Caspase 3 inactivation to suppress Fas-mediated apoptosis: identification of binding domain with p21 and ILP and inactivation machinery by p21.
(17/81452)
The death mediator caspase acts as the dominant regulator during cell death induction. The CPP32 subfamily, including caspase 3 (CPP32/Yama/Apopain), is essential for the cell death signaling. We recently reported that activation of caspase 3 is regulated by complex formation with p21 or ILP. In the present study, we investigated the binding domain with p21 and ILP to further characterize the caspase 3 inactivation machinery. Our results show that caspase 3 contains p21 binding domain in the N-terminus and ILP binding domain in the active site. Further, the caspase 3 binding domain in p21 was independent of the Cdk- or PCNA-binding domain. We also found caspase 3 protection by p21 from the p3-site cleavage serineproteinase contributes to the suppression machinery. Here, we propose the caspase 3 inactivation system by p21 and ILP as new essential system in the regulation of cell death. (+info)
B-MYB transactivates its own promoter through SP1-binding sites.
(18/81452)
B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family. (+info)
Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S.
(19/81452)
The lysosomal cysteine proteases cathepsins S and L play crucial roles in the degradation of the invariant chain during maturation of MHC class II molecules and antigen processing. The p41 form of the invariant chain includes a fragment which specifically inhibits cathepsin L but not S. The crystal structure of the p41 fragment, a homologue of the thyroglobulin type-1 domains, has been determined at 2.0 A resolution in complex with cathepsin L. The structure of the p41 fragment demonstrates a novel fold, consisting of two subdomains, each stabilized by disulfide bridges. The first subdomain is an alpha-helix-beta-strand arrangement, whereas the second subdomain has a predominantly beta-strand arrangement. The wedge shape and three-loop arrangement of the p41 fragment bound to the active site cleft of cathepsin L are reminiscent of the inhibitory edge of cystatins, thus demonstrating the first example of convergent evolution observed in cysteine protease inhibitors. However, the different fold of the p41 fragment results in additional contacts with the top of the R-domain of the enzymes, which defines the specificity-determining S2 and S1' substrate-binding sites. This enables inhibitors based on the thyroglobulin type-1 domain fold, in contrast to the rather non-selective cystatins, to exhibit specificity for their target enzymes. (+info)
Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.
(20/81452)
The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D. (+info)
Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.
(21/81452)
The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly. (+info)
Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.
(22/81452)
Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane. (+info)
Tight binding of the 5' exon to domain I of a group II self-splicing intron requires completion of the intron active site.
(23/81452)
Group II self-splicing requires the 5' exon to form base pairs with two stretches of intronic sequence (EBS1 and EBS2) which also bind the DNA target during retrotransposition of the intron. We have used dimethyl sulfate modification of bases to obtain footprints of the 5' exon on intron Pl.LSU/2 from the mitochondrion of the alga Pylaiella littoralis, as well as on truncated intron derivatives. Aside from the EBS sites, which are part of the same subdomain (ID) of ribozyme secondary structure, three distant adenines become either less or more sensitive to modification in the presence of the exon. Unexpectedly, one of these adenines in subdomain IC1 is footprinted only in the presence of the distal helix of domain V, which is involved in catalysis. While the loss of that footprint is accompanied by a 100-fold decrease in the affinity for the exon, both protection from modification and efficient binding can be restored by a separate domain V transcript, whose binding results in its own, concise footprint on domains I and III. Possible biological implications of the need for the group II active site to be complete in order to observe high-affinity binding of the 5' exon to domain I are discussed. (+info)
The amino-terminal C/H1 domain of CREB binding protein mediates zta transcriptional activation of latent Epstein-Barr virus.
(24/81452)
Latent Epstein-Barr virus (EBV) is maintained as a nucleosome-covered episome that can be transcriptionally activated by overexpression of the viral immediate-early protein, Zta. We show here that reactivation of latent EBV by Zta can be significantly enhanced by coexpression of the cellular coactivators CREB binding protein (CBP) and p300. A stable complex containing both Zta and CBP could be isolated from lytically stimulated, but not latently infected RAJI nuclear extracts. Zta-mediated viral reactivation and transcriptional activation were both significantly inhibited by coexpression of the E1A 12S protein but not by an N-terminal deletion mutation of E1A (E1ADelta2-36), which fails to bind CBP. Zta bound directly to two related cysteine- and histidine-rich domains of CBP, referred to as C/H1 and C/H3. These domains both interacted specifically with the transcriptional activation domain of Zta in an electrophoretic mobility shift assay. Interestingly, we found that the C/H3 domain was a potent dominant negative inhibitor of Zta transcriptional activation function. In contrast, an amino-terminal fragment containing the C/H1 domain was sufficient for coactivation of Zta transcription and viral reactivation function. Thus, CBP can stimulate the transcription of latent EBV in a histone acetyltransferase-independent manner mediated by the CBP amino-terminal C/H1-containing domain. We propose that CBP may regulate aspects of EBV latency and reactivation by integrating cellular signals mediated by competitive interactions between C/H1, C/H3, and the Zta activation domain. (+info)