Biology 311 Exam 2 - Binghamton University

Problems of Prokaryotic Cells

Transient Transfection

- Short term

- Non-chromosomal Plasmin

= Diluted out with cell division

Stable transfection

- Long Term

- No dilution with cell division

- Integrated into the host genome

Epitope Tagging

Add an epitome to the POI because you have a mAb

PCR (polymerase chain reaction)

- Amplified DNA

- Heal/Cooling cycle

- DNA Polymerase ok @ 95° celsius

- Thermus aquaticus "extremophile"

RT-PCR

Low abundance mRNA in cells

- Converts RNA into cDNA & amplifies DNA targets

- Used for gene expression analysis and viral detection

Transgenic Animals Example

Jackson Laboratory

- developed transgenic wooly mammoth mouse

- used wooly mammoth genes

- first engineered animal to exhibit cold climate adaptation genes

shRNA/siRNA | What is it?

- antisense RNA/DNA

- translation of target mRNA (binds to it)

siRNA

- simple to make

- transient effect (lasting for a short time)

- cleaves mRNA (divide/split/sever)

shRNA

- difficult to make

- more potent than siRNA

- cleaves mRNA

-long lasting

RISC

- shRNA

Cleaves target mRNA more effectively than antisense

Huntington's Disease

- Patients with this have a lower chance of cancer

- Contain Huntington siRNA

Huntington siRNA Characteristic

Toxic to nerve cells & cancer cells

CRISPR-Cas9

Gene editing Tool

Cell Membrane History:

1839 - Schleiden (Plants) + Schwann (Animals)

Suggested a "barrier" between cells

Cell Membrane History:

1895 - Overton (Living cells)

- Lipid soluble dyes would stain living cells

- Lipid like

Cell Membrane History:

Langmuir

Made a trough with phospholipids

Cell Membrane History:

1925 - Gorter and Gendel

Question: Is it a monolayer or bilayer?

- Had to determine the surface area of red blood cells (under-calculated)

- Extract the phospholipids (under-calculated)

Answer: 2x bilayer

Cell Membrane History:

Mudd and Mudd

- Oil/h20 mixture

--> RBCs & WBCs

- WBCs have more cellular proteins

Cell Membrane History:

Davson - Danielli model

- Protein -phospho -phosphoprotein "sandwich"

- I.D. Robertson

Freeze Fracture

Study ultrastructure via freezing and fracturing samples to reveal internal structures

Cell Fusion

- Heterokaryon

--> single cell with 2 nuclei

--> Proteins are free to move laterally in the membrane plane

Cell-Capping/Patching Cell

- mAbs

--> have 2 identical FAB regions

--> Proteins are free to move laterally in the membrane prane

FRAP // Definition & What it's doing

Fluorescence Recovery after Photobleaching

- "Con A" = concanavalin A

- Binds to all proteins via their carbohydrate groups

- Half of proteins are Mobile

- Other half of proteins are not mobile, they are stuck

Fluid Mosaic Model

- Describes structure of cell membranes

- Includes phospholipids, proteins & carbohydrates

Preferred Cell Type & Why

Red Blood cells:

- Plentiful and easy to get

- Pure cell population

- Few contaminating organelles or membranes

- Few proteins

- Right side out and INside out RBC vesicles

Spectrin // What is it + Benefits

- Cytoskeletal Protein

- Fibroud

- Cytoskeleton biconcave disc shape

Vernon Ingrame

- Father of molecular medicine

- Known for his research on sickle cell anemia

- Discovered basis of sickle cell was single amino acid change

Phospholipids/Lipids Categories

- Phosphoglycerides

- Sphingolipids

- Sterols

Phosphoglycerides // Example

Phospholipid Category

- AKA phosphatidylserine

Sphingolipids // Example + Characteristic

Phospholipid Category

- Example: sphingomyelin

- increase in electrical resistance

Sterols // Example Characteristic

Phospholipid Category

- Example: Cholesterol

--> 50% - 90% in cell (Cell membranes)

- Can be an issue with LDL (low density lipoprotein)

--> if LDL is defective, there will be huge levels of cholesterol

Questions: How/where are nascent (new) phospholipids generated?

Answer:

- In the endoplasmic reticulum

--> can flip-flop from one leaflet to the other

--> flippase

Cholesterol in membranes // Characteristic/What it does in membrane

- Alters Fluidity

- Provides structure

Phosphatidylethanolamine

- a glycerophospholipid with an ethanolamine head group

- curves

Integral Membrane Proteins

- Most are transmembrane

--> meaning: they go across the entire membrane

Peripheral Membrane Proteins

Steps:

- Take cells and wash them

- Shake it up

- Proteins that are left are bound to the outside

Cell Membrane Proteins:

Channels : Fastest

- Non-gated

- Ligand-gated

- Voltage-gated

Cell Membrane Proteins:

Transporters

- No ATP Required

Chloeratoxin

- Toxin that causes dirrhea and dehydration

Solution: drink na+ and glucose

Hydropathy Plot

- Score each amino acid of a protein based on its own hydrophobicity

Cell Membrane Junctions:

Adherenes

- Encircled cells in apical region

- Cell adhesion

- E-cadherin -> Ca^+2 dependent

- If NaCa+2 -> no E-Cadherin Function

E-Cadherin

1. Cis-Configuration

2. Trans-Configuration

3. F-Actin

Cell Membrane Junctions:

Desmosome

- Cell adhead "spot" welds

- Skin (epidermis)

- Cadherins

- INtermediate FIlaments

-Hemidesomone

Phemigus (Autoimmune disorder)

Pemphigus

- Autoimmune disorder that attacks desmosomes

Phemigus Vulgaris

--> Antibodies attack desmoglein 3

Phempigus Foliceus

--> Antibodies attack demoglein 1

Cell Membrane Junctions:

Tight Junction: Location // Characteristics

Found in:

- kidney, instinal ep cells, bladder

- separate liquid

Characteristics:

- Sodium Fluorescin Leakage Assay

- Lanthanum Hydroxide

Cell Membrane Junctions:

Gap Junction

- AKA electrical junction

- Pass small molecules (ions) between cells

- Connexin

- Rectifying (pass ions in only one direction)

- Non-rectifying (pass ions in 2 directions)

Cell Membrane Junctions:

Neurochemical Synapse

- Communication through neurotransmitters

- Transmission of signals across the nervous system

Ferrotransferin System:

Clathrin

- Triskelion

- Self Assembles

- Little to no energy required

- "basket"

Ferrotransferin System:

Dynamin

- Pinches off the pit

- GTP dependent

Ferrotransferin System:

Fibroblasts

- Fibroblasts @ 37 degrees celsius

- 290 degree surface

- Clathrin Basket

Transferrin

1. Receptor Clustering

2. Coasted pit

- Adaptor Complex

--> Clathrin Binds to the adapter complex (no ATP required)

3. Coasted Vesicle

4. Early Endosome

5. Late Endosome

5-6. Return Pathway

- Cell membrane

- Apotranferrin now released

LDL SyStem

LDL - Low densiy lipoprotein

a. LDL lipoorotein "particle"

b. LDL - transport cholesterol

- high LDL = atherosclerosis

c. ApoB - apolipoprotein

- belt (binds to the receptor)

- late endosome (release of the LDL -> lysosome

d. receptor recycles

e. Acidic pH is required

--> Change in the ligand binding arm

LDL System: Tau Protein

- Axon

- Microtubules --> Structure

GPCRs

- Most important drug pharmacologically

- Not really involved with cancer

GPCRs importance

- 800+ functional GPCRs which constituted about 4% of all identified human proteins

- 35% of all drugs work through GPCRs

- 134+ GPCRs are targets of drugs

G-Proteins

1. Trimeric - GPCRs

- α β ɣ

- if GDP - inactive

- if GTP - active

2. Monomeric G-Proteins

--> GTK pathway

G-Proteins: Receptor

"Serpentine"

- 7 pass membrane

- once G protein dissociates then either the monomeric of the dimer binds to an effector

Two Examples of GPCRs

1. Muscarinic Acetylcholine Receptor

2. [K+]Potassium Channel

--> Effector

GPCRs and Visual Transduction

- Photons can be transduced into ΔVm in the rod photoreceptor

Rod Receptor

Outer Segment

- Disks, mitochondria

Inner Segment

- Light activates rhodopsin (GPCR)

--> Isomerization (change) in the retinal molecule)

- (Before light) Rhodopsin -> Rhodopsin* (After Light)

Typical Action Portential

- G protin - "transducin"

- "effector" - cGMP PDE

- Phosphodisterase

PDE

- Tetraner = no work

- Y subunits = inhibitory

Summary:

- In the dark -> high concentration of [cGMP] -> Na+/Ca+2 -> -30mc (CHANNELS ARE OPEN)

- In the light -> low concentration of [cGMP[-> Na+/Ca++-> hyperpolarization

CHANNELS ARE CLOSED

phosphodiesterase PDE

- effector enzymes responsible for cellular signaling by breaking down cyclic second messengers (cAMP and cGMP) an converting active to inactive to regulate duration and intensity of intracellular signals.

What about Visual Adaptations?

1. Rhodopsin Kinase -> phosph Rhodopsin

2. Arrestin

a. Binds to phosph Rhodopsin

3. Ransducin -

a. Translocates from outer to inner segment

4. Arrestin

a. Translocates from inner to outer segment

Answer:

Cytokines Receptors

Receptor tyrosine kinase

Ligand (single) binding

Dimerization happens when bound

- Then triggers phosphorylation (triggered by JAK kinase)

- Tyrosines are phosphorylated

Jak Kinases

- Target for Drugs

Cancers that can occur from lack of JAK:

- Leukemias

- Lymphomas

Receptor Tyrosine Kinases (RTKS)

Process:

1. Two molecules of ligand

2. Activates the receptor

3. Dimerization

4. Autophosphorylation

5. Occurs on tyrosine

Example:

- Simulate cell division

- Epidermal Growth Factor (EGF)

- Nerve Growth Factor (NGF)

- Insulin

Autophosphorylation

- Looking at cytoplasmic domain of RTK

RAS - Monomeric G proteins

- Signals -> Cell division

- 50% of ALL cancers have defective RAS

RTK Problem

- Overproduction of defective RTKs

HER Receptors // Normal Cells vs. Cancer

Normal Cells: 20,000 HER2 Receptors

Cancer Positives: 1,000,000+ Her2 Receptors

Kadcyla

- Used to treat Her2 Positive Breast Cancer

-Herceptim + Dm1

--> Dm1 Blocks cell division

Herceptin

- Old Her2 Treatment

- mAb

--> Binds to HER2 Receptor & decreases the #

Insulin/Insulin Receptors

Insulin:

- First protein to be sesquenced

- First protein to be chemically synthesized

- First recombinant Protein

Insulin Receptors:

- First receptor of any type to be purified

--> via affinity chromatography

Insulin // What does it do?

1. Internalize Glucose

--> insuits GLUT4

2. Cell division

3. Cell differentiation

3T3-L1 Cells

- When you add insulin, they become large

Second Messengers

1. cAMP

2. cGmp

3. Calcium/Calmodulin System

4. Protein Kinae C // DAG System // IP3 Pathway

5. Nitric Oxide // NO

Second Messengers:

cAMP

- Ubiquitous (omnipresent)

- GPCR System

- Dependent Protein Kinase (PKA)

Amplification:

- 4x

- can be regulated; increased or decreased using G Proteins

s = stimulatory

i = inhibitory

Regulatory Unit:

- Photoaffinity Analogs

--> 9-Azido-cAMP^32

Catalitic Unit

- Measure Activity

- PKA: controls glycolysis + glycogen storage

Feedback Repression

- GPCRs

--> Phosphorylation of receptors

--> Arrestin binds to phosphorylated GPCRs

--> Endocytosis of the receptor

Second Messengers:

cGMP

- Similar to cAMP

- Visual Transducion System

Question: Is the change in cell physiology due to ⬆️ cAMP

(What can you use to answer the question?:

1. dbCAMP

--> Membrane soluable analog of cAMP

2. Forskolin

--> Stimulates adenyl cyclase

3. GMP - PNP

--> Non-hydrolyzable GTP analog

4. Choleratoxin

--> Inhibits GTP hydrolysis

Caffeine // Theophylline

--> Blocks PDE

---> cAMP (active) 🔄 5AMP (inactive) : Causes an increase in cAMP

Second Messengers:

Calcium//Calmodulin System

Functions

1. Cofactor for enzymes

2. E-cadherin require CA^+2

3. Remember the voltage gates calcium

4. CA^+2 can bind to calcium binding proteins: Ex. Calmodulin

Calcium Homeostasis

- Glutamete

--> Naturally occuring in homeostasis

--> Neurotransparent

--> INcreases intracellular calcium concentration Ca^+2

-CA^+2: Second messenger

Prevagen

- Jellyfish content medication for neuro health

- Calcium Binding Protein

- Medication with science basing but doesn't even work

- Doesn't even get through the GI system

- Based on science but doesn't actually target neuro health

Second Messengers:

IP3 Pathway // Dag System // Protein Kinase

Dag

- Binds to PKC: Doesn't activate it

IP3 steps:

1. Goes to endoplasmic reticulum

2. Releases CA+

3. Binds to PKC 1+2

4. PKC is active

Second Messengers:

Nitric Oxide // NO

1. It is a gas

2. Is membrane soluable

3. Famous for regulating blood pressure

--> Increases of this (The second messenger) means there is a decrease in blood pressure

4. Is a breakdown product of nitroglycerin

Protein Trafficking:

Fun Facts:

1. 20,000 genes that code for proteins 1.5% of total DNA)

2. 1,000,000 possible proteins to make

3. Typical # of proteins in a cell is +/- 10,000

Protein Trafficking:

1. Exported/Secreted

- Ex. insulin, collagen, EGF

2. Soluble in the cytoplasm (stay in the cytoplasm & isn't secreted)

- Ex. glycolytic enzymes

3. Embedded in the outer cell membrane

- Ex. RTKs, GPCRs, glycophorin

4. Organelles

- Ex. Lysosomes, nucleus, endoplasmic reticulum

- Ex. Chloroplasts (Have their own DNA but most/almost all proteins are nuclear endocded

- Ex. Mitochondria ( Have their own DNA // 37 genes in miDNA --> 13 Proteins)

Where does all protein synthesis begin?

All protein synthesis begins with polyribosomes

--> they are free floating in the cytoplasm

Protein Trafficking:

Places of Synthesis?

1. Polyribosomes

- All proteins start here & some stay here

2. Rough Endoplasmic Regiculum

- Secreted

- Membrane Receptor

- Lysosomes

3. Cytoplasm only

- Soluable enzymes

4. DNA containing organelles

- Chloroplasts & Mitochondria

SER vs. RER

RER // Rough Endoplasmic Reticulum : Features

- Ribosomes

- Connected to the nuclear envelope

- Protein Synthesis

SER vs. RER

SER// Smooth Endoplasmic Reticulum : Features & Functions

Features:

- Connected to the RER

- Tubular

- Not involved in protein synthesis

Functions:

1. Resivoir for calcium PKC activation

2. Phospholipid + fatty acid synthesis

3. Detoxification

- Hepatocytes

4. Cytoskeleton

5. Transport Molecules

6. Seroid Synthesis

7. Differentiated

- Ex. Sarcoplasmic Reticulum

Protein Cotranslation Translocation:

Descriptive Analysis

1. Proteins are synthesized on polyribosomes "nascent proteins"

- 16-30 Nh2 end amino acids hydrophobic

- Signal sequence

2. SRP - Signal recognition Particle

- particle - 6 proteins + 300 nuclotide DNA

- Binds to the signal sequence

- Prot synth stops

3. SRP/Ribosome nascent protein complex

- Docks to the RER - SRP receptor

4. Nascent Protein

- Transferred to the translocon & Protein synthesis starts up again

5. Signal Peptidase

- Cleaves the signal sequence

6 - 8.

- nascent protein is extruded into the RER lumen

Protein Cotranslation Translocation:

Investigative Approach

Question: Is there an SRP Receptor?

Yes - Potease

Protein Cotranslation Translocation:

Investigative Approach

Question: Is GTP hydrolysis required for SRP Recycling?

GTP Hydrolysis is required

GMP - PNP - nonhydrolyzable of GTP

Protein Cotranslation Translocation:

Investigative Approach

Question: Is there a signal peptidase?

Yes

Protein Cotranslation Translocation:

Investigative Approach

Question: Is the secretory protein totally inside the RER?

Yes

Protein Cotranslation Translocation:

Investigative Approach

Question: How did we discover the Translocon?

Ohms Law V = IR

Protein Cotranslation Translocation:

Investigative Approach

Question: Where are multimeric proteins assmebled?

The RER (rough endoplasmic reticulum)

Protein Cotranslation Translocation:

Investigative Approach

Question: What if something goes wrong in the RER? -> Proteins are abnormal

UPR Pathway is Activated

Post-Translation Translocation:

Characteristics

- No SRP or SRP receptor

- Key role: Bip

- Proteins are already synthesizd in cytoplasm but will end up in the same place

Post-Translation Translocation:

Type 1: Single Pass protein

- Involve SRP + SRP receptor

- Has a signal sequence/peptidase

- STOP transfer anchor sequence

--> Hydrophobic amino acids