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PMCOL 412 > Intro into Cellular Degradation > Flashcards

Intro into Cellular Degradation Flashcards

1
Q

Proteostasis

A

Maintenance of all proteins (proteome)
in the correct and specific conformation,
concentration, and location to be
functional

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2
Q

Why is proteostasis important

A
  • it is vital for the dynamic changes
    required for a cell to respond to a given stimulus
  • essential to maintain normal cellular metabolism
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3
Q

How is proteostasis achieved?

A

through the coordinated action of the Proteostasis Network (degrade and excrete proteins that are no longer needed/or are damaged)

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4
Q

Proteostasis Network (PN)

A 
• Acts as a quality control department
• PN components have immediate roles in:
–Biogenesis
–Conformational Maintenance
--Degradation (our focus)
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5
Q

Biogenesis includes:

A
  • Protein synthesis (by ribosomes)
  • Initial folding (by chaperones)
  • Trafficking
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6
Q

Conformational Maintenance includes

A
  • Refolding
  • Remodeling
  • Disaggregation
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7
Q

Consequences of PN Deficiency

A

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

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8
Q

The ___ proteasome degrades over __%

of the proteome by _______

A

26S; 90%; UPS and UIPS

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9
Q

T/F UIPS can degrade folded proteins

A

FALSE
The UIPS can effectively degrade intrinsically disorder proteins (IDPs), but
not folded proteins

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10
Q

Folded proteins can undergo UIPS after

A

Cellular stress (PTMs, oxidative damage) can partially unfold proteins making them a substrate of UIPS

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11
Q

Insoluble aggregates

A

Partially unfolded proteins and IDPs
can self-associate forming insoluble aggregates
for this reason partially unfolded proteins are more likely to form aggregates

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12
Q

How are aggregates cleared?

A

Aggregates cannot be cleared by the
proteasome and are predominantly
cleared by the autophagy-lysosomal pathway

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13
Q

UIPS acronym meaning

A

Ubiquitin-independent proteosome

Doesn’t have ubiquitination to take substrate unlike UPS

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14
Q

NDDs and proteostasis

A
  • 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
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15
Q

NDDs are characterized by…

A

by the progressive structural and functional

impairment of neurons, resulting in neuronal death

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16
Q

Despite their different symptoms, one commonality across NDDs is

A

that they are all associated with
the accumulation of aggregated proteins
ex. AD, PD, HD, Creutzfeldt-Jakob’s disease

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17
Q

Different NDDS attack…

A 
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
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18
Q

Common pathological mechanism linking clinically distinct diseases:

A

Failure of cells to cope with excess misfolded proteins
in health–proteostasis is balance
in NDDs–imbalance occurs

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19
Q

Degrons–what is it

A
  • Degradation signal

- minimal element within a protein that is sufficient for recognition and degradation by a proteolytic apparatus

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20
Q

Degron structure

A
  • short amino acid sequences

- structural motifs and exposed amino acids (often Lysine or Arginine) located anywhere in the protein

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21
Q

2 proteosome systems

A

UPS (Ubiquitin Proteasome System)

UIPS (Ubiquitin-independent Proteasome System)

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22
Q

20S proteasome structure

A

barrel-shaped complex comprised of four heptameric rings: two stacked β-rings that are sandwiched by two α-rings

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23
Q

__ of the ___ __- subunit proteases hydrolysize peptide bonds of the substrates

A

three of seven; beta-subunits

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24
Q

Proteosome structure–active site region and why

A

β subunits active sites are sequestered in the interior of the 20S chamber, such that substrates must first traverse through the exterior α-rings

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25
Q

Closed state of proteosome

A

In its closed state, the α-rings have a narrow pore that occludes the entry of most proteins

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26
Q

How substrates are granted access to proteolytic chamber

A

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

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27
Q

Proteasome activators (PA)

A
  • 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
28
Q

Proteasome activators (PA) in UIPS

A

Pa28 and pa200

29
Q

Proteasome activators (PA) in UPS

A

PA700 (aka 19s)

19s (PA) + 20S (core) = 26s

30
Q

Protein degradation via the UPS pathway involves

A

– ubiquitylation of the substrate

–then degradation of the tagged protein by the proteasome

31
Q

Canonical regulatory particle of the UPS

A

PA700 (or 19S)

19S + 20S to create the 26S proteasome (26S)

32
Q

Proteasome in UPS: 19s subunit

A 
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
33
Q

Forms of the proteosome in UPS

A

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
34
Q

Ubiquitin (Ub)

A

an 8kDa (small) polypeptide

35
Q

Ubiquitylation

A

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

36
Q

T/F: Ub can be ubiquitylated

A

TRUE
Ub can itself be ubiquitinated on any of its seven lysine residues or its N-terminal methionine
allows formation of long UB chains

37
Q

Why have Ub binding

A

Ubiquitin-based degrons are recognized and bound by a class of ubiquitin-binding domains (UBDs) of specific adaptors

38
Q

Ubiquitylation Cascade

A

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

39
Q

When Ub is translated it is is the form of

A

Precursors (either long or bound to other proteins)

Need to be monomers for post-translational modification (to be attached to protein)

40
Q

In what ways is the Ubiquitylation Cascade hierarchical

A

E1 (2 enzyme) activates a few E2 enzymes and E2 activate several hundred E3s

41
Q

E1s

A

Two common E1 enzymes activates Ub

for all cellular polyubiquitylation networks

42
Q

E3s

A
  • E3s catalyze ubiquitylation of substrates in a targetspecific manner
  • E3s are responsible for target recognition through specific physical interactions with substrate
43
Q

Ub signal and how fate is determined

A
  • Various types of ubiquitin signals are generated based on the linkage type
  • type of ubiquitination determines its fate (to proteosome, vs autophagic degradation, trafficking)
44
Q

UBDs function

A
  • 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
45
Q

Substrate Recognition and Degradation

by the 26S Proteasome: binding

A

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

46
Q

T/F: 26S proteosome recognizes non-modified substrates

A

FALSE

26S proteasome does not recognize non- modified substrates

47
Q

Following binding to 26s proteosome

A

Binding of substrate to the proteasome is followed by protein unfolding by half-dozen
ATPases that encircle the pore of proteasome

48
Q

Once 26s is bound and unfolded

A

Proteasome-associated deubiquitylating enzymes (DUBs) remove polyUb
chains

49
Q

Unfolding and deubiquitylation allow…

A 
- 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
50
Q

Substrate Recognition and Degradation

by the 26S Proteasome: process overview

A

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

51
Q

Deubiquitylation/Deubiquitylating

Enzymes (DUBs)

A
  • Deubiquitylation is a highly regulated process
  • Implicated in numerous cellular functions
  • ~95 DUBs subdivided into 5 categories based on homology at the catalytic domain
52
Q

Altered DUB

A
  • 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
53
Q

Cellular functions related to DUBs

A

cell cycle regulation, gene expression, DNA repair, kinase activation, microbial pathogenesis

54
Q

DUB mechanism in UPS

A

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)

55
Q

What happens if deubiquitination is too early/premature

A

premature by deubiquitination USP14 can
prevent protein degradation
as the proteosome will not recognize the protein

56
Q

USP14 is responsible for

A

for trimming Ub chains on substrates
destined for degradation and thus
recycling mono-Ub

57
Q

Role of other DUBs

A
  • 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
58
Q

Ub-Independent-Proteasome System (UIPS)–component parts

A 
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
59
Q

Substrates for UIPS

A

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

60
Q

PA28 structure

A

PA28 is composed of multiple,
different subunits (alpha, beta, and
gamma)

61
Q

Free 20s (without additional PAs)

A

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

62
Q

How do UIPS recognize proteins

A

Recognition of proteins by UIPS is mediated, in part, by disordered regions that act as signals (or degrons)

63
Q

Substrates of UIPS

A

IDPs and IDR-containing proteins, which
lack the three-dimensional structure, are
thought to readily traverse the α-ring gate

64
Q

The substrate pool of the UIPS is _______

A

considerably large
• 20% of cellular proteins are classified as IDPs
• ~ 41% of the eukaryotic proteome is predicted to contain IDRs

65
Q

Over __% of human proteome is regulated by the UPS, including:

A 
90%
• structured (or folded) proteins
• intrinsically disordered proteins (IDPs)
• proteins containing intrinsically
disordered regions (IDRs).
66
Q

Structured proteins can only be degraded by the ____ because …

A

UPS

because they must be unfolded prior to their degradation

67
Q

NDD adn UIPS

A

The proteins that accumulate in NDDs, such as amyloid beta, tau, TDP-43 and α-synuclein are potential substrates for the UIPS

PMCOL 412 (35 decks)

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