Design, Heterologous Expression, and Application of an Immobilized Protein Kinase.

Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the "central kinase". An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.

Activation of Nrf2 Is Required for Normal and ChREBPα-Augmented Glucose-Stimulated ß-Cell Proliferation.

Patients with both major forms of diabetes would benefit from therapies that increase ß-cell mass. Glucose, a natural mitogen, drives adaptive expansion of ß-cell mass by promoting ß-cell proliferation. We previously demonstrated that a carbohydrate response element-binding protein (ChREBPα) is required for glucose-stimulated ß-cell proliferation and that overexpression of ChREBPα amplifies the proliferative effect of glucose. Here we found that ChREBPα reprogrammed anabolic metabolism to promote proliferation. ChREBPα increased mitochondrial biogenesis, oxygen consumption rates, and ATP production. Proliferation augmentation by ChREBPα required the presence of ChREBPß. ChREBPα increased the expression and activity of Nrf2, initiating antioxidant and mitochondrial biogenic programs. The induction of Nrf2 was required for ChREBPα-mediated mitochondrial biogenesis and for glucose-stimulated and ChREBPα-augmented ß-cell proliferation. Overexpression of Nrf2 was sufficient to drive human ß-cell proliferation in vitro; this confirms the importance of this pathway. Our results reveal a novel pathway necessary for ß-cell proliferation that may be exploited for therapeutic ß-cell regeneration.

The Transcription Factor ATF5 Mediates a Mammalian Mitochondrial UPR.

Mitochondrial dysfunction is pervasive in human pathologies such as neurodegeneration, diabetes, cancer, and pathogen infections as well as during normal aging. Cells sense and respond to mitochondrial dysfunction by activating a protective transcriptional program known as the mitochondrial unfolded protein response (UPR(mt)), which includes genes that promote mitochondrial protein homeostasis and the recovery of defective organelles [1, 2]. Work in Caenorhabditis elegans has shown that the UPR(mt) is regulated by the transcription factor ATFS-1, which is regulated by organelle partitioning. Normally, ATFS-1 accumulates within mitochondria, but during respiratory chain dysfunction, high levels of reactive oxygen species (ROS), or mitochondrial protein folding stress, a percentage of ATFS-1 accumulates in the cytosol and traffics to the nucleus where it activates the UPR(mt) [2]. While similar transcriptional responses have been described in mammals [3, 4], how the UPR(mt) is regulated remains unclear. Here, we describe a mammalian transcription factor, ATF5, which is regulated similarly to ATFS-1 and induces a similar transcriptional response. ATF5 expression can rescue UPR(mt) signaling in atfs-1-deficient worms requiring the same UPR(mt) promoter element identified in C. elegans. Furthermore, mammalian cells require ATF5 to maintain mitochondrial activity during mitochondrial stress and promote organelle recovery. Combined, these data suggest that regulation of the UPR(mt) is conserved from worms to mammals.