Characterization and Inhibitor Development of the Mammalian N-end Rule Pathway PACCON 2013 Jan. 24,...
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Characterization and Inhibitor Development of the Mammalian N-end Rule Pathway
PACCON 2013Jan. 24, 2013
Min Jae Lee
Department of Applied Chemistry
College of Applied Science
Kyung Hee University, Korea
Protein Synthesis vs Degradation
Foreign vs Body Protein degradation
Extracellular vs Intracellular Protein degradation
Autophagy-Lysosomal System (ALS) vs
Ubiquitin-Proteasome System (UPS)
Ubiquitin (Ub)
C-terminal Gly-Gly-COO-
Ubiquitin (Ub) functions as a degradation signal
Lys-NH3+
Isopeptide bond
- Ub structure
Target Protein
Ubiquitin
Ubiquitins
(poly)ubiquitinated
Proteasome
(poly)ubiquitinated target substrate
peptide fragments
free Ub
The Ubiquitin-Proteasome System (UPS)
Conjugation of poly-Ub chain to the substrates
UPS (A Little More Details)
E3 ubiquitin ligases
Deubiquitinating enzymes
Degradation of the tagged protein by the
26S proteasome
A Case Study: Proteasome Inhibitor as Cancer Drug
- MG132 (Z-Leu-Leu-Leu-al), a proteasome
inhibitor
- Bortezomib, Velcade®
• FDA approved for multiple myeloma in
2003; survival from 10% to 40~50%
• First new therapy for MM since the early
60s
• Sales ~$2 billion/yr; >100,000 treated
• Price: $1548.0 per 3.5 mg vial
NH
HN B
OH
OH
O
O
N
N
OHN
NH
HN
H
O
O
O
O
Nature Review Cancer (2011)
O
CH3
NH
O
Screening for Small Molecule Inhibitors of Usp14
Ub-AMC
Ub
-AM
C h
ydro
lysi
s ac
tivi
ty
Ub Ub Usp14
Inhibitor hits
O
CH3
H2N O
free AMC
Usp14: endogenous Proteasome
inhibitor
Where is the decision to degrade a protein made?
rate-determinant
“Passive”- Traditional view
- Our view
“Active” role of proteasome on Ub
chain dynamics
Lee et al, Nature 2010
Inhibitor (IU1) of inhibitor (Usp14) enhanced the activity of proteasome
IU1 can potentially be used to eliminate toxic proteins
Rhomboidal Structure of the Ubiquitin-Proteasome System
Met-
A Degradation Signal: the N-terminal Amino Acid
Rapid proteolysis(= short half-lives)
Ub
Ub
UbUBR
protein X substrate
…
Stable(= long half-lives)
UBR proteins: N-end rule E3 ubiquitin ligases
Start codon
vs
N-terminal methionine
Some N-terminal residues
In Vitro Model System of the N-end Rule Pathway
Ubiuqitin / Protein / Reference (UPR) technique
Stabilization by dipeptide inhibitors
Two types of primary destabilizing residues
35S labeling
Autoradiography
Dipeptide inhibitors
Effect of D-form Dipeptides on UBR Protein Inhibition
with or without dipeptides
Functional Proteomic Screening for Mammalian N-end Rule Substrate
Lee et al, PNAS 2005
Regulator of G-protein Signaling proteins(RGS4, RGS5, and RGS16)
High-Throughput Screening (HTS) from 18,000 cDNAs
Collaboration with “Meso Scale Discovery”42 candidates
Lee et al, PNAS 2008Yanxialei et al, submitted
Physiological Functions of the N-end Rule Pathway
Lee et al, JBC 2012
Lee et al, PNAS 2005
Inhibitor development
Cardiomyocyte proliferation
Endogenous substrates
Classification of N-terminal Destabilizing Residues
Quantification of in vitro degradation assay
In silico Computational Docking Analysis (Type 1)
In silico Computational Docking Analysis (Type 2)
Rationally Designed Intraheterovalent Inhibitors
Chemical structures
Conceptual model of a bivalent inhibitor
Controls of RF-C11
Arg Phe
Lys
C11 UBR box ClpS box
In Vitro Effects of Bivalent Inhibitors
Comparison of IC50
RF-C11: 15.5 mMLys-Ala: 148 mM Arg-Ala: 283 mM
RF-C11: 2.74 mMTrp-Ala: 21.39 mM Phe-Ala: 72.37 mM
Type 1 Type 2
• Bivalent inhibitors can be used for the study of biochemical
mechanisms of UBR box proteins and physiological function of the N-
end rule pathway
• RF-C11 provides chemical insight for developing better rationally
designed N-end rule inhibitors
RF-C11, a Rationally-designed Intraheterovalent Inhibitior
RF-C11 showed ~ 20 times better inhibitory effects
Lee et al, PNAS 2008
Optimization is on-going
Optimizing RF-Cn Linker Length
Free a-Amine and Peptide Bond Are Essential for N-degron Recognition
Monomeric Phe-derivatives Inhibited N-end Rule Pathway
Chloro-Amphetamine Delays Phe-nsP4 Degradation
• UBR1 and its evolutionarily descendent UBR2 may have different specificity to N-degrons
• Recognition of N-degron is stereo-specific
• The optimal linker length of RF-Cn is n=5
• Heterovalent N-end rule inhibitors are successfully applied to mammalian cells.
• alpha-Amine should be intact for effective inhibition
• Peptide bonds are not essential for inhibition (Phe-amide exhibits similar activity to Phe-Ala)
• Chloro-amphetamine has been identified as an effective N-end rule inhibitor
Summary of Biochemical Understanding of the N-end Rule Pathway
Funded by
- AHAF (American Health Assistant Foundation)
postdoctoral grant,
- Korea NRF (National Research Foundation) New
Researcher Grant,
- Kyung Hee University Fellow Grant
- Korea NRF International Collaboration Grant
- Korea Ministry of Health and Welfare (MHW)
Alzheimer’s Disease Research Grant
Thank you!