The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.
We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.
The substrate specificities of two human prohormone convertases, furin and PC1, were examined with a series of 7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates. Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys or Orn resulted for furin in a 538- and a 280-fold lower kcat/Km value, but only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by either Pro, Glu, or Lys does not modify significantly the kcat/Km value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the kcat/Km value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of kcat/Km was due primarily to lower kcat rather than higher Km, possibly because of the presence of a negatively charged side chain. Finally, an octapeptidyl chloromethane derivative proved to be a potent irreversible inhibitor of either PC1 and ruin. The 811-fold difference in the apparent Kapp/[I] (1.63 x 10(6) s-1 m-1), and kcat/Km determined with the corresponding peptidyl MCA substrate (2.01 x 10(3) s-1 m-1), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin.
The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like endoprotease family and is synthesized as a preproenzyme. To investigate the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculovirus-infected insect cells, rendered soluble with 6 M guanidine HCl and 20 mM dithiothreitol, and purified by gel filtration and metal-binding affinity chromatography. Two NH 2 -terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, purified, and chemically characterized. Progress curve kinetic analysis with enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin demonstrated that both fragments were potent slow tight-binding inhibitors of either enzyme with K i in the low nanomolar range. Additional cleavages at Trp residues yielded fragment 9 -71 , which no longer represents a potent inhibitor. Upon incubation at pH 5.5 in the presence of excess 71-kDa murine PC1/3, NH 2 -terminal fragment 1-98 is cleaved at two sites, as revealed through Western blotting using NH 2 -terminal-directed PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/ 3 1-98 peptide, albeit at a much lesser extent with a micromolar K i and in a strictly competitive manner. These results suggest that the proregion of PC1/3 is an important feature in regulating its activity.
The cleavage of parathyroid hormone (PTH) from its precursor proparathyroid hormone (pro-PTH) is accomplished efficiently by the proprotein convertase furin (Hendy, G. N., Bennett, H. P. J., Gibbs, B. F., Lazure, C., Day, R., and Seidah, N. G. (1995) J. Biol. Chem. 270, 9517-9525). We also showed that a synthetic peptide comprising the ؊6 to ؉7 sequence of human pro-PTH is appropriately cleaved by purified furin in vitro. The human pro-PTH processing site Lys-Ser-Val-Lys-LysArg differs from the consensus furin site Arg-Xaa-(Lys/ Arg)-Arg that is represented by Arg-Arg-Leu-Lys-Arg in the cleavage site of pro-PTH-related peptide (proPTHrP). An earlier study demonstrated that an internally quenched fluorogenic substrate bearing an O-aminobenzoyl fluorescent donor at the NH 2 terminus and an acceptor 3-nitrotyrosine near the COOH terminus was appropriately cleaved by the convertases furin and PC1 (Jean, F., Basak, A., DiMaio, J., Seidah, N. G., and Lazure, C. (1995) Biochem. J. 307, 689 -695). Here, we have synthesized a series of internally quenched fluorogenic substrates based upon the pro-PTH and pro-PTHrP sequences to determine which residues are important for furin cleavage. Purified recombinant furin and PC1 cleaved the human pro-PTH internally quenched substrate at the appropriate site in an identical manner to that observed with the nonfluorescent peptide. Several substitutions in the P 6 -P 3 sequence were well tolerated; however, replacement of the Lys at the P 6 position with Gly and replacement of the P 3 Lys by an acidic residue led to markedly compromised cleavage by furin. Furin activity was very sensitive to substitution in P positions. Replacement of Ser at P 1 with Gly and Val at P 2 with Ala generated substrates that were less well cleaved. Substitution at the P 1 position of Val for Ser in conjunction with Ala for Val at P 2 , as well as a single substitution of Lys for Val at P 2 , generated specific inhibitors of furin cleavage. The findings of this study open the way to the rational design of inhibitors of furin with therapeutic potential.Polypeptide hormones, such as parathyroid hormone (PTH) 1 which is the major regulator of extracellular calcium homeostasis (1) and many other biologically active proteins and peptides, including PTH-related peptide (PTHrP), are initially synthesized as larger inactive precursor proteins that undergo processing to release the active moiety. The related PTH gene family member, PTHrP, was originally recognized as the major pathogenic agent in the hypercalcemia of malignancy syndrome (2). Under normal physiological conditions it subserves a variety of autocrine or paracrine roles such as modulator of the growth of various cell types including cartilage, bone, skin, and breast, or regulator of vascular smooth muscle tone (3). Among the proprotein precursors, pro-PTH is unusual in that the excised pro-segment consists of only a hexapeptide (4) located NH 2 -terminal to the 84-amino acid bioactive hormone. ProPTHrP is very similar to pro-PTH at the NH 2 terminus (5...
Endoproteolytic processing of the 26-kDa protein precursor prodynorphin (proDyn) at paired and single basic residues is most likely carried out by the proprotein convertases (PCs); however, the role of PCs at single basic residues is unclear. In previous studies we showed that limited proDyn processing by PC1/PC3 at both paired and single basic residues resulted in the formation of 8- and 10-kDa intermediates. Because PC2 is colocalized with proDyn, we examined the potential role of this convertase in cleaving proDyn. PC2 cleaved proDyn to produce dynorphin (Dyn) A 1-17, Dyn B 1-13, and alpha-neo-endorphin, without a previous requirement for PC1/PC3. PC2 also cleaved at single basic residues, resulting in the formation of the C-peptide and Dyn A 1-8. Only PC2, but not furin or PC1/PC3, could cleave the Arg-Pro bond to yield Dyn 1-8. Structure-activity studies with Dyn A 1-17 showed that a P4 Arg residue is important for single basic cleavage by PC2 and that the P1' Pro residue impedes processing. Conversion of Dyn A 1-17 or Dyn B 1-13 into leucine-enkephalin (Leu-Enk) by PC2 was never observed; however, Dyn AB 1-32 cleavage yielded small amounts of Leu-Enk, suggesting that Leu-Enk can be generated from the proDyn precursor only through a specific pathway. Finally, PC2 cleavages at single and paired basic residues were enhanced when carried out in the presence of carboxypeptidase (CP) E. Enhancement was blocked by GEMSA, a specific inhibitor of CPE activity, and could be duplicated by other carboxypeptidases, including CPD, CPB, or CPM. Our data suggest that carboxypeptidase activity enhances PC2 processing by the elimination of product inhibition caused by basic residue-extended peptides.
Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediately following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84-99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, 1H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventional method of peptide-carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the case of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.
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