HBP1 (Phospho-Ser402) ELISA Kit

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PI3K-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17 similar to 92. Combined targeting of HBP1 by PI3K antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.

2-Hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44) from Pseudomonas azelaica HBP1 is an FAD-dependent aromatic hydroxylase that catalyzes the conversion of a-hydroxybiphenyl to 2,3 dihydroxybiphenyl in the presence of NADH and oxygen. The catalytic mechanism of this three-substrate reaction was investigated at 7 degrees C by stopped-flow absorption spectroscopy. Various individual steps associated with catalysis were readily observed at pH 7.5, the optimum pH for enzyme turnover. Anaerobic reduction of the free enzyme by NADH is a biphasic process, most likely reflecting the presence of two distinct enzyme forms. Binding of 2-hydroxybiphenyl stimulated the rate of enzyme reduction by NADH by 2 orders of magnitude. The anaerobic reduction of the enzyme-substrate complex involved the formation of a transient charge-transfer complex between the reduced flavin and NAD(+), A similar transient intermediate was formed when the enzyme was complexed with the substrate analog 2-sec-butylphenol or with the non-substrate effector 2,3-dihydroxybiphenyl. Excess NAD(+) strongly stabilized the charge-transfer complexes but did not give rise to the appearance of any intermediate during the reduction of uncomplexed enzyme. Free reduced 2-hydroxybiphenyl S-monooxygenase reacted rapidly with oxygen to form oxidized enzyme with no appearance of intermediates during this reaction. In the presence of 2-hydroxybiphenyl, two consecutive spectral intermediates were observed which were assigned to the flavin C(4a)-hydroperoxide and the flavin C(4a)hydroxide, respectively. No oxygenated flavin intermediates were observed when the enzyme was in complex with 2,3-dihydroxybiphenyl, Monovalent anions retarded the dehydration of the flavin C(4a)-hydroxide without stabilization of additional intermediates. The kinetic data for 2-hydroxybiphenyl S-monooxygenase are consistent with a ternary complex mechanism in which the aromatic substrate has strict control in both the reductive and oxidative half-reaction in a way that reactions leading to substrate hydroxylation are favored over those leading to the futile formation of hydrogen peroxide. NAD(+) release from the reduced enzyme-substrate complex is the slowest step in catalysis.