Rationale: Coronary artery disease (CAD) is a pervasive and critical healthcare problem. Elevated high density lipoprotein-associated cholesterol (HDL-C) is associated with improved atherosclerotic cardiovascular disease (ASCVD) outcomes on a population level, but clinical trials aimed at HDL-C elevation have not succeeded in improving ASCVD event risk. Nevertheless, human variants in the HDL receptor, encoded by SCARB1, are associated with dyslipidemia, suggesting that HDL metabolism, not HDL-C, is a suitable target for therapy. However, variants in SCARB1 have never been directly attributed to CAD by Mendelian inheritance. Objective: To determine if compound heterozygous variants in SCARB1 cause disease in two brothers with severe, early-onset CAD. Methods and Results: Using whole exome sequencing, we have identified rare, compound heterozygous variants in SCARB1 that segregate with severe, premature CAD, following patterns of Mendelian inheritance. Using induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs) from the proband, we discovered the maternal variant (c.754_755delinsC) to be the first identified SCARB1 null allele, characterized by the absence of RNA and protein expression. Further, we demonstrate that the variant on the paternal allele (c.956G>T (p.G319V)) results in decreased cholesterol uptake, decreased SR-BI:HDL binding, and increased affinity for SR-BI dimerization. Finally, we generated a p.G319V knock-in mouse model that displays nearly 100% homozygous lethality and elevated plasma cholesterol in heterozygous animals, confirming pathogenicity of this variant. Conclusions: In summary, our data provide the first molecular mechanism to show the Mendelian inheritance of CAD as a result of human SCARB1 variants. The rarity of these variants supports pathogenicity in this family. Furthermore, SR-BI p.G319V, which has previously been reported benign in the context of heterozygosity, was uniquely presented alongside a null allele, demonstrating the disease-contributing capability of loss-of-function SCARB1 variants within the population.
FOXO transcription factors are regulators of cellular homeostasis linked to increased lifespan and tumor suppression. FOXOs are activated by diverse cell stresses including serum starvation and oxidative stress. FOXO activity is regulated through post-translational modifications that control shuttling of FOXO proteins to the nucleus. In the nucleus, FOXOs upregulate genes in multiple, often conflicting pathways including cell-cycle arrest and apoptosis. How cells control FOXO activity to ensure the proper response for a given stress is an open question. Using quantitative immunofluorescence and live-cell imaging we found that the dynamics of FOXO nuclear shuttling are stimulus dependent and correspond with cell fate. H2O2 treatment leads to an all-or-none response where some cells show no nuclear FOXO accumulation, while other cells show strong nuclear FOXO signal. The time that FOXO remains in the nucleus increases with dose and is linked with cell death. In contrast, serum starvation causes low amplitude pulses of nuclear FOXO and predominantly results in cell-cycle arrest. The accumulation of FOXO in the nucleus is linked with low AKT activity for both H2O2 and serum starvation. Our findings suggest the dynamics of FOXO nuclear shuttling is one way in which the FOXO pathway dictates different cellular outcomes. [Media: see text] [Media: see text] [Media: see text]
The p53 and FOXO transcription factors (TFs) share many similarities despite their distinct evolutionary origins. Both TFs are activated by a variety of cellular stresses and upregulate genes in similar pathways including cell-cycle arrest and apoptosis. Oxidative stress from excess H2O2activates both FOXO1 and p53, yet whether they are activated at the same time is unclear. Here we found that cells respond to high H2O2levels in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 levels remain low. In the second phase FOXO1 exits the nucleus and p53 levels rise. We found that other oxidative stress induced TFs are activated in the first phase with FOXO1 (NF-κB, NFAT1), or the second phase with p53 (NRF2, JUN) but not both following H2O2stress. The two TF phases result in large differences in gene expression patterns. Finally, we provide evidence that 2-Cys peroxiredoxins control the timing of the TF phases in response to H2O2.
Oxidative stress from excess H2O2 activates transcription factors (TFs) that restore redox balance and repair oxidative damage. Though many TFs are activated by H2O2, it is unknown whether they are activated at the same H2O2 concentration or time after H2O2 stress. We found TF activation is tightly coordinated over time and dose dependent. We first focused on p53 and FOXO1 and found that in response to low H2O2, p53 is activated rapidly while FOXO1 remains inactive. In contrast, cells respond to high H2O2 in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 remains inactive. In the second phase FOXO1 shuts off and p53 levels rise. Other TFs are activated in the first phase with FOXO1 (NF-κB, NFAT1), or the second phase with p53 (NRF2, JUN), but not both. The two phases result in large differences in gene expression. Finally, we provide evidence that 2-Cys peroxiredoxins control which TF are activated and the timing of TF activation.
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