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![Page 1: Mounting Evidence that Endocytosis Regulates -Synuclein Degradation Jaime Pérez Pineda Advanced Cell Biology Fall 2009.](https://reader036.fdocuments.us/reader036/viewer/2022062409/5697bfa51a28abf838c97acc/html5/thumbnails/1.jpg)
Mounting Evidence that Endocytosis Regulates -Synuclein
Degradation
Jaime Pérez Pineda
Advanced Cell Biology
Fall 2009
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Memory loss
Muscle weakness
Loss of voluntary movement
DementiaHallucinations
Seizures
Jerky body movements
Tremors
RigidityConfusion
DepressionLanguage problems
Stiff muscles
Ataxia
Personality changes
Decline in mental abilities
Symptoms ofNeurodegenerative
Diseases
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Road Map
INTRODUCTION:•Neurodegenerative Diseases•Culprit Protein in Parkinson’s•Protein Degradation Pathways•Endocytosis and ESCRT•Hypothesis and Model Organism
RESULTS:•Three Aims & Goals
DISCUSSION:•Major Findings
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Introduction to Parkinson’s Disease
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Common Problem in NDD
Parkinson’s Disease
Alzheimer’s Disease
Huntington’s Disease
Prion Disease
ALS
-Synuclein
Amyloid- peptide
Huntingtin
Prion protein
SOD1
Misfolding & Aggregation
Cell Death
Disease Culprit Protein
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Parkinson’s Disease
• 4 million people affected
• Classic symptoms:Resting tremors Muscular rigidity
• No known cure Muhammad Ali & Michael J. Fox
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SPORADIC FAMILIALNormal -synuclein
Misfolded
Accumulated
Genes:-synucleinparkinUCH-L1 DJ-1 PINK1
LRRK2
DNA
Point mutationsToxinsEnvironmental factors
A30P, A53T, E46K
Normal (Wild Type) -synuclein
CELL DEATH
Sporadic vs. Familial PD
>90% <10%
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What Happens in the PD Brain?
My Interest in -Syn:
Lewy Bodies: - Synuclein
inclusions
Basal Ganglia
Substantia Nigra
Motor Cortex
Less movement!
Muscle
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Culprit Protein: -Synuclein • 140 amino acid protein
• Associated with:
1. Cytoplasm2. Synaptic Machinery3. Extra cellular matrix4. Secretory pathway
• Loves to aggregate and bind to membrane
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Proof of -Synuclein Involvement:
PD-like symptoms
WT & familial mutants (A30P & A53T)
Feany, M., et al. (2000).
Masilah, E., et al. (2000).
Lasko, M., et al. (2003).
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What causes -synuclein toxicity?
Essential to examine -synuclein degradation routes
Alpha-synuclein
THERAPY!
Is -synuclein accumulation causing PD?
Normal -synuclein MisfoldedAggregated
-Synuclein accumulates
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Proteins1. VERY Important
2. Shape and Function
Normal Protein
MisfoldedOld
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Degradation Pathways
ProteasomeLysosome
Degrades proteins from cytoplasm or
nucleus
Degrades proteins from plasma
membrane, organelles or outside cell
-synuclein literature:
EvidenceUCHL1parkin
Knowing where -synuclein is found, which mechanism will degrade it?
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Lysosome
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Outside Cell
Inside Cell
Why Endocytosis?
Dixon et al. (2005)
Lee et al. (2008)
Sharma et al. (2006)
Lysosome
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Cell
Cytoplasm
Late Endosome
Lysosome
Early Endosome
MVB
Outside
Endocytosis
Golgi
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ESCRT Pathway
Endosomal Sorting Complexes Required for Transport
MVB
Cytoplasm
? ?
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Gap in Knowledge and Hypothesis
My hypothesis is that -synuclein interacts with the pre-ESCRT proteins while using the MVB/endosome pathway as a route to the lysosome.
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Model OrganismBudding Yeast (S. cerevisiae)
• Easy to grow• Cost effective• Genome sequence
available• ESCRT genes
conserved in humans• MVB/endosome
pathway best studied in yeast
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Experimental Design
EndocytosisIntact
No vps34 No vps27
Budding yeast with…
vs. and
CONTROL
Does -synuclein…1. aggregate more 2. accumulate more3. cause cell toxicity
…in endocytosis compromised yeasts
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Results: What I Found
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What I found:Strain:
Localization
Toxicity
Accumulation
Pre-ESCRT
vps34 vps27
Weak Weak
Non Toxic Non Toxic
Strong (WT)
None
ESCRT-I: mvb12 vps23 vps28 vps37
None Weak Weak Strong
Non Toxic Non Toxic
Weak Non Toxic
Weak Weak Weak Strong
ESCRT-II: vps25 vps36
Weak Weak
Non Toxic Non Toxic
Weak Strong
ESCRT-III: vps20 vps24
Weak Strong
Non Toxic
Toxic (A30P)
Strong None
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Localization:
GFP
Method:WT A30P E46KGFP
24 hrs
48 hrs
EndocytosisIntact
WT A30P E46KGFP
24 hrs
48 hrs
No vps34
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Accumulation:
Method:
V5
PGK
24 hrGAL
HeatSDS
Class BeadsProtein Gel
Membrane
YY
Anti-PGK
Anti-V5
BY4741vps34
WT
A3
0P
E4
6K
GF
P
anti-PGK
MW
anti-V5
WT
A3
0P
E4
6K
GF
P
Method:
V5
PGK
24 hrGAL
HeatSDS
Class Beads
Protein GelMembrane
YYAnti-V5
24 hrGLU
GLU
0 hrGLU
6 hrGLU
12 hrGLU
BY4741vps34
Ga
l
Ga
l
MW061224 0 61224
GluGlu
WT Loss of Induction
anti-PGK
anti-V5
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Cell Toxicity:
Method:
Spectrophotometer CultureGAL
Time0 3 6 12 18 24 36 48
Ab
sorb
an
ce
Abs
orba
nce
Time (hrs)
No vps34
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Mounting Evidence:Strain:
Localization
Toxicity
Accumulation
Pre-ESCRT
vps34 vps27
Weak Weak
Non Toxic Non Toxic
Strong (WT)
None
ESCRT-I: mvb12 vps23 vps28 vps37
None Weak Weak Strong
Non Toxic Non Toxic
Weak Non Toxic
Weak Weak Weak Strong
ESCRT-II: vps25 vps36
Weak Weak
Non Toxic Non Toxic
Weak Strong
ESCRT-III: vps20 vps24
Weak Strong
Non Toxic
Toxic (A30P)
Strong None
vps22
vps32vps2
Hse1
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Final Thoughts-synuclein
Hypothesis supported!
Hardships, inconvenience and costs
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Mounting Evidence that Endocytosis Regulates -Synuclein
Degradation
Jaime Pérez Pineda
Advanced Cell Biology
Fall 2009
![Page 29: Mounting Evidence that Endocytosis Regulates -Synuclein Degradation Jaime Pérez Pineda Advanced Cell Biology Fall 2009.](https://reader036.fdocuments.us/reader036/viewer/2022062409/5697bfa51a28abf838c97acc/html5/thumbnails/29.jpg)
Acknowledgements
BIOL 324: Keith, Carlos, Will, Rebecca, Alina and DannySTOCKROOM: Beth, Tasheka, Kaylyn, Jordanah, and Madhavi
Lastly: Mike and Dr. DebBurman!
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? ??
??
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?
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????
?? ?
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Two Very Important Proteinsvps27:forms complex with Hse1p;Required for recycling Golgiproteins, forminglumenal membranesand sorting ubiquitinatedproteins
vps34:Kinase responsible for the
synthesis of one
phospholipid; forms
membrane complex with
Vps15p to regulate protein
sorting
Yeast Genome Database
vps34 vps27MVB Cargo
Endosome Lumen
Off to ESCRT-I
Endosome Membrane