intracellular protein degradation- the lysosome and ubiquitin proteasome system scott wilson...
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Intracellular Protein Degradation- The lysosome and Ubiquitin Proteasome
System
Intracellular Protein Degradation- The lysosome and Ubiquitin Proteasome
System
Scott Wilson
Department Neurobiology
5-5573
Scott Wilson
Department Neurobiology
5-5573
OutlineOutline Sites of proteolysis
Gastrointestinal tract Circulatory system Intracellular proteolysis
Lysosome Biogenesis and function
Degradation of extracellular material Degradation of intracelluar components by autophagy
Ubiquitin proteasome pathway Components
Ubiquitin and UBLs Ubiquitin conjugating enzymes Ubiquitin deconjugating enzymes The proteasome- generation and activity
Sites of proteolysis Gastrointestinal tract Circulatory system Intracellular proteolysis
Lysosome Biogenesis and function
Degradation of extracellular material Degradation of intracelluar components by autophagy
Ubiquitin proteasome pathway Components
Ubiquitin and UBLs Ubiquitin conjugating enzymes Ubiquitin deconjugating enzymes The proteasome- generation and activity
Gastrointestinal tract Destruction of antigenicity
Controlled but no specificity- everything that enters gut is proteolyzed
Production of energy Remember that destruction of proteins is an energy producing
process (exergonic)
Circulatory system Blood coagulation
Conversion of prothrombin to thrombin which converts fibrinogen to fibrin and a blood clot is formed.
Process is highly controlled (1-antitrypsin deficiency)
Gastrointestinal tract Destruction of antigenicity
Controlled but no specificity- everything that enters gut is proteolyzed
Production of energy Remember that destruction of proteins is an energy producing
process (exergonic)
Circulatory system Blood coagulation
Conversion of prothrombin to thrombin which converts fibrinogen to fibrin and a blood clot is formed.
Process is highly controlled (1-antitrypsin deficiency)
The question:Is there turnover of cellular constituents? Or is food intact a function primarily for energy-providing (fuel for a car), that is independent from the structural and functional proteins of
the body?
The question:Is there turnover of cellular constituents? Or is food intact a function primarily for energy-providing (fuel for a car), that is independent from the structural and functional proteins of
the body?
• Studies on -galactosidase in E. coli indicated that there was no conclusive evidence that proteins withincells are in a dynamic state and that they are likely to be stable and static
• Without metabolic labels (ex. 35S cysteine or 3H leucine)the problem of determining protein stability was not approachable
How do you “tag” proteins to study protein dynamics?
How do you “tag” proteins to study protein dynamics?
1939 Rittenburg and Urey succeeded in generating radiolabeled Nitrogen (15N)
Schoenheimer found that following administration of 15N-labeled tyrosine to rats, they found that only ~50% of the label was found in excretions. Where was the rest?
The label was found incorporated in body proteins!
1939 Rittenburg and Urey succeeded in generating radiolabeled Nitrogen (15N)
Schoenheimer found that following administration of 15N-labeled tyrosine to rats, they found that only ~50% of the label was found in excretions. Where was the rest?
The label was found incorporated in body proteins!
Therefore the proteins of the body are in a dynamic state of synthesis and degradation! It is thought that we are degrading and resynthesizing
~3-5% of our cellular proteins daily.
Paradigm that cellular processes are controlled mainly by only transcription and translation must be changed.
Therefore the proteins of the body are in a dynamic state of synthesis and degradation! It is thought that we are degrading and resynthesizing
~3-5% of our cellular proteins daily.
Paradigm that cellular processes are controlled mainly by only transcription and translation must be changed.
Why are proteins degraded?Why are proteins degraded?
Quality control Proteins become denature/misfolded/damaged
Elevated temperatures (37°C) Proteins being synthesized are folded incorrectly
Regulation of biological pathways Cell cycle Receptor mediated endocytosis Synaptic remodeling
Quality control Proteins become denature/misfolded/damaged
Elevated temperatures (37°C) Proteins being synthesized are folded incorrectly
Regulation of biological pathways Cell cycle Receptor mediated endocytosis Synaptic remodeling
Now that we know proteins are in a “dynamic state” in cells….
Now that we know proteins are in a “dynamic state” in cells….
How are proteins degraded within cells? Is protein degradation regulated? Selective? Compartmentalized?
How are proteins degraded within cells? Is protein degradation regulated? Selective? Compartmentalized?
The discovery of the lysosomeThe discovery of the lysosome
De Duve discover the lysosome in the 1950’s Vacuolar structure that contains hydrolytic enzymes that are optimal at
acidic pH. Latency of of enzymatic activity- researcher found that hydrolyase
fractionated from rat liver were more active after they were stored in the refrigerator for several days? The latency was due to the slow breakdown of the lysosomal
membrane which protected the cells from the destructive forces of the acid hydrolyases.
This compartmentalization of the peptidases by a membrane protects cellular components from inappropriate degradation.
De Duve discover the lysosome in the 1950’s Vacuolar structure that contains hydrolytic enzymes that are optimal at
acidic pH. Latency of of enzymatic activity- researcher found that hydrolyase
fractionated from rat liver were more active after they were stored in the refrigerator for several days? The latency was due to the slow breakdown of the lysosomal
membrane which protected the cells from the destructive forces of the acid hydrolyases.
This compartmentalization of the peptidases by a membrane protects cellular components from inappropriate degradation.
Generation of a functional lysosome
Generation of a functional lysosome
Lysosomal proteases belong to the aspartic, cysteine, or serine proteinase families of hydrolytic enzymes.
contain about 40 types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases. All are acid hydrolyase that have optimal activity at pH 5.0
Lysosomal proteases belong to the aspartic, cysteine, or serine proteinase families of hydrolytic enzymes.
contain about 40 types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases. All are acid hydrolyase that have optimal activity at pH 5.0
Sorting acid hydrolyases to the lysosome is accomplished by post-translation modificationSorting acid hydrolyases to the lysosome is
accomplished by post-translation modification
Soluble lysosomal enzymes are synthesized as N-glycoslyated precursors in the ER and trafficked to the Golgi mannose 6-phosphate (M6P) groups are added to the hydrolyases The M6P groups are recognized by transmembrane M6P receptor proteins, which are present in the trans Golgi
network M6P receptors release hydrolyases when pH is below 6.0 and the M6P is removed
Soluble lysosomal enzymes are synthesized as N-glycoslyated precursors in the ER and trafficked to the Golgi mannose 6-phosphate (M6P) groups are added to the hydrolyases The M6P groups are recognized by transmembrane M6P receptor proteins, which are present in the trans Golgi
network M6P receptors release hydrolyases when pH is below 6.0 and the M6P is removed
Lysosomes use an H+ ATPase pump in the membrane to generate acidic pH
Lysosomes use an H+ ATPase pump in the membrane to generate acidic pH
Overview of lysosomal traffickingOverview of lysosomal trafficking
Proteases in the lysosomeProteases in the lysosome
Cysteine protease- cathepsins A, B
Aspartate protease- cathepsin D
Zinc protease-?
Activation of protease by removal of inhibitory segment- conversion of proprotein to protein
Cysteine protease- cathepsins A, B
Aspartate protease- cathepsin D
Zinc protease-?
Activation of protease by removal of inhibitory segment- conversion of proprotein to protein
Pathways into the Lysosomal/vacuolar System
Pathways into the Lysosomal/vacuolar System
1
2 3
4
4
Model of the mechanism for multivesicular endosome formation
Model of the mechanism for multivesicular endosome formation
How do proteins get into the lysosome for degradation?
How do proteins get into the lysosome for degradation?
Microautophagy- cytoplasm is segregated into membrane -bound compartments and are then fused to lysosome
Maroautophagy- entire organelles such as mitochondria, ER and other large cytoplamic entities are engulfed and then fused with the lysosome
Microautophagy- cytoplasm is segregated into membrane -bound compartments and are then fused to lysosome
Maroautophagy- entire organelles such as mitochondria, ER and other large cytoplamic entities are engulfed and then fused with the lysosome
Autophagy pathwayAutophagy pathway
Problems that still remainProblems that still remain
Proteins vary greatly in their stability - from minutes to days! Rates of protein degradation of specific proteins changes
with physiological conditions (nutrients and hormones) How could this happen by microautophagy
Lysosomal inhibitors have differential affects on different populations of protein
If lysosomal proteases degrade proteins in an exergonic manner, how could you explain evidence that the proteolytic machinery required energy?
Proteins vary greatly in their stability - from minutes to days! Rates of protein degradation of specific proteins changes
with physiological conditions (nutrients and hormones) How could this happen by microautophagy
Lysosomal inhibitors have differential affects on different populations of protein
If lysosomal proteases degrade proteins in an exergonic manner, how could you explain evidence that the proteolytic machinery required energy?
Poole et al were studying the mode of action anti-malaria drugs Chloroquine and other lysosomotropic (weak bases) block the
activity of lysosomal proteases by neutralizing the low pH of the lysosome.
Treat macrophages labeled with 3H-leucine with chloroquine and then feed them protein extracts that were labeled with 14C-leucine This allowed them to monitor the stability of phagocytosed
extracellular and intracellular proteins when the lysosome is blocked
Poole et al were studying the mode of action anti-malaria drugs Chloroquine and other lysosomotropic (weak bases) block the
activity of lysosomal proteases by neutralizing the low pH of the lysosome.
Treat macrophages labeled with 3H-leucine with chloroquine and then feed them protein extracts that were labeled with 14C-leucine This allowed them to monitor the stability of phagocytosed
extracellular and intracellular proteins when the lysosome is blocked
Still more data suggesting another pathway for degradation of intracellular proteins
Still more data suggesting another pathway for degradation of intracellular proteins
What did they find?What did they find?
Lysosomotropic drugs only affected the stability of the engulfed extracellular proteins and not the intracellular proteins.
This indicated that there must be a second pathway for the degradation of intracellular proteins and that the lysosome was the primary site of degradation of internalized extracellular proteins
Lysosomotropic drugs only affected the stability of the engulfed extracellular proteins and not the intracellular proteins.
This indicated that there must be a second pathway for the degradation of intracellular proteins and that the lysosome was the primary site of degradation of internalized extracellular proteins
The search for a new proteolytic pathwayThe search for a new proteolytic pathway
The new pathway must explain several things- Requirement for metabolic energy
ATP depletion inhibits proteolysis Why do you need ATP?
Need phosphorylation of substrates or enzymes? Remember proteolysis is exergonic
Differential stability of intracellular proteins Example- RNA polymerase I t1/2= 1.5 hrs
RNA polymerase II t1/2= 12 hrs
How stability of proteins can change under different environmental conditions
The new pathway must explain several things- Requirement for metabolic energy
ATP depletion inhibits proteolysis Why do you need ATP?
Need phosphorylation of substrates or enzymes? Remember proteolysis is exergonic
Differential stability of intracellular proteins Example- RNA polymerase I t1/2= 1.5 hrs
RNA polymerase II t1/2= 12 hrs
How stability of proteins can change under different environmental conditions
Cell-free proteolytic systemCell-free proteolytic system
Rabbit reticulocyte lysates Made from red blood cells (terminally differentiated
and do not have lysosomes)
New that for different hemoglobinopathies, the blood cells attempt to rid themselves of abnormal hemoglobins and therefore must have a proteolytic system that was not lysosomal based.
Found that reticulate lysates were capable of degrading proteins in an ATP dependent manner
Rabbit reticulocyte lysates Made from red blood cells (terminally differentiated
and do not have lysosomes)
New that for different hemoglobinopathies, the blood cells attempt to rid themselves of abnormal hemoglobins and therefore must have a proteolytic system that was not lysosomal based.
Found that reticulate lysates were capable of degrading proteins in an ATP dependent manner
A new paradigm for proteolysisA new paradigm for proteolysis
Biochemical characterization of reticulate lysates Divided the lysates into two fractions (DEAE cellulose, anion
exchange resin) Flow thru and high salt eluate Each fraction did not have proteolytic activity on its own. Combination of fraction I and II reconstituted proteolysis Previous work indicated that only a substrate and protease
were need for degradation. This was very important in that it suggested that there was not
a single protease that mediated degradation. This new system need a substrate, protease and something else
Activator?
Biochemical characterization of reticulate lysates Divided the lysates into two fractions (DEAE cellulose, anion
exchange resin) Flow thru and high salt eluate Each fraction did not have proteolytic activity on its own. Combination of fraction I and II reconstituted proteolysis Previous work indicated that only a substrate and protease
were need for degradation. This was very important in that it suggested that there was not
a single protease that mediated degradation. This new system need a substrate, protease and something else
Activator?
Characterization of fractions I and IICharacterization of fractions I and II
Analysis of Fraction I Found that fraction I contained only a single
factor that was heat sensitive and required ATP This factor was termed APF-1 for ATP-
dependent proteolysis factor Critical finding was that APF-1 can be
covalently attached to a target substrate
Analysis of Fraction I Found that fraction I contained only a single
factor that was heat sensitive and required ATP This factor was termed APF-1 for ATP-
dependent proteolysis factor Critical finding was that APF-1 can be
covalently attached to a target substrate
APF-1 is shifted to high molecular mass compounds following addition of ATP to the fraction I. 125I labeled fractions following gel-filtration chromatography
SDS PAGE analysis of samples run on gel-filtration
SDS PAGE analysis of samples run on gel-filtration
Lane 1- Fraction II + 125I- APF (no ATP)
Lane 2- Fraction II + 125I-AFP + ATP
Lane 3- Fraction II + 125I-AFP + ATP + unlabeled lysosome as substrate
Lane 4 & 5 - Increasing conc of lysosome
Lane 6- Fraction II + 125I-lysosome (no ATP) + unlabeled APF
Lane 7- Same as lane 6 + ATP
Lane 1- Fraction II + 125I- APF (no ATP)
Lane 2- Fraction II + 125I-AFP + ATP
Lane 3- Fraction II + 125I-AFP + ATP + unlabeled lysosome as substrate
Lane 4 & 5 - Increasing conc of lysosome
Lane 6- Fraction II + 125I-lysosome (no ATP) + unlabeled APF
Lane 7- Same as lane 6 + ATP
These experiments demonstrate that APFis covalently attached to substrate (explainsthe requirement of ATP)
Multiple APF-1’s can be added to a substrate
What is APF-1 ?What is APF-1 ?
Amino acid analysis and its known molecular mass indicated that APF-1 is ubiquitin.
Ubiquitin is a 76 aa protein found only in eukaryotes
The covalent attachment of ubiquitin to a substrate stimulates its proteolysis (but by what?)
Ubiquitin is covalently attached to a substrate by is C-terminal glycine to the -NH2 group of an internal lysine of the substrate
Amino acid analysis and its known molecular mass indicated that APF-1 is ubiquitin.
Ubiquitin is a 76 aa protein found only in eukaryotes
The covalent attachment of ubiquitin to a substrate stimulates its proteolysis (but by what?)
Ubiquitin is covalently attached to a substrate by is C-terminal glycine to the -NH2 group of an internal lysine of the substrate
Studies of fraction II defined the ubiquitin conjugation machinery
Studies of fraction II defined the ubiquitin conjugation machinery
Substrate recognitionSubstrate recognition
N-end rule: On average, a protein's half-life correlates with its N-terminal residue.
Proteins with N-terminal Met, Ser, Ala,Thr, or Gly have half lives greaterthan 20 hours.
･ Proteins with N-terminal Phe, Leu, Asp, Lys, or Arg have half lives of 3 minor less.
What about the protease?What about the protease?
Previous studies demonstrated that the activity of the protease was ATP dependent (not just ubiquitination requires ATP)
What is it composed of? Where is it located? How is it selective toward ubiquitinated
proteins? Why does it need ATP?
Previous studies demonstrated that the activity of the protease was ATP dependent (not just ubiquitination requires ATP)
What is it composed of? Where is it located? How is it selective toward ubiquitinated
proteins? Why does it need ATP?
Structure of the 26S proteasomeStructure of the 26S proteasome
Tanaka et al discovered a high-molecular mass protease that degraded ubiquitinated lysozyme but not untagged lysozyme
Required ATP for activity Protease was later called the 26S
proteasome
Similar multi-subunit proteases found in prokaryotes
Tanaka et al discovered a high-molecular mass protease that degraded ubiquitinated lysozyme but not untagged lysozyme
Required ATP for activity Protease was later called the 26S
proteasome
Similar multi-subunit proteases found in prokaryotes
Subunits of the 26S proteasomeSubunits of the 26S proteasome
19S regulatory particle- composed of approximately 20 different proteins
20S core particle- composed of 14 different subunits (1-7 and 1-7)
19S regulatory particle- composed of approximately 20 different proteins
20S core particle- composed of 14 different subunits (1-7 and 1-7)
19S Regulatory particle (RP)19S Regulatory particle (RP)
Recognition and binding of ubiquitinated proteins
Unfolding of ubiquitinated substrate to enter 20S mediated by AAA ATPases (ATP dependent)
Removal of ubiquitin side chains to allow entry into 20S ( lumen ~1.3 nm) by deubiquitinating enzymes
Activation/opening of 20S lumen
Recognition and binding of ubiquitinated proteins
Unfolding of ubiquitinated substrate to enter 20S mediated by AAA ATPases (ATP dependent)
Removal of ubiquitin side chains to allow entry into 20S ( lumen ~1.3 nm) by deubiquitinating enzymes
Activation/opening of 20S lumen
20S Core Particle (CP)20S Core Particle (CP)
Contains the endopeptidase activity The alpha subunits function is to control the
opening and closing of the 20S gate (interacts with 19S)
The beta subunits 1, 2 and 5 contain the endopeptidase activity of the proteasome.
Proteins are not degraded into amino acids but into short peptides ( very important for immune surveillance).
Contains the endopeptidase activity The alpha subunits function is to control the
opening and closing of the 20S gate (interacts with 19S)
The beta subunits 1, 2 and 5 contain the endopeptidase activity of the proteasome.
Proteins are not degraded into amino acids but into short peptides ( very important for immune surveillance).
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
Pathways controlled by regulated proteolysisPathways controlled by regulated proteolysis
Diseases of the lysosome and UPS pathways
Diseases of the lysosome and UPS pathways
Lysosomal Neimann Pick Disease- ataxia, brain degeneration and
spasticity.
Krabbe Disease- hypertonia, seizures, deafness and paralysis
Tay-Sachs Disease- cognitive disorder, deafness, paralysis
Lysosomal Neimann Pick Disease- ataxia, brain degeneration and
spasticity.
Krabbe Disease- hypertonia, seizures, deafness and paralysis
Tay-Sachs Disease- cognitive disorder, deafness, paralysis
Ubiquitin-dependent regulation of Ubp6
Ubiquitin-dependent regulation of Ubp6
Hanna, J et al Cell 127:99-1112006
Ubiquitin-dependent regulation of Ubp6 levels
Ubiquitin-dependent regulation of Ubp6 levels
Hanna, J et al Cell 127:99-1112006
Altered proteasome content in yeast expressing Ubp6C118AAltered proteasome content in yeast expressing Ubp6C118A
Hanna, J et al Cell 127:99-1112006
Cellular responses to ubiquitin deficiency and proteasomal stress
Cellular responses to ubiquitin deficiency and proteasomal stress
Hanna, J et al Cell 127:99-1112006
Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity
Proteasome inhibition increases Usp14 ubiquitin-hydrolase activity
Usp14
Uch37
Borodovsky, A et alEMBO J. 20:5187-962001
The proteasomal DUB Usp14 impairs protein degradation
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
Decrease steady-state levels of aggregate prone proteins in the absence of Usp14
Lee, BH et alNature 467:179-842010
Proteasome activity can be modulated by Uch37, Rpn11 and Usp14
Proteasome activity can be modulated by Uch37, Rpn11 and Usp14
Proteasomal DUB functions in yeast
1) Rpn11- cleaves near base of chain to remove ubiquitin chains “en bloc”
2) Usp14 - recycling of residual ubiquitin conjugates from proteins entering the proteasome, ubiquitin chain editing and regulation of proteasome activity
3) Uch37- ubiquitin chain editing
Mouse models
1- Rpn11- unknown but likely lethal
2- Usp14- KO embryonic lethal (E14) hypomorphic allele viable
3- Uch37 unknown
Ubiquitin is not the only small peptide to be covalently attached to proteins and or lipidsUbiquitin is not the only small peptide to be covalently attached to proteins and or lipids
SUMO 1/2 Nedd8 ISG15 ATG8 FAT10
Not thought to target proteins for destruction Each is thought to have its own conjugation and
deconjugation system
SUMO 1/2 Nedd8 ISG15 ATG8 FAT10
Not thought to target proteins for destruction Each is thought to have its own conjugation and
deconjugation system
