Protein Degredation Flashcards
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
The ubiquitin/proteasome pathway (UPP) is dependent on
Energy
in the UPP pathway, the targeted protein is marked for degredation by attachment to the protein
Ubiquitin
Degradation of the ubiquinated protein occurs in a compartmentalized protease called the
Proteasome
The most efficient way to terminate a biological effect is to degrade the protein that performs that
Function
Permits the removal of damaged proteins, facilitates growth and tissue remodeling, and offers a rapid response to stress and infection
Protein degradation
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)
The proteolytic enzymes involved in specific and non-specific protein degradation share which two key features?
-saves intracellular proteins
- ) expressed as zymogens
2. ) compartmentalized
Compartmentalizing the proteolytic enzymes does what?
Spares other cellular proteins
Refers to the time it takes for a protein to lose 50% of activity
Functional half-life
The hydrolytic activities of the proteasome exist inside a chambered barrel that is assembled from a stack ofq
Four seven subunit protein rings
Before substrates enter the inner sanctum of the proteasome, they are first unfolded by an
ATP-dependent unfolding machine
Unlike digestive and lysosomal proteolysis, degradation by the proteasome is
Energy-dependent
Which three properties regulate proteolysis?
- ) Zymogen precursors
- ) Compartmentilization
- ) pH
Another restriction that ensures that some proteases are active only in the correct location is their strict requirement for
Low pH for optimal activity
A compartmentalized proteolytic organelle
Lysosome
A tumor suppressor protein that is degraded by the UPP
p53
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
A sporadic and unscheduled degradation of proteins, such as the dietary proteins, by proteases with a broad cleavage specificity
Non-specific Degredation
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
A pathway for targeting proteins with a very high degree of specificity
The ubiquitin/proteasome system (UPS)
To mark them for degradation by the proteasome, substrates are post-translationally conjugated to a small protein called
Ubiquitin (Ub)
The most conserved protein known to man
-no known genetic mutations
Ubiquitin
Substrates are typically targeted by ubiquitin after a post-translational modification such as
Phosphorylation or hydroxylation
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?
- ) Ub activating enzyme (E1)
- ) Ub-conjugating enzyme (E2)
- ) Ub-protein ligase (E3)
Mediates the transfer of Ub from E1 to either E3 or substrate
E2
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
E1
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
E1
Receives Ub via a trans esterification reaction from E1
-Associate with E3 enzymes
E2
What are the two types of E3 enzymes?
- ) 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:
- ) Recognizes the substrate
- ) Degrades the substrate
- ) 19S complex
2. ) 20S complex
Contains three distinct proteolytic activities:
- ) Chymotrypsin-like
- ) Trypsin-like
- ) 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?
- ) Allosteric control
- ( Role of ATP hydolysis
- ) Recycling and peptide release
- ) 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
E3
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
