Cellular Diversity: Differential Protein Expression

Chapter 4: General Features of Cells

• DNA sequences are the same in every cell of the body with the exception of germ-line cells.
• Despite identical DNA sequences, cells structures vary depending on the structure and function of proteins they contain, this is caused by mechanisms of differential protein expression.

Mechanisms of Variation in the Proteome:

• Differential Gene Regulation varies expression based on environment.
  • Epigenetic molecules can up-regulate or down-regulate genes.
• Concentration and Stability affect protein levels and stability to change protein composition.
• Alternative Splicing modify pre-mRNA to final mRNA with introns and exons spliced out.
  • Depending on which exons are spliced out, different amino acid sequences are created, leading to different proteins and cell structures.
• Covalent Modifications alter protein function and location post polypeptide synthesis and folding.
  • Methylation of amino acids at the N-terminal and glycosylation are examples.

Cytosol, Cytoplasm, and Metabolism

• Cytosol is the region of a eukaryotic cell outside organelles but inside the plasma membrane.
  • Cytosol contains complexes like ribosomes without membranes.
• Cytoplasm is within plasma membrane including cytosol and organelles.
  • Cytosol is the central coordinating region for metabolic activities like Translation.
• Translation occurs in the cytosol at ribosome complexes.
• Transcription occurs in the nucleus.
• Translation is the anabolic process of polypeptide synthesis.
  • Information translates into amino acid sequence.
  • It requires significant energy, making it a highly endergonic process.
• Glycolysis is a catabolic process that breaks down sugars.
  • It can occur with or without oxygen.

The Cytoskeleton

• Composed of a network of three protein structures called filaments.
• The Spindle Apparatus used in mitosis/meiosis is not part of the cytoskeleton because it is not a permanent structure

Motor Proteins

• Use ATP to promote movements with Head(ATP hydrolysis), Hinge, and Tail domains.
• Walking Movement involves protein movement while cytoskeleton remains in place.
  • Like walking, filament is the ground and protein-head is the leg
• Filament Movement is where the protein stays still.
  • ATP energy is used to push the cytoskeleton along.
• Whiplash Movement is where neither move.
  • Energy transfers from head to tail, bending Molecule.

Cilia and Flagella

• Both project outward from the cell membrane.
• Flagella are longer, found singly or in pairs.
• Cilia are shorter, cover cell surface.
• Axonemal Structure is a shared structure of microtubules and dynein.
  • Nine microtubule doublets and two central microtubules (9+2)
• Basal Body- directly under the Axoneme containing microtubule triplets.

The Endomembrane System

• Network membranes surrounding nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, and vacuoles.
• Includes the plasma membrane.
• Constituent parts connect directly or through vesicles.
• Peroxisome is not a component of the Secretory System.

The Nucleus

• Organelle in eukaryotic cells housing/protecting genetic material and location of DNA transcription.
• Nucleolus synthesizes rRNA and ribosomal subunits.
• Surrounded by Nuclear Envelope with Nuclear Pores.
  • Gatekeeper Proteins keep DNA inside while allowing molecule passage.
• Nuclear Lamina supports envelope with lamin filaments.
• Contains DNA in Chromosomes.
  • Chromatin composition: DNA tightly wrapped around Histone proteins.

Ribosomes

• Complexes of RNA and Protein formed at the nucleolus.
  • Contains one large and one small subunit.
  • rRNA makes up biggest portion of both.
• RNA portion consists of three RNA types.
  • rRNA is the primary structural component.
  • mRNA carries information of the amino acid sequence.
  • tRNA assembles the amino acid sequence based on information.
• Responsible for polypeptide and protein synthesis.
  • In translation, mRNA molecule attaches to small ribosome subunit.

Golgi Apparatus

• Organelle close to and facing the ER.
• Structure: stack of membrane-bound sacs called Cisternae (same as ER tubules).
• The Golgi is a packaging and transport center, part of the cell's endomembrane and secretory systems.
• Secretory Vesicles pass substances through sacs (no diffusion).
  • They are "stamped" with carbohydrates, shipped along the cytoskeleton.
• Three subdivisions are oriented in the cell.
  • Cis Golgi is near ER membrane.
  • Trans Golgi is close to the plasma membrane
  • Medial Golgi is in the middle: site of Glycosylation and Proteolysis. Substances are packaged and sent out of the cell (Exocytosis) in trans Golgi.

Lysosomes

• Organelles found in animal cells containing Hydrolytic Enzymes
• Acid Hydrolases break down macromolecules, organelles, and various substances at pH 4.8.
• Substance breakdown: Primary Lysosome stage (pH > 4.8), then transitions to Secondary Lysosome stage (pH drops to 4.8) for breakdown.
• Autophagy: recycling of old, worn-out organelles, forms Phagosomes.

Vacuoles

• Organelles found only in plant cells and some protists.
• Tonoplast is capable of expanding and contracting.
• Central Vacuoles found in all plants.
  • Store H2O, nutrients, and waste; Enough H2O creates Turgor Pressure that becomes Hydrostatic Skeleton.
• Contractile Vacuoles are found in some protists.
  • Expand and contract for cell volume maintenance.
• Phagocytic Vacuoles are found in protists and white blood cells.
  • Used for engulfing and digesting foreign materials.

Peroxisomes

• Small organelles, not part of secretory system, found in eukaryotic cells.
• Formed when vesicles bud from the ER and combine.
• Catalyze reactions that break down molecules.
• Reactions remove/add hydrogen/oxygen, producing hydrogen peroxide (H2O2).
  • Catalase enzyme breaks down H2O2 into H2O + O.
• Microbody classification includes Glyoxisomes (in plants).

Endoplasmic Reticulum

• Network of membranes.
  • Forms flattened tubules called Cisternae.
• Divided into 2 regions: Smooth ER and Rough ER.
  • Region between them is the ER Lumen.
• Rough Endoplasmic Reticulum synthesizes/distributes proteins.
  • Inserts proteins (Glycosylation )into organelle membranes.
• Smooth Endoplasmic Reticulum detoxifies (barbiturates).
  • Functions: Carbohydrate catabolism, lipid synthesis/modification, calcium reservation.
• Carbohydrate Catabolism starts with Phosphorylation.
  • Phosphate added to starch end, forming Glucose 6-Phosphate.
• SER acts as a Calcium Reservoir.

Plasma Membrane

• Boundary between cell and extracellular environment.
• Largely Phospholipids for Semi-Permeability for hydrophobic molecules, blocking hydrophilic ones.
• Contains 3 protein types.
  • Transport Proteins for Selective Permeability.
  • Receptor Proteins for cell communication.
  • Adhesive Proteins allowing temporary attachment of cells in different tissues

Endosymbiotic Theory

• Mitochondria and Chloroplasts are semiautonomous: contain DNA, RNA, and ribosomes.
  • Cannot properly without proteins from the rest of the cell.
  • Cannot divide without nucleus.
• Chloroplasts/mitochondria are from an Endosymbiotic relationship with eukaryotic cells.
  • Cell gets energy, organelles get nutrients and place to live
  • Theory explains why organelles contain DNA

Mitochondria

• Primary role is to make ATP.
• Involved in synthesis, modification, and breakdown of cellular molecules.
  • Generate heat in brown fat cells (bears and babies).
• Divide through binary fission.
• Possess Outer and Inner Membranes, the latter is folded.
  • Space between is Intermembrane Space with protons.
  • Inside: Mitochondrial Matrix, filled with synthesized ATP.
• Along inner membrane's Cristae: Electron Transport Chains and ATP protein molecules

Chloroplasts and Other Plastids

• Chloroplasts are only in plants and algae.
  • Capture light energy and synthesize organic molecules like glucose.
  • Divide using binary fission.
• Chloroplasts possess Outer and Inner Membranes.
  • Intermembrane Space between them
  • Stroma fills inner: Calvin Cycle
  • Grana are stacks of Thylakoid Membranes (flattened tubules) connected by Lamella.
    • Pigments are along thylakoid membrane; oxygen produced in Thylakoid Lumen
• Plastids are a small organelle class where Chloroplasts are involved in photosynthesis (chlorophyll).
  • Chromoplasts store pigments.
  • Amyloplasts colorless: store root starch.

Mechanisms of Protein Sorting

• Co-translational Import starts with ER Signal Sequence picked by Signal Recognition Particle (SRP).
  • SRP binds to the ribosome, stops translation, transfers to ER, docks on ER Membrane
• Amino acid sequence threaded in ER Membrane, ER Signal Sequence cleaved by Signal Peptidase enzyme. Presence/absence of Retention Signal dictates if protein stays or goes to Glycosylation.
• Post-translational Import requires Matrix Targeting Sequence at C-terminal,
  • Chaperone Proteins prevent folding as it travels to the target organelle.
  • Proteins pulled into organelle by chaperone protein set.
  • Signal Peptidase removes the targeting sequence allowing it to fold.

Chapter 5: Membrane Structure, Synthesis, and Transport

• Biological Membranes
• Protein Insertion
• Fluid-Mosaic Model
• Types of Membrane Proteins
• Semifluidity
• Factors Affecting Fluidity
• Lateral Transport Experiment
• Exceptions to Protein Movement
• Electron Microscopy
• Phospholipid Synthesis
• Glycosylation
• Selective Permeability
• Cell Gradients
• Passive Transport
• Channels
• Carriers
• Aquaporin
• Pumps
• ATP-Driven lon Pumps
• Endocytosis and Exocytosis

Biological Membranes

• Membranes serve as formidable and flexible cell boundaries separating the interior from extracellular region.
• Facilitate intercellular communication, transport, and cell division.
• 50% lipids, 50% proteins and carbohydrates
• Phospholipids form bilayer with hydrophobic tails inside and hydrophilic heads on outside.
• Lipid Rafts, found in cell division sites, bind at specific locations that cause cell division.
• Proteins: transport, communication, adhesion and synthesis
• Carbohydrates for protection: 90% glycoproteins; 10% glycolipids.

Protein Insertion in ER Membrane

• ER signal sequences cause docking on the ER membrane.
• Ribosome translates molecules into ER membrane or Lumen.
• Ribosome translates sequence until reaching Stop Transfer Sequence, Signal peptidase removes ER signal sequence.

Fluid Mosaic Model

• Model represents membrane structure as lipids and proteins moving relative to each other.
• Proteins and carbohydrates are in a pattern.
• Asymmetrical phospholipid bilayer
  • Most proteins/carbohydrates on Extracellular Leaflet located out of cell.
  • Very few on Protoplasmic Leaflet.

Types of Membrane Proteins

• Classified by isolation/analysis breaking apart Intrinsic proteins or keeping intact Extrinsic.

Intrinsic

• Requires membrane fracture.
• Transmembrane Proteins span entire membrane; containing hydrophobic/philic regions.
• Lipid-Anchored Proteins: covalently bonded, located between fatty acid tails

Extrinsic (Peripheral)

• Isolation using opposite charged solution
• Connected to hydrophilic phospholipid heads.

Semifluidity

• Molecules easily move within membrane but stay close, as seen in membrane-bound porteins
• Biological membranes are semifluid, liquids can only move adjacent to their neighbors
• Lateral movement of lipids is energy efficient.
• Lipids can flip-flop with flippase transporter proteins and ATP hydrolysis in cell repair and cancer.

Factors Affecting Fluidity

• Optimal temperature of 37°C helps phospholipids move without assistance.
• Hotter causes moving too fast, colder too slow.
• Cholesterol stabilizes and regulates by binding/cushioning phospholipids.
• Saturated reduces movement and Hydrocarbons, Unsaturated induces double bonds.
• Inducing chain length in plant cells occurs while saturated and unsaturated.

Lateral Transport Experiment

• Frye/Edidin fused mouse and human cells for lateral protein movement verification.
• Chimera resulted in mouse and human cells.
• Experiment with mouse cells that have H-2 Antigen.
• Half cells at 0°C and half incubated at 37°C.
• With antigen stain: frozen antigen was on mouse side, incubated expressed over cell surface.

Exceptions to Protein Movement

• Not all integral membrane proteins move restricting range between 10-70%.
• Attachment/cell-compartmentalizing proteins cannot undergo lateral movement.
  • Binding to cytoskeleton or molecules limits movement.

Electron Microscopy

• Transmission Electron Microscopy (TEM)
  • Uses thin-sectioned biological sample stained with heavy metal dyes.
  • These dyes bind to polar phospholipid heads not nonpolar fatty acid bodies.
• Freeze Fracture Electron Microscopy analyzes nonpolar phospholipid bilayers.
  • Freezing sample and using knife-like tool dividing into Extracellular/Protoplasmic halves.
  • Halves covered with heavy metal for mold analysis.
    • The mold is used because it's fragile.

Phospholipid Synthesis

• Begins in cytosol, proceeds in smooth ER
• Raw materials: Fatty Acid Chains, Glycerol Phosphate, coA, Acyltransferase, Phosphatase, Choline/Serine Phosphotransferase enzymes.
• Five Steps:Activation of Fatty Acids, Formation of Fatty Acyl Glycerol Phosphate, Insertion of Fatty Acyl Glycerol Phosphate into Cytosolic Side, Removing Charged Phosphate, Attaching Choline/Serine Amino Acid. Synthesized phospholipid moves through the membrane.

Glycosylation

• Attaches carbohydrate to protein/lipid.
• Products: Glycoproteins and Glycolipids in self/non-self tissue recognition.
• Glycocalyx shields and protects membrane.
• Types: N-linked in the RER and O-linked in medial Golgi
  • Oligosaccharide attaches to the N-side in N-linked and attaches to the O side in O-Linked.
• Co-translational import embeds protein ER Lumen with enzyme Oligosaccharide Transferase detecting sequence.

Selective Permeability

• Biological membranes selectively permeable.
• Essential and waste molecules passing through.
• Nonpolar molecules pass easiest through bilayer.
  • Polars pass at concentration.
• Transports are necessary when moving through glucose as ions or ATP will never diffuse.
• Cells maintain transmembrane and ion electrochemical gradients.
• Passive transport moves solutes until transmembrane gradient evens out without energy: passive and facilitated diffusion.

Passive Transport (Diffusion)

• Moves high to low concentration without energy.
• Types are Passive Diffusion and Facilitated Diffusion from proteins
  • Protein types: rate differs in Channels or Carriers depending on barriers.

Channels

• 5 Types:
  • Ligand-gated Channels require molecule binding.
  • IRP-gated Channels needs IRP Molecule association.
  • Phosphorylation-gated Channels use covalent attachment.
  • Voltage-gated Channels and muscle tissue need charge change.
  • Mechanosensitive Channels need changes in tension and frequency change.

Carriers

• Known as transporters- for organic molecules like sugars uptake.
• Classification
  • Uniporters: carry one molecule.
  • Symporters/antiporters: carry multiple molecules in same or different direction

Active Transport (Diffusion)

• Transmmebrane goes from low to high with energy.
• Maintain homeostasis
• Differentiation by energy: primary active transport uses ATP Hydrolysis.

Ion Pumps

• Secondary active transport uses pre-existing Na*/H+ gradient.
  • Na+/K+ ATPase pump involves transporting to establish equilibrium.
  • Exports 3 Na+ and imports two K+ ions.

Electrochemical Gradients

• The resting membrane potential is -70mV
• Ion Gradients go through channels
• Negative value means it’s more negative inside cell
• Ion Electrochemical Gradients has chemical and electrical gradients.

ATP Driven Lon Pumps

• Na+/K+ ATPase pump moves with ATP: exports three +sodium and imports two +potassium results a +net charge
• It is particularly vital in neuron activity.

Endocytosis and Exocytosis

• Cargo larger than 1 kDa enters and exits. _ Exocytosis: material is carried into vesicles and excreted through the plasma membrane such as collagen and hormones. _ Starts with cargo to transport, coated transfer proteins for membrane. _ Coat proteins are removed along with the vesicle. T-snare proteins fuse to proteins along plasma.
• Endocytosis: plasma membrane invaginates for a vesicle. _ The 3 types: solid-Phagocytosis, liquid-Pinocytosis, specific proteins and receptors-Receptor-Mediated Endocytosis. _ Pinosytosis in intestine and sertoli cells. _foreign bodies contact cell and coat for snare binding with protein.

Chapter 6: Energy, Enzymes, and Metabolism

• Thermodynamics
• Spontaneity of Chemical Reactions
• Metabolism
• Energy Intermediates
• ATP Synthesis
• Regulation
• Enzymes and Ribozymes. Recycling of mRNA and Proteins.
• Influences on Enzyme Activity.

Thermodynamics

• thermodynamics refers to heat changes between different forms of energy
• the ability to promote change.
• Kinetic, Potential, Chemical (energy in the molecular bonds), Piezoelectric (pressure-based), heat transfer.
• Measure random movement
• measured in joules (J), calories (c) and kilocalories.

Laws of Thermodynamics

• Energy cannot be destroyed or created. It can only be transferred (conservation of energy).
• Entropy has a measure to increase disorder (entropy).
• 10%Rule only only 10% energy can move up food chain.

Spontaneity of Chemical Reactions

_ is determined by the change in free energy. _ free energy, temperature, enthalpy, entropy _ H = G =TS, ∆G = ∆H – T∆S

• Types: Spontaneous and NonSpontaneous. _ Exergonic vs _ Endergonic
• Exothermic vs Endothermic _ Reactants and products difference between oxidizing.

ATP

• Adenosine Triphosphate _ Electrostaic repulson-living tissue stores energy through ATP. _ energy is stored between phosphate molecules and they are negative. _ Hydrolysis release the high energy, exergonic result. _ ATP -> ADP + iP Δe -7.3kal/mol
• Indirect Synthesis is Chemiosmois

Enzyme and ribozymes

• Are catalysts
• lowers the Energy of Activation.
• bond and stretches.
• Enzyme Activity.
• Active Sites Specific-bind substrate in sites (Induced)
• Prosthetic group
• Cofactors = inorganic enzymes
• coenzymes= organic
• Metabolism
• cellular metabolism
• Enzymes Path ways and multienzyme, Anabolic/Catabolic pathways exgonic and endgoric

Other Influences on Enzyme Activity

• Prosthetic Groups: small molecules that are intertwined in the enzyme
  • Isolated through enzyme destruction.
• Cofactors: inorganic compounds forming temporary enzyme association to increases activity.
• Coenzymes: organic/non-proteinaceous molecules participating in catalyzed reaction but unchanged vitamins.
• Metabolism is the reactions in any organanism.
• Reactant energy cannot proceed without specific concentration and catalyst.
  • Rate determined by a catalyst.
• Enzyme regulation occurs Metabolic Pathways and Multienzyme saves unwanted enzymes.
• Anabolic/Catabolic requires an exergonic (breakdown) reactions.

Energy Intermediates

• Transport electrons during synthesis.
• Reduced (gain electrons) to form electron carriers from electron receptors such as NADH oxidized during ATP generation.
• Nicotinamide Adenine Dinucleotide: two nucleotides, NMP⁺ and AMP such as coenzymes.

Regulation of Metabolic Pathways

• Environmental Gene Regulation affects genes for growth.
• Cellular Regulation by cell-signaling through hormones indicated by the rate of exocytosis.
• Biochemical Regulation includes inhibitors to restrict enzyme actibity.
  • Reversible and Irreversible are how they tempoarily biond.

Recycling in the Cell

• It is crucial to recycle mRNA and protein when not functioning in the needed.
• molecule recycled if made with a defect.

MRNA degradation

• Exonuclease is an enzyme used to remove Poly-A tail
• Exosome is a multiprotein enzyme.
• Protein degradation begins when a ubiquitin protein covalent marks targeted proteins, directed to the protease.

EXTRA extra

• Amalyse breaks saliva down
• 20% proteins are atp optimal temp for enzymes Coenzyme stabilizers atp cycle 10;000

Chapter 7: Respiration, Fermentation, & Secondary Metabolism

Main Topics

• Cellular Respiration
• Glycolysis
• Pyruvate Oxidation
• Krebs Cycle

Cellular Respiration

• Hydrolysis molecules obtaining cells energy.
• Occurring with or without oxygen.
• Four metabolic: glycolysis-break down, 3citric, oxidative.

Stages

• Occurs in the Cytosol
• Without and with energy. Investment, 2 atp is hydrolyzed. Liberlation- gain net 2 ATP NET 2 NADH Is breakdown energy in NADH

Oxidation

• Products from ctosol in Mitocondrial matrix, go in intermem space to to undergo activation.
• Through channel.

1. Pyruvate Dehydrogenase: catalyses removes co2-2.
2. Dihydrolipoyltransacetylase: catalyses Acetyl COA reaction
3. Dihydrolipoyldehydrogenase:: catylaes NAD +1, 2e=

Kreb cycle

• Also AKA acid or tricaorbocyillc acid
• molecole enters and leaves with a orgnaic regenerated
• Glucose 2 acetyle, 1 molecules provides 2 revolts.

Prep

• Acetyl CoA or oxalate - Iso citrate NADH 1 ATP. FAD! Glucose 2 CO2 FAD!!