Cell Structure and Function: Protein Synthesis, Membrane Trafficking, and Extracellular Matrix

Nucleic Acids and DNA Structure

Polymerization of Nucleotides:
- Nucleic acids are polymers composed of repeating monomeric subunits called nucleotides.
- Nucleotide synthesis occurs via $2$ dehydration reactions.
- Each nucleotide consists of three structural components: a phosphate group, a pentose sugar (deoxyribose in DNA), and a nitrogenous base.
Nitrogenous Bases Classification:
- Purines (double-ring nitrogenous structures):
  - Adenine ( $A$ ): Contains $H_2N$ and $NH_2$ side groups.
  - Guanine ( $G$ ): Contains $C=O$ , $HN$ , and $H_2N$ side groups.
- Pyrimidines (single-ring nitrogenous structures):
  - Thymine ( $T$ ): Contains $C=O$ and $NH$ side groups.
  - Cytosine ( $C$ ): Contains $NH_2$ and $C=O$ side groups.
DNA Structure and Double Helix:
- Phosphate-deoxyribose backbone forms the structural scaffolding.
- Strand orientation is antiparallel, running from the $5'$ end to the $3'$ end on one strand, and from the $3'$ end to the $5'$ end on the complementary strand.
- Base pairing occurs inside the double helix via hydrogen bonds:
  - Adenine ( $A$ ) pairs specifically with Thymine ( $T$ ).
  - Guanine ( $G$ ) pairs specifically with Cytosine ( $C$ ).

Genomic Scale:

DNA stores cellular genetic information.
The human genome contains approximately $3$ billion ( $3 \times 10^9$ ) base pairs.
Encodes approximately $25,000$ proteins.

Gene is a stretch of DNA that codes a RNA

Transcription and Nuclear Pore Architecture

Transcription Mechanism (mRNA Synthesis from DNA Template):
- Primary enzyme: RNA polymerase. Transcribes messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA) from the DNA template strand.
- DNA components: Coding strand and Template strand.
- Initiation: RNA polymerase binds specifically to the promoter region of the DNA and initiates mRNA synthesis at the start point on the template strand.
- Elongation: RNA polymerase moves along the DNA sequence, unwinding the double helix, adding complementary RNA nucleotides, and generating an mRNA transcript along the length of the template strand. The DNA rewinds behind the enzyme, forming a transient DNA-RNA hybrid region.
- Termination: mRNA synthesis ends when RNA polymerase reaches the termination signal, resulting in the release of a completed mRNA transcript.
- Functional Query: What is DNA polymerase? (Distinct from RNA polymerase; functions primarily in DNA replication rather than RNA transcription).
Nuclear Pore Complex (NPC) Architecture:
- Massive protein complex embedded across the inner and outer nuclear membranes of the nuclear envelope.
- Composed of approximately $30$ distinct proteins.
- Structural Domains & Components:
  - Outer membrane and Inner membrane separated by the nuclear lumen/lumenal domain.
  - Cytoplasmic filaments extending outward into the cytosol.
  - Cytoplasmic ring moiety anchored on the outer nuclear surface.
  - Central framework creating the primary transport channel with peripheral channels.
  - Nuclear ring moiety anchored on the inner nuclear surface.
  - Nuclear basket structure projecting into the nucleoplasm, terminating at a distal ring (scale parameter: $5\,\text{nm}$ ).
  - Intranuclear filaments extending deeper into the nucleoplasm.

Protein Synthesis and Ribosome Function

Ribosome Structure and Composition:
- Granules composed of proteins and ribosomal RNA (rRNA) responsible for translating mRNA into protein sequences.
- Functional complexes include $70S$ ribosomal translation assemblies (represented as $70S\text{-rib:mRNA pept-tRNA}$ complexes).
Subcellular Locations of Ribosomes:
- Free in the cytoplasm: Responsible for synthesizing soluble, cytosolic proteins.
- Attached to membranes: Located on the Rough Endoplasmic Reticulum (RER) and the inner mitochondrial membrane.
Stages of Translation:
- Initiation Step: The ribosome binds to mRNA and locates the start codon.
- Elongation Step: The peptide chain grows as incoming aminoacyl-tRNAs bind to complementary mRNA codons.
  - Codon-Anticodon Pairing Examples: An mRNA sequence of $5'\text{-UGG-AAA-GAU-UUC-}3'$ pairs with tRNA anticodons $3'\text{-ACC-}5'$ (carrying Tryptophan, Trp), $3'\text{-UUU-}5'$ (carrying Lysine, Lys), $3'\text{-CUA-}5'$ (carrying Aspartate, Asp), and $3'\text{-AAG-}5'$ (carrying Phenylalanine, Phe).
  - Incoming tRNA bound to an amino acid enters the ribosome; outgoing empty tRNA leaves after transferring its amino acid to the growing polypeptide chain.
- Termination Step and Ribosome Recycling: Synthesis terminates at a stop codon, releasing the completed peptide chain, followed by disassembly and recycling of the ribosomal complex.

Endoplasmic Reticulum and Subcellular Protein Trafficking

Rough Endoplasmic Reticulum (RER) Protein Synthesis:
- Synthesizes specific classes of proteins:
  - Integral membrane proteins destined for the plasma membrane and organelle membranes.
  - Secreted proteins intended for exocytosis.
  - Organellar luminal proteins, such as hydrolytic enzymes inside lysosomes.

Mechanism of RER Translocation:
1. Translation initiates in the cytosol; an N-terminal ER signal sequence emerges from the ribosome.
2. Signal Recognition Particle (SRP) binds to the ER signal sequence.
3. SRP binds to the SRP receptor site located on the cytosolic face of the Rough ER membrane.
4. The growing polypeptide chain is translocated into the Rough ER cistern.
5. The signal sequence is enzymatically removed.
6. Post-translational modifications occur within the lumen, such as the addition of sugar groups (glycosylation).
7. The released protein is enclosed inside a protein-coated transport vesicle that pinches off from the RER.
Full Subcellular Pathway of Protein Synthesis and Sorting:
1. DNA is transcribed to mRNA within the nucleus.
2. mRNA exits the nucleus through nuclear pore complexes into the cytosol and attaches to ribosomes.
3. Soluble proteins are translated entirely within the cytosol.
4. Targeted proteins are translated on the surface of the RER.
5. Translation on the RER translocates the protein into the RER lumen or inserts it into the RER membrane.
6. Transport vesicles bud off the RER carrying the protein and shuttle to the Golgi apparatus.
7. Retrograde Golgi-to-ER transport recycles essential lipids and proteins back to the ER.
8. Vesicles fuse with the Golgi apparatus for further protein modification, sorting, and packaging.
9. Vesicles bud off the Golgi and are targeted to intracellular organelles (e.g., lysosomes, storage vesicles, peroxisomes, mitochondria).
10. Secretory vesicles transport integral membrane proteins to the plasma membrane or export soluble contents to the extracellular fluid via exocytosis.
  -Smooth ER is for lipid synthesis

Mitochondria helps produce a lot of ATP

Mitochondria has a double membrane and contains its own DNA

Vesicular Transport, Exocytosis, and Endocytosis

Vesicular Transport off the Golgi:
- Shipping vesicles originate from the trans face of the Golgi apparatus.
- Pathway A: Vesicle contents are packaged into secretory vesicles destined for exocytosis into the extracellular fluid.
- Pathway B: Vesicle membrane components fuse directly to be incorporated into the plasma membrane.
- Pathway C: Vesicles containing acid hydrolase enzymes mature into lysosomes (or fuse with phagosomes).
SNARE-Mediated Exocytosis Mechanism:
1. Vesicle SNARE ( $v\text{-SNARE}$ ) on the secretory vesicle interacts with Target SNARE ( $t\text{-SNARE}$ ) on the plasma membrane.
2. Interlocking of $v\text{-SNARE}$ and $t\text{-SNARE}$ pulls the vesicle tightly against the membrane.
3. A fusion pore forms through the fused lipid bilayers.
4. Vesicle contents are secreted into the extracellular fluid.
Molecular Control of SNARE Assembly and $Ca^{2+}$ -Triggering:
- Participating Molecules: Synaptotagmin-1, Munc18, Synaptobrevin/VAMP, SNAP-25, Syntaxin-1, Complexin, Munc13, RIMS, NSF, and SNAPs.
- Docking: Syntaxin-1 and SNAP-25 ( $t\text{-SNAREs}$ ) interact with Synaptobrevin/VAMP ( $v\text{-SNARE}$ ) regulated by Munc18.
- Priming I: Munc13 and RIMS facilitate partial assembly of the trans-SNARE complex (prefusion SNARE-SM protein complex).
- Priming II: Complexin activates and stabilizes the prefusion SNARE/SM protein complex.
- $Ca^{2+}$ Activation: Influx of $Ca^{2+}$ activates $Ca^{2+}$ -binding sites on Synaptotagmin-1.
- Fusion Pore Opening: $Ca^{2+}$ -triggered activation of Synaptotagmin-1 opens the fusion pore.
- Fusion Completion: The complex transitions to a fully assembled cis-SNARE complex as the fusion pore expands.
- Disassembly & Recycling: NSF (N-ethylmaleimide-sensitive factor) and SNAPs bind the cis-SNARE complex; ATP hydrolysis ( $ATP \rightarrow ADP + P_i$ ) drives disassembly and vesicle recycling.
Examples of Specialized Hydrophilic Cargo Secretion:
- Axon Terminals at Neuromuscular Junctions: Vesicles containing neurotransmitters release cargo to stimulate muscle tissues.
- Pancreatic Acinar Cells: Vesicles containing hydrolytic enzymes package and export digestive zymogens.
Endocytic Pathways:
- Extracellular substances are internalized via plasma membrane invagination into the cytoplasm, usually facilitated by a protein coat (typically clathrin).
- Phagocytosis ("cell eating"): Internalization of large particles or cellular debris to form a phagosome, which fuses with a lysosome.
- Pinocytosis ("cell drinking"): Non-specific fluid-phase uptake of extracellular fluid and small dissolved solutes.
- Receptor-Mediated Endocytosis: Selective uptake of specific extracellular ligands binding to surface receptors in clathrin-coated pits.
- Transcytosis: Sequential endocytosis on one side of a cell, vesicular trafficking across the cytoplasm, and exocytosis on the opposite side.

Extracellular Matrix (ECM) Composition

Four Most Abundant ECM Proteins:
- Collagen ( $Col(1\text{--}12)$ ): The most abundant protein in the human body.
- Elastin: Provides elastic recoil and tensile resilience.
- Fibronectin ( $Fn1$ ): Mediates cell adhesion and structural matrix organization.
- Proteoglycans: High-molecular-weight proteins covalently linked to glycosaminoglycan (GAG) chains.
Tissue-Specific Relative Abundance of ECM Proteins:
- Tendon: Predominantly Type I Collagen ( $Col1$ ); also contains Fibronectin ( $Fn1$ ), Decorin, COMP (Cartilage Oligomeric Matrix Protein), Biglycan, CILP (Cartilage Intermediate Layer Protein), Type III Collagen ( $Col3$ ), Type VI Collagen ( $Col6$ ), and other minor collagens/proteins.
- Articular Cartilage: Predominantly Type II Collagen ( $Col2$ ) and Aggrecan ( $ACAN$ ); also contains CILP1, COMP, Decorin, Type III Collagen ( $Col3$ ), Type VI Collagen ( $Col6$ ), Type IX Collagen ( $Col9$ ), Type XI Collagen ( $Col11$ ), and other structural proteins.
- Ligament: Predominantly Type I Collagen ( $Col1$ ); also contains Decorin, Fibronectin ( $Fn1$ ), Biglycan, Lumican, COMP, Type III Collagen ( $Col3$ ), Type V Collagen ( $Col5$ ), Type VI Collagen ( $Col6$ ), Type XII Collagen ( $Col12$ ), and secondary proteins.
- Skeletal Muscle: Contains Type I Collagen ( $Col1$ ), Type III Collagen ( $Col3$ ), Versican, Elastin, and minor collagens.
- Bone: Predominantly Type I Collagen ( $Col1$ ); also contains Fibronectin ( $Fn1$ ), Biglycan ( $BGN$ ), DEFA3 (Defensin Alpha 3), AHSG (Alpha-2-HS-Glycoprotein), SERPINF1, DPTJ (Dermatopontin variant), CHAD (Chondroadherin), Type III Collagen ( $Col3$ ), and other collagens.

Collagen Synthesis, Assembly, and Crosslinking

Intracellular Steps of Collagen Synthesis:
1. Transcription: Transcription of specific $\\alpha$ -chain mRNA transcripts inside the nucleus.
2. Translation: Translation of procollagen $\\alpha$ -chains on membrane-bound ribosomes of the RER.
3. Hydroxylation: Specific prolyl and lysyl residues are enzymatically hydroxylated in the RER.
4. Glycosylation: Glycosylation of specific hydroxylysine residues occurs.
5. Triple Helix Assembly: Three procollagen $\\alpha$ -chains assemble into a soluble triple-helical procollagen molecule.
6. Transport: Soluble procollagen is shuttled to the Golgi complex via transport vesicles.
7. Golgi Packaging: Soluble procollagen molecules are packaged into secretory vesicles inside the Golgi complex.
Extracellular Steps of Collagen Assembly:
1. Exocytosis: Soluble procollagen molecules are secreted into the extracellular space.
2. Cleavage of Terminal Peptides: Procollagen peptidases cleave non-helical terminal propeptides, converting soluble procollagen into insoluble collagen molecules.
3. Fibril Aggregation: Insoluble collagen molecules aggregate spontaneously to form collagen fibrils exhibiting a $67\,\text{nm}$ staggered pattern.
4. Covalent Crosslinking: Formation of covalent crosslinks between collagen molecules stabilizes the fibril matrix.
Role of Lysyl Oxidase and Essential Cofactors:
- Lysyl Oxidase: A copper-dependent enzyme that covalently crosslinks collagen molecules.
- Mechanism: Lysyl oxidase induces formaldehyde/aldehyde formation on collagen residues, promoting spontaneous covalent bond formation between neighboring collagen molecules.
- Required Cofactors: Collagen production and crosslinking strictly require both Vitamin C and copper.
Pathophysiology of Vitamin C Deficiency (Scurvy):
- Absence of Vitamin C leads to defective hydroxylation, resulting in the assembly of weak, unstable collagen.
- Pathological Consequences: Receding gums, capillary rupture causing internal bleeding, soft/weakened bones, and impaired tissue healing.

Carbohydrates, Monosaccharides, and Disaccharides

Carbohydrate Classification:
- Classified according to molecular size and carbon chain length.
- Monosaccharides: Simple sugars composed of a single chain or single ring structure containing $3$ to $7$ carbon atoms ( $C:H:O$ building blocks).
Primary Monosaccharides:
- Glucose ( $C_6H_{12}O_6$ ): Primary hexose sugar utilized for cellular energy.
- Fructose ( $C_6H_{12}O_6$ ): Hexose keto-sugar.
- Galactose ( $C_6H_{12}O_6$ ): Hexose aldo-sugar.
- Ribose ( $C_5H_{10}O_5$ ): Pentose sugar integral to RNA nucleotide structure.
Disaccharides and Chemical Reactions:
- Disaccharides are double sugars formed from two monosaccharides.
- Dehydration Synthesis: Monosaccharide + Monosaccharide $\rightarrow$ Disaccharide + $H_2O$ (water is released).
- Hydrolysis: Disaccharide + $H_2O \rightarrow$ Monosaccharide + Monosaccharide (water is consumed).
- Specific Disaccharide Synthesis Equations:
  - Glucose + Fructose $\rightleftharpoons$ Sucrose + $H_2O$
  - Glucose + Glucose $\rightleftharpoons$ Maltose + $H_2O$
  - Galactose + Glucose $\rightleftharpoons$ Lactose + $H_2O$

Proteoglycans and Glycosaminoglycans (GAGs)

Glycosaminoglycans (GAGs):
- Long-chain, unbranched polysaccharides consisting of repeating disaccharide units.
- All GAGs carry a net negative charge.
- Examples of GAGs:
  - Chondroitin sulfate
  - Keratan sulfate
  - Hyaluronic acid
Proteoglycan Architecture:
- Consists of a central core protein bound to multiple GAG chains.
- Aggrecan ( $ACAN$ ) Structure:
  - N-terminal Hyaluronan-Binding Region (HABR).
  - Central core protein attached to chondroitin sulfate chains and keratan sulfate chains.
  - Contains N-linked oligosaccharides and Epidermal Growth Factor (EGF)-like domains.
- PG-M / Versican: Major chondroitin sulfate proteoglycan involved in structural matrix support.

Connective Tissue Physiology, Injuries, and Pathology

Physiology of Poorly Vascularized Connective Tissues:
- Tendons and Ligaments: Composed of dense regular connective tissue designed to withstand tensile forces (tension).
- Fibrocartilage: Situated between bones to withstand heavy compressive forces (compression).
- Articular Cartilage (Hyaline Cartilage): Covers the articulating ends of bones to absorb compressive forces (compression).
- Tissue Properties:
  - Cartilage is considered avascular (devoid of blood vessels).
  - Cartilage and blood are not innervated (devoid of neural connections).
  - Cartilage and blood undergo substantial shape changes under physical loads.
- Nutritional Dynamics: Cells within these tissues receive nutrients and eliminate metabolic waste products via mechanical fluid movement and passive diffusion through the hydrated ECM during deformation cycles.
Ligament Traumas and Sprains:
- Anterior Cruciate Ligament (ACL): Frequently torn knee ligament.
- Ulnar Collateral Ligament (UCL): Commonly injured elbow ligament.
- Sprain Definitions:
  - Mild Sprain: Mild stretching and minor micro-tearing of the ECM.
  - Severe Sprain: Complete mechanical tear and disruption of the ECM structure.
Inherited and Degenerative Connective Tissue Disorders:
- Ehlers-Danlos Syndrome: Primary disorder affecting the skin and joints. Connective tissue becomes weak, resulting in loose, hypermobile joints and fragile, sagging skin. Caused by mutations directly in collagen genes or in processing enzymes required for collagen maturation.
- Osteogenesis Imperfecta: Causes extreme bone fragility, leading to frequent fractures under low stress. Direct mutations in collagen genes account for $90\%$ of all cases.
- Osteoarthritis: Possesses a strong genetic link, though exact causal genes are not fully identified. Women are significantly more prone to developing osteoarthritis and suffer more severe forms than men. Responsible for approximately $750,000$ knee replacement surgeries annually. Pathologically characterized by degenerated articular cartilage, joint space loss, and subchondral bone spurring on both the femur and tibia bones.

Cytoplasmic inclusions are : glycogen, lipid droplets, and pigments

Organelles are things with a membrane inside the cell

Glycolysis is a way to get ATP without oxygen anaerobic 2ATP

Aerobic with mitochondria and O2 34-36 ATP

What could go wrong - no O2, CO, cyanide, arsenic

Amino acids and fatty acids can make pyruvate for more ATP

Mitochondria is constantly fissing and fusing (splitting and merging) - communicating

Lysosomes contain digestive enzymes at pH 4.5 - the cell is at about 7

Lysosomes are the garbage disposal of the cell

Microfilaments are made from actin and give structure to cells

Intermediate filaments are tough fibers made from 70 different proteins - nuero cells

Microtubules determine cell shape and are kinda like a train track