HOXA1 binds RBCK1/HOIL-1 and TRAF2 and modulates the TNF/NF-κB pathway in a transcription-independent manner.

HOX proteins define a family of key transcription factors regulating animal embryogenesis. HOX genes have also been linked to oncogenesis and HOXA1 has been described to be active in several cancers, including breast cancer. Through a proteome-wide interaction screening, we previously identified the TNFR-associated proteins RBCK1/HOIL-1 and TRAF2 as HOXA1 interactors suggesting that HOXA1 is functionally linked to the TNF/NF-κB signaling pathway. Here, we reveal a strong positive correlation between expression of HOXA1 and of members of the TNF/NF-κB pathway in breast tumor datasets. Functionally, we demonstrate that HOXA1 can activate NF-κB and operates upstream of the NF-κB inhibitor IκB. Consistently, we next demonstrate that the HOXA1-mediated activation of NF-κB is non-transcriptional and that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1. We also identify an 11 Histidine repeat and the homeodomain of HOXA1 to be required both for RBCK1 and TRAF2 interaction and NF-κB stimulation. Finally, we highlight that activation of NF-κB is crucial for HOXA1 oncogenic activity.

Endoplasmic reticulum stress does not contribute to steatohepatitis in obese and insulin-resistant high-fat-diet-fed foz/foz mice.

Non-alcoholic fatty liver (steatosis) and steatohepatitis [non-alcoholic steatohepatitis (NASH)] are hepatic complications of the metabolic syndrome. Endoplasmic reticulum (ER) stress is proposed as a crucial disease mechanism in obese and insulin-resistant animals (such as ob/ob mice) with simple steatosis, but its role in NASH remains controversial. We therefore evaluated the role of ER stress as a disease mechanism in foz/foz mice, which develop both the metabolic and histological features that mimic human NASH. We explored ER stress markers in the liver of foz/foz mice in response to a high-fat diet (HFD) at several time points. We then evaluated the effect of treatment with an ER stress inducer tunicamycin, or conversely with the ER protectant tauroursodeoxycholic acid (TUDCA), on the metabolic and hepatic features. foz/foz mice are obese, glucose intolerant and develop NASH characterized by steatosis, inflammation, ballooned hepatocytes and apoptosis from 6 weeks of HFD feeding. This was not associated with activation of the upstream unfolded protein response [phospho-eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme 1α (IRE1α) activity and spliced X-box-binding protein 1 (Xbp1)]. Activation of c-Jun N-terminal kinase (JNK) and up-regulation of activating transcription factor-4 (Atf4) and CCAAT/enhancer-binding protein-homologous protein (Chop) transcripts were however compatible with a 'pathological' response to ER stress. We tested this by using intervention experiments. Induction of chronic ER stress failed to worsen obesity, glucose intolerance and NASH pathology in HFD-fed foz/foz mice. In addition, the ER protectant TUDCA, although reducing steatosis, failed to improve glucose intolerance, hepatic inflammation and apoptosis in HFD-fed foz/foz mice. These results show that signals driving hepatic inflammation, apoptosis and insulin resistance are independent of ER stress in obese diabetic mice with steatohepatitis.

Protein interactions of the transcription factor Hoxa1.

BACKGROUND: Hox proteins are transcription factors involved in crucial processes during animal development. Their mode of action remains scantily documented. While other families of transcription factors, like Smad or Stat, are known cell signaling transducers, such a function has never been squarely addressed for Hox proteins. RESULTS: To investigate the mode of action of mammalian Hoxa1, we characterized its interactome by a systematic yeast two-hybrid screening against ~12,200 ORF-derived polypeptides. Fifty nine interactors were identified of which 45 could be confirmed by affinity co-purification in animal cell lines. Many Hoxa1 interactors are proteins involved in cell-signaling transduction, cell adhesion and vesicular trafficking. Forty-one interactions were detectable in live cells by Bimolecular Fluorescence Complementation which revealed distinctive intracellular patterns for these interactions consistent with the selective recruitment of Hoxa1 by subgroups of partner proteins at vesicular, cytoplasmic or nuclear compartments. CONCLUSIONS: The characterization of the Hoxa1 interactome presented here suggests unexplored roles for Hox proteins in cell-to-cell communication and cell physiology.

Pentapeptide insertion mutagenesis of the Hoxa1 protein: mapping of transcription activation and DNA-binding regulatory domains.

The mode of action of Hoxa1, like that of most Hox proteins, remains poorly characterized. In an effort to identify functional determinants contributing to the activity of Hoxa1 as a transcription factor, we generated 18 pentapeptide insertion mutants of the Hoxa1 protein and we assayed them in transfected cells for their activity on target enhancers from the EphA2 and Hoxb1 genes known to respond to Hoxa1 in the developing hindbrain. Only four mutants displayed a complete loss-of-function. Three of them contained an insertion in the homeodomain of Hoxa1, whereas the fourth loss-of-function mutant harbored an insertion in the very N-terminal end of the protein. Transcription activation assays in yeast further revealed that the integrity of both the N-terminal end and homeodomain is required for Hoxa1-mediated transcriptional activation. Furthermore, an insertion in the serine-threonine-proline rich C-terminal extremity of Hoxa1 induced an increase in activity in mammalian cells as well as in the yeast assay. The C-terminal extremity thus modulates the transcriptional activation capacity of the protein. Finally, electrophoretic mobility shift assays revealed that the N-terminal extremity of the protein also exerts a modulatory influence on DNA binding by Hoxa1-Pbx1a heterodimers.

MALDI-TOF MS identification and antimicrobial susceptibility testing directly from positive enrichment broth.

A rapid preparation procedure was validated for MALDI-TOF MS identification followed by an antimicrobial susceptibility testing directly from a non-selective enrichment broth of sterile fluid samples associated with negative classical cultures. This method can be easily integrated in the laboratory routine allowing precious time gain for microbe identification and subsequent adequate treatment initiation.

Hox protein interactions: screening and network building.

Understanding the mode of action of Hox proteins requires the identification of molecular and cellular pathways they take part in. This includes to characterize the networks of protein-protein interactions involving Hox proteins. In this chapter we propose a strategy and methods to map Hox interaction networks, from yeast two-hybrid and high-throughput yeast two-hybrid interaction screening to bioinformatic analyses based on the software platform Cytoscape.

Peritoneal dialysis in an ageing population: a 10-year experience.

BACKGROUND: Chronic kidney disease (CKD) is becoming increasingly prevalent and there are increasing numbers of older patients with advanced CKD. Peritoneal dialysis (PD) is a potential treatment. This study aims to compare PD outcomes in age-defined populations in the largest PD centre in the Republic of Ireland over 10 years. METHODS: We retrospectively identified all adult patients, over the age of 50 years, who commenced PD as their first modality of renal replacement therapy (RRT) between 1 January 1998 and 31 December 2008 at our institution. Primary outcome was patient survival; secondary outcomes were technique failure, peritonitis-free survival, transplantation and hospitalisations. RESULTS: One hundred and forty-eight patients with a mean age of 63 years were included. Twenty-two patients were on assisted PD, the majority of whom were aged 70 years or over (P = 0.001). There were no differences in patient survival or technique failure by age group, Charlson Co-Morbidity Index (CCI), modified-CCI or adjusted CCI. Renal transplantation occurred predominantly in younger patients (P = 0.001) with lower m-CCI (P = 0.001) and a-CCI (P = 0.002) who performed PD independently (P = 0.004). Older patients required longer hospital stays to initiate PD (P = 0.004). Assisted PD was not associated with an increase in early complications or technique failure but death rates were higher (P = 0.002). CONCLUSION: This study shows PD to be an acceptable modality of renal replacement therapy in elderly patients, with no observed differences in survival, technique survival or complication rates. Co-morbidities appear to play a stronger role in predicting survival than age alone. Assisted PD is a viable option in those unable to undergo PD independently.

The nuclear factor kappaB-activator gene PLEKHG5 is mutated in a form of autosomal recessive lower motor neuron disease with childhood onset.

Lower motor neuron diseases (LMNDs) include a large spectrum of clinically and genetically heterogeneous disorders. Studying a large inbred African family, we recently described a novel autosomal recessive LMND variant characterized by childhood onset, generalized muscle involvement, and severe outcome, and we mapped the disease gene to a 3.9-cM interval on chromosome 1p36. We identified a homozygous missense mutation (c.1940 T-->C [p.647 Phe-->Ser]) of the Pleckstrin homology domain-containing, family G member 5 gene, PLEKHG5. In transiently transfected HEK293 and MCF10A cell lines, we found that wild-type PLEKHG5 activated the nuclear factor kappa B (NF kappa B) signaling pathway and that both the stability and the intracellular location of mutant PLEKHG5 protein were altered, severely impairing the NF kappa B transduction pathway. Moreover, aggregates were observed in transiently transfected NSC34 murine motor neurons overexpressing the mutant PLEKHG5 protein. Both loss of PLEKHG5 function and aggregate formation may contribute to neurotoxicity in this novel form of LMND.