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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. - PowerPoint PPT Presentation
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The Inner Life of the Cell
http://www.youtube.com/watch?v=2-p-QajenM0&feature=related
Structural study of cell-cycle control proteins
: Structural basis of ubiquitylation
Current Opinion in Structural Biology 2002, 12:822–830
:Ubiquitin ligases: cell-cycle control and cancer
NATURE Reviews Cancer 2006, 6:369-381
The control of the cell cycle
Growth factor (mitogen)
Anti-proliferative signals
(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 divisionare periodic1. This is the result of a constant syntheticrate coupled with a defined window in the cycle ofspecific proteolysis, which is executed by the ubiquitinproteasomesystem (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 targetedfor degradation by the UPS.
Three-layer regulation of the cell cycle
Therefore, the cell cycle is predominantly regulated by two types of post-
translational protein modification — phosphorylation and ubiquitylation.
Overview of the ubiquitin-proteasome pathway
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.
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.
D. The mechanism by which they promote ubiquitination has not been well understood.
Ubiquitin ligase (E3) enzyme complex
HECT-type E3s catalyse ubiquitination
by first forming an E3–ubiquitin
thioester intermediate.
RING-type E3s do not appear to form
such an intermediate. They are
characterized by the presence of a RING
zinc finger domain that binds the E2.
Two distinct types of E3s
A. The SCF complexes are RING-type E3s
B. The largest family of ubiquitin–protein ligases.
The SCF (Skp1–Cullin–F-box protein) complexes
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.
SCFSkp2 : Cdk-inhibitor p27Kip1
SCFFbw7 : cyclinE
SCFb-TrCP : b-catenin and IkB
Human SCF complexes with demonstrated E3 activity
The SCF complexes are RING-type E3s that consist of
A. Cul1 (776 residues),
B. Rbx1 (108 residues),
C. Skp1 (163 residues) and
D. F-box protein family (430 to.1,000 residues).
The composition of SCF complexes
Rbx1, which contains the RING domain, and Cul1 form a catalytic core
complex
that recruits a cognate E2F-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.
invariable
variable
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 ubiqutinate various substrate?
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.
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
The schematic structures of SCF
Cell-cycle regulation by the SCF complex and APC/C
SCF
APC
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
The Cul1 residues that contact Rbx1 are shown in light green, and the Rbx1 residues in pink.
Intermolecular b-sheet formed by Rbx1 and Cul1 C-terminal domain
The zinc-finger (RING) domain of Rbx1
Rigidity of Cul1 scaffold required for SCF function
The Cul1 linker mutant retains the ability to bind phosphorylated p27, in a manner dependent on the presence of Skp1, Skp2 and Cks1.
The SCFSkp2 complex with the wild-type (WT)
Cul1 (lane 1) but not the linker mutant Cul1
(lane 3) promotes the Cks1-dependent
polyubiquitination (Ubn ) of p27 in an in vitro
ubiquitination assay reconstituted with purified
components.
To startinvestigating the importance of the rigid architecture of the Cul1scaffold, we sought to construct a Cul1 mutant where the NTD andCTD interface is disrupted, and where the two domains are linkedby a flexible linker (Fig. 5a).
Model of the SCFSkp2–E2 complex
1 2 3
4 5 6
1 : prophase, 2 : pro-metaphase 3 : metaphase4, 5, 6 : early, mid, and late anaphase, respectively
Fixed HeLa cells were stained for DNA (blue), microtubules (green) and kinetochores (red)
The principal stages of mitosis in human cells and chromosome segregation
Regulation of mitosis by ubiqutin ligase APC (anaphase promoting complex)
(cullin-like)
(RING-finger domain)
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.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 APCwhose identity was confirmed by
immunoblotting (not shown)
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
Characterization of Native Human APC
Native electrophoreses of APC
Electron Microscopy of Negatively Stained APC
Diameter of 15 nm
140A° X 140A° x135A° in size
3D Model of the APC Obtained by Cryo-Electron Microscopy
Using this procedure, a 3D model of the APC with
a final resolution of 24 A° was generated.
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.
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