Judy K. Cundiff

The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress.

This study shows that the eIF2 kinase PERK is required not only for translational control but also for activation of ATF6 and its target genes in the unfolded protein response. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the endoplasmic reticulum to the Golgi for intramembrane proteolysis and activation of ATF6.

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.

The eIF2 kinase GCN2 is essential for the murine immune system to adapt to amino acid deprivation by asparaginase.

Amino acid starvation by asparaginase (ASNase) enhances phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible 2 (GCN2) kinase, leading to reduced global mRNA translation rates. This conserves energy and allows cells time to reprogram stress-related gene expression to alleviate cell injury. This study addressed the importance of GCN2 for the immune system to adapt to amino acid starvation by ASNase. GCN2(+/+) and GCN2(-/-) mice were injected once daily with ASNase or saline for up to 7 d. In both thymus and spleen, activation of amino acid stress response genes to ASNase, such as asparagine synthetase and CAAT enhancer binding protein homologous protein, required GCN2. ASNase reduced food intake and body weight in both genotypes, but spleen and thymus wet weights and total cell numbers in thymus, spleen, bone marrow, and mesenteric lymph nodes were less in GCN2(-/-) mice treated with ASNase (genotype x ASNase, P < 0.05). In the thymus, GCN2(-/-) mice treated with ASNase demonstrated enhanced apoptosis and fewer cells in all subpopulations examined (CD3+, CD4-8-, CD4+8+, CD4+8-, CD4-8+) compared with GCN2(+/+) mice treated with ASNase (genotype x ASNase, P < 0.05). In the spleen, GCN2 deletion magnified ASNase-induced reductions in CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ leukocytes (genotype x ASNase, P < 0.05). These results indicate that loss of GCN2 enhances immunosuppression by ASNase and that this eIF2 kinase is broadly required for amino acid stress management in the immune system.

Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

Anti-STAT6 CTL activity in Stat6−/− mice

The generation of germline gene mutations in mice has been an invaluable tool for experimental biology. However, studying immune responses that develop in the absence of a specific protein that could alter thymic selection complicates experimental interpretations. We observed that CD8+ T cells from Stat6−/− mice displayed “autoreactivity” to STAT6-expressing cells, associated with specific STAT6 peptides binding to MHC class I molecules. These results suggest caution in interpreting experiments where STAT6-expressing cells are transferred into Stat6−/− mice, or where adoptive transfer of Stat6−/− lymphocytes is performed. Our results further highlight additional considerations when studying immune responses involving cell transfer into gene-deficient mice.

Anti-STAT6 CTL activity in Stat6−/− mice: A cautionary tale

The generation of germline gene mutations in mice has been an invaluable tool for experimental biology. However, studying immune responses that develop in the absence of a specific protein that could alter thymic selection complicates experimental interpretations. We observed that CD8+ T cells from Stat6−/− mice displayed “autoreactivity” to STAT6-expressing cells, associated with specific STAT6 peptides binding to MHC class I molecules. These results suggest caution in interpreting experiments where STAT6-expressing cells are transferred into Stat6−/− mice, or where adoptive transfer of Stat6−/− lymphocytes is performed. Our results further highlight additional considerations when studying immune responses involving cell transfer into gene-deficient mice.

Anti-STAT6 CTL activity inStat6−/−mice: A cautionary tale

The generation of germline gene mutations in mice has been an invaluable tool for experimental biology. However, studying immune responses that develop in the absence of a specific protein that could alter thymic selection complicates experimental interpretations. We observed that CD8+ T cells from Stat6−/− mice displayed “autoreactivity” to STAT6-expressing cells, associated with specific STAT6 peptides binding to MHC class I molecules. These results suggest caution in interpreting experiments where STAT6-expressing cells are transferred into Stat6−/− mice, or where adoptive transfer of Stat6−/− lymphocytes is performed. Our results further highlight additional considerations when studying immune responses involving cell transfer into gene-deficient mice.