is dolichol, and the phosphate group is a diphosphate. Dolichol-PP-sugars are synthesized in the cytoplasm, flipped into the ER, and added to the nascent peptide chain co-translationally. Glycosylation occurs before folding, directing and helping folding. Oligosaccharyl transferase (OST) is a complex of many proteins involved in this process.

O-Linked Glycosylation Process

O-linked sugars are added to the terminal OH group of serine and threonine residues of many proteins, and to hydroxyproline and hydroxylysine of collagen. Unlike N-linked glycosylation, O-linked does not involve a lipid-linked oligosaccharide precursor. It starts with the addition of a single sugar residue like N-acetylgalactosamine (GalNAc).

Glycosylation Inhibition

Inhibiting N-glycosylation completely by blocking dolichol-PP-GlcNAc formation leads to an increase in misfolded proteins, severe ER stress, cytotoxicity, and cell death.

Glycosylphosphatidylinositol (GPI) Anchor Proteins

Proteins attached to GPI via their carboxyl termini face the ER lumen and extracellular environment. GPI proteins are enriched in lipid rafts and are functionally diverse (e.g., hydrolytic enzymes, adhesion molecules).

GPI Biosynthesis

1. Preassembly of a GPI precursor in the ER membrane.
2. Cleavage of a carboxy-terminal GPI signal peptide.
3. Attachment of GPI to newly synthesized protein in the ER lumen by GPI transamidase.

GPI proteins can be shed from cells in soluble form in response to phospholipase action in the plasma membrane.

Lysosomes: Cellular Digestion and Recycling

Lysosome Functions

Lysosomes serve as the digestive system of the cell. They degrade extracellular material from endocytosis and intracellular components that are no longer needed. Lysosomes are crucial for cellular digestion and recycling. Proper functioning relies on specific signaling and enzyme activity. Dysfunction can lead to serious diseases, highlighting the importance of lysosomal health.

Hallmarks of Cancer

Cancer Cell Behavior

Cancer cells break fundamental rules of cell behavior, exploiting opportunities to grow and survive. Mutations lead to the development of cancer hallmarks.

Tumor Types

Tumors can be benign or malignant. Specific types include:

• Sarcoma: Muscle, fat, bone.
• Lymphoma: Lymphatic system.
• Leukemia: Blood (or immune) cells.
• Carcinoma: Organs (e.g., stomach, pancreas, lung, ovary, skin).
• Other types: Melanoma, glioma, etc.

Cancer is a clonal, genetic disease. Daughter cells accumulate more mutations and advantages. Cancers progress through stepwise changes: cells become more abnormal with more mutations.

Cellular Changes in Cancer Progression

• Hypertrophy: Cells increase in size without dividing.
• Hyperplasia: Cells divide more but remain the same size.
• Metaplasia: Cells change type/function/identity.
• Dysplasia: Cells become abnormal in size and lose organization.
• Neoplasia: Abnormal growth of cells.

Key Hallmarks of Cancer

• Resist Cell Death: Avoid apoptosis.
• Sustain Proliferative Signaling: Grow without normal growth signals.
• Evade Growth Suppressors: Grow despite inhibitory signals.
• Limitless Replication: Proliferate indefinitely.
• Invade and Metastasize: Spread and establish colonies in other body parts.
• Induce Angiogenesis: Attract blood vessels for nutrients and waste removal.

Oncogenes and Tumor Suppressor Genes

Oncogenes: Abnormal Accelerators

Oncogenes are mutated proto-oncogenes that act as abnormal accelerators, driving cell growth and division. All oncogene mutations are “gain of function,” leading to more accelerators. Cooperation of mutations can lead to faster cancer progression.

Tumor Suppressor Genes: Cellular Brakes

Tumor suppressor genes act as brakes on the cell cycle. Their loss of function, where protein activity is reduced or lost, contributes to cancer development.

Types of Tumor Suppressor Genes

• Gatekeeper Genes: Control entry to the cell cycle (e.g., p53, RB).
• Caretaker Genes: Involved in DNA repair and genome stability.
• Landscaper Genes: Modify interactions with the extracellular environment.

Mitogens and Cell Cycle Regulation

Mitogens drive mitosis (e.g., Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF)).

Kinases in Cell Signaling

• Receptor Tyrosine Kinases: EGF Receptor, PDGFR, HER2.
• Cytoplasmic Kinases: Tyrosine and serine kinases.

C-myc: A Key Oncogene

C-myc is an oncogene first identified in chicken virus-related cancer. It functions as a gene regulatory protein that:

• Increases expression of ribosomal RNA and proteins.
• Accelerates S-phase entry.
• Changes metabolism to glycolysis.
• Activates telomerase.

RB and the Cell Cycle

The RB protein acts as a brake, preventing the cell from moving from G1 to S phase. When stimulated by mitogens, RB is inhibited, allowing progression. CDK drives progression through the cell cycle by phosphorylating RB, leading to its inactivation.

Mitogens interact with cell-surface molecules to signal progression from G1 to S phase. E2F proteins are released when RB is phosphorylated, allowing transcription of S-phase cyclins.

Philadelphia Chromosome

The Philadelphia chromosome results from chromosomal rearrangement, leading to new kinases that inhibit apoptosis and drive cell division.

HPV and p53 Tumor Suppressor

p53: The Stress Sensor and Gatekeeper

p53 is a stress sensor and transcription factor. When a cell is stressed, p53 gets phosphorylated and can halt the cell cycle. This gives the cell time to repair damage or induce apoptosis if repairs fail. Different stresses are mediated by different kinases that activate p53 through phosphorylation.

p53 is the most well-known gene in cancer research. Approximately 50% of cancers have a mutation in p53, while many others lose its function.

Consequences of p53 Loss

• Loss of cell cycle arrest.
• Loss of apoptosis induction.
• Loss of damage repair ability.

Human Papillomavirus (HPV) and Cancer

Human Papillomavirus (HPV) causes cervical cancers. Vaccination against HPV can prevent most cervical cancers.

Cancer Development with HPV

HPV can lead to abnormal cell growth (hyperplasia) and potentially to high-grade lesions (dysplasia). Diagnosis involves detecting viral DNA/RNA in abnormal cervical cells.

Key Viral Proteins

Expression of viral proteins E6 and E7 is crucial for cancer formation. E7 expression weakens cytotoxic CD8 T-cells, making them ineffective against tumors.

Prevention and Early Detection

• Vaccination: Against high-risk strains of HPV (e.g., Gardasil-9 protects against 9 strains).
• Early Detection: Cervical cancer screening involves taking a smear of cervical epithelial cells. HPV self-screening checks for the presence of the virus in the vagina.

Cancer Treatment and p53

Treatment for cancer has variable success rates with surgery, radiation, chemotherapy, and experimental therapies. DNA damaging agents have over 80% efficacy, while microtubule disrupting agents have around 10%.

Efficacy of Cancer Therapy

Successful therapy requires intact apoptosis pathways. DNA damaging therapy relies on a functional DNA damage response. More p53 increases the likelihood of cell cycle arrest and apoptosis.

Treatment for p53-Mutated Cancers

Half of all cancers have lost the p53 gene due to mutations. Restoring p53 function is difficult, but there may be ways to exploit its loss in treatment strategies.