👤 Natasha L Grimsey

The melanocortin-4 receptor (MC4R) plays a critical role in regulating energy homeostasis. Studies on obesogenic human MC4R (hMC4R) variants have not yet revealed how hMC4R maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q hMC4R variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP) response element (CRE)-driven transcription, and calcium mobilization but not phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating hormone-induced CRE-driven transcription but not impaired α-melanocyte-stimulating hormone-induced AC, calcium, or pERK1/2. This profile was not observed for transfected H158R, a constitutively active hMC4R variant associated with overweight but not obesity. We concluded that there is potential for α-melanocyte-stimulating hormone-induced CRE-driven transcription in HEK293 cells transfected with obesogenic hMC4R variants to be the key predictive tool for determining whether they exhibit loss of function. Furthermore, in vivo, α-melanocyte-stimulating hormone-induced hMC4R CRE-driven transcription may be key for maintaining body weight. Show less

Insufficiencies of the micronutrient thiamine (Vitamin B1) have been associated with inducing Alzheimer's disease (AD)-like neuropathology. The hypometabolic state associated with chronic thiamine insufficiency (TI) has been demonstrated to be a contributor towards the development of amyloid plaque deposition and neurotoxicity. However, the molecular mechanism underlying TI induced AD pathology is still unresolved. Previously, we have established that TI stabilizes the metabolic stress transcriptional factor, Hypoxia Inducible Factor-1α (HIF1α). Utilizing neuronal hippocampal cells (HT22), TI-induced HIF1α activation triggered the amyloidogenic cascade through transcriptional expression and increased activity of β-secretase (BACE1). Knockdown and pharmacological inhibition of HIF1α during TI significantly reduced BACE1 and C-terminal Fragment of 99 amino acids (C99) formation. TI also increased the expression of the HIF1α regulated pro-apoptotic protein, BCL2/adenovirus E1B 19 kDa protein-interacting protein (BNIP3). Correspondingly, cell toxicity during TI conditions was significantly reduced with HIF1α and BNIP3 knockdown. The role of BNIP3 in TI-mediated toxicity was further highlighted by localization of dimeric BNIP3 into the mitochondria and nuclear accumulation of Endonuclease G. Subsequently, TI decreased mitochondrial membrane potential and enhanced chromatin fragmentation. However, cell toxicity via the HIF1α/BNIP3 cascade required TI induced oxidative stress. HIF1α, BACE1 and BNIP3 expression was induced in 3xTg-AD mice after TI and administration with the HIF1α inhibitor YC1 significantly attenuated HIF1α and target genes levels in vivo. Overall, these findings demonstrate a critical stress response during TI involving the induction of HIF1α transcriptional activity that directly promotes neurotoxicity and AD-like pathology. Show less

The sorting of G protein-coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain-containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs. Show less