Extracellular Matrix (ECM)

• The extracellular matrix (ECM) is a network of macromolecules providing structural and biochemical support to surrounding cells.

Main components of the ECM:

• Collagen provides tensile strength.
• Proteoglycans are large molecules of proteins and glycosaminoglycans (GAGs) involved in hydration and cushioning.
• Fibronectin is a glycoprotein responsible for cell adhesion and migration.
• Laminin is a key component of the basal lamina, that functions in cell attachment and signaling.
• Elastin is a protein for elasticity to tissues like skin, lungs, and blood vessels.
• Matrix Metalloproteases (MMPs) are enzymes that degrade ECM components allowing remodeling.

Key ECM Components

• Collagen is a fibrous protein with a triple-helix structure.
• Collagen is the most abundant ECM protein and provides tensile strength.
• Collagen supports tissue structure and integrity and prevents excessive stretching.
• Proteoglycans consist of a core protein with covalently attached glycosaminoglycans (GAGs).
• Proteoglycans are highly hydrated, forming a gel-like matrix.
• Proteoglycans provide compressive strength, regulate signaling molecules, and retain water to maintain tissue hydration.
• Fibronectin is a glycoprotein with multiple binding domains for ECM components and integrins.
• Fibronectin exists as a dimer and helps link ECM to cells.
• Fibronectin guides cell adhesion, migration, and differentiation, especially during embryonic development.
• Laminin is a large glycoprotein found in the basal lamina.
• Laminin comprises three chains (α, β, γ) forming a cross-shaped structure.
• Laminin facilitates cell attachment, tissue organization, and signaling for differentiation.

Contribution of Collagen and Proteoglycans to ECM Strength

• Collagen provides tensile strength by forming strong, fibrous networks.
• Triple-helical structure of collagen resists stretching forces, making tissues like tendons and bones durable.
• Proteoglycans offer compressive strength by attracting and retaining water.
• The gel-like nature of proteoglycans, especially those rich in glycosaminoglycans like hyaluronic acid, resists compressive forces in cartilage and intervertebral discs.
• Together, collagen and proteoglycans withstand tensile and compressive forces, ensuring structural stability in tissues.

Role of Fibronectin and Laminin in Embryonic Development and Cell Migration

• Fibronectin guides cell migration by interacting with integrins on cell surfaces.
• Fibronectin forms a scaffold that directs cells to specific locations during embryogenesis.
• Fibronectin helps establish tissue boundaries and promote organogenesis.
• Laminin is crucial in the formation of the basal lamina, providing a foundation for developing tissues.
• Laminin provides adhesion sites for migrating cells, influencing differentiation and tissue organization.
• Laminin plays a key role in neuronal development by guiding axon outgrowth.

Roles of Matrix Metalloproteases (MMPs) in ECM Remodeling

• Matrix metalloproteases (MMPs) are enzymes that degrade ECM proteins, facilitating tissue remodeling.
• MMPs help shape organs and tissues in embryogenesis by tissue remodeling during development.
• MMPs clear damaged ECM and facilitate new tissue growth during wound healing.
• MMPs degrade ECM barriers to allow cell movement during cell migration, crucial in immune responses and angiogenesis.
• MMPs contribute to cancer metastasis by breaking down ECM to allow cancer cell invasion, during tumor progression.
• MMPs degrade proteins like collagen, laminin, and fibronectin in ECM degradation and turnover.
• MMPs clear damaged ECM and promote new matrix deposition during wound healing.
• MMPs enable blood vessel formation by modifying the ECM to support endothelial cell migration during angiogenesis.
• MMPs degrade ECM barriers, allowing cancer cells to invade surrounding tissues during tumor invasion and metastasis.
• Integrins are transmembrane receptors connecting the ECM to the cytoskeleton.
• Integrins affect cell behavior through outside-in and inside-out signaling.

Integrins activate:

• FAK (focal adhesion kinase) and PI3K-AKT pathways prevent apoptosis during cell survival.
• Cells undergo anoikis (programmed cell death) if integrins lose ECM contact.

Malignant Transformation:

• Cancer cells alter integrin expression to avoid anoikis.
• Integrins degrade ECM and promote metastasis by working with MMPs.
• Abnormal integrin signaling causes uncontrolled proliferation and angiogenesis.
• The ECM connects to the intracellular space through integrins, linking ECM proteins to the actin cytoskeleton via adaptor proteins.
• Collagen, fibronectin, and laminin bind integrins, influencing cell adhesion and migration.
• Proteoglycans (e.g., Perlecan) regulate signaling by binding growth factors.
• Integrins transmit mechanical and chemical signals, affecting cell survival and differentiation.

Experiment Showing Integrins Bind RGD Sequences

• The RGD sequence (Arg-Gly-Asp) is a motif in fibronectin that binds integrins.
• Scientists synthesized peptides containing the RGD sequence and competed with fibronectin for integrin binding.
• Cells failed to adhere when RGD peptides were present, thus RGD is required for integrin interaction.

Differences Between Focal Adhesions and Hemidesmosomes

• Focal adhesions connect ECM to actin cytoskeleton; hemidesmosomes connect ECM to intermediate filaments.
• Key proteins in focal adhesions are integrins (α5β1), talin, and vinculin.
• Key proteins in hemidesmosomes are integrin α6β4 and plectin.
• Focal adhesions facilitate cell migration and signaling.
• Hemidesmosomes are important for strong adhesion in epithelial cells (e.g., skin).
• Fibronectin is the ECM ligand for focal adhesions, while laminin is the ligand for hemidesmosomes.

Cell-Cell Adhesion Molecules Comparison

• Cadherins are calcium-dependent glycoproteins that mediate homophilic binding between cells.
• E-cadherin (epithelia) and N-cadherin (neurons) are examples of Cadherins
• Selectins bind carbohydrates on other cells, mediating temporary adhesion in immune response.
• P-selectin (platelets) and L-selectin (leukocytes) are examples of selectins
• Ig-Superfamily CAMs are immunoglobulin-like domains for cell-cell recognition and weaker interactions.
• NCAM (neurons) and ICAM (inflammation) are examples of Ig-Superfamily CAMs.
• Integrins are heterodimeric receptors that bind ECM proteins and transmit signals.
• α5β1 (fibronectin) and α6β4 (hemidesmosomes) are Integrins examples

Cell Adhesion in Inflammation and Cancer

• In inflammation selectins recruit white blood cells (WBCs) to inflamed areas.
• In inflammation integrins (LFA-1, VLA-4) strengthen WBC adhesion to endothelial cells, allowing extravasation.
• Loss of E-cadherins promotes metastasis in cancer by weakening cell-cell adhesion.
• Overexpression of MMPs degrades the ECM, aiding invasion in cancer.

Role of ECM & Adhesion Proteins in Cancer

• MMPs promote cancer invasion by degrading collagen and laminin.
• Disorganized collagen fibers create paths for cancer cells to spread during breast cancer.
• E-cadherins normally maintain epithelial integrity, whose loss increases motility and invasiveness.

Organelles of the Endomembrane System & Transport Pathways

• ER (rough & smooth) involved in protein & lipid synthesis.
• Golgi apparatus modifies, sorts, and packages proteins.
• Lysosomes degrade macromolecules.
• Vesicles transport materials.

Biosynthetic/Secretory Pathway

• ER → Golgi → Plasma Membrane/Lysosomes.

Endocytic Pathway

• Plasma Membrane → Endosomes → Lysosomes.

Tracking Protein Transport using Autoradiography vs. GFP

• Autoradiography tracks movement of proteins labeled with radioactive amino acids.
• GFP tracks protein movement by tagging proteins with green fluorescent protein.
• Temperature-sensitive mutants: At low temperatures, proteins become stuck in one compartment; upon warming, transport resumes, helping map pathways.

The final destination of a protein depends on

• Whether is synthesized on free ribosomes or ribosomes bound to the rough endoplasmic reticulum (RER).

Proteins Synthesized on Free Ribosomes

• These proteins remain in the cytosol or are targeted to organelles that are not part of the endomembrane system.
• Final destinations:*
• Cytosolic proteins (e.g., glycolysis enzymes, cytoskeletal proteins).
• Mitochondrial proteins (imported via TOM/TIM complexes).
• Nuclear proteins (contain a nuclear localization signal (NLS) and enter through nuclear pores).
• Peroxisomal proteins (contain a peroxisomal targeting signal (PTS) for import).

Free ribosomes

• Synthesis of protein in free ribosomes
• Proteins Synthesized on RER-Bound Ribosomes*
• These proteins enter the ER during translation and follow the biosynthetic/secretory pathway.
• Final destinations:*
• Secreted proteins (e.g., hormones, antibodies).
• Plasma membrane proteins (e.g., ion channels, receptors).
• Lysosomal enzymes (e.g., hydrolases tagged with Mannose-6-Phosphate).
• Golgi-resident proteins (e.g., glycosyltransferases).
• ER-resident proteins (e.g., chaperones like BiP, retained via a KDEL sequence).

Predicting a Protein's Final Location Based on Signal Sequences

• No signal sequence Proteins location:*
• Location of Translation: Free ribosomes
• Final Destination: Cytosol
• Mitochondrial signal Sequence Proteins location:*
• Location of Translation: Free ribosomes
• Final Destination: Mitochondria
• Nuclear localization signal (NLS) location:*
• Location of Translation: Free ribosomes
• Final Destination: Nucleus
• Peroxisomal targeting sequences location:*
• Location of Translation: Free ribosomes
• Final Destination: Peroxisome
• ER signal sequence location:*
• Location of Translation: RER-bound ribosomes
• Final Destination: Secretory pathway (ER → Golgi → PM,lysosome, secretion)

Co-Translational Translocation

• Co-translational translocation: proteins are synthesized and inserted into the ER membrane or lumen simultaneously.
• Key Components and Steps:*
• Signal Sequence: hydrophobic sequence (usually at the N-terminus) emerges from the ribosome during translation.
• Signal Sequence: Targets the ribosome to the ER.
• Signal Recognition Particle (SRP) is a ribonucleoprotein complex binds that to the signal sequence and pauses translation.
• SRP guides the ribosome to the ER by interacting with the SRP receptor on the ER membrane.
• SRP Receptor, a membrane protein complex, recognizes the SRP-ribosome complex
• Once the ribosome is docked, SRP is released and translation resumes.
• Translocon (Sec61 complex):*
• A protein channel in the ER membrane,it allows the growing polypeptide to enter the ER.
• The ribosome binds to the translocon, and the growing peptide is threaded into the ER lumen or membrane.
• Signal Peptidase:*
• If the protein is soluble (not membrane-bound), signal peptidase cleaves the signal sequence.
• The protein is fully released into the ER lumen.
• Co-Translational Summary Notes:*
• Signal sequence emerges and targets protein to ER.
• SRP binds and pauses translation, directs ribosome to ER.
• SRP receptor binds and anchors ribosome to ER.
• Translocon(Sec61 Complex) opens and channels protein into ER.
• Signal peptidase cleaves and removes signal sequence if protein is sooluble.
• Result: The protein is inserted into the ER membrane (if it has transmembrane domains) or into the ER lumen (if it is a secretory protein).
• Signal peptidase cleaves the signal sequence from the nascent protein once it enters the ER lumen.

Synthesis of Integral Membrane Proteins

• Integral membrane proteins are synthesized by ribosomes bound to the rough ER and inserted into the membrane via the translocon.
• Proteins destined for the membrane contain hydrophobic transmembrane domains (TMDs) that stop translocation and embed them in the ER membrane.
• As translation continues, additional TMDs are inserted into the membrane, ensuring proper orientation.
• These proteins may undergo modifications such as glycosylation before being transported to their final destination (Golgi, lysosomes, or plasma membrane).

Glycosylation of Proteins in the ER

• Glycosylation :addition of carbohydrate groups to proteins.
• In the ER: (N-linked glycosylation), oligosaccharide is attached to the amide nitrogen of an asparagine residue.
• A 14-sugar oligosaccharide (Glc3Man9GlcNAc2) is assembled on a lipid carrier (dolichol phosphate).
• The oligosaccharide is transferred to a specific asparagine (Asn) in the growing polypeptide by oligosaccharyltransferase.
• The sugar chain is modified by the removal of three glucose residues, is important for quality control.
• Glycosylation Steps:*
• Lipid Carrier: Dolichol Phosphate:
• Functions as a membrane-bound platform for assembling the oligosaccharide before transfer to the nascent protein.
• Located in the ER membrane, flipping between the cytosolic and luminal sides.
• Glycosyltransferases:*
• Enzymes that sequentially add sugar residues (N-acetylglucosamine, mannose, glucose) to the growing oligosaccharide chain on dolichol phosphate.
• Asparagine (ASN) Residue:*
• Glycosylation occurs at a specific Asn (N) residue in the consensus sequence Asn-X-Ser/Thr, where X can be any amino acid except proline.
• The oligosaccharide is transferred from dolichol phosphate to this residue by the enzyme oligosaccharyltransferase (OST).
• Process of N-Linked Glycosylation in the ER:*
• Oligosaccharide Assembly on Dolichol Phosphate:
• Sugar residues are sequentially to dolichol phosphate by glycosyltransferases; occurs on the cytosolic side of the ER membrane.

Flipping into the ER Lumen:

• The partially assembled oligosaccharide is flipped into the ER lumen by a flippase enzyme.
• Final Oligosaccharide Assembly:*
• Additional sugars are added inside the ER lumen, completing the core oligosaccharide (Glc3Man9GlcNAc2).
• Transfer to Asn Residue:*
• The completed oligosaccharide is transferred to the Asn-X-Ser/Thr motif on the growing polypeptide chain.
• The enzyme oligosaccharyltransferase (OST) catalyzes this transfer.
• Processing and Quality Control:*
• The glycoprotein undergoes trimming and folding with the help of calnexin and calreticulin before being transported to the Golgi for further modifications.

ER Quality Control Mechanism

• The ER quality control system ensures that only properly folded glycoproteins move forward in the secretory pathway.
• Calnexin : Chaperone protein ; binds to glycoproteins containg a single remaining glucose residue as aid proper folding.
• UGGT (UDP-glucose:glycoprotein glucosyltransferase) :If protein is misfolded, UGGT adds a glucose residue back to the oligosaccharide allowing it to rebind calnexis for attempt at folding.
• If protein fails to fold correct ; target for ER-associated degradation (ERAD) and send to proteasome for degradation. `###Unfolded Protein Response (UPR)
• The UPR is activated when misfolded proteins accumulate in the ER. The response involves three major sensors being a cell survival mechanism.
• IRE1: Splices mRNA encoding XBP1, which activates genes involved in protein folding and degradation.
• PERK: Phosphorylates eIF2a, reducing overall protein translation while increasing translation of stress-related proteins.
• ATF6: Moves to the Golgi, it's cleaved to release a transcription factor that upregulates chaperones and ERAD components.
• If the stress is severe, UPR can trigger apoptosis to prevent further damage.

Protein Glycosylation in the ER vs. Golgi

• Featur in the ER:*
• Sugar addition: Pre-assembled Glc3Man9GlcNAc2 is transferred to Asn
• Modification Glucose & mannose trimmed for quality control
• Function assists in protein folding
• Features in Golgi:*
• Sugar addition: Sugars are sequentially modified and O-linked sugars are added to Ser/Thr
• Modification Mannose removed, the addition of complex sugars are added (e.g., sialic acid, fucose)
• Function final sorting and functional modification

Diagram of RER and Golgi Network

• Rough ER (RER).
• Cis-Golgi network (CGN) - receiving side.
• Trans-Golgi network (TGN) - sorting and shipping side.
• COPII-coated vesicles - move(forward) proteins from ER to Golgi: Proteins with “ER export:" signals:
• Enzymes are in the later stages of the biosynthetic/secretory pathway.
• Glycosyltransferases of the Golgi complex, Ion channels in the plasma membrane.
• COPI-coated vesicles – return (backward) ER-resident proteins from Golgi to ER.
• Arrows show indicate two ways of transport*
• Anterograde transport (COPII vesicles) – ER to Golgi.
• Retrograde transport (COPI vesicles) - Golgi to ER.
• KDEL Signal Sequence*
• KDEL(Lys-Asp-Glu-Leu) retrieval signal for ER-resident proteins.
• If a protein with KDEL is mistakenly sent to the Golgi, KDEL receptors recognize in the cis-Golgi and retrieve it via COPI-coated vesicles.
• This ensures ER chaperones (e.g., BiP) remain in the ER.
• Lysosomal Enzyme Targeting*
• Lysosomal enzymes are tagged in the Golgi with mannose-6-phosphate (M6P) to ensure they are sent to lysosomes and not secretory vesicles.
• In the cis-Golgi, a phosphotransferase enzyme adds a phosphate group to mannose residues, forming M6P.
• In the trans-Golgi, M6P receptors (M6PRs) find the tagged enzymes and package them into clathrin-coated vesicles.
• These vesicles fuse with late endosomes; the acidic pH causes M6PRs to release their cargo.
• The enzymes are then delivered to the lysosome to carry out their degradative functions.