ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells.

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.

Identification, characterization and mapping of the human ZIS (zinc-finger, splicing) gene.

From a human fetal brain cDNA library, we isolated two transcripts (ZIS-1 and ZIS-2) corresponding to the human ZIS gene, an ortholog of the rat Zis (zinc finger, splicing). A comparison of base sequences of the cDNA and its corresponding genomic DNA (a P1-derived artificial chromosome clone) revealed that both transcripts have an ORF of 1011bp and encodes 337 amino acids, but ZIS-1 has 10 exons and ZIS-2 contains 11 exons. Although both transcripts share the first nine exons, exon 10 of ZIS-2 is lacking in ZIS-1, and instead, exon 11 (10th exon) of ZIS-1 is larger in size, leading to the longer 3'-UTR. Thus, the two transcripts result from differential splicing. A Northern blot analysis on various adult and fetal tissues revealed that 5.2- and 3.2-kb transcripts were ubiquitously expressed, and 3.9- and 1.9-kb transcripts were highly expressed in the fetal brain and kidney, respectively. There were several other transcripts that may be alternatively processed forms of the human ZIS. Considering the ZIS gene size, the 3.2-kb transcripts most likely corresponds to ZIS-1 and may act as a major transcript of ZIS. The human ZIS has a high homology to the rat Zis for the coding DNA sequence with 91% identity and for the amino acid sequence with 87% identity. ZIS and Zis contain the same numbers of exons and introns. Both genes have unusually long 3'-UTR, and their encoding proteins contain similar components, i.e. a zinc finger domain, a nuclear localization signal, an Asp-Glu region, and a Ser-Arg-rich region. Furthermore, the expression patterns of the two genes in tissues are similar each other. Thus, the human ZIS may act as a transcriptional factor to regulate transcription and/or splicing, as does the rat Zis.

Human ESP1/CRP2, a member of the LIM domain protein family: characterization of the cDNA and assignment of the gene locus to chromosome 14q32.3.

The LIM domain is present in a wide variety of proteins with diverse functions and exhibits characteristic arrangements of Cys and His residues with a novel zinc-binding motif. LIM domain proteins have been implicated in development, cell regulation, and cell structure. A LIM domain protein was identified by screening a human cDNA library with rat cysteine-rich intestinal protein (CRIP) as a probe, under conditions of low stringency. Comparison of the predicted amino acid sequence with several LIM domain proteins revealed 93% of the residues to be identical to rat LIM domain protein, termed ESP1 or CRP2. Thus, the protein is hereafter referred to as human ESP1/CRP2. The cDNA encompasses a 1171-base region, including 26, 624, and 521 bases in the 5'-noncoding region, coding region, and 3'-noncoding regions, respectively, and encodes the entire ESP1/CRP2 of 208 amino acids (M(r), 22,496). Human ESP1/CRP2 protein has two LIM domains, and each shares 35.1% and 77 or 79% identical residues with human cysteine-rich protein (CRP) and rat CRIP, respectively. Northern blot analysis of ESP1/CRP2 in various human tissues showed distinct tissue distributions compared with CRP and CRIP, suggesting that each might serve related but specific roles in tissue organization or function. Using a panel of human-rodent somatic cell hybrids, the ESP1/CRP2 locus was assigned to chromosome 14. Fluorescence in situ hybridization, using cDNA and a genome DNA fragment of the ESP1/CRP2 as probes, confirms this assignment and relegates regional localization to band 14q32.3.

Heme requirement for production of active endothelial nitric oxide synthase in baculovirus-infected insect cells.

We have cloned cDNAs encoding endothelial nitric oxide synthase (ecNOS) from a human fetal liver cDNA library. Overproduction of ecNOS in a baculovirus/insect cell expression system in conventional medium yielded a large amount of ecNOS protein localized in particulate components, but ecNOS activity was low. This activity was increased by addition of hemin to 2.5-fold. While a precursor for heme biosynthesis increased the activity, inhibitors of heme biosynthesis reduced the ecNOS activity to 50% without affecting the level of enzyme. After extraction of cells with 1% Triton X-100, ecNOS protein was purified by column chromatography. The resultant ecNOS required supplementation with cofactors for activity, but it did not require hemin. Binding of a protoporphyrin IX heme was confirmed by a pyridine hemochrome assay.

Isolation and characterization of mutations in the human holocarboxylase synthetase cDNA.

Holocarboxylase synthetase (HCS) plays an essential role in biotin utilization in eukaryotic cells and its deficiency causes biotin-responsive multiple carboxylase deficiency in humans. We have cloned the human HCS cDNA and show that antiserum against the recombinant protein immunoprecipitates human HCS. A one base deletion resulting in a premature termination and a missense mutation (Leu to Pro) were found in cells from siblings with HCS deficiency. Human HCS shows homology to BirA, which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. The human HCS gene maps to chromosome 21q22.1.