Jean Cappello

Attenuation of AMPK signaling by ROQUIN promotes T follicular helper cell formation

T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN–AMPK metabolic signaling nexus essential for selectively promoting Tfh responses. DOI: http://dx.doi.org/10.7554/eLife.08698.001

Identification of a Pathogenic Variant in TREX1 in Early-Onset Cerebral Systemic Lupus Erythematosus by Whole-Exome Sequencing

Systemic lupus erythematosus (SLE) is a chronic and heterogeneous autoimmune disease. Both twin and sibling studies indicate a strong genetic contribution to lupus, but in the majority of cases the pathogenic variant remains to be identified. The genetic contribution to disease is likely to be greatest in cases with early onset and severe phenotypes. Whole‐exome sequencing now offers the possibility of identifying rare alleles responsible for disease in such cases. This study was undertaken to identify genetic causes of SLE using whole‐exome sequencing.

Deletion of Glu155 causes a deficiency of glutathione transferase Omega 1-1 but does not alter sensitivity to arsenic trioxide and other cytotoxic drugs.

The Omega class glutathione transferase GSTO1-1 can catalyze the reduction of pentavalent methylated arsenic species and is responsible for the biotransfomation of potentially toxic alpha-haloketones. We investigated the cause of GSTO1-1 deficiency in the T-47D breast cancer cell line and found that the cell line is hemizygous for a polymorphic allele that encodes the deletion of Glu155. Northern and Western blots show that T-47D cells contain GSTO1 mRNA but no GSTO1-1 protein suggesting that the deletion of Glu155 causes GSTO1-1 deficiency in vivo. In further support of this contention we found that lymphoblastoid cell lines from subjects who are heterozygous for the deletion of Glu155 have only 60% of normal activity with the GSTO1-1 specific substrate 4-nitrophenacyl glutathione. Pulse-chase studies showed that the deletion of Glu155 causes increased turnover of GSTO1-1 in T47-D cells. These data establish the fact that the polymorphic deletion of Glu155 can cause GSTO1-1 deficiency in vivo. GSTO1-1 expression is elevated in some cell lines that are resistant to the cytotoxic cancer drugs adriamycin, etoposide and cisplatinum but its specific contribution to multi drug resistance has not been evaluated. In this study GSTO1-1 deficient T47-D cells were used to determine if GSTO1-1 contributes directly to arsenic and drug resistance. We established stable expression of normal GSTO1-1 in T-47D cells and found that this did not alter sensitivity to arsenic trioxide, cisplatinum daunorubicin or etoposide.

The impact of glutathione transferase kappa deficiency on adiponectin multimerisation in vivo.

Glutathione transferase Kappa (GSTK1-1) also termed disulfide bond-forming oxidoreductase A-like protein (DsbA-L) has been implicated in the post-translational multimerization of adiponectin and has been negatively correlated with obesity in mice and humans. We investigated adiponectin in Gstk1−/− mice and surprisingly found no difference in the levels of total serum adiponectin or the level of high molecular weight (HMW) multimers when compared with normal controls. Non-reducing SDS-polyacrylamide gel electrophoresis and western blotting also showed a similar distribution of low, middle and HMW multimers in normal and Gstk1−/− mice. Variation in adiponectin has been correlated with glucose tolerance and with the levels of phosphorylated AMP-kinase but we found similar glucose tolerance and similar levels of phospho 5-AMP-activated protein kinase in normal and Gstk1−/− mice. Consequently, our findings suggest that GSTK1-1 is not absolutely required for adiponectin multimerization in vivo and alternate pathways may be activated in GSTK1-1 deficiency.

Phenylalanine-induced leucopenia in genetic and dichloroacetic acid generated deficiency of glutathione transferase Zeta

Glutathione transferase Zeta (GSTZ1-1) is identical to maleylacetoacetate isomerase and catalyses a significant step in the catabolism of phenylalanine and tyrosine. Exposure of GSTZ1-1 deficient mice to high dietary phenylalanine causes a rapid loss of circulating white blood cells (WBCs). The loss was significant (P<0.05) after 2 days and total WBCs were reduced by 60% after 6 days. The rapid loss of WBCs was attributed to the accumulation of the catabolic intermediates maleylacetoacetate or maleylacetone (MA) in the circulation. Serum from GSTZ1-1 deficient mice treated with phenylalanine was cytotoxic to splenocytes from normal BALB/c mice and direct incubation of normal splenocytes with MA caused a rapid loss of viability. Dichloroacetic acid (DCA) has been used therapeutically to treat lactic acidosis and is potentially of use in cancer chemotherapy. Since DCA can inactivate GSTZ1-1 there is a possibility that long-term treatment of patients with DCA could cause GSTZ1-1 deficiency and susceptibility to oxidative stress and phenylalanine/tyrosine-induced WBC loss. However, although we found that DCA at 200mg/(kg day) causes a severe loss of hepatic GSTZ1-1 activity in BALB/c mice, it did not induce WBC cytotoxicity when combined with high dietary phenylalanine.

Glutathione transferase kappa deficiency causes glomerular nephropathy without overt oxidative stress

Glutathione transferase kappa (GSTK1-1) is a highly conserved, mitochondrial enzyme potentially involved in redox reactions. GSTK1-1-deficient mice were generated to further study the enzymes biological role. Reduced and total glutathione levels in liver and kidney were unchanged by GSTK1-1 deficiency and NADPH quinone oxidoreductase 1 expression was not elevated indicating that there is no general underlying oxidative stress in Gstk1−/− mice. Electron microscopy of liver and kidney showed no changes in mitochondrial morphology with GSTK1-1 deficiency. The death of a number of Gstk1−/− males with urinary tract problems prompted close examination of the kidneys. Electron microscopy revealed glomerular basement membrane changes at 3 months, accompanied by detectable microalbuminuria in male mice (albumin:creatinine ratio of 2.66±0.83 vs 1.13±0.20 mg/mmol for Gstk1−/− and wild-type (WT), respectively, P=0.001). This was followed by significant foot process effacement (40–55% vs 10% for Gstk1−/− and WT, respectively) at 6 months of age in all Gstk1−/− mice examined. Kidney tubules were ultrastructurally normal. Compared with human disease, the Gstk1−/− kidneys show changes seen in glomerulopathies causing nephrotic syndrome. Gstk1−/− mice may offer insights into the early development of glomerular nephropathies.

Dichloroacetic acid up-regulates hepatic glutathione synthesis via the induction of glutamate-cysteine ligase

Dichloroacetic acid (DCA) has potential for use in cancer therapy and the treatment of metabolic acidosis. However, DCA can create a deficiency of glutathione transferase Zeta (GSTZ1-1). Gstz1 knockout mice have elevated oxidative stress and low glutathione levels that increases their sensitivity to acetaminophen toxicity. As it is highly likely that patients that are treated with DCA will develop drug induced GSTZ1-1 deficiency we considered they could be at risk of elevated toxicity if they are exposed to other drugs that cause oxidative stress or consume glutathione (GSH). To test this hypothesis we treated mice with DCA and acetaminophen (APAP). Surprisingly, the mice pre-treated with DCA suffered less APAP-mediated hepatotoxicity than untreated mice. This protection is most likely due to an increased capacity for the liver to synthesize GSH, since DCA increased the expression and activity of glutamate-cysteine ligase GCL, the rate-limiting enzyme of GSH synthesis. Other pathways for acetaminophen disposal were unchanged or diminished by DCA. Pre-treatment with DCA may be of use in other settings where the maintenance of protective levels of GSH are required. However, DCA may lower the efficacy of drugs that rely on oxidative stress and the depletion of GSH to enhance their cytotoxicity or of drugs that are detoxified by GSH conjugation. Consequently, as the use of DCA in the clinic is likely to increase, it will be critical to evaluate the interactions of DCA with other drugs to ensure the combinations retain their efficacy and do not cause enhanced toxicity.

Glutathione transferase M2 variants inhibit ryanodine receptor function in adult mouse cardiomyocytes

Release of Ca(2+) from the sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential step in cardiac excitation-contraction coupling. Excess Ca(2+) release due to overactive RyR2 can cause arrhythmia that can lead to cardiac arrest. Fragments derived from the carboxy-terminal domain of human glutathione transferase M2 (GSTM2C) specifically inhibit RyR2 activity. Our aim was to further improve this inhibition by mutagenesis and to assess the therapeutic potential of GSTM2C based peptides to treat Ca(2+) release-based arrhythmia. We generated several mutant variants of the C-terminal fragment GSTM2C H5-8 and from those mutant proteins we identified two (RM13 and SM2) that exhibited significantly greater inhibition of cardiac SR Ca(2+) release and single RyR2 channel activity. Flow cytometry analysis showed that these two mutant proteins as well as GSTM2C H5-8 are taken up by isolated adult mouse cardiomyocytes without the aid of any additional compounds, Ca(2+) imaging and isolated cell contraction measurements revealed that GSTM2C H5-8, SM2 and RM13 reduce the SR Ca(2+) release rate and the fractional shortening of adult mouse cardiomyocytes, while importantly increasing the rate of Ca(2+) removal from the sarcoplasm. These observations indicate that peptides derived from GSTM2C inhibit RyR2 at a cellular level and thus they may provide the basis for a novel therapeutic agent to treat arrhythmia and heart attack.

Identification of a pathogenic variant in TREX1 in early-onset cerebral SLE by whole-exome sequencing

S S47 NOVEL GENETIC DISEASES ASSOCIATED WITH ATOPY