Protein ubiquitination in eukaryotic cells is mediated by diverse E3 ligase enzymes that each target specific substrates. The cullin E3 ligase complexes are the most abundant class of E3 ligases; they contain various cullin components that serve as scaffolds for interaction with substrate-recruiting adaptor proteins. SPOP is a BTB-domain adaptor of the cullin-3 E3 ligase complexes; it selectively recruits substrates via its N-terminal MATH domain, whereas its BTB domain mediates dimerization and interactions with cullin-3. It has recently been recognized that the high-order oligomerization of SPOP enhances the ubiquitination of substrates. Here, a dimerization interface in the SPOP C-terminus is identified and it is shown that the dimerization interfaces of the BTB domain and of the C-terminus act independently and in tandem to generate high-order SPOP oligomers. The crystal structure of the dimeric SPOP C-terminal domain is reported at 1.5 Å resolution and it is shown that Tyr353 plays a critical role in high-order oligomerization. A model of the high-order SPOP oligomer is presented that depicts a helical organization that could enhance the efficiency of substrate ubiquitination.

A randomised phase III trial was conducted to assess the role of interferon-alpha (INFa) 2a as maintenance therapy following surgery and/or chemotherapy in patients with epithelial ovarian carcinoma. Patients were randomised following initial surgery/chemotherapy to interferon-alpha 2a as 4.5 mega-units subcutaneously 3 days per week or to no further treatment. A total of 300 patients were randomised within the study between February 1990 and July 1997. No benefit for interferon maintenance was seen in terms of either overall or clinical event-free survival. We conclude that INF-a is not effective as a maintenance therapy in the management of women with ovarian cancer. The need for novel therapeutics or strategies to prevent the almost inevitable relapse of patients despite increasingly effective surgery and chemotherapy remains.

SUMMARYThe POZ/BTB domain is an evolutionarily conserved motif found in approximately forty zinc-finger transcription factors (POZ-ZF factors). Several POZ-ZF factors are implicated in human cancer, and POZdomain interaction interfaces represent an attractive target for therapeutic intervention. Miz-1 is a POZ-ZF factor that regulates DNA-damage-induced cell cycle arrest, and plays an important role in human cancer by virtue of its interaction with the c-Myc and Bcl6 oncogene products. The Miz-1 POZ domain mediates both self-association and the recruitment of non-POZ partners. POZ-ZF factors generally function as homo-dimers, although higher-order associations and heteromeric interactions are known to be physiologically important; crucially, the interaction interfaces in such large complexes have not been characterised. We report here the crystal structure of the Miz-1 POZ domain to 2.1Å resolution. The tetrameric organisation of Miz-1 POZ reveals two types of interaction interface between subunits; an interface of alpha-helices resembles the dimerisation interface of reported POZ domain structures, whereas a novel beta-sheet interface directs the association of two POZ domain dimers. We show that the beta-sheet interface directs tetramerisation of the Miz-1 POZ domain in solution, and therefore represents a newly described candidate interface for the higherorder homo-and hetero-oligomerisation of POZ-ZF proteins in vivo. Keywords:The POZ/BTB (poxvirus and zinc finger/bric-à-brac, tramtrack and broad complex) domain mediates Higher-order oligomers 12 and heteromeric interactions 13 between different POZ-ZF factors are thought to be physiologically important, although the interaction interfaces in these complexes have not been characterised.We report here the crystal structure of the tetrameric Miz-1 POZ domain. The oligomeric organisation of Miz-1 POZ reveals a novel beta-sheet interaction interface that directs the association of two POZ domain dimers. Weshow that this region mediates the association of Miz-1 POZ dimers in solution, and therefore represents a newly described candidate interface for the higher-order homo-and hetero-oligomerisation of POZ-ZF proteins in vivo. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 4General Organisation of the Miz-1 POZ DomainThe entire Miz-1 POZ domain (Miz-1 residues 2 -115) was expressed in E.coli, purified and crystallized. The crystal structure was solved by molecular replacement and refined to R = 18.1%, R free = 22.9% at 2.1 Å resolution (Table 1 and Figure 1A), with a single tetramer per asymmetric unit. All residues were built in the model.The Miz-1 POZ tetramer may be described as an association of two dimers, each of which resembles the reported PLZF, BCL6 and LRF POZ structures ( Figure 1). The assembly has two distinct types of interface between subunits: the A:B and C:D interfaces resemble those described in PLZF, BCL6 and LRF, whereas A:D The Miz-1 POZ Domain Core and Dimerisation Interface POZ domains of the POZ-ZF factors are highly conserved, an...

The POZ domain is an evolutionarily conserved protein-protein interaction domain that is found in approximately 40 mammalian transcription factors. POZ domains mediate both homodimerization and the heteromeric interactions of different POZ-domain transcription factors with each other. Miz1 is a POZ-domain transcription factor that regulates cell-cycle arrest and DNA-damage responses. The activities of Miz1 are altered by its interaction with the POZ-domain transcriptional repressors BCL6 and NAC1, and these interactions have been implicated in tumourigenesis in B-cell lymphomas and in ovarian serous carcinomas that overexpress BCL6 and NAC1, respectively. A strategy for the purification of tethered POZ domains that form forced heterodimers is described, and crystal structures of the heterodimeric POZ domains of Miz1/BCL6 and of Miz1/NAC1 are reported. These structures will be relevant for the design of therapeutics that target POZ-domain interaction interfaces.

PDB Reference: Nac1 POZ domain, 3ga1, r3ga1sf.Nac1 is a POZ-domain transcription factor that is involved in the self-renewal of embryonic stem cells. It is overexpressed in ovarian serous carcinoma and targeting the interactions of its POZ domain is a potential therapeutic strategy. Nac1 lacks a zinc-finger DNA-binding domain and thereby differs from most other POZ-domain transcription factors. Here, the crystal structure of the Nac1 POZ domain at 2.1 Å resolution is reported. The Nac1 POZ domain crystallized as a dimer in which the interaction interfaces between subunits resemble those found in the POZ-zinc finger transcription factors. The organization of the Nac1 POZ-domain core resembles reported POZ-domain structures, whereas the C-terminus differs markedly. The C-terminal -helix of the Nac1 POZ domain is shorter than that observed in most other POZ-domain transcription factors; variation in the organization of this region may be a general feature of POZdomain structures.