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There are three prolyl hydroxylases (PHD1, 2 and 3) that regulate the hypoxia-inducible factors (HIFs), the master transcriptional regulators that respond to changes in intracellular O2 tension. In high O2 tension (normoxia) the PHDs hydroxylate two conserved proline residues on HIF-1?, which leads to binding of the von Hippel-Lindau (VHL) tumour suppressor, the recognition component of a ubiquitin-ligase complex, initiating HIF-1? ubiquitylation and degradation. However, it is not known whether PHDs and VHL act separately to exert their enzymatic activities on HIF-1? or as a multiprotein complex. Here we show that the tumour suppressor protein LIMD1 (LIM domain-containing protein) acts as a molecular scaffold, simultaneously binding the PHDs and VHL, thereby assembling a PHD-LIMD1-VHL protein complex and creating an enzymatic niche that enables efficient degradation of HIF-1?. Depletion of endogenous LIMD1 increases HIF-1? levels and transcriptional activity in both normoxia and hypoxia. Conversely, LIMD1 expression downregulates HIF-1 transcriptional activity in a manner depending on PHD and 26S proteasome activities. LIMD1 family member proteins Ajuba and WTIP also bind to VHL and PHDs 1 and 3, indicating that these LIM domain-containing proteins represent a previously unrecognized group of hypoxic regulators. [ABSTRACT FROM AUTHOR]

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1--2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1--ER--E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER--ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance. [ABSTRACT FROM AUTHOR]

ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be ‘rescued’ by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. [ABSTRACT FROM AUTHOR]