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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.

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the inner life of the cell
The Inner Life of the Cell

http://www.youtube.com/watch?v=2-p-QajenM0&feature=related

slide2

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

slide4

Anti-proliferative

signals

Growth factor

(mitogen)

(CDK activating kinase)

slide6

Cell cycle control by ubiquitylation

(Structural study of SCF and APC)

slide7

Ubiquitin

The three dimensional structure of ubiquitin: contains 76 amino acids

slide10

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).

slide11

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.

slide12

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.

slide14

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.

slide15

Two distinct types of E3s

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.

slide16

The SCF (Skp1–Cullin–F-box protein) complexes

  • 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.

slide17

Human SCF complexes with demonstrated E3 activity

SCFSkp2 : Cdk-inhibitor p27Kip1

SCFFbw7 : cyclinE

SCFb-TrCP : b-catenin and IkB

slide18

The composition of SCF complexes

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).

invariable

variable

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.

slide19

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.

slide20

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.

slide27

The N-terminal domain of Cullin1

N-terminal tip of repeat 1 that is the Skp1-F boxSkp2 binding site

slide29

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.

slide31

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) 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.

slide33

1

2

3

4

5

6

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)

slide35

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)

slide36

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

slide39

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