Jennifer M. Lewis

XIAP inhibition of caspase-3 preserves its association with the Apaf-1 apoptosome and prevents CD95- and Bax-induced apoptosis.

Ligation of death receptors or formation of the Apaf-1 apoptosome results in the activation of caspases and execution of apoptosis. We recently demonstrated that X-linked inhibitor-of-apoptosis protein (XIAP) associates with the apoptosome in vitro. By utilizing XIAP mutants, we now report that XIAP binds to the ‘native’ apoptosome complex via a specific interaction with the small p12 subunit of processed caspase-9. Indeed, we provide the first direct evidence that XIAP can simultaneously bind active caspases-9 and -3 within the same complex and that inhibition of caspase-3 by the Linker-BIR2 domain prevents disruption of BIR3-caspase-9 interactions. Recent studies suggest that inhibition of caspase-3 is dispensable for its anti-apoptotic effects. However, we clearly demonstrate that inhibition of caspase-3 is required to inhibit CD95 (Fas/Apo-1)-mediated apoptosis, whereas inhibition of either caspase-9 or caspase-3 prevents Bax-induced cell death. Finally, we illustrate for the first time that XIAP mutants, which are incapable of binding to caspases-9 and -3 are completely devoid of anti-apoptotic activity. Thus, XIAPs capacity to maintain inhibition of caspase-9 within the Apaf-1 apoptosome is influenced by its ability to simultaneously inhibit active caspase-3, and depending upon the apoptotic stimulus, inhibition of caspase-9 or 3 is essential for XIAPs anti-apoptotic activity.

Uncoupling of the Signaling and Caspase-inhibitory Properties of X-linked Inhibitor of Apoptosis

In addition to its well described function as an enzymatic inhibitor of specific caspases, X-linked inhibitor of apoptosis (X-linked IAP or XIAP) can function as a cofactor in Smad, NF-κB, and JNK signaling pathways. However, caspases themselves have been shown to regulate the activity of a number of signaling cascades, raising the possibility that the effect of XIAP in these pathways is indirect. Here we examine this question by introducing point mutations in XIAP predicted to disrupt the ability of the molecule to bind to and inhibit caspases. We show that whereas these mutant variants of XIAP lost caspase-inhibitory activity, they maintained their ability to activate Smad, NF-κB, and JNK signaling pathways. Indeed, the signaling properties of the molecule were mapped to domains not directly involved in caspase binding and inhibition. The activation of NF-κB by XIAP was dependent on the E3 ubiquitin ligase activity of the RING domain. On the other hand, the ability of XIAP to activate Smad-dependent signaling was mapped to the third baculoviral IAP repeat (BIR) and loop regions of the molecule. Thus, the anti-apoptotic and signaling properties of XIAP can be uncoupled.

Xaf1 can cooperate with TNFα in the induction of apoptosis, independently of interaction with XIAP

XIAP-associated factor 1 (Xaf1) binds XIAP and re-localizes it to the nucleus, thus inhibiting XIAP activity and enhancing apoptosis [1]. Xaf1 expression is reduced or absent in tumor samples and cell lines suggesting it may function as a tumor suppressor [2–5]. To further study Xaf1 function we generated Xaf1 inducible cells in the osteosarcoma cell line Saos-2. Despite Xaf1 inducing apoptosis that is dramatically enhanced by TNFα we find no evidence for an interaction between Xaf1 and XIAP. Furthermore, Xaf1 expression sensitized XIAP−/− fibroblasts to TNFα, demonstrating the existence of a novel mechanism of Xaf1 induced apoptosis distinct from antagonizing XIAP. Xaf1 expression promotes cytochrome c release that cannot be blocked by inhibition of caspase activity. This implicates a role for the mitochondrial apoptotic pathway, consistent with the ability of Bcl2 to block Xaf1 induced apoptosis. The data indicate that in Saos2 cells Xaf1 activates the mitochondrial apoptotic pathway to facilitate cytochrome c release, thus amplifying apoptotic signals from death receptors.

Use of Hyperosmolar Stress to Measure Stress-Activated Protein Kinase Activation and Function in Human HTR Cells and Mouse Trophoblast Stem Cells

Embryo growth is inversely correlated with hyperosmolar stress-induced stress-activated protein kinase/jun kinase (SAPK/JNK) induction. To examine whether stress has similar effects in stem cells derived from the embryo, the authors test trophoblast stem cells. The stress response of human placental and mouse trophoblast stem cell lines are tested here. Peak phosphorylated SAPK/JNK was induced by 400 mM sorbitol at 0.5 hours. At this dose, there is an SAPK/JNK-dependent decrease in mitogenic, phosphorylated cMyc at 0.5 hours preceding an SAPK/JNK-dependent decrease in cell cycle entrance at 24 hours. At 0.5 hours, SAPK/JNK decreases terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis at sorbitol doses from 50 mM to 400 mM and induces phosphorylated cJun prior to an SAPK/JNK-dependent, approximate 8-fold increase in apoptosis by 24 hours at 400 mM. SAPK/JNK phosphorylation peaked at 0.5 to 4 hours and largely subsided by 12 hours. Thus, total SAPK/JNK exists before stress and mediates rapid, homeostatic molecular responses that become biologic consequences after phosphorylated SAPK/JNK ends. This suggests continuity in the homeostatic mechanisms and functions of SAPK/JNK in placental lineage cells during implantation, in which SAPK/JNK is completely responsible for cell cycle arrest and largely responsible for apoptosis.

Reading between the lines: Teaching linear algebra

This paper compares lessons on linear equations from the same curriculum materials taught by two teachers of different levels of mathematical knowledge for teaching (MKT). The analysis indicates that the mathematical quality of instruction in these two classrooms appears to be a function of differences in MKT. Although the two teachers were teaching from the same curriculum materials, the teacher with higher MKT had more complete and concise ways to describe key concepts, had multiple ways to represent ideas about linear equations, could move nimbly among different mathematical expressions of linear relationships, and gave students a larger role in articulating the mathematical ideas of the lesson. However, curriculum materials seem to have moderated what would otherwise have been larger disparities in the quality of instruction between the two teachers. The lower-MKT teacher made minor mathematical errors, stayed on topic, and defined concepts in reasonably accurate ways when he followed the curriculum materials closely.

Strategies for Assessing Classroom Teaching: Examining Administrator Thinking as Validity Evidence

ABSTRACT All 50 states use observations to evaluate practicing teachers, but we know little about how administrators actually reason when they use those observation protocols. Drawing on think-aloud and stimulated recall data, this study describes the types of strategies and warrants practicing administrators used when rating with their district’s observation protocol. Administrators in a large urban district used an observation protocol aligned to Danielson’s Framework for Teaching to rate a brief lesson clip. Administrators’ thinking was recorded, clarified, and inductively coded. Findings suggest administrator thinking and justification is complex even for short lengths of instruction. Administrators used a range of reasoning strategies, many of which were not sanctioned by their training. Exploratory analyses suggest strategy use was not related to the accuracy of ratings. Implications for the validity of teacher observation scores in high-stakes settings are considered.