Ubiquitination

Barford Group

Section: Section of Structural Biology

Similar to protein phosphorylation, ubiquitination is a reversible processes, regulated by the opposing activities of E3 protein ubiquitin ligases which function to covalently attach ubiquitin molecules to target proteins, and de-ubiquitinating enzymes (DUBs) that removal ubiquitin from target proteins. We are studying a variety of E3s and DUBs involved in cell cycle control and regulation of the TNF-NFκB pathways.

Anaphase Promoting Complex

Regulated cell cycle progression is dependent upon controlled ubiquitin dependent proteolysis, mediated by the SCF and the anaphase promoting complex (APC), coupled to reversible protein phosphorylation. The APC is a multiprotein complex that is active during mitosis and G1 of the cell cycle, and is responsible for the metaphase to anaphase transition and the eventual exit from mitosis. 

The budding yeast APC contains 13 subunits, including the tetratricopeptide repeat (TPR) proteins Cdc16, Cdc23, and Cdc27, which form a complex with a molecular mass in excess of 800 κDa (Table 1). Substrate specificity is dictated by the association of the APC with two WD40-repeat containing regulatory subunits, Cdc20 and Cdh1. In yeast, Cdc20 is responsible for destruction of securin at the metaphase to anaphase transition, allowing sister chromatid separation, while Cdh1 is responsible for destruction of mitotic cyclins, facilitating mitotic exit. Inactivation of the APC in response to the spindle checkpoint occurs by inhibition of Cdc20 function via its sequestration with Mad2.

Currently little is known about the assembly of the APC, and the molecular mechanism of how the APC recognises substrates in a cell-cycle programmed manner, and the basis for its regulation and catalysis. To address these questions, we are applying the techniques of X-ray crystallography and electron microscopy to determine the structures of the intact APC. We plan to correlate the biological and biochemical properties of the APC at different stages of the cell cycle with its 3-dimensional structure. The structure of Apc10/Doc1 was solved by X-ray crystallography (Au et al., 2002), however, attempts to express other APC subunits have not been successful.

Using genetic approaches to TAP-tag one of the endogenous APC subunits, we have developed a system to isolate and purify to homogeneity sufficient quantities of the APC from Saccharomyces cerevisiae to perform structural, biochemical and functional studies of the complex. The yields and purity of APC are sufficient for cryo-electron microscopy studies and also to undertake future crystallisation screening. We have now obtained cryo-electron microscopy images of the APC which will lead to a medium resolution structure. Selected subunits of the APC have been His-tagged to allow their identification within the structure using gold-labelled NTA beads. Approaches have been developed to arrest the cell cycle at defined transitions (G1 and S) to isolate the APC from synchronised cells for structural and biochemical studies. Moreover, the quality of the APC purified has revealed the presence of hitherto unidentified and unknown APC subunits, whose identities and functions are now being characterised. Our aim is to obtain a high-resolution structure of the APC by cryo-electron microscopy and X-ray crystallography via use of Fluidigm micro-fluidics chips.

Biochemical and Functional Analysis of APC

The isolated wild type and mutant forms of the APC from yeast has allowed us to undertake a number of biochemical and functional studies, including ubiquitination reactions and APC-binding interactions using defined substrates, subunits and co-activators. These investigations have revealed that specific APC subunits play critical roles in substrate recognition. Specifically the DOC1 subunit was shown to be required for the optimal activity of the APC, and this results from the role the DOC1 plays to mediate APC-substrate interactions (Passmore et al., 2003).

 

Publications:

Passmore LA, Barford D. (2004). Getting into position: the catalytic mechanisms of protein ubiquitylation. Biochem J., 379, 513-525.

Passmore LA, McCormack EA, Au SW, Paul A, Willison KR, Harper JW, Barford D. (2003). Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition. EMBO J., 22, 786-796.

Au SW, Leng X, Harper JW, Barford D. (2002). Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit.  J Mol Biol., 316, 955-968.

Table 1 Subunits of the Anaphase Promoting Complex 

Subunit	Structural Features	Mass (kDa)
Core Subunits
APC1	Rpn1/Rpn2 homology (LRR)	196
APC2	Cullin domain	96
APC4	Unknown	73
APC5	HEAT motifs	77
APC9	Unknown	30
APC11	RING-H2 finger	18
CPC16	Tetratricopeptide repeats	94
CPC23	Tetratricopeptide repeats	70
CPC26	Heat shock protein, acidic	14
CPC27	Tetratricopeptide repeats	85
DOC1/APC10	Doc homology domain	26
APC13 (Swm1)	Unknown	19
Mnd2	Unknown	43
APC Co-activators
CDC20	7 WD40 repeats, mitotic co-activator	68
CDCH1	7 WD40 repeats, G1 co-activator	64
AMA1	7 WD40 repeats, mitotic co-activator	66

 

Cylindromatosis

Signalling downstream of the TNF receptor involves activation of NF-kB, and stimulation of gene expression in a process involving coupled phosphorylation and ubiquitination. In this pathway, the multi-subunit kinase, IKK, phosphorylates an inhibitory subunit (I-kB) of NF-kB leading to its ubiquitination via the SCF and subsequent proteosome dependent degradation. IKK is itself activated by an upstream kinase consisting of a complex of the kinase subunit TAK1 together with TAB1 and TAB2. This complex is assembled and activated following the generation of Lys63-linked poly-ubiquitin chains attached to TRAF E3 ligases that are activated in response to TNF signalling.

Cylindromatosis is a rare benign human skin cancer affecting the head and neck region that is genetically linked to mutations in a tumour suppressor gene termed CYLD. Recent data indicates that CYLD encodes a de-ubiquitinating activity (DUB) with specificity for Lys63-linked polyubiuitin chains. The normal role of CYLD is to antagonize TNF signalling by hydrolysing poly-ubiquitin chains and loss of CYLD activity in cylindromatosis results in hyperactivity of the TNF-NF-kB signalling pathway.

Our interests are to determine the structure of CYLD and understand the basis for its specificity for Lys63-linked polyubiquitin chains.

A20

A20 is a dual functional enzyme possessing both deubiquitinating activity towards Lys63-linked poly-ubiquitin chains and Lys48 E3 ligase activity. The enzyme is an inhibitor of TNF signalling and acts towards RIP (receptor interacting protein). Lys63-linked poly-ubiquitin chains attached to RIP are recognized by A20, cleaved via its DUB activity, and replaced by a Lys48-linked poly-ubiquitin chain, thereby targeting A20 for degradation.