The DWNN family of proteins

DWNN domains

DWNN is a novel 76 residue domain recently identified in our lab by promotor-trap mutagenesis of CHO cells. Knock-out of the gene confers resistance to cytotoxic T-cell killing (CTL) and to staurosporine-induced apoptosis. Close homologues have been identified in all eukaryotic genomes for which sequence data is available, including plants. The domain occurs at single-copy number in most completely-sequenced genomes, although there are two copies in Arabidopsis:

Neighbour-joining tree from an alignment of sequenced DWNN domains, showing the early evolution of the domain, and its high level of conservation.

Domain structure of DWNN-containing proteins

DWNN homologues are associated with a number of other conserved domains, including a zinc finger (CCHC), a Ring finger (C3HC4) and a p53-associated domain. Ring fingers have recently been shown to have a role in the regulation of proteins such as p53 by facilitating the attachment of ubiquitin, which then targets them for degradation by the proteosome. The p53-associated domain is known to associate with p53 in mouse and with Rb in human.

Predicted structure of DWNN domains

BLAST searches find no significant homology between DWNN and any protein of known structure. The PSI-PRED[3] and JPred[2] secondary structure prediction algorithms both predict a central alpha helix flanked by beta sheets:

Secondary structure prediction by the PSI-Pred server

Based on these predictions, the 3D-PSSM (ICRF) and BIOINBGU[1] (Ben Gurion) servers both confidently predict a ubiquitin-like fold for the DWNN domain:

The b-grasp fold of ubiquitin

Predicted function of DWNN-containing proteins

In the past few years a number of ubiquitin-like proteins have been shown to play a role in the regulation of important proteins such as p53 and Rb, by modulation of the ubiquitin-mediated protein degradation pathway. Proteins are tagged for degradation by the covalent addition of ubiquitin at specific sites by enzymes known as E2/E3 ubiquitin ligases, which typically contain Ring finger domains. Ring fingers have recently been shown to possess intrinsic ubiquitin-ligase activity [1]. The predicted ubiquitin-like structure of the domain, coupled with the associated Ring finger domains and the association of the p53-associated domain with p53 and Rb, opens the possibility that DWNN-containing proteins operate as E2/E3 ubiquitin ligases.

Current research activities

Our research aims to answer some or all of the following questions:

• Is the DWNN domain a structural homologue of ubiquitin?
• Does it have a similar function?
• How do the different domains interact with each other?
• Do DWNN proteins interact with tumour-suppressors?
• Do mutations in DWNN proteins lead to cancer?
  

We are currently producing expression constructs for DWNN domains from the following organisms:

NMR work on the human DWNN domain is underway in collaboration with the group of Prof Iain Campbell in Oxford, UK, and X-ray analysis will begin shortly in collaboration with the group of Prof Tom Blundell in Cambridge, UK. BIACore studies of interactions between DWNN and p53 and Rb are underway at UWC, and screens for additional interactions are being conducted using the yeast-2-hybrid system.

Collaborators

• Prof Jasper Rees, Biotechnology Department, UWC
• Dr Monde Ntwasa, Department of Molecular and Cellular Biology, University of the Witwatersrand, South Africa
• Prof Iain Campbell, Oxford Centre for Molecular Sciences, Oxford University, U.K.
• Prof Tom Blundell, Biochemistry Department, Cambridge University, U.K.

References

[1] Fischer, D. Hybrid Fold Recognition: Combining Sequence Derived Properties with Evolutionary Information. Pacific Symp. Biocomputing, Hawaii, 119-130, January 2000. World Scientific

[2] Enhanced Genome Annotation using Structural Profiles in the Program 3D-PSSM. Kelley LA, MacCallum RM & Sternberg MJE (2000). J. Mol. Biol. 299(2), 501-522

[3] Jones, D. T. (1999) Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292: 195-202

[4] Huang Hk, Joazeiro CA, Bonfoco E, Kamada S, Leverson JD, Hunter T, J Biol Chem. 2000;275 (35):26661-4