The transcription factor SP3 drives TNF-α expression in response to Smac mimetics.

The controlled production and downstream signaling of the inflammatory cytokine tumor necrosis factor-α (TNF-α) are important for immunity and its anticancer effects. Although chronic stimulation with TNF-α is detrimental to the health of the host in several autoimmune and inflammatory disorders, TNF-α-contrary to what its name implies-leads to cancer formation by promoting cell proliferation and survival. Smac mimetic compounds (SMCs), small-molecule antagonists of inhibitor of apoptosis proteins (IAPs), switch the TNF-α signal from promoting survival to promoting death in cancer cells. Using a genome-wide siRNA screen to identify factors required for SMC-to-TNF-α-mediated cancer cell death, we identified the transcription factor SP3 as a critical molecule in both basal and SMC-induced production of TNF-α by engaging the nuclear factor κB (NF-κB) transcriptional pathway. Moreover, the promotion of TNF-α expression by SP3 activity confers differential sensitivity of cancer versus normal cells to SMC treatment. The key role of SP3 in TNF-α production and signaling will help us further understand TNF-α biology and provide insight into mechanisms relevant to cancer and inflammatory disease.

Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts.

BACKGROUND: SIFD (Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay) is a novel form of congenital sideroblastic anemia associated with B-cell immunodeficiency, periodic fevers, and developmental delay caused by mutations in the CCA-adding enzyme TRNT1, but the precise molecular pathophysiology is not known. RESULTS: We show that the disease causing mutations in patient-derived fibroblasts do not affect subcellular localization of TRNT1 and show no gross morphological differences when compared to control cells. Analysis of cellular respiration and oxidative phosphorylation (OXPHOS) complexes demonstrates that both basal and maximal respiration rates are decreased in patient cells, which may be attributed to an observed decrease in the abundance of select proteins of the OXPHOS complexes. CONCLUSIONS: Our data provides further insight into cellular pathophysiology of SIFD.

Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD).

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation.

In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways) or counteract these signals (anti-apoptotic pathways). We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia). Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56) content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia) whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase). The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor.

Coping with the stress: expression of ATF4, ATF6, and downstream targets in organs of hibernating ground squirrels.

Perturbation of the endoplasmic reticulum (ER) protein folding apparatus via any one of several environmental or metabolic stresses rapidly triggers a complex program of cellular responses that is termed the unfolded protein response (UPR). Stresses that trigger this response in mammals can include low temperature, hypoxia, ischemia, and oxidative stress. All of these can be natural features of mammalian hibernation, and hence the UPR might be integral to long term survival in a state of cold torpor. The present study analyzes changes in gene and/or protein expression of multiple markers of the UPR in tissues of euthermic (control) versus hibernating ground squirrels, Spermophilus tridecemlineatus. Immunoblot analysis of ATF4 protein expression revealed strong increases of 1.9- to 2.5-fold in brown adipose tissue, skeletal muscle, and brain during hibernation. However, transcript levels of atf4 were unchanged or lowered which suggests that ATF4 protein levels were regulated at the translational level. Subcellular localization studies showed that ATF4 translocated into the nucleus during hibernation, as did its cofactor, the phosphorylated form of CREB-1, which rose by 25- to 39-fold in nuclear extracts of brain and skeletal muscle of torpid animals. The responses of other proteins involved in the UPR including p-PERK, ATF6, GADD153, and GADD34 were also evaluated. The data suggest that ATF4 up-regulation may play an important role in coordinating gene expression responses that support the hibernating phenotype.

Up-regulation of the endoplasmic reticulum molecular chaperone GRP78 during hibernation in thirteen-lined ground squirrels.

Hibernating mammals endure conditions of low body temperature and oxidative stress that would be highly injurious to humans and most other mammals. Stress conditions frequently trigger the production of molecular chaperones; in the endoplasmic reticulum the glucose-regulated protein-78 (GRP78) helps to minimize protein misfolding under stress. The present study evaluated the GRP78 response in seven organs of hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Transcript levels of grp78, assessed by RT-PCR, were significantly higher (3.5- to 4.1-fold) in brown adipose tissue and brain of hibernating squirrels compared with euthermic control animals but remained low or stable in all other tissues. GRP78 protein content, assessed by Western blotting, was also elevated in brown adipose and brain during hibernation by 1.4-1.6 fold. A 2490 bp cDNA sequence was retrieved that contained the full length open reading frame of ground squirrel grp78 and the translated protein sequence of 654 amino acids shared 98-99% identity with GRP78 from other mammalian sources. Selected specific amino acid substitutions were found in the ground squirrel sequence that may aid GRP78 function under the near 0 degrees C body temperatures of the hibernating state. Electrophoretic mobility shift and supershift assays showed that the activating transcription factor, ATF4, binds to the promoter region of the grp78 gene in ground squirrel brain and may be responsible for grp78 up-regulation during hibernation. Changes in grp78 gene and protein expression appear to aid stress tolerance in two highly oxygen-dependent organs that are critical to whole animal survival during hibernation.