Confinement of caspase-12 proteolytic activity to autoprocessing.

Caspase-12 is a dominant-negative regulator of caspase-1 (IL-1beta-converting enzyme) and an attenuator of cytokine responsiveness to septic infections. This molecular role for caspase-12 appears to be akin to the role of cFLIP in regulating caspase-8 in the extrinsic cell death pathway; however, unlike cFLIP/Usurpin, we demonstrate here that caspase-12 is catalytically competent. To examine these catalytic properties, rat caspase-12 was cloned, and the recombinant enzyme was used to examine the cleavage of macromolecular and synthetic fluorogenic substrates. Although caspase-12 could mediate autoproteolytic maturation of its own proenzyme, in both cis and trans, it was not able to cleave any other polypeptide substrate, including other caspase proenzymes, apoptotic substrates, cytokine precursors, or proteins in the endoplasmic reticulum that normally undergo caspase-mediated proteolysis. The dearth of potential substrates for caspase-12 also was confirmed by whole-cell diagonal-gel analysis. Autolytic cleavage within the caspase-12 proenzyme was mapped to a single site at the large-small subunit junction, ATAD(319), and this motif was recognized by caspase-12 when incorporated into synthetic fluorogenic substrates. The specific activity of caspase-12 with these substates was several orders of magnitude lower than caspases-1 and -3, highlighting its relative catalytic paucity. In intact cells, caspase-12 autoproteolysis occurred in the inhibitory complex containing caspase-1. We propose that the proteolytic activity of caspase-12 is confined to its own proenzyme and that autocleavage within the caspase-1 complex may be a means for temporal limitation of the inhibitory effects of caspase-12 on proinflammatory cytokine maturation.

Biochemical characterization of cholesteryl ester transfer protein inhibitors.

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

Caspase-7 expanded function and intrinsic expression level underlies strain-specific brain phenotype of caspase-3-null mice.

Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain-dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3-/-) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with lack of DNA fragmentation in 129-Casp3-/- apoptotic precursor neurons, whereas B6-Casp3-/- cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation. The amount of caspase-7 activation in apoptotic precursor neurons is independent of the presence of caspase-3. Together, our findings demonstrate for the first time a strong correlation between caspase-7 activity, normal brain development, and apoptotic DNA fragmentation in Casp3-/- mice.

Identification of genes affecting apolipoprotein B secretion following siRNA-mediated gene knockdown in primary human hepatocytes.

OBJECTIVE: Genome-wide association studies (GWAS) are useful in studying the complex pathways underlying diseases such as atherosclerosis; however, additional testing is often necessary to identify the disease causal genes linked to GWAS loci. We used siRNA-mediated gene knockdown in primary human hepatocytes (PHuH) to identify potential GWAS causal genes affecting the hepatic secretion of apolipoprotein B (ApoB), ApoA1, and proprotein convertase subtilisin/kexin type 9. MATERIALS AND METHODS: Candidate causal genes within GWAS loci affecting human plasma levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were identified from the literature; 191 genes were selected from 74 loci. A functional siRNA screen was performed using PHuH. RESULTS: Four genes: poly (ADP-ribose) polymerases member 10, haptoglobin, fucosyltransferase 1, and lysophosphatidic acid receptor 2 were identified and confirmed. Knocking down these genes reduced cell-associated and secreted ApoB levels. CONCLUSION: Modification of these four genes may affect plasma lipids through modulation of ApoB secretion.

Hippi is essential for node cilia assembly and Sonic hedgehog signaling.

Hippi functions as an adapter protein that mediates pro-apoptotic signaling from poly-glutamine-expanded huntingtin, an established cause of Huntington disease, to the extrinsic cell death pathway. To explore other functions of Hippi we generated Hippi knock-out mice. This deletion causes randomization of the embryo turning process and heart looping, which are hallmarks of defective left-right (LR) axis patterning. We report that motile monocilia normally present at the surface of the embryonic node, and proposed to initiate the break in LR symmetry, are absent on Hippi-/- embryos. Furthermore, defects in central nervous system development are observed. The Sonic hedgehog (Shh) pathway is downregulated in the neural tube in the absence of Hippi, which results in failure to establish ventral neural cell fate. Together, these findings demonstrate a dual role for Hippi in cilia assembly and Shh signaling during development, in addition to its proposed role in apoptosis signal transduction in the adult brain under pathogenically stressful conditions.

The cell adhesion molecule CEACAM1-L is a substrate of caspase-3-mediated cleavage in apoptotic mouse intestinal cells.

The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms.