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.
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
(CDK activating kinase)
(Structural study of SCF and APC)
The three dimensional structure of ubiquitin: contains 76 amino acids
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
Cell cycle control of SCF ubiquitin ligase by proteolysis of Cdk inhibitor protein
CKIs, negative-regulators of cyclin–CDK kinase complexes, are also targeted
for degradation by the UPS.
Therefore, the cell cycle is predominantly regulated by two types of post-translational protein modification — phosphorylation and ubiquitylation.
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.
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.
HECT-type E3s catalyse ubiquitination by first forming an E3–ubiquitin thioester intermediate.
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
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).
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.
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.
N-terminal tip of repeat 1 that is the Skp1-F boxSkp2 binding site
30 A° -wide groove
The Cul1 residues that contact Rbx1 are shown in light green, and the Rbx1 residues in pink.
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) promotes the Cks1-dependent polyubiquitination (Ubn ) of p27 in an in vitro ubiquitination assay reconstituted with purified components.
The Cul1 linker mutant retains the ability to bind phosphorylated p27, in a manner dependent on the presence of Skp1, Skp2 and Cks1.
1 : prophase, 2 : pro-metaphase
3 : metaphase
4, 5, 6 : early, mid, and late anaphase, respectively
The principal stages of mitosis in human cells and chromosome segregation
Fixed HeLa cells were stained for DNA (blue), microtubules (green) and kinetochores (red)
Regulation of mitosis by ubiqutin ligase APC (anaphase promoting complex)
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 APC
whose 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
Diameter of 15 nm
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