Mutations of genes encoding isocitrate dehydrogenase (IDH1 and IDH2) have been recently described in acute myeloid leukemia (AML). Serum and myeloblast samples from patients with IDH-mutant AML contain high levels of the metabolite 2-hydroxyglutarate (2-HG), a product of the altered IDH protein. In this prospective study, we sought to determine whether 2-HG can potentially serve as a noninvasive biomarker of disease burden through serial measurements in patients receiving conventional therapy for newly diagnosed AML. Our data demonstrate that serum, urine, marrow aspirate, and myeloblast 2-HG levels are significantly higher in IDH-mutant patients, with a correlation between baseline serum and urine 2-HG levels. Serum and urine 2-HG, along with IDH1/2-mutant allele burden in mar- IntroductionMutations in genes encoding isocitrate dehydrogenase (IDH1/2) were recently discovered in acute myeloid leukemia (AML). 1,2 Their prognostic significance remains under investigation. [3][4][5][6][7][8][9] IDH proteins catalyze the oxidative decarboxylation of isocitrate to ␣-ketoglutarate (␣-KG). IDH mutations reside in the active site 10 and include missense alterations affecting arginine-132 (R132) in IDH1, and the analogous arginine residue (R172), or one at arginine-140 (R140), in IDH2. [1][2][3]5,6,8,11 The altered IDH proteins instead catalyze reduction of ␣-KG to the metabolite 2-hydroxyglutarate (2-HG). 12 2-HG is normally present at low levels in cells, 13 readily interconverted by 2-HG dehydrogenase to ␣-KG, 14,15 but IDH mutations promote its accumulation in myeloblasts and sera of affected patients. 10 No prior studies have prospectively measured 2-HG in IDHmutant AML during treatment. We focused on the utility of 2-HG as a potential biomarker of disease burden and sought to assess the effect of treatment on the trajectory and kinetics of 2-HG levels. To accomplish this, we serially measured serum, urine, marrow aspirate, and myeloblast 2-HG during conventional therapy for newly diagnosed AML. MethodsAdult patients at Massachusetts General Hospital, eligible for treatment of newly diagnosed AML, as defined by World Health Organization criteria, were enrolled. Samples were obtained through a protocol approved by our institution, Dana-Farber Harvard Cancer Center, its institutional review board, and the scientific review committees, with the approved protocol number 11-121. Informed consent was obtained per the Declaration of Helsinki.Serum, urine, and marrow samples were obtained for 2-HG measurement before therapy. Mononuclear cells from blood and marrow aspirate were isolated using density gradient centrifugation with Ficoll-Hypaque (GE Healthcare). 2-HG measurement was performed by Agios Pharmaceuticals, with methods previously described. 13,16 Serum and myeloblast 2-HG levels were considered elevated if Ͼ 1000 ng/mL or 1000 ng/2 ϫ 10 6 cells, respectively, as per previous reports. 10 In those with elevated baseline 2-HG, serum and urine were serially obtained for 2-HG measurement, at days 7, 14, 30, 60, and re...
SUMMARY Transcription factor activity and turnover are functionally linked, but the global patterns by which DNA-bound regulators are eliminated remain poorly understood. We established an assay to define the chromosomal location of DNA-associated proteins that are slated for degradation by the ubiquitin-proteasome system. The genome-wide map described here ties proteolysis in mammalian cells to active enhancers and to promoters of specific gene families. Nuclear-encoded mitochondrial genes in particular correlate with protein elimination, which positively affects their transcription. We show that the nuclear receptor corepressor NCoR1 is a key target of proteolysis and physically interacts with the transcription factor CREB. Proteasome inhibition stabilizes NCoR1 in a site-specific manner and restrains mitochondrial activity by repressing CREB-sensitive genes. In conclusion, this functional map of nuclear proteolysis links chromatin architecture with local protein stability and identifies proteolytic derepression as highly dynamic in regulating the transcription of genes involved in energy metabolism.
Loss of protein quality is a driving force of aging. The accumulation of misfolded proteins represents a vulnerability for long-lived cells, such as haematopoietic stem cells. How these cells, which have the ability to reconstitute all haematopoietic lineages throughout life, maintain their regenerative potential and avert the effects of aging is poorly understood. Here, we determined the protein content in haematopoietic stem and progenitor cells to identify prevalent chaperones that support proteome integrity. We identified Peptidyl-Prolyl Isomerase A (PPIA or Cyclophilin A) as the dominant cytosolic foldase in this cell population. Loss of PPIA accelerated aging in the mouse stem cell compartment. In an effort to define targets of PPIA, we found that RNA- and DNA-binding proteins are common substrates of this chaperone. These proteins are enriched in intrinsically disordered regions (IDRs), which can catalyse protein condensation. Isomerized target prolines are almost exclusively located within IDRs. We discovered that over 20% of PPIA client proteins are known to participate in liquid-liquid phase separation, enabling the formation of supramolecular membrane-less organelles. Using the poly-A binding protein PABPC1 as an example, we demonstrate that PPIA promotes phase separation of ribonucleoprotein particles, thereby increasing cellular stress resistance. Haematopoietic stem cell aging is associated with a decreased expression of PPIA and reduced synthesis of intrinsically disordered proteins. Our findings link the ubiquitously expressed chaperone PPIA to phase transition and identify macromolecular condensation as a potential determinant of the aging process in haematopoietic stem cells.
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