Intracellular Protein Degradation

Chris Weihl MD/PhDDepartment of Neurology

How is trash handled?

Protein Degradation in the Cell

UPS

Aggresome

Autophagy

Endocytosis

Nucleus

Ub

Ub

Ub

Ub

Consequence of impaired protein degradation• Protein aggregates• Ubiquitinated inclusions• Vacuolation• Damaged organelles• Secondary impairment in other cellular processes• Cell Death

• Underlying pathogenesis of degenerative disorders (neurodegeneration, muscle degeneration, liver degeneration, lung disease, aging)

Protein DegradationTurnover of protein is NOT constantTurnover of protein is NOT constant

Half lives of proteins vary from minutes to infinityHalf lives of proteins vary from minutes to infinity

““Normal” proteins – 100-200 hrsNormal” proteins – 100-200 hrs

Short-lived proteinsShort-lived proteinsregulatory proteinsregulatory proteins

enzymes that catalyze committed stepsenzymes that catalyze committed stepstranscription factorstranscription factors

Long-lived proteinsLong-lived proteinsSpecial cases (structural proteins, crystallins)Special cases (structural proteins, crystallins)

Protein Degradation

Example: Lactic Acid DehydrogenaseTissue Half-lifeHeart 1.6 daysMuscle 31 daysLiver 16 days

• May depend on tissue distribution

• Protein degradation is a regulated processExample: Acetyl CoA carboxylase

Nutritional state Half-lifeFed 48 hoursFasted 18 hours

Protein Degradation Ubiquitin/Proteasome Pathway

80-90%Most intracellular proteins

• Lysosomal processes10-20%

Extracellular proteinsCell organellesSome intracellular proteins

How are proteins selected for degradation?

UBIQUITIN

KK

GG

Small peptide that is a “TAG” 76 amino acids C-terminal glycine - isopeptide

bond with the -amino group of lysine residues on the substrate

Attached as monoubiquitin or polyubiquitin chains

Ubiquitination of proteins is a FOUR-step process

First, Ubiquitin is activated by forming a link to “enzyme 1” (E1).

Then, ubiquitin is transferred to one of several types of “enzyme 2” (E2).

Then, “enzyme 3” (E3) catalizes the transfer of ubiquitin from E2 to a Lys -amino group of the “condemned” protein.

Lastly, molecules of Ubiquitin are commonly conjugated to the protein to be degraded by E3s & E4s

AMP

The UPS is enormous!

The genes of the UPS constitutes ~5% of the genome

• E1’s- 1-2 activating enzymes

• E2’s- 10-20 conjugating enzymes

• E3’s- 500-800 ubiquitin ligase- drives specificity

• DUBs- 100 ubiquitin specific proteases- regulators of pathway

The UPS is enormous!The UPS is enormous!

The genes of the UPS constitutes ~5% of the genome

E1’s- 1-2 activating enzymes E2’s- 10-20 conjugating enzymes E3’s- 500-800 ubiquitin ligase- drives specificity DUBs- 100 ubiquitin specific proteases- regulators of pathway

The genes of the UPS constitutes ~5% of the genome

E1’s- 1-2 activating enzymes E2’s- 10-20 conjugating enzymes E3’s- 500-800 ubiquitin ligase- drives specificity DUBs- 100 ubiquitin specific proteases- regulators of pathway

PROTEASOME COMPONENTS

20S Proteasome

19S Particle

26S Proteasome

ATP

Hydrolysis peptide bonds after:Hydrolysis peptide bonds after:

hydrophobic a.a.hydrophobic a.a. = = CHYMOTRYPSIN-CHYMOTRYPSIN-LIKE - LIKE - 55

acidic a.a.acidic a.a. = (-) = (-)CASPASE-LIKE CASPASE-LIKE --11

basic a.a.basic a.a. = (+) = (+)TRYPSIN-LIKE TRYPSIN-LIKE --22

DEUBIQUITINATIONDEUBIQUITINATION

De-ubiquitinatingDe-ubiquitinating

Pathways controlled by regulated proteolysisPathways controlled by regulated proteolysis

Mechanism of muscle atrophy

MURF/Atrogin

Knockout of Atrogin Rescues atrophy

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ub-ub-ub-ubub-ub-ub-ubub-ub-ub-ub0

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200000

300000

400000

500000

600000

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Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity

Usp14

Uch37

Borodovsky, A et alEMBO J. 20:5187-962001

The proteasomal DUB Usp14 impairs protein degradation

Lee, BH et alNature 467:179-842010

Decrease steady-state levels of aggregate prone proteins in the absence of Usp14

Lee, BH et alNature 467:179-842010

Lyosomal degradation

• Autophagy

Autophagy

• Lysosomal degradation of proteins and organelles• Occurs via three routes

• Macroautophagy• Microautophagy (direct uptake of cellular debris via the

lysosome)• Chaperone mediated autophagy (selective import of

substrates via Hsc70 and Lamp2a)

Yeast Genetics meets Human Genetics

• Identification of >50 autophagy essential proteins with mammalian homologs

Macroautophagy

Autophagosome

InductionmTOR

BeclinATG7

SequestrationPhagophoreATG5-ATG12-ATG16L1

Nucleation

Lysosome

Autolysosome

Degradation

FOXO3

Trafficking Fusion

“Autophagic Flux”

& Cargo loading

Genetic knockout of autophagy initiating proteins

Complete loss of ATG5 leads to lethality

Tissue specific knockout of autophagy

• Degeneration of CNS tissue; Hara et al 2006

• Hepatomegaly in Liver; Komatsu et al 2005

• Atrophy and weakness of skeletal muscle; Masiero et al 2009

• Pathologic similarities • Ubiquitinated inclusions• Aberrant mitochondria• Oxidatively damaged protein

Basal Autophagy

• Autophagy has a “housekeeping” role in the maintenance of cellular homeostasis

• Autophagy is responsible for the clearance of ubiquitinated proteins

Selective Autophagy

• Aggregaphagy– p62/SQSTM1, Nbr1• Mitophagy – Parkin, Nix• Reticulophagy – endoplasmic reticulum• Ribophagy – translating ribosomes• Xenophagy – e.g. Salmonella via optineurin• Lipophagy – autophagy mediated lipolysis

• Performed by an expanding group of ubiquitin adaptors

p62 as an autophagic tool• p62 associates with ubiquitinated proteins and LC3• p62 is an autophagic substrate

LC3 as an autophagic tool

LC3-I (18kD)LC3-II (16kD)

GFP-LC3

starved

IBMPFD myopathy

0

1

2

LC

3II

pro

tein

levels

(A

.U)

Con WT RH9 RH12

p62

pro

tein

levels

(A

.U)

0

1

2

Con WT RH9 RH12

Ju et al, JCB 2009

Ju et al, JCB 2009

Upregulation of functional autophagosomes

Decrease in autophagosome degradation or “autophagic flux” Phagophore closure Autophagosome-lysosome fusion Absence of functional lysosomes

VCP

Ju et al, JCB 2009

Nucleus

Ub

Immunosuppressant used to treat transplant rejection

Inhibits the mTOR pathway mTOR integrates extrinsic growth signals

and cellular nutrient status and energy state

Active mTOR Protein synthesis and cell growth

Inactive mTOR (or rapamycin treatment) Inhibition of protein synthesis and increased

autophagic degradation of protein

Nucleus

Ub

Increase autophagic stimulus

Ub

Depending upon the disease, stimulating or inhibiting autophagy may be appropriate.

Identifying drugs that “facilitate” autophagy.