Jessica Wiwczar

BAD-Dependent Regulation of Fuel Metabolism and KATP Channel Activity Confers Resistance to Epileptic Seizures

Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses.

Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts

Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt tq209). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe–2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe–2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe–2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.

A Peptidomics Strategy to Elucidate the Proteolytic Pathways that Inactivate Peptide Hormones

Proteolysis plays a key role in regulating the levels and activity of peptide hormones. Characterization of the proteolytic pathways that cleave peptide hormones is of basic interest and can, in some cases, spur the development of novel therapeutics. The lack, however, of an efficient approach to identify endogenous fragments of peptide hormones has hindered the elucidation of these proteolytic pathways. Here, we apply a mass spectrometry (MS) based peptidomics approach to characterize the intestinal fragments of peptide histidine isoleucine (PHI), a hormone that promotes glucose-stimulated insulin secretion (GSIS). Our approach reveals a proteolytic pathway in the intestine that truncates PHI at its C-terminus to produce a PHI fragment that is inactive in a GSIS assay, a result that provides a potential mechanism of PHI regulation in vivo. Differences between these in vivo peptidomics studies and in vitro lysate experiments, which showed N- and C-terminal processing of PHI, underscore the effectiveness of this approach to discover physiologically relevant proteolytic pathways. Moreover, integrating this peptidomics approach with bioassays (i.e., GSIS) provides a general strategy to reveal proteolytic pathways that may regulate the activity of peptide hormones.

Phospho-BAD BH3 Mimicry Protects β Cells and Restores Functional β Cell Mass in Diabetes

Strategies that simultaneously enhance the survival and glucose responsiveness of insulin-producing β cells will greatly augment β cell replacement therapies in type 1 diabetes (T1D). We show that genetic and pharmacologic mimetics of the phosphorylated BCL-2 homology 3 (BH3) domain of BAD impart β-cell-autonomous protective effects in the face of stress stimuli relevant to β cell demise in T1D. Importantly, these benefits translate into improved engraftment of donor islets in transplanted diabetic mice, increased β cell viability in islet grafts, restoration of insulin release, and diabetes reversal. Survival of β cells in this setting is not merely due to the inability of phospho-BAD to suppress prosurvival BCL-2 proteins but requires its activation of the glucose-metabolizing enzyme glucokinase. Thus, BAD phospho-BH3 mimetics may prove useful in the restoration of functional β cell mass in diabetes.

Alternative Electron Acceptors for Photosystem II

Photosystem II (PSII) is conserved in all oxygenic photosynthetic organisms and is important for its unique ability to use energy from light to split water, generate molecular oxygen in the Earth’s atmosphere and drive electrons into the photosynthetic electron transport chain by reducing the plastoquinone (PQ) pool in the thylakoid membrane. The focus of this chapter is on alternative electron-transfer pathways on the acceptor side of PSII. Upon close examination of the literature there is evidence of exogenous electron acceptors that are reduced directly by the primary PQ electron acceptor (QA), bypassing the canonical terminal PQ-reduction (QB) site. These herbicide-insensitive electron-acceptor molecules include but are not limited to ferricyanide, synthetic cobalt coordination complexes, and cytochrome c. We also discuss experimental treatments to PSII such as cation exchange and herbicide treatment that have been shown to alter the redox midpoint potential (Em) of QA and impact electron transfer from QA to QB. The results described in this chapter provide a platform for understanding how electrons generated in PSII by photochemical water oxidation can be extracted from the electron-acceptor side of PSII for energy applications.

Corrigendum: Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts

This corrects the article DOI: 10.1038/nature11536

Erratum: Corrigendum: Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts

This corrects the article DOI: 10.1038/nature11536

Erratum: Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts (Nature (2012) 491 (608-612) doi:10.1038/nature11536)

This corrects the article DOI: 10.1038/nature11536