The endoplasmic reticulum (ER) transmembrane proteins, ATF6␣ and ATF6␤, are cleaved in response to ER stress, which can be induced by tunicamycin. The resulting N-terminal fragments of both ATF6 isoforms, which have conserved basic leucine-zipper and DNA binding domains but divergent transcriptional activation domains, translocate to the nucleus where they bind to ER stress-response elements (ERSE) in ER stress-response genes (ERSRG), such as GRP78. Although it is known that ATF6␣ is a potent activator of ERSRGs, the transcriptional potency and functions of ATF6␤ remain to be explored. Accordingly, N-terminal fragments of each ATF6 isoform (N-ATF6␣ and N-ATF6␤) were overexpressed in HeLa cells and the effects on GRP78 induction were assessed. When expressed at similar levels, N-ATF6␣ conferred ϳ200-fold greater GRP78 promoter activation than N-ATF6␤. Because ER stress activates nuclear translocation of both ATF6␣ and ␤ and because both bind to ERSEs, the effect of co-expressing them on GRP78 induction was assessed. Surprisingly, N-ATF6␤ inhibited N-ATF6␣-mediated GRP78 promoter activation in a dominant-negative manner. Moreover, N-ATF6␤ inhibited TN-mediated GRP78 promoter activation, which requires endogenous ATF6␣. ATF6 isoform-specific small inhibitory RNAs were used to show that, as expected, endogenous ATF6␣ was required for maximal ERSRG induction; however, endogenous ATF6␤ moderated ERSRG induction. These results indicate that compared with ATF6␣, ATF6␤ is a very poor activator of ERSRG induction and it represses ATF6␣-mediated ERSRG induction. Thus, ATF6␤ may serve as a transcriptional repressor functioning in part to regulate the strength and duration of ATF6␣-mediated ERSRG activation during the ER stress response.

Abstract-␣B-Crystallin (␣BC), a small heat shock protein expressed in high levels in the heart, is phosphorylated on 45, and 59 after stress. However, it is not known whether ␣BC phosphorylation directly affects cell survival.In the present study, constructs were prepared that encode forms of ␣BC harboring Ser to Ala (blocks phosphorylation) or Ser to Glu (mimics phosphorylation) mutations at positions 19, 45, and 59. The effects of each form on apoptosis of cultured cardiac myocytes after hyperosmotic or hypoxic stress were assessed. Compared with controls, cells that expressed ␣BC with Ser to Ala substitutions at all three positions, ␣BC(AAA), exhibited more stress-induced apoptosis. Cells expressing either ␣BC(AAE) or (EEE) exhibited 3-fold less apoptosis than cells expressing ␣BC(AAA), indicating that phosphorylation of Ser-59 confers protection. ␣BC is known to bind to procaspase-3 and to decrease caspase-3 activation. Compared with cells expressing ␣BC(AAA), the activation of caspase-3 was decreased by 3-fold in cells expressing ␣BC(AAE). These results demonstrate that mimicking the phosphorylation of ␣BC on Ser-59 is necessary and sufficient to confer caspase-3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress. These findings provide direct evidence that ␣BC(S59P) contributes to the cardioprotection observed after physiologically relevant stresses, such as transient hypoxia. Identifying the targets of ␣BC(S59P) will reveal important details about the mechanism underlying the cytoprotective effects of this small heat shock protein.

Overexpression studies have shown that the small heat shock proteins (sHSP) protect the myocardium from ischemia-reperfusion (I/R)-induced damage. However, gene deletion studies are necessary to demonstrate whether sHSPs are required for protection. The genes for alphaB-crystallin (alphaBC) and HSPB2, two sHSPs that are expressed in high levels in the heart, are in close proximity to one another; as a result, both genes were disrupted in a recently generated knockout (KO) mouse line. The alphaBC/HSPB2 KO mouse line is currently the only model that features disruption of sHSPs normally expressed in the heart. Accordingly, we examined the cardiac morphology, function, and response to I/R-induced stress in alphaBC-HSPB2 KO mice. Initial gross, light microscopic and echocardiographic characterization showed that the morphological and functional properties of hearts from adult KO mice were indistinguishable from age-matched wild-type (WT) mice. Electron microscopy showed that, compared with WT mouse hearts, KO mouse heart sarcomeres were relatively normal. Isolated perfused KO mouse hearts displayed normal contractility; however, when compared with WT, after I/R, KO mouse hearts exhibited a twofold reduction in contractile recovery, as well as increased necrosis and apoptosis. Additionally, when compared with WT, KO mouse hearts exhibited 43% less reduced glutathione, which is known to protect from I/R-induced damage. Thus, whereas neither alphaBC nor HSPB2 is essential for myocardial development and function under nonstressful conditions, one or both are required for maximal functional recovery and protection from I/R-induced necrosis and apoptosis.

ATF6 is a 670-amino acid endoplasmic reticulum (ER) transmembrane protein that is cleaved in response to ER stress. The resulting N-terminal fragment of ϳ400 amino acids translocates to the nucleus and activates selected ER stress-inducible genes, such as GRP-78 and sarco/endoplasmic reticulum ATPase, which are required for cell survival. In studying the mechanism of ATF6-activated transcription, we found that when HeLa cells were transfected with a plasmid encoding ATF6-(1-373), ER stress-inducible reporter gene activation was high, but ATF6-(1-373) expression was low, unless a proteasome inhibitor was added. In contrast, transfection with a plasmid encoding ATF6-(94 -373) resulted in low reporter activation and high expression of ATF6-(94 -373), which was independent of the proteasome inhibitor. Thus, the information responsible for transcriptional activation and proteasomal degradation must lie within the N-terminal 93 amino acids of ATF6. This portion of ATF6 was found to be homologous to the herpes simplex viral protein, VP16. One 8-amino acid domain of particular interest in this region of ATF6 is 75% identical to the VN8 region in VP16. VN8 is required for VP16-mediated transcription, as well as rapid degradation of VP16 by proteasomes. Point mutations in the VN8-like region of ATF6 caused a loss of transcription, increased expression levels, and an increase in half-life. Thus, the potent transcriptional activities and rapid degradation of ATF6 and VP16 require the VN8 domains in each protein. Homology searches indicate that ATF6 is the only eukaryotic protein known that possesses an active VN8 domain, raising questions about how this domain evolved and the functional importance underlying its appearance in only these two transcription factors.

Phosphoinositide-3-kinase δ (PI3Kδ) is a critical regulator of cell growth and transformation and has been explored as a therapeutic target for a range of diseases. Through the exploration of the thienopyrimidine scaffold, we have identified a ligand-efficient methylation that leads to remarkable selectivity for PI3Kδ over the closely related isoforms. Interrogation through the Free–Wilson analysis highlights the innate selectivity the thienopyrimidine scaffold has for PI3Kδ and provides a predictive model for the activity against the PI3K isoforms.