×
Log in to StudySoup
Get Full Access to TCU - BIOL 30603 - Study Guide
Join StudySoup for FREE
Get Full Access to TCU - BIOL 30603 - Study Guide

Already have an account? Login here
×
Reset your password

TCU / Biology / BIOL 30603 / What is glycobiology?

What is glycobiology?

What is glycobiology?

Description

School: Texas Christian University
Department: Biology
Course: Molecular, Cellular, and Developmental Biology
Professor: Misamore dr. akkaraju
Term: Spring 2016
Tags: Cell Biology and Biology
Cost: 50
Name: Cell bio study guide EXAM 3
Description: This study guide is everything that is needed for exam 3. Every lecture has been relistened to and large topics are underlined while important terms are in bold. While this material correlates heavily with the slides shown in class, these notes have additional comments to allow for full understanding without slides. Harder concepts and experiments are explained in large detail.
Uploaded: 04/02/2016
28 Pages 41 Views 12 Unlocks
Reviews


Cell Bio Study Guide Exam 3


What is glycobiology?



Glycobiology  

Glycobiology: Field of biology that studies the structure and function of glycoconjugates and  the proteins that specifically interact with them

Monosaccharide cannot be hydrolyzed into a simpler unit (CH2O)

-found in a ring structure

Sugar derivatives are formed when the hydroxyl groups of simple monosaccharides are  replaced by other groups

-like an amine group  

-glucosamine, glucuronic acid

Disaccharide is 2 monosaccharides joined together

oligosaccharides: short chains of sugars (2-10)

complex oligosaccharide: non-repetitive sugar sequence  

-branching can occur in oligosaccharides or polysaccharides  


How does glycosylation help with protein folding?



-branching occurs because monosaccharides have several free hydroxyl groups  that can form links We also discuss several other topics like How corporate bonds finance restructuring?

Polysaccharides are negatively charged so they attract water  

-gel-like consistency (main component of mucus)

Glycoconjugate: covalent addition of one or more sugars to a protein or lipid  -glycoprotein or glycan (basically means its on the outside of the cell)

-1/2 of eukaryotic proteins are glycosylated

-glycoconjugates are anything that has a sugar attached to it

Glycoprotein vs Proteoglycan  

both have sugars attached to a protein  

Glycoprotein is much smaller

-so… its an oligosaccharide linked to protein  If you want to learn more check out What is basal ganglia?

-little bit of sugar on your protein  


Why bird flu achieve human to human transmission?



Proteoglycan is much bigger

-contains polysaccharide chains called GAG chains

-little bit of protein on your sugar

GAG chain = glycosaminoglycan chains  

-long, unbranched sugar chains composed of repeating disaccharide units one  of them HAS to be an amino sugar  Don't forget about the age old question of What is the definition of colonialism?

-often sulfated; highly negatively charged

GAGS form gel-like matrix because of their negative charge  

-GAGs occupy a huge volume relative to their mass.

-form porous hydrated gels that helps the body withstand compressive  forces  

-found in joints (extracellular matrix)

Proteoglycans = GAG chain + protein

-if the GAG chains is attached to a protein it was assembled in the golgi

You can have a GAG chain that isn’t attached to a protein

-example: hyaluronan is a gag chain that is assembled in the plasma membrane  -its found in beauty products because when you put it under your skin, the  negative charge attracts water and this is filler and no wrinkles

Focus is now on GLYCOPROTEINS

Functions

-protect a protein from degradation

-hold it in the ER until protein is properly folded

-transport signal for protein trafficking

-form glycocalyx  

sugar coating involved in cell to cell recognition and protection

Protein Glycosylation in ER

-sugars are covalently added to proteins in ER

-pre-formed precursor oligosaccharide is transferred to proteins in ER -N-linked because the sugar hooks onto the NH2 side chain on asparagine  We also discuss several other topics like What is the meaning of hippocampus?

Trigger sequence is either: Asn-X-Ser or Asn-X-Thr

Formation of the precursor oligosaccharide (occuring in lipid bilayer of ER membrane)  1. it is assembled sugar by sugar onto the carrier lipid dolichol

2. the first sugar is put on and uses a high energy pyrophostate bond  3. then 2GlcNac and 5 Man are added which causes it to flip into the membrane  4. 4 more mannose and 3 more glucose are added inside the membrane  Final pdt: 3 glucose 9 mannose 2 GlcNAc

Oligosaccharyl transferase is hanging out near where the protein is being brought in the ER -it is scanning the sequence of protein looking for Asn  

-it will attach the oligosaccharide onto protein using pyrophosphate bond  energy  

-location of glycoslyation doesn’t really matter

How glycosylation helps with protein folding:

last two glucose will be trimmed off of the oligosaccharide  

-the remaining glucose will attach to calnexin and allow the protein to be held  in the ER membrane while it gets properly folded

-glucosidase: remove a last glucose and let properly folded protein leave ER -if not properly folded glucosyl transferase will add a glucose to it again and it  will go bind to a chaperone protein againIf you want to learn more check out What legal and medical rights are denied to gays and lesbians?

going to golgi after ER

no diversity in protein glycosylation in the ER

-only in golgi

gets really complicated because not all proteins are going to interact with the same sugar  groups

Two different routes  

-high mannose oligsacharide: its trimmed a lil in the golgi but no sugars are  added there

-complex oligosaccharide: trimmed and sugars ARE added in golgi

Process of putting together complex oligosaccharides

-all 3 glucose came off in ER along with 1 mannose  

-in golgi now some mannose are trimmed (3)

-if it leaves now its a high mannose oligo  

Exposure to enzyme Endo H (way to see if it is high mannose or complex oligosaccharide) -endo H can degrade the sugar if its just the mannose but if it has the  

additional sugar on it, it cannot  

-high mannose oligo = Endo H sensitive  

-complex oligosaccharide = Endo H resistant

Glycosylation that occurs in golgi is called O-linked  

-when the sugar is added to the Oxygen on threonine or serine  

- not just one sequence like in N linked

proteoglycans are always o linked

glycosylation is so important in making sure proteins leaving the golgi go where they are  supposed to go  Don't forget about the age old question of What is planning?

example: mannose 6-phosphate tag is added to lysosomal hydrolyases in golgi  (M6P tag directs them to lysosome)

mannose got added in the ER (mannose 6-phosphate tag)

the picture is showing that a protein with a specific sugar tag came to golgi and interacted with  the specific protein (GlcNAc phosphotransferase) and a GlcNAc with a phosphate tag is added  and then theres an additional enzyme that comes and removes the GlcNAc -GlcNAc just used to add phosphate

Transport of lysosomal Hyrolases

when in the lysosome, the mannose 6 phosphate dissociates because it was only used to get  the protein there (lysosome hydrolase)

I-cell disease

Consequence: tags can’t be added to mannose

lysosome doesn’t work because it has no lysosomal hydrolases and you get protein aggregates  in the cell

-so the proteins get secreted outside the cell and into the blood (because they  don’t know where to go , no tags)

-fatal by 6 years old

Bird Flu (influenca and sugar linkages)

Why it achieve human to human transmission  

the receptor that the virus recognizes is glycosylated  

-for the flu receptor the last two sugar in the complex oligosaccharide are  -SIALIC ACID

-GALACTOSE  

-it matters about the linkage between these two…

the sialic acid can link in a 2,6 linkage or a 2,3 linkage  

human flu recognizes 2,6 linkage

Bird flu: recognizes 2,3 linkage

In birds all of their lung cells express sugar chains have a 2,3 linkage between the sialic acid  and galactose so they have evolved to recognize that  

in humans the upper respirator tract cells expressed 2,6 linkages and only in the very lower  parts were 2,3 linkages detected

Glycobiology II 

Sialic acids and Glycans  

Glycoproteins are a lot smaller than proteoglycans  

Question: protein is glycosylated, it doesn’t have to have begun in the ER, can happen only in  golgi

-if it is O linked, it starts in the golgi  

-if you had serine, it has to have happened in the golgi  

-sequence in the ER is Asn-X-Ser, Asn-X-Thr

Sialic acids are the most common terminal sugar in mammalian cells (not found in plants or  anything else)

-sequences can vary but ALWAYS last is sialic acid

-Negatively charged

NOT found in plants, invertebrates or prokaryotes EXCEPT for some human pathogens -human pathogens hide themselves by coating themselves in siliac acids -if you delete genes coding for these you get embryonic development issues

Kinds of sialic acids:

Neu5Ac found in humans

Neu5Gc found in all other mammals

The reason humans have different than mammals is because Neu5Ac is the precursor to  Neu5Gc

-CMAH is the gene that converts Neu5Ac to Neu5Gc

-humans lost this gene so we don’t make Nue5Gc

Which type would you expect to see in?…

-Beef you expect to see both types because yes its a mammal so Gc but ALSO Ac is the  precursor to Gc so it can have both

-carrots have neither  

Humans lost the CMAH gene as a way to avoid infection by Plasmodium species that cause  Malaria

Malaria: caused by plasmodium sp (parasites) and it invades the red blood cells -synchronized lysing of RBCs is what causes host to have alternating chills/fever -spread by anopheles mosquito

Plasmodium binding to RBC critical point in lifecycle

-if this doesn’t happen, nothing will happen (won’t get disease)

1. Plasmodium enters through mosquito bite

2. Plasmodium travels to liver and then to bloodstream where it infects RBCs 3. Plasmodium gametocytes ingested by mosquito

4. Lifecycle completed in mosquito

no human to human transmission

There is plasmodium that will only infect humans and some that will only infect apes -Plasmodium falciparum — infects humans

-Plasmodium reichenowi — infects great apes

NO CROSSING OVER OF THESE

-even though Plasmodium species seem nearly identical

BUT Aotus monkey is susceptible to Plasmodium falciparum like humans -Why?

-because Aotus monkey’s Red blood cells have peak at Neu5Ac peak  (fluorescence)

Malaria and RBC binding

Plasmodium protein EBA-175 — binds to glycoprotein on RBCs

Pf-EBA175 from P. falciparum (infects humans)

Pr-EBA175 from P. reichenowi (great apes)

Hypothesis for why we don’t have crossing over of the infectious disease malaria: Pf EBA175 will preferentially bind Neu5Ac coated RBCs and Pr-EBA175 to Neu5Gc RBCs

-this supports the hypothesis, there is preference

Plasmodium event — when plasmodium split into the Pf and Pr

(fig 1)

Our biochemical pathways cannot tell the difference between Neu5Ac and Neu5Gc -so if you eat food that has Neu5Gc your body will use that

-this means our cells have Neu5Gc incorporated in their glycans and therefore on their  surface

But our immune system can tell the difference  

-it makes anti-Neu5Gc antibodies

Neu5GC sugars on human endothelial cells are labeled with red fluorescent antibodies -when are these being made  

-looked at babies and mothers

-babies were being breast fed so they didn’t have any Neu5Gc antibodies -when they started eating real solid food, they had them on their RBCs

antibody: protein produced by B-cells that binds “foreign” molecules — actually bind to antigen  because antigens are usually foreign

Antigen: molecule that induces immune response (usually foreign)

once you have an antibody bound to an antigen on bacterium it is a big kill me sign  -the antibodies do not kill microbes themselves, if an antigen binds to antibody  it will phagocytose and send signals to immune system

Response to tissue injury/infection

-Recruitment of WBCs and serum proteins (antibodies, complement)

-Microbial destruction through phagocytosis, complement mediated killing and  release of antimicrobial chemicals (peroxide)

Acute inflammation: initial inflammatory response  

Chronic inflammation: prolonged inflammation

-there can be damage to host cell

-when macrophages are being recruited to the same space and they shouldn’t  be

Chronic inflammation has been linked to cancer and atherosclerosis which is the build up of  plaque in the arteries

Why don’t our cells make Neu5Gc?

-loss of CMAH

Where is Neu5Gc coming from?

-food

Why do our cells incorporate Neu5Gc?

-because they can, they don’t tell the difference  

Why do we make antibodies against it?

-because they can tell the difference

-what are the consequences of this? making it against your own cells

The worry is that making anti bodies against self is causing chronic inflammation (linked to build  up of plaque)

Why do we make antibodies against it?

-what are the consequences of this? making it against your own cells

Hypothesis: Endothelial cells with Neu5Gc on their surface would be more likely to attract  phagocytic cells and complement due to antibodies against Neu5Gc.

-THIS TEST IS SHOWING HOW THIS IS RELATED TO CAUSING HEART DISEASE  OR ATHEROSCLEROSIS

Growing cells in dishes

-fed cells different sugars (Ac or Gc)

-then added Neu5Gc antibodies — which should recognize only the Gc sugars  -if their hypothesis is correct you should get a higher recruitment of phagocytic cells with  Gc sugars

Human serum= 20% antibodies

-how you get antibodies

They took the blood of people and their levels of Gc antibodies varied a lot -this can be caused by what they eat, meat or not

-they took somebody with high levels and low levels

-they took their endothelial cells that were fed Gc or Ac

-and they looked at phagocytic recruitment in their specific cells

If the hypothesis were correct and you had Ac-fed exposed to +S30 you still have low levels of  phagocytic cells

-ITS NOT ENOUGH TO JUST HAVE THE GC SUGARS INCORPORATED IN YOUR  CELLS

-you need the high level of Gc antibodies to have the high levels of recruitment of  phagocytic cells

Does this prove that the presence of Neu5GC on endothelial cells contributes to atherosclerotic  plaques?

-no (because its not just the presence)

-previous research has shown an association between phagocytic cells and plaque Now this test is about how sialic acids can cause or not cause cancer

you have a Wild Type mouse

and a -/- mouse — meaning you have a knock out of both CMAH genes

WT mouse will make Nue5Gc because its a mammal and has CMAH gene

Which mouse will make αNeu5Gc antibodies if exposed to Neu5Gc?

A) WT

B) CMAH -/-

C) Both

D) Neither

NOT BOTH because WT normally makes Neu5GC so it won’t make antibodies against your  own stuff

but if you inject Neu5Gc into a CMAH -/- they will  

-because -/- didn’t have Gc before and now they are exposed to it so it  

is considered foreign and will make antibodies against it

-AGAIN, WT mouse will NOT make antibodies because its normal self is  Nue5Gc

Whole Idea of study:  

Antibodies —> chronic inflammation —> cancer  

If this is true, which mouse, WT or Cmah -/-, would be more likely to develop inflammation and  eventually cancer after exposure to Neu5GC?

-the CMAH -/-

To test it:

they put tumor cells expressing Nue5Gc in mice and looked at the size of the tumor growth -the bigger tumor was found in -/- mouse due to that mouse making anti bodies  causing inflammation and cancer

Exposure to Neu5Gc in animals that lack CMAH leads to production of αNeu5Gc antibodies  

Hypothesis: In animals lacking the CMAH gene, exposure to Neu5Gc rich foods in the presence  of αNeu5Gc antibodies will lead to increased inflammation and development of spontaneous  cancer.

Data said that the mouse that was injected with Neu5Gc antibodies and was fed Neu5Gc rich  foods, had way higher levels of inflammation.

Babies levels of Nue5Gc are none until they start eating real food (not breastfeeding) so if we  don’t eat Nue5Gc food we won’t make antibodies against them and we would reduce our risk for  cancer.

Metabolism and Mitochondria 

You release the same amount of energy when you directly burn it or a stepwise oxidation  -cells do a stepwise release so that it is able to capture the energy along the way

3 stages of how the cell metabolizes food

first stage- breaking down foods to different subunits, amino acids, glycerol  second stage- glucose goes through glycolysis and makes pyruvate (turned into acetal CoA) third stage- break down of acetal CoA and oxidative phosphorylation

Oxygen is not required for glycolysis

-you only need that during oxidative phosphorylation (citric acid cycle)

2 mechanisms for ATP generation

-1. Substrate-level phosphorylation - when a phosphate from a molecule is added to  ADP

happens during glycolysis

-2.) oxidative phosphorylation - through electron transport chain

Glycolysis

1 molecule of glucose -> 2 molecules of pyruvate

2 ATP consumed

4 ATP produced

2 NADH produced

Net gain: 2 ATP and 2 NADH and 2 molecules of pyruvate

Substrate level phosphorylation (occurs at steps 6 and 7 of glycolysis)

-(6) first NADH is made

-(6)then a high energy phosphate bond is made and when it is broken…

-(7)then the cell can transfer the phosphate to ADP to make ATP

This addition to ATP (from the precursor of pyruvate) is energetically favorable

You should know information presented in the last 3 slides on glycolysis, but you do not need to  know detailed steps in panel 13-1  

-whats happening to sugars

-where ATP is being consumed and produced

At the end of glycolysis 2 molecules of pyruvate have been generated

-then pyruvate can go into mitochondrial matrix and go through citric acid  cycle (but have to have oxygen)

-if none is present then it undergoes fermentation

Lactic acid is produced (in muscles when you are running out of oxygen) from pyruvate

Yeast

-turning pyruvate into ethanol

Is ATP generated during these fermentation steps (i.e. pyruvate -> lactate or pyruvate ->  ethanol)?

-NO

-In glycolysis? YES just not in fermentation step

T or F- the reason cells undergoes fermentation instead of just discarding pyruvate as a waste  product is because fermentation products such as lactate shown here can later be broken down  for energy once oxygen is present?

FALSE- that is not the reason fermentation happens

-the reason is to regenerate the NAD+

-during glycolysis NADH is made and fermentation regenerates NAD+

-allows glycolysis to continue

BUT USUALLY O2 is present

-pyruvate is pumped into mitochondria and converted to acetyl CoA

Pyruvate -> Acetyl CoA

-one of the carbons of pyruvate leaves as CO2

generate:

1 CO2

1 NADH

Note: This is not the only way for the cell to generate Acetyl CoA. Can also generate if from  fatty acids—see Fig. 13.11 in textbook.

Glucose is fully oxidized by the end of the citric acid cycle and released as CO2

2 molecules of CO2

3 NADH molecules

1 FADH2

1 GTP  

are all generated

for every one molecule of glucose you get 2 molecules of pyruvate and then 2 molecules of  acetal coA

Glycogen is a long polymer of glucose and stores energy  

or fatty acids can be converted to acyl coA

T or F the cell undergoes glycolysis and the citric acid cycle for the sole purpose of producing  ATP and NADH and other electron carriers that will eventually be used during oxidative  phosphorylation?

-FALSE  

-Ex: pyruvate is the substrate for several enzymes

-nucleotides are derived from glucose 6 phosphates

Final stage: Oxidative Phosphorylation

-requires a membrane  

-in eukaryotic cells: inner mitochondrial membrane

-in prokaryotes: just their membrane

Chemiosmotic coupling

Where are these electrons originally derived from?

-from glucose (being oxidized)  

Energy of electron transport is used to pump protons across membrane  -this creates a gradient and the H’s come back in and go through ATP synthase THEN energy in the proton gradient is harnessed by ATP synthase to make ATP

Mitochondria

-Location of oxidative phosphorylation

electron transfer + ATP formation

-Evolved from bacteria engulfed by ancestral cells

Allows for MUCH more efficient use of glucose

-Glycolysis + fermentation -> 2 ATP

-Glycolysis + oxidative phosphorylation -> 30 ATP

Without mitochondria, unlikely that complex multicellular organisms could have evolved! The inner membrane is just like a cell membrane is selectively permeable

Oxidative Phosphorylation

-Consumption of O2

-final thing that accepts those electrons is oxygen (turns into H2O) -Addition of phosphate group to ADP to form ATP

Electron Transport System (ETS)

-NADH dehydrogenase complex: accepts electrons from NADH and pumps out  H+

-Cytochrome C reductase complex: uses energy received to pump out more H+ -Cytochrome C oxidase complex: pumps out H+ but also produces water by  transferring electrons to oxygen  

ETS is really important because it powers ATP synthase  

-100 ATP being made per second

2 gradients being made

-charge gradient (voltage gradient)

-pH gradient (lower concentration of H to Higher)

Both will contribute to the proton motive force

-the stronger this is, the more ATP can be generated

This is also used for coupled transport to bring things in the mitochondrial matrix

pyruvate and phosphate molecules are negatively charged

so naturally they don’t want to come in the mitochondrial matrix (which is already  negatively charged)

-as hydrogen ions are coming in the matrix down their gradient is used to bring these in this isn’t very common in mitochondria but it is in prokaryotes

Reduction is gaining an electron  

oxidizing is losing an electron  

look at which species lost or gained an H

Each electron transfer in ETS is a redox reaction

NAD+ is oxidized

NAD+ will have a low affinity for electrons because it wants to give up electrons so it will have a  lower affinity than the complex its giving it to

oxygen has highest affinity of them all

we don’t go from NADH to oxygen because its too big of a voltage difference (1140) that it would  almost be explosive, cell can’t handle it

cytochrome c oxidase complex holds the oxygen that the electrons will go to very tightly  because the oxygen needs 4 electrons to attach to 2 hydrogens for it to form water and if it gets  released before this, you get super oxide forming and thats bad, causes damage to  mitochondria

Cytochrome C oxidase removes electrons from cytochrome C and transfers to O2 (4e- +4H+ +  O2-> 2 H2O

Complex must bind very tightly to O2 until it has received all 4 electrons otherwise superoxide  can form—highly reactive

-important that oxygen is held tightly until process is finished

Formation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2) leads to oxidative damage of mitochondrial proteins, membranes, DNA

-if you have high levels of ROS being produced in mitochondria there is lots of damage =  BAD

-this may be associated with cell aging

-sometimes cytochrome c can get released from the cell which can trigger  apoptosis

-hydrogen peroxide can be formed from superoxide

prokaryotes can undergo anaerobic respiration  

-diffference: O2 not only molecule (prokaryotes use some other molecule) that can serve  as terminal electron acceptor in ETS.

-However O2 most efficient—more ATP can potentially generated

BUT Benefits of anaerobic respiration

Able to grow in environments w/out O2

No reactive oxygen species are generated

Prokaryotes that use anaerobic respiration generally very slow growing but have unique  evolutionary niche.

Cell Signaling 

hydrophobic signals use intercellular receptors

Extracellular signals can do two things:

-alter protein function —> alter cytoplasmic machinery —> alter cell behavior -alter protein synthesis —> alter cytoplasmic machinery —> alter cell behavior

Which will be faster—altered protein function or altered protein synthesis? protein function - almost immediately, phosphorylation example  

Intracellular receptors

-when extracellular signal molecules are small enough or hydrophobic enough to cross  plasma membrane

-Ex: Steroid hormones

Ex: Dissolved gasses

Intracellular Receptors: Nuclear Receptors

-they bind steroid hormones  

-Huge impact on physiology and development

Pass through the membrane and meet receptor in the cytosol, if its unbound it is inactive -binds and is active and then moves to nucleus and activates gene expression (binds to specific promoter sequences to activate transcription)

nuclear receptors usually make transcription happen

Intracellular Receptors: intracellular enzymes

Some dissolved gases can diffuse across the membrane and directly regulate the activity of  intracellular proteins

example: NOS makes a gas (NO) which goes and binds to the enzyme guanylyl cyclase which makes cyclic GMP which then goes and stimulates other proteins within the cell, in the  case of muscle cells it can cause cell relaxation

FROM PICTURE

Which are the extracellular signaling molecules in this figure?

-nitric oxide and acetylcholine

Which are the intracellular signaling molecules in this figure?

-cyclic GMP (made inside the cell and affects other things inside the cell)

Which are the receptors in this figure?

-acetocholine and guanylyl cyclase

example:

Viagra blocks the enzyme that degrades cyclic GMP.  

Why is Viagra effective in treating erectile dysfunction?

-no relaxation of the smooth muscle cell so that creates vasodilation and more blood  flow to penis

Extracellular Receptors

Majority of signaling molecules are extracellular receptors  

-after it binds on the outside

-Intracellular signaling molecules pass the signal

-Effector proteins carry out the action to produce a cellular response

-another name for this is a signal transduction pathway

Signal transduction is just when its passing signals to the next

Intracellular Signaling Pathways (after the signal is received by extracellular receptor) May have multiple functions

1.Relay a signal

2.Amplify a signal

3.Integrate multiple signals from more than one signaling pathway (can have excitatory signals  and inhibitory signals)

4.Distribute the signal to more than one effector protein

Molecular Switches

Intracellular signaling proteins have to be able to turn on/off in order to control signaling

2 major classes  

signaling by protein phosphorylation  

signaling by GTP binding proteins

Two cells that express the same receptor will respond in the same manner when the receptor is  stimulated?

-FALSE

-due to different signaling pathways

Eukaryotic cell signaling IS REALLY complex

3 major classes of cell surface receptors

-ion-channel-coupled receptors

-g-protein-coupled receptors

-enzyme-coupled receptors  

The number of different types of receptors is greater than the number of different extracelullar  signal molecules

-true, there is more receptors than there is signaling molecules

-ex: acetylcholine activates different receptors in heart pacemaker cell and salivary gland  cell  

g-protein coupled receptors are used for smell

-mice have more of these

G-protein-coupled receptors

-Huge receptor class—more than 700 GPCRs in humans alone

-All have similar structure—7 pass transmembrane receptor

-All are coupled to G-proteins

-receptor interacts with g protein

. G-proteins

3 subunits-α, β, γ

tethered to membrane

α subunit bound to GDP when unstimulated

-then when a ligand binds it allows for a conformational change that  allows for the exchange of GDP with GTP

Process of G-coupled protein receiving a signal

-Signal molecule binds GPCR

-GPCR undergoes conformational change

-G-protein alters conformation

-Alteration of α−subunit changes affinity for GDP and GDP dissociates -GTP exchanged for GDP

-Conformation changes of g protein

-α and βγ subunits now active (you often times have the disassociation of the  Beta and Gamma parts) (can go on to interact with target protein) - Alpha subunit Interacts with target proteins

-This can activate or inactivate the target protein—depends on G protein Gs—stimulates target protein

Gi—inhibits target protein

-GTP hydrolyzed to GDP

-Subunits dissociate from target protein

-α,β,γ subunits reassociate forming inactive G protein that is ready to couple  once again with GPCR

she said the g protein is shut off by intrinsic ability of g protein

G protein Targets: ion channels example

-Ach released by nerve cells

-Ach binds GPCR on heart pacemaker cells

-Activated βγ complex binds K+ channel

-K+ channel opens

-Increases membrane permeability to K+

-Makes it more difficult to electrically activate cells

-Heartbeat slows

G protein Targets: Enzymes

Activated enzymes make second messenger signaling molecules What’s the first messenger? (ligand)

Two major classes of enzymes:

Adenylyl cyclase-> cAMP (second messenger)

Phospholipase C-> inosital triphosphate & diacylglycerol (second messenger) Adenylyl Cyclase & cyclic AMP

Usually the α subunit of G-protein switches on adenylyl cyclase which makes cAMP

cAMP activates other enzymes, often protein kinase A (PKA) which in turn phosphorylates  different target proteins

-cAMP phosphodiesterase converts cylic AMP -> AMP

cAMP Signaling

Many cellular responses to extracellular signals are mediated by cAMP (you can make lots of  cAMP AMPLIFYING SIGNAL HERE)

-PKA is a kinase that adds a phosphate to a target protein

-cAMP activates PKA

Ex: adrenaline binds adrenergic receptor

Consequences of this activation will vary depending on cell type

Ex. Adrenaline acting on muscle cell

-it resulted in glycogen break down because you will be needing lots of glucose for that  muscle

cAMP Signaling (another example)

Different cell type

Same extracellular signal

Same extracellular receptor

Same second messenger signal

Same second messenger target

Different PKA target

-> transcription factor

What will the cellular response be?

-the outcome will be different

-activating transcription, so cell will be producing proteins

-changing protein synthesis

Which response will be faster? This one or glycogen breakdown?

-glycogen breakdown (because everything already made)

-almost immediate (altering enzyme activity) there

-here we are changing protein synthesis  

Cholera: Acute bacterial infection caused

by ingestion of water contaminated

with Vibrio cholerae.

occurs when you don’t have access to clean water

 Sudden watery diarrhea and vomiting can result in severe dehydration.

 Left untreated, death may occur rapidly, especially in young children.

Cholera Signaling

B subunit binds to a receptor on the cell of the gut and then endocytosis and once in the cell the  A subunit breaks free of B and goes and binds Gs-alpha which stimulates adenylate cyclase

Cholera toxin (CT) is produced by the bacterium Vibrio cholerae (which stays in the lumen of  intestine)

-the reason you get such a strong response is because the CT modified a G protein so it can’t hydrolyze GTP

-can’t hydrolyze the GTP, so it is going to stay on (no way for protein to turn  off)

-constantly activating adenylate cyclase —> tons of cAMP

-this causes Cl- to leave the cell (lots of it) and this causes water to rush  

with it

-you have lots of water rushing into the lumen of intestine (causees the  

extreme diarrhea  

-This is also how the V. Cholerae to spread because people have diarrhea and it gets in  the water there (this is why watery diarrhea is beneficial to V. cholerae

G protein Targets: Phospholipase C

-another example but the beta gamma g protein activates phospholipase C -will cleave inositol phospholipid into two things (diacylglyercol and inositol  1,4,5 triphosphate)

-inositol 1,4,5 triphosphate goes and opens Ca2+ channels in the endoplasmic  reticulum  

-eventually Ca2+ and diacylglercerol will activate PKC

Now Enzyme-coupled receptors

Cytoplasmic domain acts as an enzyme or forms a complex with another protein that acts as an  enzyme

-causes dimerization  

-then autophosphorylation

-once the dimers tails are phosphorylated they can interact with intracellular  signaling pathways (phosphorylate signaling proteins)

-they serve as a scaffold for lots of things to be stimulated at one time  

Largest class: Receptor tyrosine kinases (RTKs)

(example)

Tyrosine phosphorylation allows many signaling molecules to bind to cytosolic tails of receptor Simultaneously can signal along multiple pathways  

Different RTKs recruit different intracellular signaling proteins

Two common ones PLC and Ras

Ras Monomeric GTPases - just one subunit

Monomeric GTPases: large family of small GTP binding proteins of which Ras is a  member

-proteins that bind GTP  

Ras becomes activated by:

GEF: Guanine nucleotide exchange factor  

GAP: GTPase activating proteins

they help activate the monomeric GTPases

RTK and Ras signaling

Ras can activate the MAPK signaling pathway (last kinase in chain)

-often see a change in gene expression but can be change in protein activity  

MAP kinase kinase kinase phosphorylates  

MAP kinase kinase which phosphorylates  

MAP kinase  

30% of cancers contain mutations in Ras that inactivate the GTPase activity

What happens to Ras if there is no GTPase activity? (check this)

-Cellular survival and/or proliferation common response  

Ras can activate PI-3-kinase-Akt signaling

PI-3-kinase-Akt can help suppress apoptosis

When Akt is activated it goes and causes phosphorylation of Bad

AKT promotes cell survival

-Bad indirectly stimulates apoptosis by binding and inactivating Bcl2

-Bcl2 suppresses apoptosis

Bad and inactive Bcl2 are bound together

-then get survival signal from active Akt

when you phosphorylate Bad is lets go of Bcl2 and Bcl2 is now active and can go inhibit  apoptosis

Cellular Crosstalk

When the target protein is fully phosphorylated, it triggers a cellular response fig 16.43

What would happen if only signal A were present? no response

What would happen if A and D were both present? yes response  

What would happen if B and C were both present? no response

~switching powerpoints to Cell Signaling - Disrupting the Signal~

Going to talk about the Huntington’s disease paper and go through how they figured this out

Huntington’s Disease (HD)

Onset 35-45 years

Neurodegenerative disease

Muscle coordination affected

 Cognitive decline-> dementia

 Lethal: life expectancy 55-65 yrs

Autosomal dominant disease

1:10,000 people in US

No cure; treatments just help relieve symptoms

Huntingtin Gene (HTT)

Trinucleotide repeat (CAG) is expanded in the coding region of HTT gene resulting in expanded  string of glutamines (Q)  

if you are Healthy: 10-26 repeats

if you have HD: >37

Longer the expansion; earlier onset of HD symptoms  

mutHTT adopts pathogenic conformation

resistant to degradation; accumulates in cell

Huntingtin (HTT)

Medium-sized spiny neurons primarily affected

Aggregates of mutHTT forms inclusion bodies (orange) in nucleus

Molecular Impacts

1. Transcriptional dysregulation

2. Impaired protein degradation

3. Altered protein folding

4. Disrupted neuronal circuitry

5. Mitochondrial dysfunction

Grew GFP-HTT-25Q in petri dish  

-then you put gene on a plasmid, then you clone these(GTP-HTT) into  the plasmid and you put plasmid into the cell by transfection -collect the cel lysates (get ride of the cell membranes and collect  

proteins in the cell)

How would scientists separate out only the GFP-HTT-25Q proteins?

-then you can do immunopreciptitation (IP) using an GFP specific  

antibody (alpha GFP)  

-at the bottom of the anti body it will have a little bead

-it will pull down GFP-HTT-25Q and whatever it is bound to (in  

centerfuge)

-you would pull down any proteins its interacting with

If HTT is in the nucleus, what is the green protein likely to be?  

-a transcription factor, makes sense because they have done staining to know that Htt is  in the nucleus so it would be interacting with a protein that is in the nucleus which  most are transcription factors

-and at some point they are going to bind to DNA

 What type of protein?

What do all transcription factors bind?

-DNA

-this is not random, very specific sequences this is important because this is how they  could tell which transcription factor it was

Transcription factors bind specific DNA response elements. These response elements are  known for most transcription factors  

-they had an array of lots of DNA sequences, all containing known response sequences -where is this green protein binding? they found that it is binding to a response element  recognized by PPAR-delta  

Peroxisome Proliferator Activated Receptor (PPAR)

3 types

PPAR-α

PPAR-δ

PPAR-γ

these ligands can be fatty acids, etc.

then PPAR protein goes into the nucleus and will heterodimerize with RXR (retinoid X receptor)  and then activate transcription after it recognizes its specific sequence

-PPAR is a nuclear receptor, its not a steroid receptor but acts similar to them

PPAR + RXR Bind DNA response elements w/ sequence AGGTCANAGGTCA (don’t have to  memorize_

after that it can activate transcription

They think that HTT interacts with PPAR delta  

to prove this:

-they put GFP in the petri dish

-also put Flag-PPAR-delta

-they collect the cell lysates

-they do IP GFP

What would you expect to see?

-just GFP (because thats the one with the bead and it wouldn’t interact with PPAR or  anything else)

Because …

you would not see B (GFP and PPAR interacting) because

-GFP interacts with Htt first THEN PPAR will interact with GFP

-in this example no Htt was added so no interaction

Now what? How do you actually “see” which proteins are in the tube? What technique can you  use?

-Now since they added Htt and that attaches to GFP and the bead, in the tube you will  see GFP AND PPAR because the Htt is interacting with the PPAR and it is pulled to  the bottom of the tube

-NOW you can use western blot technique

Western Blotting - for protein separation (by size)

After running total cellular protein and proteins after IP on a gel

-you will see all proteins plus target proteins so to see the target proteins… -you add primary antibodies against PPAR delta (immunoblot for PPAR)

-you add a second antibody with a tag that lights up (detect that signal)

-then you will get bands that correspond with your target protein

Protein we are interested in detecting is PPAR delta  

All steps :

1. collect protein lysates  

2. IP GFP

3. western

Why immunoblot with Flag antibody

-flag with ultimately recognize the PPAR delta because it recognizes flag and flag is  attached to PPAR (Flag-PPAR-delta)

In this actually setup they ran three conditions

1. GFP + Flag-PPAR-delta

2. GFP-HTT 25 (glutamine repeats or Q) + Flag-PPAR-delta

3. GFP-HTT 104 Q + Flag-PPAR-delta

KEY:

input is going to be everything (all proteins before you do IP) (called input) the right half (IP: GFP) after

positions:

1 = GFP + FLAG-PPAR-delta

2 = GFP-HTT25Q + FLAG-PPAR-delta

3 = GFP-HTT104Q + FLAG-PPAR-delta

On the left half (input), Why are there bands with all 3 GFP proteins?

-there are bands by IB: FLAG for each trial condition because it is detecting the  presence of FLAG-PPAR-delta which was added in each and had not yet been separated out by  IP: GFP bead and brought to the bottom

After IP: GFP what positions should have bands?

-2,3 showed bands

-this is because IP: GFP separates out GFP by bringing it to the bottom (due to  the bead) and in position 1 there is no band because the band signifies the presence of PPAR  and as the key above shows, position 1 has only GFP with no HTT so it would not interact with  PPAR

-positions 2 and 3 do have bands because GFP is attached to HTT which allows  for the interaction between GFP and PPAR and IB:FLAG is detecting PPAR that is separated  out with GFP and is lighting up (showing bands)

So now they have proved there is a specific interaction between HTT and PPAR -and this extra long repeating CAG sequence in the HTT gene causes PPAR to not  function properly

-If you are not allowing PPAR to work the way its supposed to you are going to inhibit  PPAR transcription

Formal Hypothesis: Mutant HTT (poly-Q-expanded) binds PPARδ and prevents it from  activating gene transcription.  

So another test to test this hypothesis ^^^

Knock-in mice expressing full length mouse HTT protein with different polyQ lengths

ST-HdhQ7/Q7 non-expanded glutamines (WT) —- normal

ST-HdhQ111/Q111 Expanded glutamines (HD) —- HTT

They compare expression of genes in both  

-the genes that they know are regulated by PPAR

-Which mouse should have higher levels of PPARδ dependent gene expression? -the Hdh Q7/Q7 because this is the WT mouse and PPAR in this mouse is carrying out  its normal function

-in Hdh Q111/Q111 they have Htt so PPAR can’t function normally and they have  LOWER levels of expression

Hypothesis: Mutant HTT (poly-Q-expanded) is interacting with PPARδ and preventing it from  activating gene transcription.  

Loss of PPARδ gene expression is contributing to mitochondrial abnormalities,  neurodegeneration, and motor dysfunction

If this is true, what would you expect to see in mice that lost PPARδ activity through other  mechanisms?

-you should get the same phenotypes as Htt (mitochondrial abnormalities,  neurodegeneration, and motor dysfunction)

Testing this:

Knock-in mice expressing dominant negative PPARδ

-Dominant negative PPAR (DN PPAR) is another thing that prevents PPAR from  functioning properly

PPAR + RXR —> activate gene expression

PPAR + DN PPAR does not bind to RxR —> no gene expression

looking at weight

-the dominant WT is heavier

-DN: is smaller

motor function (ledge test)

-dominant negative: lost balance more

Closing score

-Dominant negative: clasped their hind legs more

Kyphosis (hunch back)

-dominant negative: more

prone to falling  

-dominant negative: happens more quickly (respective to time)

Hypothesis: Loss of PPARδ gene expression is contributing to mitochondrial abnormalities,  neurodegeneration, and motor dysfunction

supports the hypothesis!

Measured size of mitochondria (mitochondrial abnormalities)

dominant neg: have smaller mitochondria and less ATP production

Counted neuron numbers (neurodegeneation)

dominant neg: decreased numbers of nuerons  

Conclusion: Interfering with PPARδ function in mice can phenocopy HD. Therefore PPARδ dysregulation is a key in HD pathogenesis

“We theorized that if impaired PPAR-δ function is contributing to HD pathogenesis, then an  attractive treatment option would be to agonize PPAR-δ.”

-from study

-agonize PPAR - means to stimulate PPAR

-maybe if you add in a ligand for PPAR delta then you can stimulate PPAR transcription  genes and help hunting tons disease

“Of the various possible PPAR-δ agonists, we opted to use KD3010, as this PPAR-δ agonist is  potent and specific, crosses the blood brain barrier and was approved for use in humans in a  Phase1b metabolic disease safety trial in which no incidences of side effects were reported.” -This is important because you can’t have the agonist making side effects occur

KD3010 PPAR-δ Agonist

HD = has huntingtons disease

Non-Tg control = non HD mouse

-just a normal mouse like WT

What is the vehicle control?

-whatever your drug is dissolved in

-showing that just injecting a saline solution is not having an effect

What can they conclude? How effective was KD30101?

-not 100% effective but it does bring the neurological dysfunction score down from non treated

What did these scientists discover that was novel?

The big thing they didn’t know before: huntingtons gene interacts with PPAR delta They showed that if you inhibited PPAR you got a similar phenotypes as huntingtons showed you can help it through adding an agonist

~switching to Cytoskeleton~

Cytoskeleton  

-cell division (forming spindle apparatus)

-stable structures (cilia micro villi)

-polarization of the cell (assymmetrical shape)

-mechanical strength

-Motility and phagocytosis

-ability to fight off bacterial infection

Cytoskeleton filaments

3 major families

-Microtubules:

intracellular transport

-Actin:

Cell shape/locomotion

-Intermediate Filaments:

mechanical strength  

Accessory proteins also contribute

The cytoskeleton function as highways in the cell but are NOT permanent structures in the cell,  more like an ant trail, it is able to move and adapt if something is put in its way it will just go  around

Cytoskeleton Organization

The reason you have this dynamic ability in your cell is because of small soluble subunits that  make up cytoskeletal structure. they go back in forth from being individual units and being large  filamentous polymers  

-disadvantage: this takes energy

Advantages:  

rapid diffusion of subunits

rapid structural reorganization

Tubulin forms Microtubules

Tubulin subunit= heterodimer made of α and β subunits (these stack up to make the large  filaments mentioned above)

Tubulin heterodimers = microtubule subunit  

-Binds GTP

-plus/minus ends (polarity) (due to the alpha end and beta other end)

-Microtubule = 13 protofilaments

-protofilaments are a straight line of alternating alpha and beta subunits

-then 13 of those get side by side to make microtubule  

-hollow inside

Actin - thin and flexible  

Monomer

-but still have plus and minus end because the differing shape of ends  

Binds ATP instead of GTP

Actin filament -> 2 protofilaments wind together, tight bonding

Filaments twist around each other in right-handed helix

Crosslinking between filaments

actin is thin and flexible

microtubules are stiff

different cells will express different types of tubular and actin

ex: alpha actin only found in muscle cells

Tubulin binds GTP

-alpha subunits hides GTP deep in its center so it never gets hydrolyzed to GDP -beta binds it on the outside so it DOES get hydrolyzed  

-this is important for overall stability of filaments

Actin binds ATP

—ATP in the center but can be hydrolyzed  

When new subunits are getting added on, it will add when it is in the GTP or ATP state

Filament Growth

-you can add subunits to either + or - end but

-PLUS (+) end has faster growth of subunits than minus (-) end (because of polarity) -the plus end has the beta subunits bound to GTP

-After the subunit has been added, you are going to have hydrolysis of GTP to GDP soluble subunits are in T form

T form: ATP/GTP bound

D form: ADP/GDP bound  

-polymers are a mixture of T form and D form  

PLUS side there will be a ATP/GTP cap (which favors growth)

-because the ATP/GTP bound subunits are getting added so fast that the hydrolysis isn’t  keeping up

MINUS side hydrolysis will happen before a T form subunit can be added  

When it goes from GTP to GDP, you don’t get as strong of a bond, you start to see bending of  the subunits

-this is why the GTP cap is good for growth  

-if they are curving away from each other, called shrinking or a “catastrophe”

Dynamic instability

-When you have rapid growth, the addition starts to lag, the hydrolysis will catch up and then  you have rapid shrinking  

filaments continuously grow and shrink

Catastrophe: Loss of GTP cap and rapid shrinking

In actin its called Treadmilling

-can have an equilibrium between addition and loss so the overall length stays the same

Dynamic instability = microtubules

Tread milling = actin

—Both allow for spatial and temporal flexibility in filament formation

Spindle Formation

3 classes of dynamic microtubules:

Kinetochore microtubules (blue)

Interpolar microbutules (red)

Astral microtubules (green)

Microtubules contribute to the polarization of the cell because they are polarized so charged  things will move one way on one microtubule and the other way on another -directional transport

Example: Neuron

-All microtubules in the axon point in the same direction—plus ends toward axon  terminal

Intermediate Filaments (IF)

-you have a alpha helical region of monomer (NH2 at one end and COOH on the other) -that monomer interacts with another to form a coiled coil dimer

-then two coiled coils get in a staggered tetramer then lateral bonding eventually to be  like a rope  

-because of this there is no polarity  

-tetramer is the same thing as tublin subunits or actin subunits

It is like a rope and very hard to break because of the strong lateral bonds  -found in cell subject to mechanical stress example: epithelial cells

Keratin is an intermediate filament found in epithelial cells

-you can have mutations in these

Example of mutation: Epidermolysis bullosa simplex

intermediate filaments aren’t making the right connection  

-any stress on the skin, get huge blisters  

-can lead to high risk of infection

Nuclear lamins - found in nuclear envelope

-Nuclear lamina (blue) formed of IFs called lamins

-Strengthen inside surface of the inner nuclear membrane

-Provide attachment points for chromosomes

-Phosphorylation of lamins causes disassembly during mitosis

Progeria - advanced aging (hair loss, joint problem)

-caused by defect in lamin processing

-Nuclear lamina does not form properly

-Nuclear envelope does not have enough structural support—abnormal  shape

-Nuclear instability -> impaired cell division, increased cell death and  

diminished tissue repair

Bacterial Homologues

-Originally thought bacteria did not have a cytoskeleton

-but FtsZ is tubular like

-and there is also actin homologues that determine cell shape in bacteria

Accessory Proteins - help regulate the function of cytoskeletal parts

-can regulate length, stability, geometry and therefore function of cytoskeletal filaments  through accessory proteins

roles:  

Nucleation

Elongation

Stabilization

Disassembly

Cross-linking

Page Expired
5off
It looks like your free minutes have expired! Lucky for you we have all the content you need, just sign up here