The Inner Life of the Cell
http://www.youtube.com/watch?v=2-p-QajenM0&feature=related
Structural study of cell-cycle control proteins
Current Opinion in Structural Biology 2002, 12:822–830
: Structural basis of ubiquitylation
NATURE Reviews Cancer 2006, 6:369-381
:Ubiquitin ligases: cell-cycle control and cancer
The control of the cell cycle
Anti-proliferative
signals
Growth factor
(mitogen)
(CDK activating
kinase)
Cell cycle control by ubiquitylation
(Structural study of SCF and APC)
Ubiquitin
The three dimensional structure of ubiquitin: contains 76 amino acids
Simplified view of the cell-cycle control system
Levels of cyclin expression during cell division
are periodic1. This is the result of a constant synthetic
rate coupled with a defined window in the cycle of
specific proteolysis, which is executed by the ubiquitinproteasome
system (UPS).
Cell cycle control of SCF ubiquitin ligase by proteolysis of Cdk inhibitor protein
P27 (CIP)
CKIs, negative-regulators of cyclin–CDK kinase complexes, are also targeted
for degradation by the UPS.
Three-layer regulation of the cell cycle
Therefore, the cell cycle is predominantly regulated by two types of posttranslational protein modification — phosphorylation and ubiquitylation.
Overview of the ubiquitin-proteasome pathway
Ubiquitin ligase (E3) enzyme complex
A. Ubiquitin-protein ligases (also known as E3s) act at the last step of a three-enzyme
cascade involving the ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes.
B. The E3 mediates the transfer of ubiquitin from the E2 to the substrate protein by
promoting the formation of an isopeptide bond between the Ub carboxy-terminus and
specific lysine side chains on the substrate.
C. E3s bind both the protein target and a cognate E2 and have a central role in conferring
specificity to the ubiquitination pathway.
D. The mechanism by which they promote ubiquitination has not been well understood.
Two distinct types of E3s
HECT-type E3s catalyse ubiquitination
RING-type E3s do not appear to form
by first forming an E3–ubiquitin thioester
such an intermediate. They are
intermediate.
characterized by the presence of a RING
zinc finger domain that binds the E2.
The SCF (Skp1–Cullin–F-box protein) complexes
A. The SCF complexes are RING-type E3s
B. The largest family of ubiquitin–protein ligases.
C. ubiquitinate a broad range of proteins involved in cell cycle progression,
signal transduction and transcription.
D. Deregulation of SCF-dependent proteolysis can contribute to neoplastic
transformation.
Human SCF complexes with demonstrated E3 activity
SCFSkp2 :
Cdk-inhibitor p27Kip1
SCFFbw7 : cyclinE
SCFb-TrCP : b-catenin and IkB
The composition of SCF complexes
The SCF complexes are RING-type E3s that consist of
A. Cul1 (776 residues),
invariable
B. Rbx1 (108 residues),
C. Skp1 (163 residues) and
variable
D. F-box protein family (430 to.1,000 residues).
Rbx1, which contains the RING domain, and Cul1 form a catalytic core complex
that recruits a cognate E2
F-box proteins are characterized by an amino-terminal 40-residue F-box motif
that binds Skp1 followed by protein–protein interaction modules such as leucine
rich repeats or WD-40 repeats that bind substrate.
How is it possible to ubiqutinate various substrate?
E3 components in the UPS are thought to be primarily responsible for the specific
recognition of a large number of target proteins. This requires both specificity and
versatility, which are provided by the existence of 500–1,000 different E3 ligases.
How is it possible to make various SCFs to ubiqutinate various substrate?
A. The large number of F-box proteins in eukaryotic genomes (at least 38 in
human) allows for the specific ubiquitination of a large number of functionally and
structurally diverse substrates
B. In addition to multiple F-box proteins, most higher eukaryotes also contain
multiple homologues of the other SCF subunits, including two Rbx1 and five cullin
family members (paralogues) conserved from C. elegans to humans.
The schematic structures of SCF
Cell-cycle regulation by the SCF complex and APC/C
APC
SCF
Functions of the SKP1–CUL1–F-box-protein (SCF) complex
Cell-cycle regulation by the SCF complex and APC/C
The structure of Skp1 and Skp2 complex
Overall structure of the SCFskp2
The N-terminal domain of Cullin1
N-terminal tip of repeat 1 that is the
Skp1-F boxSkp2 binding site
The C-terminal domain of Cullin1 and Rbx1
30 A° -wide groove
Intermolecular b-sheet formed by Rbx1 and Cul1 C-terminal domain
The Cul1 residues that contact Rbx1 are shown in light green, and the Rbx1 residues in
pink.
The zinc-finger (RING) domain of Rbx1
Rigidity
of Cul1 scaffold required for SCF function
To start
investigating the importance of the rigid architecture of the Cul1
scaffold, we sought to construct a Cul1 mutant where the NTD and
CTD interface is disrupted, and where the two domains are linked
by a flexible linker (Fig. 5a).
The SCFSkp2 complex with the wild-type (WT) Cul1
(lane 1) but not the linker mutant Cul1 (lane 3)
The Cul1 linker mutant retains the ability to bind
phosphorylated p27, in a manner dependent on the
presence of Skp1, Skp2 and Cks1.
promotes the Cks1-dependent polyubiquitination
(Ubn ) of p27 in an in vitro ubiquitination assay
reconstituted with purified components.
Model of the SCFSkp2–E2 complex
The principal stages of mitosis in human cells and chromosome segregation
1
2
3
4
5
6
1 : prophase, 2 : pro-metaphase
3 : metaphase
4, 5, 6 : early, mid, and late anaphase, respectively
Fixed HeLa cells were stained for DNA (blue), microtubules (green) and kinetochores (red)
Regulation of mitosis by ubiqutin ligase APC (anaphase promoting complex)
Isolation of Native Human APC
APC was immunoprecipitated from extracts of
HeLa cells using CDC27 peptide antibodies.
Bound complexes were subsequently eluted in
their native form with an excess of antigenic peptide.
(cullin-like)
The peptide was subsequently separated from the eluted
protein by gel filtration chromatography
SDS–PAGE and silver staining analysis of the resulting
fractions revealed all known 11 subunits of human APC
whose identity was confirmed by immunoblotting
(not shown)
(RING-finger domain)
Characterization of Native Human APC
In the presence of purified ubiquitin, E1
and E2 enzymes, and ATP, the APC
fractions were able to ubiquitinate a
radiolabeled fragment of cyclin B in a
dose-dependent manner
Native electrophoreses of APC
Electron Microscopy of Negatively Stained APC
Diameter of 15 nm
3D Model of the APC Obtained by Cryo-Electron Microscopy
Purified APC samples were imaged using liquid
nitrogen temperature electron microscopy.
About 13,000 molecular images of randomly
orientated APC particles were interactively collected
from digitized micrographs.
A first set of characteristic APC views was obtained by
multivariate statistical analysis and automatic classification.
After angular reconstitution, a preliminary low resolution
3D structure was derived.
Subsequently, the resolution of the structure was reiteratively
improved by generating large number of reference images
and performing multiple cycles of multireference alignment,
automatic classification, and angular reconstitution.
Using this procedure, a 3D model of the APC with a final
resolution of 24 A° was generated.
140A° X 140A° x135A° in size