XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.     

Fission yeast Sop2p: a novel and evolutionarily conserved protein that interacts with Arp3p and modulates profilin function.

Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. Here, we report the identification and characterization of an essential gene from Schizosaccharomyces pombe, sop2+, a mutation in which rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and act1-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the beta-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species, and we have isolated a human cDNA highly related to sop2+, SOP2 Hs, which functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba beta-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex which also contains the actin-related protein, Arp3p. Immunofluorescence studies reveal the presence of Sop2p in (i) punctate structures distributed throughout the cell, (ii) cables that extend the length of the cell, and (iii) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin.     

DNA of Epstein-Barr virus. III. Identification of restriction enzyme fragments that contain DNA sequences which differ among strains of Epstein-Barr virus.

Previous kinetic and absorption hybridization experiments had demonstrated that the DNA of the B95-8 strain of Epstein-Barr virus was missing approximately 10% of the DNA sequences present in the DNA of the HR-1 strain (R.F. Pritchett, S.D. Hayward, and E. Kieff, J. Virol. 15:556-569, 1975; B. Sugder, W.C. Summers, and G. Klein, J. Virol. 18:765-775, 1976). The HR-1 strain differs from other laboratory strains, including the B95-8 and W91 strains, and from virus present in throat washings from patients with infectious mononucleosis in its inability to transform lymphocytes into lymphoblasts capable of long-term growth in culture (P. Gerber, Lancet i:1001, 1973; J. Menezes, W. Leibold, and G. Klein, Exp. Cell. Res. 92:478-484, 1975; G. Miller, D. Coope, J. Niederman, and J. Pagano, J. Virol. 18:1071-1080, 1976; G. Miller, J. Robinson, L. Heston, and M. Lipman, Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974). In the experiments reported here, the restriction enzyme fragments of Epstein-Barr virus DNA which contain sequences which differ among the HR-1, B95-8, and W91 strains have been identified. The DNA of the HR-1, B95-8, and W91 strains each differed in complexity. The sequences previously shown to be missing in the B95-8 strain were contained in the EcoRI-C and -D and Hsu I-E and -N fragments of the HR-1 strain and in the EcoRI-C and Hsu I-D and -E fragments of the W91 strain. The HR-1 strain was missing DNA contained in EcoRI fragments A and J through K and Hsu I fragment B of the B95-8 strain and in the EcoRI-A and Hsu I-B fragments of the W91 strain. The relationship of these data to the linkage map of restriction enzyme fragments of the DNA of the B95-8 and W91 strains (E. Kieff, N. Raab-Traub, D. Given, W. King, A.T. Powell, R. Pritchett, and T. Dambaugh, In F. Rapp and G. de-The, ed., Oncogenesis and Herpesviruses III, in press; D. Given and E. Kieff, submitted for publication) and the possible significance of the data are discussed.     

Identification within the simian virus 40 genome of a chromosomal loop attachment site that contains topoisomerase II cleavage sites.

We demonstrate that the simian virus 40 genome contains a single MAR (matrix association region) that maps within a large T-antigen coding region (nucleotides 4071 to 4377). This region contains topoisomerase II cleavage sites, exhibits sequence similarity with cellular MARs, and recognizes the same evolutionarily conserved, abundant nuclear binding sites seen by cellular MARs.     

Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases

Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes. Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity). The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid. We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation. We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences. Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.     

Amino acid sequences around exofacial proteolytic cleavage sites of band 3 from bovine and porcine erythrocytes

• 1.1. Amino acid sequences of bovine and porcine band 3, an erythrocyte anion transporter, were determined.
• 2.2. The sequence of bovine band 3 was positioned to residues 519–599 (the numbering is based on human band 3), in which probably 6 residues were unidentified.
• 3.3. Binding site of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a potent anion transport inhibitor, was identified as Lys-539 in the bovine case.
• 4.4. A loop (residues 551–567), which provides exofacial proteolytic cleavage sites, contains only 53% homology between human and bovine, whereas the residues flanking it on either side are >84% homologous.
• 5.5. Furthermore, the loop of porcine band 3 was indicated to consist of a 6 or 7-residues short peptide as compared with those of other species.

     

(dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.     

Chimeric polioviruses that include sequences derived from two independent antigenic sites of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies against FMDV in guinea pigs.

Five poliovirus recombinants containing sequences corresponding to foot-and-mouth disease virus (FMDV) antigenic sites were constructed. Viable virus was recovered from four of these plasmids, in which the VP1 beta B-beta C loop (antigenic site 1) of poliovirus type 1 Sabin had been replaced with sequences derived from the VP1 beta G-beta H loop (antigenic site 1) of FMDV O1 Kaufbeuren (O1K), chimera O1.1 (residues 141 to 154), chimera O1.2 (residues 147 to 156), and chimera O1.3 (residues 140 to 160) or from the beta B-beta C loop of VP1 (antigenic site 3) in chimera O3.1 (residues 40 to 49). One chimera (O1.3) was neutralized by FMDV-specific polyclonal serum and monoclonal antibodies directed against antigenic site 1 of FMDV. Chimeras O1.3 and O3.1 induced site-specific FMDV-neutralizing antibodies in guinea pigs. Chimera O1.3 was capable of inducing a protective response against FMDV challenge in some guinea pigs.     

Huntington disease-linked restriction fragment length polymorphism localized within band p16.1 of chromosome 4 by in situ hybridization.

A 5.5-kilobase (kb) single sequence DNA fragment (G8) reveals the DNA polymorphic locus D4S10 on Southern blot analysis. This locus is closely linked to Huntington disease and has been mapped to chromosome 4 short arm using human-mouse somatic cell hybrids, and specifically to chromosome 4 band p16 using DNA from individuals with deletions of chromosome 4 short arm who exhibit Wolf-Hirschhorn syndrome. With in situ hybridization techniques, we have confirmed the location of D4S10 on chromosome 4 and further localized it within band p16 utilizing five patients, four with overlapping chromosome 4 short-arm aberrations. The DNA segment G8 was hybridized to the mataphase chromosomes of the five patients. Two of them have different interstitial deletions of one of the chromosome 4 short arms (TA and BA), two have different chromosome 4 short-arm terminal deletions (RG and DQ), and one has a normal male karyotype. By noting the presence or absence of hybridization to the partially deleted chromosomes with known precise breakpoints, we were able to more accurately localize probe G8 to the distal half of band p16.1 of chromosome 4.     

Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences

In mammals, the esterification of sterols by ACAT plays a critical role in eukaryotic lipid homeostasis. Using the predominant isoform of the yeast ACAT-related enzyme family, Are2p, as a model, we targeted phylogenetically conserved sequences for mutagenesis in order to identify functionally important motifs. Deletion, truncation, and missense mutations implicate a regulatory role for the amino-terminal domain of Are2p and identified two carboxyl-terminal motifs as required for catalytic activity. A serine-to-leucine mutation in the (H/Y)SF motif (residues 338-340), unique to sterol esterification enzymes, nullified the activity and stability of yeast Are2p. Similarly, a tyrosine-to-alanine change in the FYxDWWN motif of Are2p (residues 523-529) produced an enzyme with decreased activity and apparent affinity for oleoyl-CoA. Mutagenesis of the tryptophan residues in this motif completely abolished activity. In human ACAT1, mutagenesis of the corresponding motifs (residues 268-270, and 403-409, respectively) also nullified enzymatic activity. On the basis of their critical roles in enzymatic activity and their sequence conservation, we propose that these motifs mediate sterol and acyl-CoA binding by this class of enzymes.