Amino acid sequence homology between rat prostatic steroid binding protein and rabbit uteroglobin

Using a computer program designed to detect evolutionary relationships between proteins, I find that the polypeptide chain of rabbit uteroglobin has amino acid sequence homology with the C1 and C2 polypeptide chains of rat prostatic steroid binding protein. Using this finding I suggest several interesting approaches for studying the biology of these proteins.     

Intestinal caveolin-1 is important for dietary fatty acid absorption

How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound ³H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the ³H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the ³H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with ³H-oleate or ³H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed ³H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.     

Protease signalling in cell death: caspases versus cysteine cathepsins

Proteases were, for a long time, mainly considered as protein degrading enzymes. However, in the last decade this view has changed dramatically, and the focus is now on proteases as signalling molecules. One of the best examples is apoptosis, the major mechanism used by eukaryotes to remove superfluous, damaged and potentially dangerous cells, in which a number of proteases have been found to play a central role. Of these the caspases have been considered to be the major players. However, more recently, other proteases have been increasingly suggested as being important in apoptosis, in particular the cysteine cathepsins. In this review the roles of caspases and cysteine cathepsins in apoptosis signalling are compared and discussed.     

The inhibitor of apoptosis protein family and its antagonists in acute leukemias

The Inhibitor of Apoptosis Protein family (IAP) functions as inhibitors of apoptotic pathways, both death receptor- and mitochondrial mediated. We detail the current body of knowledge for the IAP family with regard to their structure and function, their expression in normal and leukemic cells, and their prognostic importance in acute leukemia. Although there is some evidence that IAPs play an important role in the chemoresistance of leukemia cell lines, little is known about their influence on this phenomenon in acute leukemia cells of human origin. IAPs are also explored as a specific target for new antitumor strategies, including antisense oligonucleotides of XIAP (X-chromosome-linked IAP) or survivin and small molecules of polyphenylurea-based XIAP inhibitors. Several proteins negatively regulate the function of the IAP family. One of those antagonists is Smac/DIABLO. Short peptides of Smac were found to enhanced apoptosis, induced by chemo- or immunotherapy, in the leukemic cells in vitro. Moreover, small-molecule agents, resembling Smac/DIABLO in function, were shown to potentiate cytotoxicity of chemotherapy in different malignancies. IAPs, exhibiting downstream influence on both external and intrinsic pathways as well as on some caspase-independent mechanisms of apoptosis, are potentially attractive target for anti-tumor therapy, although their role in the pathology and prognosis of acute leukemia has to be further elucidated.     

Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism

We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.     

Paracrine repercussions of preconditioning on angiogenesis and apoptosis of endothelial cells

The mechanisms of cytoprotection conferred by stress preconditioning remain largely uncharacterized in endothelial cells (EC). We report that stress preconditioning of EC with serum starvation induces the release of soluble mediator(s) that confer resistance to apoptosis, increase proliferation, and enhance angiogenesis in a second set of "non-preconditioned" EC. Preconditioning was found to target specifically the mitochondrial control of apoptosis in EC with increased protein levels of Bcl-2, decreased protein levels of Bax, and decreased cytosolic release of cytochrome c. Regulators of apoptosis acting upstream and downstream of the mitochondria such as p53, cIAP-1, cIAP-2, and XIAP were not altered. Mediators classically associated with preconditioning in other cell types such as adenosine, opioids, and nitric oxide are not implicated in this cytoprotective loop. Blockade of protein kinase C-dependent signaling inhibited cytoprotection of EC. Further characterization of this paracrine pathway should provide insights into the molecular regulation of preconditioning in endothelial cells.     

Regulators of IAP function: coming to grips with the grim reaper

Inhibitor of apoptosis proteins (IAPs) are a conserved class of proteins that control apoptosis in both vertebrates and invertebrates. They exert their anti-apoptotic function through inhibition of caspases, the principal executioners of apoptotic cell death. Recent advances in vertebrates and Drosophila have demonstrated that IAPs use ubiquitin conjugation to control the stability, and thus the activity, of select target proteins. The Drosophila IAP1 gene is an instructive example: it employs at least two distinct ubiquitin-dependent mechanisms of protein destruction. The apoptosis-inducing genes grim, reaper and hid modulate these mechanisms, and determine the outcome.     

Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes

PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20 q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3′ end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3′ end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitiative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5′ flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.     

The BIR and BIR-like domains of Bombyx mori nucleopolyhedrovirus IAP2 protein are required for efficient viral propagation

The baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) possesses two genes, iap1 and iap2, which encode inhibitor of apoptosis (IAP) proteins. We previously showed that although both genes are dispensable for viral propagation, iap2 is required for efficient viral propagation in cultured cells. BmNPV IAP2 contains three putative functional domains: a baculovirus IAP repeat (BIR), a BIR-like (BIRL) domain, and a RING finger domain. To identify the domain affecting viral growth, we generated a series of BmNPV bacmids expressing iap2 derivatives lacking one or two domains, or possessing a single amino acid substitution to abolish IAP2 ubiquitin ligase activity. We examined their properties in both cultured cells and B. mori larvae. We found that either the BIR or BIRL domain of IAP2 plays an important role in BmNPV infection, and that the RING finger domain, which is required for ubiquitin ligase activity, does not greatly contribute to BmNPV propagation. This is the first study to identify functional domains of the baculovirus IAP2 protein.     

Recognition of Smac-mimetic compounds by the BIR domain of cIAP1

Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.