Regulation of the Alr1 transporter by Mg supply

Magnesium is required for many essential processes and pathways, including protein synthesis and energy metabolism. In the yeast Saccharomyces cerevisiae, Mg2+ uptake across the plasma membrane primarily occurs via the magnesium transporter Alr1. Preliminary data had shown that Alr1-HA accumulation was decreased by Mg. Here I show that the epitope tag was responsible for this differential accumulation of Alr1, as native or N-terminally tagged Alr1 displayed much less differential accumulation. Immunoblotting and Ni2+ uptake assays indicated that both Mg-deficiency and the mnr2 mutation increased Alr1 activity, but under conditions where native protein accumulation was little affected. Since Mg-dependent regulation of Alr system activity and Alr1 protein accumulation did not correlate, I investigated the importance of the post-translational modification of Alr1 to regulation. These studies suggested that the post-translational modification of Alr1 and the C-terminal domain of the protein may be important for regulation

Regulation of Alr1 Mg Transporter Activity by Intracellular Magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni2+, Mn2+, Zn2+ and Co2+. Using Ni2+ uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms

Assessment of an automated approach for variant interpretation in screening for monogenic disorders: A single‐center study

Abstract Background Automation has been introduced into variant interpretation, but it is not known how automated variant interpretation performs on a stand‐alone basis. The purpose of this study was to evaluate a fully automated computerized approach. Method We reviewed all variants encountered in a set of carrier screening panels over a 1‐year interval. Observed variants with high‐confidence ClinVar interpretations were included in the analysis; those without high‐confidence ClinVar entries were excluded. Results Discrepancy rates between automated interpretations and high‐confidence ClinVar entries were analyzed. Of the variants interpreted as positive (likely pathogenic or pathogenic) based on ClinVar information, 22.6% were classified as negative (variants of uncertain significance, likely benign or benign) variants by the automated method. Of the ClinVar negative variants, 1.7% were classified as positive by the automated software. On a per‐case basis, which accounts for variant frequency, 63.4% of cases with a ClinVar high‐confidence positive variant were classified as negative by the automated method. Conclusion While automation in genetic variant interpretation holds promise, there is still a need for manual review of the output. Additional validation of automated variant interpretation methods should be conducted

Circulating CD1c+ myeloid dendritic cells are potential precursors to LCH lesion CD1a+CD207+ cells.

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder that is characterized by the inflammatory lesions with pathogenic CD1a+CD207+ dendritic cells (DCs). BRAFV600E and other somatic activating MAPK gene mutations have been identified in differentiating bone marrow and blood myeloid cells, but the origin of the LCH lesion CD1a+CD207+ DCs and mechanisms of lesion formation remain incompletely defined. To identify candidate LCH CD1a+CD207+ DC precursor populations, gene-expression profiles of LCH lesion CD1a+CD207+ DCs were first compared with established gene signatures from human myeloid cell subpopulations. Interestingly, the CD1c+ myeloid DC (mDC) gene signature was most enriched in the LCH CD1a+CD207+ DC transcriptome. Additionally, the BRAFV600E allele was not only localized to CD1a+CD207- DCs and CD1a+CD207+ DCs, but it was also identified in CD1c+ mDCs in LCH lesions. Transcriptomes of CD1a+CD207- DCs were nearly indistinguishable from CD1a+CD207+ DCs (both CD1a+CD207low and CD1a+CD207high subpopulations). Transcription profiles of LCH lesion CD1a+CD207+ DCs and peripheral blood CD1c+ mDCs from healthy donors were compared to identify potential LCH DC-specific biomarkers: HLA-DQB2 expression was significantly increased in LCH lesion CD1a+CD207+ DCs compared with circulating CD1c+ mDCs from healthy donors. HLA-DQB2 antigen was identified on LCH lesion CD1a+CD207- DCs and CD1a+CD207+ DCs as well as on CD1c+(CD1a+CD207-) mDCs, but it was not identified in any other lesion myeloid subpopulations. HLA-DQB2 expression was specific to peripheral blood of patients with BRAFV600E+ peripheral blood mononuclear cells, and HLA-DQB2+CD1c+ blood cells were highly enriched for the BRAFV600E in these patients. These data support a model in which blood CD1c+HLA-DQB2+ mDCs with activated ERK migrate to lesion sites where they differentiate into pathogenic CD1a+CD207+ DCs

Alternative genetic mechanisms of BRAF activation in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing pathologic CD20

RAF/MEK/extracellular signal-related kinase pathway suppresses dendritic cell migration and traps dendritic cells in Langerhans cell histiocytosis lesions

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal-related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)-mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death