研究生课 泛素化介导的蛋白质降解 含NCB和NM文章 分子生物学课

C3HC4 (RING-HC)
c-Cbl Siah BRCA1 BARD1 MURF PML IAP1 IAP2 IAP3
CKICAENDKD------------------ VKIEP CGHLMCTSCLTSWQE SEG--QG CPFC CPVCFDYVLP-----------------PILQ CQAGHLVCNQCRQKL-------SC CPTC CPICLELIKE------------------PVSTK CDHIFCKFCMLKLLNQKKGPSQ CPLC CSRCANILKE-----------------PVCLGG CEHIFCSGCISDCVGS-----G CPVC CPICLEMFSK-----------------PVVILP CQHNLCRKCANDVFQ[18]RFR CPSC CQQCQAEA KC------------------PKLLP CLHTLCSGCLEASGM------Q CPIC CKVCMDKEVS-----------------IVFIP CGHLVVCKDCAPSLRK------- CPIC CKVCMDKEVS-----------------VVFIP CGHLVVCQECAPSLRK------- CPIC CKICMDRNIA-----------------IVFVP CGHLVTCKQCAEAVDK------- CPMC
Two major scientific issues central to our understandings of E3 ligase
1. Mechanisms controlling the assembly and activity of E3 ligases
2. Molecular nature of E3 ligases and their substrates
p300 102-222
RB 273-321
ARF 210-244
438-491
Mdm2/MdmX
412-437
Gankyrin
1 19
102
Mdm2 p53 binding
Ubc9 Smurf1
40-59 75-114
Akt
S166/S186 221
ATM
274 305 322 S395
* * acidic
p53 can be activated by multiple stresses
DNA damage
Oncogenic insults
Kinases
CDK inhibitors b-catenin
myc
transcription
c-jun c-fos
. ..
.
......
. . .
. .
.....
.
Combinatorial nature of UPS
Rad23
Adaptors (a few)
E3s: next wave of drug targets
16
E1:
16
E2:
53
E3:
>1000
Cullin-ROC complexes 300 - 500
RING proteins
450
HECT proteins
~40
U-box proteins
10 - 20
Ubl ligases
?
Single-subunit RING type E3: Mdm2
MDM2 structure and interacting proteins
X(1-3) Zn ++ X2
C2 C5
C/H3 C78
X(9-27)
RING finger
X2
C1
C4
X2 Zn++ X2
C2 H3
C5
C8
X5 Zn ++ X2
C6 C7
X17
X17
Zinc finger
RING Finger Genes
Yeast:
39
Worm:
154
Fly:
135
Human:
➢ Proteasome inhibitor Velcade: multiple myeloma ➢ UPS: targets in anti-cancer therapy ➢ E3s: determine substrate specificity, better targets
Cancer, Huntington’s disease, spinocerebellar ataxias, Paget’s disease of bone, AIDS-related cancer, muscle wasting, cystic fibrosis, Alzheimer’s disease, Parkinson’s disease, hypertension, etc.
NLS NES
181-185 191-205
Zn
*
L5/L11/L26
284-374
438 478 491
RING
NoLS
464-471
MDM2 ubiquitinates p53
UbcH5 + + MDM2 - + +
++ -++
poly ubiquitinated p53
12 3
456
Honda et al. (1997) FEBS Letter 420:25
Lingqiang Zhang, Ph.D Professor, Principal Investigator Dept. Proteomics & Genomics (P&G) Beijing Inst. Radiation Medicine (BIRM) Tel: 66931216 Email: zhanglq@nic.bmi.ac.cn
2) protein quality control: misfolded or damaged proteins (e.g. CFTR, prion, huntingtin)
Different types of Lysine modifications (Ubiquitination and ubiquitin-like modifications)
NF-κB signalling pathways
canonical pathway Non-canonical pathway
Ubiquitin
Lys-48
Gly-76
Human Ub: MQIFVKTLTGKTITLEVEPNDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG Yeast Ub: MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
Ubiquitin-proteasome system (UPS)
Daniela Hoeller et al. Nat. Rev. Cancer, 2006, 6, 776
Protein Ubiquitination
RING E3 HECT E3
Functions of the Ub system
Daniela Hoeller et al. Nat. Rev. Cancer, 2006, 6, 776
Ubiquitin and ubiquitin-like proteins
Ubiquitin-mediated Proteolysis
E1 -S-Ub
Lys-48
E2 -S-Ub
E3 ?
C terminus
Two families of E3 ubiquitin ligases
E3
? substrate
? E2 -S-Ub
Ub UbUbUbUb
RING family E3 (1000s)
HECT family E3 (~40)
Cullin
adaptor
ROC
substrat
Ube
Ub Ub UbUb
E2
Ub
MDM2 RING
p53
Ub Ub Ub UbUb
E2
Ub
E6AP HECT
substrate
Ub Ub Ub UbUb
E2
Ub
RING Finger Genes
RING: Really Interesting New Gene
X(4-48)
C1
C6
C/H4 C67
X2 Zn ++ X2
X2
signal transduction developmental signaling tumor suppression tumor suppression signal transduction t r an s c ri p t io n a p op t o si s a p op t o si s a p op t o si s
• Non-proteolytic (mono-Ub or K63 Ub chain) DNA repair, transcription, kinase activation.
• Proteolytic (K48 or K29 linked Ub chains)
1) concentration control: key regulators (e.g. p53, cyclins, HIF)
substrates Ub Ub Ub Ub Ub
misfolded proteins
many
antigen
signal
cell
processing transduction cycle
tumor suppression oncogenes
viruses bacterial
IkBa
cyclins
p53
C2H2C4
HDM2
CVICQGRP KN---------------GCIV HGKTG HLMACFTCAKKLKKRNK---P CPVC control p53
How Many E3 Ligases?
Proteases:
来自百度文库
550
Kinases:
520
Phosphatses:
150
DNA polymerases:
The Nobel Prize in Chemistry, 2004
“for the discovery of ubiquitin-mediated protein degradation”
Aaron Ciechanover
Avram Hershko
Irwin Rose
Wolf, D.H. “Proteasomes: The World of Regulatory Proteolysis”
Cell Cycle by Ubiquitination
securin
B
CDC2
M
G2
D
KE4/26F
SLBP
G1
p21
p53
S
A K2
E K2
E2F
Rb
Different modification states of p53 and MDM2
Daniela Hoeller et al. Ubiquitin and ubiquitin-like proteins in cancer pathogenesis. Nat. Rev. Cancer, 2006, 6, 776
350
Arabidopsis: 490
C3H2C3 (RING-H2)
ROC1 ROC2 APC11 PRAJA1 PARKIN
CAICRNHIMD LCIECQA NQASATSE ECTVAWG VCNHAFHFHCISRWLKTRQ---V CPLD cell cycle control CAICRVQVMD ACLRCQA EN---KQE DCVVVWG ECNHSFHNCCMSLWVKQNN---R CPLC unknown CGICRMAFNG CCPDCKV P-----GD DCPLVWG QCSHCFHMHCILKWLHAQQVQQH CPMC mitotsis CPICCSEYVK---------------GEVATELP CHHYFHKPCVSIWLQKSG---T CPVC unknown CITCTDVRSP-----------VLVFQCNSRHVI CLDCFHLYCVTRLNDRQ [11]P CVAGC parkison disease
Functions of Ubiquitination-mediated protein degradation
• 细胞周期和细胞分裂、分化 • 发育的调节 • 细胞对外环境的应激反应的调节 • 神经网络的形态发生 • 细胞表面受体和离子通道的下调 • DNA修复 • 免疫和感染反应的调节 • 细胞器的生物生成 • ……