The malate shuttle detoxifies ammonia in exhausted T cells by producing 2-ketoglutarate.

The malate shuttle is traditionally understood to maintain NAD+/NADH balance between the cytosol and mitochondria. Whether the malate shuttle has additional functions is unclear. Here we show that chronic viral infections induce CD8+ T cell expression of GOT1, a central enzyme in the malate shuttle. Got1 deficiency decreased the NAD+/NADH ratio and limited antiviral CD8+ T cell responses to chronic infection; however, increasing the NAD+/NADH ratio did not restore T cell responses. Got1 deficiency reduced the production of the ammonia scavenger 2-ketoglutarate (2-KG) from glutaminolysis and led to a toxic accumulation of ammonia in CD8+ T cells. Supplementation with 2-KG assimilated and detoxified ammonia in Got1-deficient T cells and restored antiviral responses. These data indicate that the major function of the malate shuttle in CD8+ T cells is not to maintain the NAD+/NADH balance but rather to detoxify ammonia and enable sustainable ammonia-neutral glutamine catabolism in CD8+ T cells during chronic infection.

A novel autosomal recessive TERT T1129P mutation in a dyskeratosis congenita family leads to cellular senescence and loss of CD34+ hematopoietic stem cells not reversible by mTOR-inhibition.

The TERT gene encodes for the reverse transcriptase activity of the telomerase complex and mutations in TERT can lead to dysfunctional telomerase activity resulting in diseases such as dyskeratosis congenita (DKC). Here, we describe a novel TERT mutation at position T1129P leading to DKC with progressive bone marrow (BM) failure in homozygous members of a consanguineous family. BM hematopoietic stem cells (HSCs) of an affected family member were 300-fold reduced associated with a significantly impaired colony forming capacity in vitro and impaired repopulation activity in mouse xenografts. Recent data in yeast suggested improved cellular checkpoint controls by mTOR inhibition preventing cells with short telomeres or DNA damage from dividing. To evaluate a potential therapeutic option for the patient, we treated her primary skin fibroblasts and BM HSCs with the mTOR inhibitor rapamycin. This led to prolonged survival and decreased levels of senescence in T1129P mutant fibroblasts. In contrast, the impaired HSC function could not be improved by mTOR inhibition, as colony forming capacity and multilineage engraftment potential in xenotransplanted mice remained severely impaired. Thus, rapamycin treatment did not rescue the compromised stem cell function of TERTT1129P mutant patient HSCs and outlines limitations of a potential DKC therapy based on rapamycin.

The thrombopoietin receptor P106L mutation functionally separates receptor signaling activity from thrombopoietin homeostasis.

The interaction between thrombopoietin (THPO) and its receptor c-Mpl regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and thrombocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpl P106L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F104S, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis.