Unit 4 - Bio 102

3 ways cells transport proteins

• nuclear pores

• translocators

• vesicles

how proteins get to the nucleus

go through nuclear pores

• need an nuclear localization signal/sequence

NLS

• nuclear localization signal

• code that says to bring the proteins to the nucleus

importin

protein that recognizes a NLS and protein and brings it through the nuclear pore

how proteins get into the mitochondria

protein translocators/transporters

how proteins get through translocators

• presequence goes on the N terminus and threads through either one or both membranes

• protein needs to be unfolded (down to just primary)

what happens inside the membranes after the translocator is used

• chaperone proteins wait to fold

• presequence signal is removed

how proteins get to the ER and onward

they are enclosed in transport vesicles

contents that need to live outside of the cell

• digestive enzymes

• toxins

• outer membrane proteins

• neurotransmitters

signal sequences

direct proteins to their correct locations in the cell

where signal sequences generally attach

at the N-terminus

when some proteins are transported to their location using the signal sequence

during translation

two characteristics of signal sequences

necessary and sufficient for localization

what it means for a signal sequence to be necessary for localization

• direct the ER protein to the ER

• without it, the protein would stay in the cytosol

what it means for a signal sequence to be sufficient for localization

it is capable enough to move a cystolic protein into the ER

3 types of endocytosis

• phagocytosis

• pinocytosis

• receptor mediated endocytosis

phagocytosis

eating large particles and breaking them down

pseudopodium

• used for phagocytosis

• used to grab the large molecules or particles

• brings the particles in and closes it up to create a vesicle or vacuole with food in it

example of specimen that do phagocytosis

macrophages

pinocytosis

bringing fluid/liquid into the cell

• also sometimes small particles like ions within the liquids (salty water)

how pinocytosis works

• membrane bubble forms and fills with liquid

• bubble pinches off and forms a vesicle with stuff in it

receptor-mediated endocytosis

involves a receptor and a coated pit (dw other cards go into more depth)

receptor

molecule receiving a signal

aspects of the pit in RME

• receptors line the pit

• made with clathrin (this is a protein and it is why it is called a “coated pit”)

what happens when a ligand binds to a receptor during RME

it signals for more clathrin to come in and form spheres around the membrane bubble

• creates a deeper bubble

dynamin

a protein that works like a piece of string that pulls the membrane to make it break off and form a circle separate from the rest of the membrane during RME

stages of receptor-mediated endocytosis

• initiation

• assembly

• maturation

• scission

RME main purpose

ligand transportation

familial hypercholesterolemia

genetic disease that states that there is too much cholesterol

negative impacts of too much cholesterol

• heart attacks

• atherosclerosis

• blocks arteries which reduces oxygen getting to heart and brain

job of the liver

• creates very low density lipoprotein to be delivered to cells

• coverts high density lipoprotein into bile salts which removed cholesterol from the body

what VLDL turns into

LDL

LDL’s job

delivers cholesterol to cells

HDL’s job

removes excess cholesterol

lipoprotein job

carries cholesterol

normal cholesterol levels

<200 mg

heterozygous FH average cholesterol levels

350-550 mg/dL

homozygous FH average cholesterol levels

as high as 1000 mg/dL

“bad” cholesterol

LDL - low density

“good” cholesterol

HDL - high density

HMG CoA Reductase

catalyzes the rate-limiting step of cholesterol synthesis

• determines how quickly we made LDL because it is the slowest part of the process

how HMG CoA reductase is regulated

feedback inhibition

pathway of cholesterol

• liver makes LDL using reductase

• LDL goes into the bloodstream

• LDL goes to the first cell and binds to the LDL receptors on it which signals receptor-mediated endocytosis

• when the receptors are filled, the cell knows to stop making receptors, and the excess LDL either goes back and inhibits the HMG CoA reductase or it gets turned into HDL which goes back into the liver and gets turned to bile salts

Goldstein and Brown

studied the LDL pathway using cell culture

cell culture

cells growing on a plate in medium

two changes Goldstein and Brown made to observe LDL from liver cells

• removed LDL

• added LDL

what removing LDL SHOULD do

create less LDL to inhibit the HMG CoA reductase, so the reductase activity should increase

what adding LDL SHOULD do

add more LDL to inhibit the HMG CoA reductase, so it decreases HMG CoA reductase activity

what Goldstein and Brown observed when they added and removed LDL in FH cells

HMG CoA reductase was independent of LDL levels (it was not affected)

Goldstein and Brown’s two hypotheses for why LDL levels did not affect HMG CoA reductase activities in FH cells

• enzyme defect

• receptor defect

how Goldstein and Brown tested if the enzyme was working properly. what was the result?

they added the cholesterol directly into the cells to bypass the LDL receptor

• this DID lower the HMG CoA reductase which showed that the enzyme worked fine and it was the receptor that was defected

how Goldstein and Brown tested if the receptor was working properly. what was the result?

• radiolabeled LDL and tracked it

• the LDL stayed on the cell surface and was not taking into the cell

• since the receptors don’t work properly, RME won’t work

how LDL usually gets into the cells

• binds to a receptor and gets brought in with a clathrin coated vesicle

• vesicle fuses with an endosome

• endosome delivers LDL to a lysosome and after it delivers, the endosome buds off and returns the LDL receptors to the plasma membrane

statins

cholesterol-lowering drugs

• inhibit HMG CoA reductase

signal transduction

converting one type of signal to another

broad overview of signal transduction

cells send out signals → extracellular signal molecule binds to a receptor on target cell → extracellular signal is transduced into an intracellular signal → target cell undergoes a response

kinds of signal transduction

• endocrine

• paracrine

  • autocrine

• contact-dependent

• neuronal

endocrine signaling

• most wide reaching and long distances

• when cells release hormones and they diffuse all around the body and lead to signals within the body

paracrine signaling

• more local with local signals (growth factors, nitric oxide) being released

• evenly distributed by concentration gradient - so farther cells will get less signal

autocrine signaling

• subset of paracrine

• signal disperses and goes back to itself and binds to its own receptor

• some of the signals released can be used by other cells

contact-dependent signaling

• no extracellular molecule involved

• short ranged

• signal on surface binds to target cell receptor

neuronal signaling

• can deliver signals long distances but not widely

• uses neurotransmitters - delivered quickly and specifically

• more 1 on 1 than endocrine

what makes an extracellular signal fast

• effector proteins are already present

• work by altering an existing protein

• secs → mins

what makes an extracellular signal slow

• requires a change in gene expression - new RNA and protein required

• mins → hours

ways intracellular signals get passed in the cells

• relaying

• amplifying

• integrating

• distributing

relaying in intracellular signaling

• signals goes from A→ B → C

• just sends it onward

• usually done on scaffold

amplification of intracellular signals

• makes the signal stronger

• C makes 20 copies of D

integrating intracellular signals

• sends one cohesive signal onward

• need multiple D to make one E

distribution of intracellular signals

• signal goes to more than one effector protein

• one molecule activates a variety of others

• H makes signals I and J and K

positive feedback

products of the pathway increase the activity of the components of the pathway

negative feedback

products of the pathway decrease the activity of the components of the pathway

2 states signaling molecules can exist in

on and off

2 general types of molecular switches

• protein phosphorylation

• GTP-binding proteins

protein phosphorylation

ATP releases the phosphate group which controls on/off

protein kinase job in protein phosphorylation

take the phosphate from ATP and add it to the protein to turn it on

protein phosphatase job in protein phosphorylation

removes a phosphate group to turn the protein off

GTP binding

activation based on GTP and GDP

GTP

activates protein during GTP binding

GDP

inactivates protein during GTP binding

GTPase activity of proteins during GTP binding

leads to the hydrolysis of GTP to turn it into GDP

qualifications of extracellular signal molecules

• large

• hydrophilic

• require cell surface receptors

qualifications of intracellular signal molecules

• small

• hydrophobic

how are steroid hormones different from from other signals

they can simply go through the membrane and bind to an intracellular/nuclear receptor

3 types of cell surface receptors

• ion channel coupled receptors

• G-protein coupled receptors

• enzyme-coupled receptors

ion-channeled receptors

• aka ligand gated channels

• rapid transmission

• neurotransmitters bind to ion-coupled receptors on target cell

• ions rush into and out of cell driven by electrochemical gradient

• changes membrane potential

• receives a signal and opens a channel

G-protein coupled receptors

• guanine nucleotide protein coupled receptor

• largest family

• over 700 GPCRs in humans

• mediate responses to hormones, local mediators, and neurotransmitters

enzyme coupled receptors

two kinds:

• signal molecule is a dimer - binding of signal molecule dimerizes the receptor - dimerized receptor has enzyme activity

• receptor itself is an enzyme

general structure of GPCRs

• spot for ligand to bind on the extracellular space

• spot for G-protein to bind on cytosol side

3 subunits of a G-protein

• alpha

• beta

• gamma

what happens when a signal molecule binds to an inactive receptor protein

the 3 subunits of a G-protein comes over and binds to the now active receptor protein

• this activates alpha, beta, gamma by knocking off the GDP

what happens when the three subunits binds to the active receptor protein

alpha separates from beta and gamma

• GTP binds to alpha and activates it

two things GPCRs can do

• open or close ion channels

• set off cascade events

what happens when activated alpha binds to an inactivated enzyme

• enzyme activates

• enzyme releases adenylyl cyclase

adenylyl cyclase

converts ATP to cAMP

• does this by removing 2 phosphates and making it a circle

phosphodiesterase

breaks down cAMP into AMP

cAMP

can activate protein kinase A (PKA)

PKA

activates phosphorylase kinase

phosphorylase kinase

activates glycogen phosphorylase

glycogen phosphorylase

breaks down glycogen

chlolera toxin

binds to alpha subunit of G protein and prevents hydrolysis of GTP to GDP