Protein Degredation Flashcards by Joel Glotfelty

Besides just mutated or erroneous proteins, the degradation of cellular proteins that are no longer required, and the elimination of certain regulatory proteins is essential for normal

Cellular function

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The ubiquitin/proteasome pathway (UPP) is dependent on

Energy

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in the UPP pathway, the targeted protein is marked for degredation by attachment to the protein

Ubiquitin

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Degradation of the ubiquinated protein occurs in a compartmentalized protease called the

Proteasome

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The most efficient way to terminate a biological effect is to degrade the protein that performs that

Function

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Permits the removal of damaged proteins, facilitates growth and tissue remodeling, and offers a rapid response to stress and infection

Protein degradation

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The breakdown of proteins that generates amino acids for use in new protein synthesis and intermediates for the synthesis of other metabolites

Proteolysis (protein turnover)

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The proteolytic enzymes involved in specific and non-specific protein degradation share which two key features?

-saves intracellular proteins

) expressed as zymogens

2. ) compartmentalized

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Compartmentalizing the proteolytic enzymes does what?

Spares other cellular proteins

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Refers to the time it takes for a protein to lose 50% of activity

Functional half-life

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The hydrolytic activities of the proteasome exist inside a chambered barrel that is assembled from a stack ofq

Four seven subunit protein rings

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Before substrates enter the inner sanctum of the proteasome, they are first unfolded by an

ATP-dependent unfolding machine

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Unlike digestive and lysosomal proteolysis, degradation by the proteasome is

Energy-dependent

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Which three properties regulate proteolysis?

) Zymogen precursors
) Compartmentilization
) pH

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Another restriction that ensures that some proteases are active only in the correct location is their strict requirement for

Low pH for optimal activity

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A compartmentalized proteolytic organelle

Lysosome

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A tumor suppressor protein that is degraded by the UPP

p53

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Proteases that always degrade the same class of protein. The key feature is that the protein was not damaged, but it simply was no longer required by the cell

Specific degradation

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A sporadic and unscheduled degradation of proteins, such as the dietary proteins, by proteases with a broad cleavage specificity

Non-specific Degredation

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Since we want some proteins to only be active for a short amount of time, we could only express them when they are needed, or when they are not needed we could mark them for degradation with a

Post translational modification

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A pathway for targeting proteins with a very high degree of specificity

The ubiquitin/proteasome system (UPS)

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To mark them for degradation by the proteasome, substrates are post-translationally conjugated to a small protein called

Ubiquitin (Ub)

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The most conserved protein known to man

-no known genetic mutations

Ubiquitin

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Substrates are typically targeted by ubiquitin after a post-translational modification such as

Phosphorylation or hydroxylation

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Substrate-specificity in the UPS is accomplished by forming unique combinations of targeting factors. The substrate-targeting factors are known as

Ub-conjugating enzymes (E2) and Ub-protein ligases (E3)

Refers to the distinct targeting complexes that can be assembled from this large set of proteins. -Each unique E2/E3 complex is likely to have only one or a few substrates to which they attach Ub

Combinatorial Diversity

Has a highly compact structure (fist w/ extended thumb) and 7 lysine residues on the surface -a highly reactive carboxy terminus is accessible

Ubiquitin

Ubiquitin is always expressed as a fusion protein (Ub's in tandem, or fused to specific ribosomal proteins) and is inactive until it is

Hydrolyzed to mono-Ub

Ubiquitin has a compact structure with a reactive carboxy terminus that participates in all

Covalent interactions

The lysine residues on the surface of Ub can be ligated to additional

Ub's

When multiple Ub’s are attached to a substrate in the form of a chain, it can form a high-affinity hydrophobic interaction with the

Proteasome

A single Ub does not bind the proteasome because the hydrophobic patch on its surface is not sufficient to form a

High-affinity interaction

Neighboring ubiquitins form intramolecular hydrophobic interactions that yield a highly compact multi-Ub chain with a large hydrophobic surface that forms a high-affinity interaction with the

Proteasome

A significant reason for expression Ub as a fusion protein is to block the

Reactive carboxy terminus

What are the three key enzymes that promote the attachment of ubiquitin to to proteolytic substrates?

1. ) Ub activating enzyme (E1) 2. ) Ub-conjugating enzyme (E2) 3. ) Ub-protein ligase (E3)

Mediates the transfer of Ub from E1 to either E3 or substrate

The Ub-protein ligase (E3) attaches the Ub to substrate via an

Isopeptide bond

The carboxy terminus of Ub is generated by proteolysis and ends with the covalent residues

RGG

Ub is released from the fusion protein following hydrolysis by

Ub-carboxy terminal hydrolases (UCH)

In the formation of a multi-Ub chain, a lysine residue in the first Ub becomes covalently linked to the carboxy-terminus of the second Ub by an

Isopeptide bond

The formation of the hydrophobic stripe, which is required to interact with the proteasome, requires a minimum of

4 linked Ubs

Ub contains 7 lysine residues. The resulting chain of ubiquitins can target a substrate to the proteasome, if the linkage involves -If it is attached to a different residue, the potential conformation and biological effect could be very different

Lysine-48

Free mono-Ub is activated in an ATP dependent reaction by an enzyme called

The bond between Ub and E1 is a

Thioester bond

Ub is transferred from E1 to E2 and then to E3 in a -Only the ubiquination of E1 requires ATP

Thioester cascade

The difference between a peptide and an isopeptide bond is that while a peptide bond involves an α-amino group, an isopeptide bond involves an

ε-amino group

Can cleave and release Ub from a substrate

Ub processing enzymes

Activates the carboxy terminus of Ub, forms a noncovalent interaction with Ub-adenylate, and forms a thioester bond with Ub

Receives Ub via a trans esterification reaction from E1 -Associate with E3 enzymes

What are the two types of E3 enzymes?

1. ) Ring E3's | 2. ) Hect E3's

Differ from RING E3's because they have the ability to form a thioester bond with Ub and so they don't have to bind E2

Hect E3's

Can cleave the isopeptide bonds that link Ub to protein substrates, and Ub that is assembled into multiubiquitin chains

Ub-processing proteases (Ubp)

The bond between Ub and substrate is an

Isopeptide bond

Following interaction with the proteasome, multi-ubiquinated proteins are de-ubiquinated and unfolded. This reaction is coupled, since failure to remove the Ub results in

Inhibition of degradation

The formation of a hydrophobic stripe is the primary determinant for promoting interaction with the

Proteasome

Important for DNA repair, but does not promote degradation by the proteasome

Multi-ubiquitination through lysine 63

Short chains assembled through Lysine 63 promote translocation of plasma membrane proteins to the

Lysosome for degradation

The recognition of the substrate and the degradation of the substrate are spatially and temporally separable events in the

UPS

A multisubunit and multicatalytic chambered protease in which substrate binding and substrate degradation are carried out by specialized sub-complexes

Proteasome

In the Proteasome, which complex: 1. ) Recognizes the substrate 2. ) Degrades the substrate

1. ) 19S complex | 2. ) 20S complex

Contains three distinct proteolytic activities: 1. ) Chymotrypsin-like 2. ) Trypsin-like 3. ) PGPH

20S subunit (The catalytic core particle)

A barrel -like structure generated by a stack of four rings (α on top and bottom, 2β in middle)

20S subunit

Identical, and contain the catalytic subunits

β -subunits

Form a densely packed mat of overlapping loops that prevent entry into the 20S catalytic chamber

α-subunits

Three proteins in the β-subunits have the hydrolytic activity and exist as

Zymogens

Cellular proteins are not hydrolyzed unless they are first recognized by the -unfolds and translocates the proteins (requires ATP)

19S subunit

The α and β rings dimerize. One α/β dimer associates with another α/β dimer in a reaction that is mediated by a chaperone called

Ump1 (degraded upon activation of proteolytic sites)

One of the key properties of the 19S particle is that it contains receptors that enabled it to only bind to particles containing -Prevents unmarked proteins from being degraded

Multi-Ub chains

The affinity between the proteasome and multi-Ub chains increases from

4Ub to 8UB (but not 8Ub to 16Ub)

Associated with deubiquitinating enzymes that can dismantle multi-Ub chains from substrates

19S particle

The 19S particle contains a ring of 6 non-redundant ATPases that

Unfold substrates to be degraded

Regulates the axial channel and binds regulatory proteins

19S particle

Two 19S particles attach to each end of the 20S complex to form a dumbbell-shaped complex called the

26S proteasome

The efficiency of multi-step events is often improved by physically restricting the path of the reactants and products, through a mechanism termed

Channeling

The specificity of the proteasome is conferred entirely by the

19S subunit

A DNA repair protein that binds XPC in the nucleotide excision repair pathway -mutations can lead to XP

Rad23

Ub proteins do not bind the 20S particle. Instead, they bind the

19S regulatory particle

Immune cells express a specialized proteasome, which generates fragments of proteins that can be expressed on MHC class I and II molecules, forming antigens. These are called

Immunoproteasome

In the immunoproteasome, the 19S particle is replaced with -contributes to recognition of distinct types of substrates

PA28

In the immunoproteasome, the three beta with the hydrolytic activity are

Replaced

Generates peptides of the correct length and hydrophobicity to be placed in a cleft present in the extracellular domain of MHC molecules.

Immunoproteasomes altered cleavage properties

What are the four mechanisms of regulation from protein degradation?

1. ) Allosteric control 2. ( Role of ATP hydolysis 3. ) Recycling and peptide release 4. ) Combinatorial diversity

The process of protein hydrolysis by the proteasome has been termed

'bite-and-chew'

Polypeptide chains that enter the 20S chamber are first attacked by the -generates large fragments

Chymotryptic activity

High concentration of the large chymotryptic fragments allosterically inhibits further translocation of the polypeptide into the

Catalytic chamber

During this allosteric inhibition, what begins to chew up the chymotryptic fragments?

Trypsin and PGPH

The process whereby the 26S proteasome dissociates into the 19S and 20S subunits after each cycle of protein degradation, releasing the degradation products

'chew-and-spew'

p53 tummor suppressor is degraded by the -prevents cells from exercising proper checkpoint control during the cell cycle

Human Papilloma Virus (HPV) E6 protein

The variant of HPV that causes cancer does so because it expresses an altered viral E6 protein that can alter the substrate targeting properties of

E6AP is termed a

Hect E3 ligase

Form a thioester linkage with Ub, and also interacts with an E2 enzyme

Hect E3 ligase

Constitutively degraded in normal oxygenated tissues

Hif1 transcription factor

Required for activating expression of a number of genes that respond to hypoxia -degraded in normal cells becayse the tissues are well oxygenated

Hif1

Mutations in the F-box protein of the SCF E3 Ligase (a RING E3 ligase) can prevent

Hif1 degradation

A major fraction of solid sarcomas in the kidney and pancreas are due to the stabilization of

Hif1 due to F-box substitution

The inflammatory response defines a cellular reaction to insult and injury. The central player in this response is the transcription factor -initially synthesized as a large precursor

NFκB

The first role for the proteasome is to process NFκB. The resulting processed and mature polypeptide (p65) assembles into a dimer with another protein p50/p65, to form the active

NFκB transcription factor

However, p50/p65 can be sequestered in the cytoplasm by the inhibitory molecule

IκBα

Cell injury results in the activation of a kinase, called the Iκ kinase (IκK), in a mechanism that requires a functional ubiquitin-conjugation pathway. The activated IκK phosphorylates IκBα, which prevents it from

Sequestering p50/p65

Specific E2/E3 factors recognize phosphorylated IκBα and promote its degradation by the

Proteasome

Specific E2/E3 factors recognize phosphorylated IκBα and promote its degradation by the proteasome. This results in the release of p50/p65, which can now enter the nucleus and activate transcription of

Stress-responsive genes

Required for activating a DNA damage response

Proteasome

A proteasome subunit that can bind multiubiquitin chains

Ataxin-3

If ataxin-3 undergoes trinucleotide repeats, the result is

Machodo-Joseph disease

Failure to degrade hypoxia inducible transcription factor Hif1 causes

Von hippel-Lindau (VHL) syndrome

Under normoxia conditions, Hif1 is targeted by -requires Rbx1

VHL