Intro into Cellular Degradation Flashcards
Proteostasis
Maintenance of all proteins (proteome)
in the correct and specific conformation,
concentration, and location to be
functional
Why is proteostasis important
- it is vital for the dynamic changes
required for a cell to respond to a given stimulus - essential to maintain normal cellular metabolism
How is proteostasis achieved?
through the coordinated action of the Proteostasis Network (degrade and excrete proteins that are no longer needed/or are damaged)
Proteostasis Network (PN)
• Acts as a quality control department • PN components have immediate roles in: –Biogenesis –Conformational Maintenance --Degradation (our focus)
Biogenesis includes:
- Protein synthesis (by ribosomes)
- Initial folding (by chaperones)
- Trafficking
Conformational Maintenance includes
- Refolding
- Remodeling
- Disaggregation
Consequences of PN Deficiency
deficient proteostasis –> newly synth proteins fold inefficiently –> Metastable proteins lose their functionally active conformations (specially under cell stress) AND cytosolic protein aggregates accumulate –> misfolding proteins challenge proteostasis –> cycles
NET = inability to restore proteostasis leads to disease
The ___ proteasome degrades over __%
of the proteome by _______
26S; 90%; UPS and UIPS
T/F UIPS can degrade folded proteins
FALSE
The UIPS can effectively degrade intrinsically disorder proteins (IDPs), but
not folded proteins
Folded proteins can undergo UIPS after
Cellular stress (PTMs, oxidative damage) can partially unfold proteins making them a substrate of UIPS
Insoluble aggregates
Partially unfolded proteins and IDPs
can self-associate forming insoluble aggregates
for this reason partially unfolded proteins are more likely to form aggregates
How are aggregates cleared?
Aggregates cannot be cleared by the
proteasome and are predominantly
cleared by the autophagy-lysosomal pathway
UIPS acronym meaning
Ubiquitin-independent proteosome
Doesn’t have ubiquitination to take substrate unlike UPS
NDDs and proteostasis
- NDDs are Proteinopathies characterized by the accumulation of misfolded and/or aggregation-prone proteins
- The imbalance attributed, in part, to decay in the capacity of protein turnover–loss of proteostatic balance
NDDs are characterized by…
by the progressive structural and functional
impairment of neurons, resulting in neuronal death
Despite their different symptoms, one commonality across NDDs is
that they are all associated with
the accumulation of aggregated proteins
ex. AD, PD, HD, Creutzfeldt-Jakob’s disease
Different NDDS attack…
very specific subsets of neurons ex. bvFTLD--ACC, FL. HD--straitum MSN neurons AD--LC, HP pyramidal neurons ALS--motor neurons PD--DA neurons in SNpc, DMV and OB
Common pathological mechanism linking clinically distinct diseases:
Failure of cells to cope with excess misfolded proteins
in health–proteostasis is balance
in NDDs–imbalance occurs
Degrons–what is it
- Degradation signal
- minimal element within a protein that is sufficient for recognition and degradation by a proteolytic apparatus
Degron structure
- short amino acid sequences
- structural motifs and exposed amino acids (often Lysine or Arginine) located anywhere in the protein
2 proteosome systems
UPS (Ubiquitin Proteasome System)
UIPS (Ubiquitin-independent Proteasome System)
20S proteasome structure
barrel-shaped complex comprised of four heptameric rings: two stacked β-rings that are sandwiched by two α-rings
__ of the ___ __- subunit proteases hydrolysize peptide bonds of the substrates
three of seven; beta-subunits
Proteosome structure–active site region and why
β subunits active sites are sequestered in the interior of the 20S chamber, such that substrates must first traverse through the exterior α-rings
Closed state of proteosome
In its closed state, the α-rings have a narrow pore that occludes the entry of most proteins
How substrates are granted access to proteolytic chamber
β subunits active sites are sequestered in the interior of the 20S chamber, and therefore must pass through alpha rings first
narrow alpha rings = closed
widen to grant access
Proteasome activators (PA)
- multi-protein complexes that facilitate degradation by the 20S
- Specific PAs determine whether that 20S
proteasome is coupled to the UPS or UIPS - therefore PAs differ between UPS and UIPS
Proteasome activators (PA) in UIPS
Pa28 and pa200
Proteasome activators (PA) in UPS
PA700 (aka 19s)
19s (PA) + 20S (core) = 26s
Protein degradation via the UPS pathway involves
– ubiquitylation of the substrate
–then degradation of the tagged protein by the proteasome
Canonical regulatory particle of the UPS
PA700 (or 19S)
19S + 20S to create the 26S proteasome (26S)
Proteasome in UPS: 19s subunit
The lid contains subunits that bind to • polyUb chains • deubiquitinating enzymes (DUBs) that regulate association with the particle The base contains; • motifs that interact with the α-rings • DUB( • ATPases that unfold the substrate
Forms of the proteosome in UPS
Combo of the 20s core and 19s subunit can take on a variety of forms
- can be double capped (19S on either end of core)
- singly capped
- or a 26S hybrid proteosome
Ubiquitin (Ub)
an 8kDa (small) polypeptide
Ubiquitylation
Post-translational modification wherein Ub covalently modifies protein substrates
– Formation of isopeptide bond between a lys residue on the protein and the carboxyl terminus of ubiquitin
T/F: Ub can be ubiquitylated
TRUE
Ub can itself be ubiquitinated on any of its seven lysine residues or its N-terminal methionine
allows formation of long UB chains
Why have Ub binding
Ubiquitin-based degrons are recognized and bound by a class of ubiquitin-binding domains (UBDs) of specific adaptors
Ubiquitylation Cascade
1) Ub monomerized
2) . Ub is covalently conjugated to E1 (ubiquitin-activating enzyme) (ATP-dependent)
3) Ub is transferred to the E2 (ubiquitin-conjugating enzyme)
4) The target protein exposes a degron
5) E3 (ubiquitin–protein ligases) assists or directly catalyses the transfer of ubiquitin from the E2 to a substrate (this process repeats in subsequent rounds)
6) In subsequent rounds, Ub molecules can be conjugated to Ub itself to form chains
7) Deubiquitylating enzymes (DUBs) remove ubiquitin molecules from substrates or process ubiquitin precursors to generate free ubiquitin pools
When Ub is translated it is is the form of
Precursors (either long or bound to other proteins)
Need to be monomers for post-translational modification (to be attached to protein)
In what ways is the Ubiquitylation Cascade hierarchical
E1 (2 enzyme) activates a few E2 enzymes and E2 activate several hundred E3s
E1s
Two common E1 enzymes activates Ub
for all cellular polyubiquitylation networks
E3s
- E3s catalyze ubiquitylation of substrates in a targetspecific manner
- E3s are responsible for target recognition through specific physical interactions with substrate
Ub signal and how fate is determined
- Various types of ubiquitin signals are generated based on the linkage type
- type of ubiquitination determines its fate (to proteosome, vs autophagic degradation, trafficking)
UBDs function
- Ubiquitin linkages are recognized and interpreted by Ub-binding domains
(UBDs) of specific adaptors - Effector molecules decipher the Ub code (the linkage type and length of ubiquitin chains) and link the substrates to downstream processes
Substrate Recognition and Degradation
by the 26S Proteasome: binding
Proteins with at least four Ubs can bind either:
-directly to intrinsic Ub receptors in the 19S
regulatory complex
OR
- to adaptor proteins that contain both poly ubiquitin-binding and proteasome-binding domains
T/F: 26S proteosome recognizes non-modified substrates
FALSE
26S proteasome does not recognize non- modified substrates
Following binding to 26s proteosome
Binding of substrate to the proteasome is followed by protein unfolding by half-dozen
ATPases that encircle the pore of proteasome
Once 26s is bound and unfolded
Proteasome-associated deubiquitylating enzymes (DUBs) remove polyUb
chains
Unfolding and deubiquitylation allow…
- translocation of the protein into the central proteolytic chamber, where it is cleaved into short peptides - Peptides are further degraded into free aa by cytosolic amino and carboxypeptidases
Substrate Recognition and Degradation
by the 26S Proteasome: process overview
1) Binding–most have at least 4 Ubs and can bind to 19s or adaptor protein
2) Protein is unfolded by ATPases around the pore
3) DUBs remove polyUB chains
4)the protein enters the central proteolytic chamber, where it is cleaved into short peptides
5) Peptides are further degraded
into free aa by cytosolic amino and carboxypeptidases
Deubiquitylation/Deubiquitylating
Enzymes (DUBs)
- Deubiquitylation is a highly regulated process
- Implicated in numerous cellular functions
- ~95 DUBs subdivided into 5 categories based on homology at the catalytic domain
Altered DUB
- Pathogenic microorganisms have acquired genes encoding DUBs: disruption of ubiquitylation in the host confers a selective advantage
- Mutations in several DUBs have been linked to diseases
Cellular functions related to DUBs
cell cycle regulation, gene expression, DNA repair, kinase activation, microbial pathogenesis
DUB mechanism in UPS
1) USP14 and UCHL5 associate reversibly to the 19S lid
2) UCHL5 trims poly-Ub chain at their distal end and release mono-Ub for re-utilization (deubiquitinates and regenerates free pull of UB)
3) UCHL5 stimulates ATP hydrolysis and 20S gate opening (helps gate open)
4) PSMD14/POH1 is a constitutive component of the base of the19S regulatory subunit (similar effect to UCHL5–regenerates pull of free Ub)
What happens if deubiquitination is too early/premature
premature by deubiquitination USP14 can
prevent protein degradation
as the proteosome will not recognize the protein
USP14 is responsible for
for trimming Ub chains on substrates
destined for degradation and thus
recycling mono-Ub
Role of other DUBs
- can deubiquitinate and rescue proteins from proteasomal degradation
- can enhance degradation of a few substrates by editing poly-Ub chains for better recognition by and access to the proteasome
Ub-Independent-Proteasome System (UIPS)–component parts
UIPS is coordinated by the 20S and may be amplified with UIPS-specific PAs, including PA200 and PA28 UIPS-PAs open the α-ring gate through a binding-induced conformational change
Substrates for UIPS
UIPS substrates are unfolded proteins that can fit into the channel without an active
unfoldase
because of this UIPS-specific PAs typically lack unfolding activity
PA28 structure
PA28 is composed of multiple,
different subunits (alpha, beta, and
gamma)
Free 20s (without additional PAs)
The free 20S (no PA) is likely to be a contributor to the UIPS
• It has relatively low enzymatic activity in the absence of Pas
• some small or unfolded substrates may traverse the closed gates and be degraded by the minimal machine
How do UIPS recognize proteins
Recognition of proteins by UIPS is mediated, in part, by disordered regions that act as signals (or degrons)
Substrates of UIPS
IDPs and IDR-containing proteins, which
lack the three-dimensional structure, are
thought to readily traverse the α-ring gate
The substrate pool of the UIPS is _______
considerably large
• 20% of cellular proteins are classified as IDPs
• ~ 41% of the eukaryotic proteome is predicted to contain IDRs
Over __% of human proteome is regulated by the UPS, including:
90% • structured (or folded) proteins • intrinsically disordered proteins (IDPs) • proteins containing intrinsically disordered regions (IDRs).
Structured proteins can only be degraded by the ____ because …
UPS
because they must be unfolded prior to their degradation
NDD adn UIPS
The proteins that accumulate in NDDs, such as amyloid beta, tau, TDP-43 and α-synuclein are potential substrates for the UIPS
