TMEM27 Suppresses Tumor Development by Promoting Ret Ubiquitination, Positioning, and Degradation

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma

Inhibition of IKKβ by celastrol and its analogues – an in silico and in vitro approach

Context: Alzheimer’s disease (AD) is the most common form of dementia affecting the aged population and neuroinflammation is one of the most observed AD pathologies. NF-κB is the central regulator of inflammation and inhibitor κB kinase (IKK) is the converging point in NF-κB activation. Celastrol is a natural triterpene used as a treatment for inflammatory conditions. Objective: This study determines the neuroprotective and inhibitory effect of celastrol on amyloid beta1-42 (Aβ1-42) induced cytotoxicity and IKKβ activity, respectively. Materials and methods: Retinoic acid differentiated IMR-32 cells were treated with celastrol (1 μM) before treatment with Aβ1-42 (IC30 10 μM) for 24 h. The cytotoxicity and IKK phosphorylation were measured by MTT and western blotting analysis, respectively. We screened 36 celastrol analogues for the IKKβ inhibition by molecular docking and evaluated their drug like properties to delineate the neuroprotective effects. Results: Celastrol (1 μM) inhibited Aβ1-42 (10 μM) induced IκBα phosphorylation and protected IMR-32 cells from cell death. Celastrol and 25 analogues showed strong binding affinity with IKKβ as evidenced by strong hydrogen-bonding interactions with critical active site residues. All the 25 analogues displayed strong anti-inflammatory properties but only 11 analogues showed drug-likeness. Collectively, molecule 15 has highest binding affinity, CNS activity and more drug likeness than parent compound celastrol. Discussion and conclusion: The decreased expression of pIκBα in celastrol pretreated cells affirms the functional representation of inhibited IKKβ activity in these cells. The neuroprotective potentials of celastrol and its analogues may be related to IKK inhibition

Inhibition of IKKβ by celastrol and its analogues – an <i>in silico</i> and <i>in vitro</i> approach

<p><b>Context:</b> Alzheimer’s disease (AD) is the most common form of dementia affecting the aged population and neuroinflammation is one of the most observed AD pathologies. NF-κB is the central regulator of inflammation and inhibitor κB kinase (IKK) is the converging point in NF-κB activation. Celastrol is a natural triterpene used as a treatment for inflammatory conditions.</p> <p><b>Objective:</b> This study determines the neuroprotective and inhibitory effect of celastrol on amyloid beta_1-42 (Aβ_1-42) induced cytotoxicity and IKKβ activity, respectively.</p> <p><b>Materials and methods:</b> Retinoic acid differentiated IMR-32 cells were treated with celastrol (1 μM) before treatment with Aβ_1-42 (IC₃₀ 10 μM) for 24 h. The cytotoxicity and IKK phosphorylation were measured by MTT and western blotting analysis, respectively. We screened 36 celastrol analogues for the IKKβ inhibition by molecular docking and evaluated their drug like properties to delineate the neuroprotective effects.</p> <p><b>Results:</b> Celastrol (1 μM) inhibited Aβ_1-42 (10 μM) induced IκBα phosphorylation and protected IMR-32 cells from cell death. Celastrol and 25 analogues showed strong binding affinity with IKKβ as evidenced by strong hydrogen-bonding interactions with critical active site residues. All the 25 analogues displayed strong anti-inflammatory properties but only 11 analogues showed drug-likeness. Collectively, molecule 15 has highest binding affinity, CNS activity and more drug likeness than parent compound celastrol.</p> <p><b>Discussion and conclusion:</b> The decreased expression of pIκBα in celastrol pretreated cells affirms the functional representation of inhibited IKKβ activity in these cells. The neuroprotective potentials of celastrol and its analogues may be related to IKK inhibition.</p