Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.

Dichloroacetate Prevents Cisplatin-Induced Nephrotoxicity without Compromising Cisplatin Anticancer Properties.

Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin.

Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition.

The skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(-)RyR1 ratio. The results suggest that the reduced expression of ASI(-)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(-) RyR1, having a higher gain of in Ca(2+) release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers.

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 enzyme's 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.

Mouse strains with point mutations in TAP1 and TAP2.

We report two new mouse strains: Jasmine (C57BL/6J/Apb-Tap2jas/Apb), with a point mutation in the transporter associated with antigen processing (TAP)2 ; and Rose, (C57BL/6J/Apb-Tap1rose/Apb), with a point mutation in TAP1. These strains were detected as the result of ethyl nitroso urea (ENU) screens for recessive point mutations affecting the immune system. As expected in cases of defective TAP expression, the mice have very low major histocompatibility complex (MHC)-I cell-surface expression, and few CD8(+) T cells. The Rose strain has an A to T substitution in exon 10 of TAP1, resulting in an asparagine to valine substitution at position 643. Jasmine has an A to C transversion in exon 5 of TAP2, resulting in a threonine to proline substitution at position 293 of the protein. The mutation does not affect mRNA levels, but results in a very severe reduction in TAP2 protein. TAP1 protein levels are also decreased in Jasmine mice, demonstrating a new role for mouse TAP2 in stabilizing TAP1 protein expression. Jasmine is the first strain available with defective TAP2. The two mouse strains provide additional animal models for the human condition Bare Lymphocyte syndrome type 1, and identify new residues important for TAP function.

Splice variants of the condensin II gene Ncaph2 include alternative reading frame translations of exon 1.

Condensins I and II are five-protein complexes that are important for the condensation of chromatin. They are essential for mitosis and important for regulating gene expression during interphase. Here, we investigated the transcription and translation of the mouse Ncaph2 gene, which encodes a subunit of condensin II. We identified three splice variants within the first exon, a NAGNAG splice variant at the beginning of exon 16 and alternative 3'-UTRs. In total, Ncaph2 is potentially capable of generating 12 unique mRNA transcripts and six unique proteins. We confirm that Ncaph2 can generate three different N-termini, all encoded by exon 1, one of which is translated from an alternative reading frame. This alternative reading frame splice variant appears to be a novel outcome of splicing. If this is applicable to other genes, it would account for a previously unappreciated level of eukaryotic protein diversity.

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.

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.

A mutation in a chromosome condensin II subunit, kleisin beta, specifically disrupts T cell development.

Condensins are ubiquitously expressed multiprotein complexes that are important for chromosome condensation and epigenetic regulation of gene transcription, but whose specific roles in vertebrates are poorly understood. We describe a mouse strain, nessy, isolated during an ethylnitrosourea screen for recessive immunological mutations. The nessy mouse has a defect in T lymphocyte development that decreases circulating T cell numbers, increases their expression of the activation/memory marker CD44, and dramatically decreases the numbers of CD4(+)CD8(+) thymocytes and their immediate DN4 precursors. A missense mutation in an unusual alternatively spliced first exon of the kleisin beta gene, a member of the condensin II complex, was shown to be responsible and act in a T cell-autonomous manner. Despite the ubiquitous expression and role of condensins, kleisin beta(nes/nes) mice were viable, fertile, and showed no defects even in the parallel pathway of B cell lymphocyte differentiation. These data define a unique lineage-specific requirement for kleisin beta in mammalian T cell differentiation.