Cell BIology - BIOL 4100 - Exam 5 - Dr. S

True or False: We generate garbage inside the cell every day!

True

What types of things make up cellular garbage?

Proteins (misfolded, unused, turnover), metabolic products, organelles

List the consequences (6) of impaired protein degradation:

Protein aggregates, ubiquitinated inclusions, vacuolation (paraptosis), damaged organelles, impairment of cellular processes, cell death

___________ __________ ______________ is the underlying pathogenesis of degenerative disorders such as neurodegeneration, muscle and liver degeneration, lung disease and aging.

Impaired protein degradation

Degenerative diseases lead to:

cell death

Explain impaired protein degradation

Cells lose ability to get rid of cellular garbage

True or False: Turnover of protein is constant

FALSE! Turnover of protein is not constant; we get rid of proteins we don't need (defective); not a scheduled mechanism

What are the two categories for the half life of proteins:

Short-lived proteins and long lived proteins

Describe short-lived proteins and give an example:

Regulatory proteins; enzymes that catalyze committed steps in cellular processes; Ex. Transcription Factors; we only need it for the period of time that we need it

Describe long-lived proteins and give an example:

Structural proteins; nuclear pore proteins; Ex. Histones; we use them all the time

True or False: The half-life of a protein can depend on its location in the body.

True; heart beats all the time so proteins in the heart would have a short half-life bc we use them all the time

True or False: Protein degradation is independent of nutritional state.

False; Protein degradation can be regulated by availability of nutrients

______________ Pathway contributes to 80-90% of protein degradation.

Ubiquitin/Proteasome

Ubiquitin/Proteasome Pathway deals with most ___________________ proteins.

intracellular

____________________ Pathway contributes to 10-20% of protein degradation.

Endosomal/Lysosomal

Endosomal/Lysosomal Pathway deals with most ___________________ proteins and cellular _______________. It does also deal with some intracellular proteins.

extracellular; organelles

True or False: Endosomal/Lysosomal is more non-specific than the Ubiquitin/Proteasome Pathway.

True

Describe the structure of ubiquitin:

Ubiquitin is a highly conserved (96%) 76 amino acid polypeptide with a molecular weight of 8.5 kD; characterized by one α-helix and five β-sheets and a four amino acid tail domain (LRGG)

All molecules of ubiquitin have what it known as the ________ at the C-Terminus.

G76

What is the function of ubiquitin?

It is a protein that you put on other proteins to identify them for degradation

How many lysine residues are in every ubiquitin?

7

On which residue on ubiquitin does attachment occur to substrate protein?

G76

True or False: Ubiquitin is always added a lysine residue.

True

If lysines are on the interior of the substrate protein, will the substrate become ubiquitinated?

No, the lysines must be on the outside of the tertiary structure of the substrate protein in order for the protein to become ubiquitinated

Describe homologous ubiquitin chains:

When ubiquitin is added to the same residue in each ubiquitin molecule (Ub is added to K48 on one which was previously added to K48, etc.)

Mono-ubiquitination is said to be involved in:

Signaling pathways

Which ubiquitin chain was the first one found?

K48

Describe a mixed polyubiquitinated chain:

Ubiquitin can add at different lysine residues on each ubiquitin

Describe branched polyubiquitinated chains:

Ubiquitin can bind to two molecules of ubiquitin at different lysine residues on the same ubiquitin

Where does ubiquitin gets its name?

Because it is ubiquitous (found everywhere) due to its role in tagging proteins for degradation

The shape of the polyubiquitinated chain gives rise to its:

functionality

If a certain polyubiquitinated chain has similar structure to another chain, then it will most likely half a similar __________.

function

Describe the function mono-ubiquitination:

transcription, endocytosis, trafficking (some type of cell regulation)

Describe the function of K48, K11, K27, and K29:

mostly proteasomal targeting (degradative pathway)

Describe the function of K63 polyubiquitination:

signaling, DNA repair, endocytosis

What determines the "K number" of each ubiquitin?

Determined by the lysine that the next molecule of ubiquitin binds to

How many molecules of ubiquitin does it take for a polyubiquitinated chain to becomes biologically active?

4

Name the three enzymes involved in the Ubiquitin pathway:

E1 (Ubiquitin Activating Enzyme), E2 (Ubiquitin Conjugating Enzyme), and E3 (Ubiquitin Protein Ligase)

True or False: A protein can be ubiquitinated using only E1 and E2.

False; you must have all three enzymes in order for the process to work

Give the steps for E1:

1. E1 activates the C-terminus of ubiquitin by forming acyl-adenylate intermediate.

2. Catalytic cysteine residue of E1 uses ATP to add ubiquitin to the E1 via formation of an E1-ubiquitin thioester intermediate.

3. Reaction is repeated as another ubiquitin is adenylated and the charged ubiquitin is transferred to an E2 forming another thioester bond.

True or False: E1 is an extremely efficient enzyme.

True

Which enzyme is the first to interact with ubiquitin?

E1

Give the steps for E2:

1. Carries activated ubiquitin from the E1 to the substrate.

2. Each UBC domain has a Cys residue located in a shallow groove at its active site which will form a thioester bond with the ubiquitin molecule being transferred from the E1.

3. Responsible for the linkage specificity and chain assembly.

Which enzyme determines which type of chain the ubiquitin will become?

E2

E2 is a large family of proteins. Which structure is common among all E2's?

UBC domain

Give the steps for E3:

1. Transfer ubiquitin from E2 enzymes to substrates.

2. Involved in final target selection and specificity of the reaction.

3. Degrons -- any motif or structure on the substrate that targets proteins for degradation.

E3 is looking to find ___________ on the substrate which are places where ubiquitin can bind.

degrons

True or False: All proteins have degrons.

True (although they are not always accessible)

For HECT domain E3's, the N-lobe binds to _______ and the C-lobe binds to __________.

E2 (which is carrying ubiquitin); ubiquitin

Describe HECT domain E3's:

1. Comprises ~350 amino acids located at the C-terminus of most of these enzymes.

2. Forms a thioester bond with ubiquitin prior to transferring it to the substrate.

3. The HECT domain is bi-lobed with the N-terminus interacting with the E2 and C-terminus containing the active site cysteine to bind with ubiquitin.

4. The last ~60 amino acids of the C-lobe infer substrate binding.

Describe RING Domain E3's:

1. Conserved Cys and His residues coordinate to orient two Zn2+ ions to form a cross-brace structure.

2. DOES NOT form catalytic intermediate with ubiquitin, instead interacts directly with the E2 to form a scaffold allowing E2 and substrate interaction.

3. Can function as monomers, dimers, or as part of multi-protein subunits to achieve ubiquitination.

What does RING domain E3's stand for?

Really Interesting New Gene

What two amino acids residues do RING domains require in order to react?

Histidine and cysteine

True or False: RING domain E3's bind to ubiquitin.

False: RING domain E3's only bind to E2, NOT ubiquitin

Explain the different ways that E3's can add ubiquitin to a substrate:

1. Two different E3's can add to two different substrates.

2. Two different E3's can add ubiquitin chains at two different sites on the same substrate.

3. Two different E3's can work together to build a single chain at a specific site on a single substrate.

Describe Deubiquitinating Enzymes (DUBs):

1. Thiol proteases or Zinc-dependent metalloproteases.

2. Removes ubiquitin molecules from polyubiquitin chains.

3. Regenerates monomeric ubiquitin protein.

List the processes that DUBs play a role in:

Proteolysis, regulation of signaling pathways in the cell, endocytosis and cargo sorting, histone regulation and chromatin dynamics, and DNA repair mechanisms

Out of the proteins involved in the UPS (E1, E2 and E3), which is the most diverse and specific?

E3

Genes of the UPS constitute approximately ______ of the entire eukaryotic genome.

5%

Out of the proteins involved in the UPS (E1, E2 and E3), which is the smallest group?

E1

What is the job of the UbL - UBA protein family?

Take ubiquitinated proteins to the proteosome

What are the two parts of the UbL-UBA proteins?

The Ubiquitin-Like Domain and the Ubiquitin Associated Domain (where these proteins associate with ubiquitin)

How do the UbL-UBA proteins move throughout the cell?

They move along microtubules using motor proteins

What are the parts of the 26S Proteasome?

1 20S particle and 2 19S particles on either end

Describe the structure of the 20S core particle:

It is made up of 4 stacked heptameric rings (each ring has seven subunits); two α rings (structural) and 2 β rings (catalytic)

True or False: The 20S particle is the location of the enzymatic activity of the proteasome.

True

Give the parts (2) of the 19S particle of the proteasome:

Lid (10 subunits) and the base (9 subunits)

What are the functions of the subunits of the 19S particle of the proteasome?

1. Rpt 1-6 (ATPase subunits): recognize Ub-substrates, unfold Ub-substrates (ATP), opens gate in 20S CP (ATP), and translocates substrates into CP

2. Rpn 1-2 (non-ATPase subunits): structure of base

3. Rpn 13 (ubiquitin receptor): where ubiquitin chain binds into base structure

4. Rpn 10 (Hinge protein): where lid is built/allows motion to reach out and grab ubiquitin

5. Rpn 5-6: bind to base

6. Rpn 3, 7-9, 12: structural

7. Rpn 11: de-ubiquitin (DUB activity); have to remove ubiquitin to get protein into 20S

8. Sem1: assembly

True or False: In order for the proteasome to form, you must have 1 20S particle and 2 19S particles.

False; you can have any combination of 20S, 19S, and 11S

Degradation happens in which particle of the proteasome?

20S

What is the role of 11S particle?

Short peptide degradation; could be protein that has already been broken down a little

Explain the wobbling effect of the 19S particle of the proteasome:

2-4 ATPase domains can bind ATP simultaneously resulting in a "wobble" of the 19S base structure; this allows the proteasome to maximize stability and flexibility

_____________ is the passage of the unfolded substrate into the core of the proteasome.

Translocation

True or False: Substrates must be at least partially unfolded prior to entering the proteasome.

True

True or False: ATP hydrolysis is required for protein translocation into the proteasome.

FALSE: ATP hydrolysis is required for protein unfolding but not for translocation

Give the three different proteases found in the 20S particle and their functions:

1. PGPH-Like Activity (β1 - Acidic amino acids): cuts amino acid chain after every acidic residue.

2. Trypsin-Like Activity (β2 - Basic amino acids): cuts amino acids chain after every basic residue.

3. Chymotrypsin-Like Activity ( β5 - hydrophobic amino acids): cuts amino acid chain after every hydrophobic amino acid.

True or False: With a 19S particle on each end of the proteasome, proteins can be degraded in both directions at each end.

True

Explain the signaling process for the proteasome:

1. Ligand comes in and binds.

2. Causes ubiquitination of TRAF2 and TRAF3.

3. Those become degraded.

4. Nik is still around so it can activate IKK, which initiates a signaling cascade.

How can the proteasome help regulate signaling pathways?

It can control what happens in a signal transduction pathway by using proteasome to make a transcription factor

List the pathological implications of the Proteasome:

1. Malignancies: defects in the UPS can causes deregulation of suppressors.

2. Neurodegenerative Disease: Alzheimer's, Parkinson's (death of nerve cells; causes issues if proteins cannot be gotten rid of).

3. Genetic Disease: Angelman's Syndrome (defects in E6-AP synthesis), Cystic Fibrosis (CFTR expression differences), breakdown of UPS causes changes in the DNA itself.

_________________ was the scientist who first visualized autophagy and came up with the term.

Christian de Duve

George Palade discovered Palade particles or ____________.

ribosomes

What was the main experiment that scientists did to first visualize autophagy?

They studied liver cells in rats by treating them with glucagon (breaks down glycogen stores and floods the system with glucose); this basically induced starvation and forced the cells to take a bite of themselves

Define: Autophagy

A ubiquitous process in all eukaryotic cells in response to some stress applied to the cell that results in breakdown of cytoplasmic components in a struggle for survival and environmental adaptation

Autophagy is a _____________ mechanism.

survival

Most of what we know about autophagy was developed in ___________ cells.

yeast

Why would yeast be a better system to study autophagy than rats?

Yeast grow much faster than rats

Define: Macroautophagy

Process in which cellular components are degraded by lysosomes and recycled; take the biggest bite of something; mostly non-specific

If someone refers to plain autophagy, they are most likely referring to:

macroautophagy

Define: MIcroautophagy

Type of autophagic pathway which is mediated by direct lysosomal (mammals) or vacuolar (plants and fungi) engulfment of the cytoplasmic cargo; much smaller bite; also mostly non-specific

Define: Chaperone-mediated Autophagy

Chaperone-dependent selection of soluble cytosolic proteins that are then targeted to lysosomes for degradation; single protein getting degraded; most specific type

Which is the most specific type of autophagy?

Chaperone Mediated Autophagy

Define: Autophagosome

Completed double membrane bound compartment which sequesters cytoplasmic cargoes (finished product); vesicle we are forming

Define: Phagophore

Double membrane structure that functions in the initial sequestering of cargo (isolation membrane); beginning of the autophagosome

Define: Phagophore Assembly Site (PAS)

Often a peri-lysosomal location or compartment where nucleation of the phagophore initiates; can be spread throughout the cell

The PAS is often near the ________.

MTOC

True or False: Autophagy primarily occurs under normal nutrient conditions.

False: it is a result from starvation; survival mechanism

Give the steps of the Initiation stage of macroautophagy under normal conditions:

1. AMPK is inactive which means raptor is active.

2. Turns on mTOR which phosphorylates ULK1 which turns it off.

Give the steps of the Initiation stage of macroautophagy under starvation conditions:

1. AMPK becomes active as a response to the starvation.

2. Raptor gets turned of and TSC gets turned on (bc we turned off raptor, we also turn off mTOR).

3. AMPK phosphorylates ULK1, turning it on.

Give the steps for the Nucleation stage of macroautophagy:

1. Active ULK1 (double phosphorylated) phosphorylates Beclin (triumverant of proteins: ATG14L, Beclin, and VPS34).

2. VPS34 (PI3K) then phosphorylates PI at the 3rd carbon forming PI3P