Anti-RNF34 monoclonal antibody Made to order

Background/Aims: To identify the regulator of nucleotide binding oligomerization domain-containing protein 1 (NOD1) and its regulatory function. Methods and Results: We performed a yeast two-hybrid screening assay and identified the E3 ligase RNF34 as a candidate partner of NOD1. Using co-immunoprecipitation (co-IP) and glutathione S transferase (GST)-pull down assays, we further confirmed that RNF34 is associated with NOD1. Western blotting showed that RNF34 downregulated the stability of NOD1 and promoted its ubiquitination. Functional analysis demonstrated that RNF34 overexpression inhibited NOD1-dependent activation of nuclear factor-kappa B (NF-kappa B), whereas knockdown of RNF34 using small interfering RNA increased NF-kappa B activation following stimulation from NOD1 overexpression or transfection of y-D-glutamyl-meso-diaminopimelic acid. Conclusion: These findings confirm that RNF34 is a negative regulator of the NOD1 pathway through direct interaction and ubiquitination of NOD1, and suggest a novel regulatory mechanism of NOD1. Copyright (C) 2014 S. Karger AG, Basel

The transcriptional coactivator PGC-1 alpha is a master regulator of energy metabolism and adaptive thermogenesis in the brown fat cell. PGC-1 alpha is a short-lived protein, and the molecular components that control PGC-1 alpha turnover and their functional importance in energy metabolism are largely unknown. Here we performed a luciferase-based overexpression screen and identified a Ring-finger-containing protein, RNF34, as a specific E3 ubiquitin ligase for PGC-1 alpha. RNF34 is a nuclear protein that interacts with and ubiquitinates PGC-1 alpha to promote its turnover. Interestingly, RNF34 binds to the C-terminal half of PGC-1 alpha and targets it for degradation independently of the previously identified N-terminal phosphodegron motif. In brown fat cells, knockdown of RNF34 increases the endogenous PGC-1 alpha protein level, uncoupling protein 1 (UCP1) expression, and oxygen consumption, while the opposite effects are observed in brown fat cells ectopically expressing wild-type RNF34 but not in cells expressing the ligase activity-defective mutant. Moreover, cold exposure and beta 3-adrenergic receptor signaling, conditions that induce PGC-1 alpha expression, suppress RNF34 expression in the brown fat cell, indicating a physiological relevance of this E3 ligase in thermogenesis. Our results reveal that RNF34 is a bona fide E3 ubiquitin ligase for PGC-1 alpha and negatively regulates brown fat cell metabolism.