Apoptosis inducing factor (AIF) is a mediator of caspase-independent cell death that is also necessary for mitochondrial energy production. How these seemingly opposite cellular functions of AIF are controlled is poorly understood. X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspases that also regulates several caspase-independent signaling pathways. The RING domain of XIAP possesses E3 ubiquitin ligase activity, though the importance of this function to signal regulation remains incompletely defined. XIAP binds and ubiquitinates AIF, and in this study, we determined the functional consequences of XIAP-mediated AIF ubiquitination. Unlike canonical ubiquitination, XIAP-dependent AIF ubiquitination did not lead to proteasomal degradation of AIF. Experiments using ubiquitin mutants demonstrated that the XIAP-dependent ubiquitin linkage was not formed through the commonly used lysine 48, suggesting a noncanonical ubiquitin linkage is employed. Further studies demonstrated that only lysine 255 of AIF was a target of XIAP-dependent ubiquitination. Using recombinant AIF, we determined that mutating lysine 255 of AIF interferes with the ability of AIF not only to bind DNA but also to degrade chromatin in vitro. These data indicate that XIAP regulates the death-inducing activity of AIF through nondegradative ubiquitination, further defining the role of XIAP in controlling AIF and caspase-independent cell death pathways.

NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47phox. NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1β PX.

The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and nonnative). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. V C 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60-68, 2017.

Polymers are ubiquitous and essential to modern society, which is why the American Chemical Society has mandated inclusion of polymer chemistry in the undergraduate curriculum. To meet this requirement, we have chosen to weave a polymeric theme through multiple laboratory courses beginning with organic chemistry, where students use aspartic acid to synthesize poly(aspartic acid), an ecofriendly alternative to nonbiodegradable poly(carboxylates). Subsequently, these student-synthesized polymers serve as substrates for the enzyme poly(aspartic acid) hydrolase-1 in our biochemistry course. This experiment introduces the concept of biodegradation through a gel assay that allows students to visualize enzyme-mediated polymer degradation. Students learn the difference between mono-and polydisperse polymers, how biodegradation affects the size of a polymer through analysis of mobility shifts in an agarose gel, and how to use densitometry software to calculate enzyme activity. Finally, keeping the same polymeric theme provides a source of continuity in our curriculum while expanding students' understanding of polymer chemistry from the viewpoint of different chemistry disciplines.

NOXO1β is a cytosolic protein which, in conjunction with NOXA1, regulates generation of reactive oxygen species (ROS) by the Nox1 enzyme complex. NOXO1β is targeted to membranes through an N-terminal PX domain. We have used nuclear magnetic resonance spectroscopy to solve the structure of the NOXO1β PX domain and surface plasmon resonance to assess phospholipid specificity. The solution structure of the NOXO1β PX domain shows greatest similarity to the PI3K-C2α PX domain with regard to the positions and types of residues that are predicted to interact with phosphatidylinositol phosphate (PtdInsP) head groups. Surface plasmon resonance experiments identify PtdIns(4,5)P2 and PtdIns(3,4,5)P3 as preferred targets of NOXO1β PX. These findings contrast with previous lipid overlay experiments which show strongest binding to monophosphorylated PtdInsP and phosphatidylserine. Our data suggest that localized membrane accumulation of PtdIns(4,5)P2 or PtdIns(3,4,5)P2 may serve to recruit NOXO1β and activate Nox1.