Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.

Phosphorylation of SNAP-23 by the Novel Kinase SNAK Regulates t-SNARE Complex Assembly

The docking and fusion of cargo-containing vesicles with target membranes of eukaryotic cells is mediated by the interaction of SNARE proteins present on both vesicle and target membranes. In many cases, the target membrane SNARE, or t-SNARE, exists as a complex of syntaxin with a member of the SNAP-25 family of palmitoylated proteins. We have identified a novel human kinase SNAK (SNARE kinase) that specifically phosphorylates the nonneuronal t-SNARE SNAP-23 in vivo. Interestingly, only SNAP-23 that is not assembled into t-SNARE complexes is phosphorylated by SNAK, and phosphorylated SNAP-23 resides exclusively in the cytosol. Coexpression with SNAK significantly enhances the stability of unassembled SNAP-23, and as a consequence, the assembly of newly synthesized SNAP-23 with syntaxin is augmented. These data demonstrate that phosphorylation of SNAP-23 by SNAK enhances the kinetics of t-SNARE assembly in vivo.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named “nocturnin” (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein–protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor β stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Characterization of Two Novel Type I Ribosome-Inactivating Proteins from the Storage Roots of the Andean Crop Mirabilis expansa1

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

Differential Expression of a Novel Gene in Response to Coronatine, Methyl Jasmonate, and Wounding in the Coi1 Mutant of Arabidopsis1

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit 1

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF1) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF1. Both the 64-kD protein and the 61-kD CF1 α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF1 α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m−2 s−1) and declined in response to low irradiance (80 μmol photons m−2 s−1) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF1 α-subunit found only in monocots. Analysis of purified CF1 complexes showed that the 64-kD protein represented up to 15% of the total CF1 α-subunit.

The origin of 15R-prostaglandins in the Caribbean coral Plexaura homomalla: Molecular cloning and expression of a novel cyclooxygenase

The highest concentrations of prostaglandins in nature are found in the Caribbean gorgonian Plexaura homomalla. Depending on its geographical location, this coral contains prostaglandins with typical mammalian stereochemistry (15S-hydroxy) or the unusual 15R-prostaglandins. Their metabolic origin has remained the subject of mechanistic speculations for three decades. Here, we report the structure of a type of cyclooxygenase (COX) that catalyzes transformation of arachidonic acid into 15R-prostaglandins. Using a homology-based reverse transcriptase–PCR strategy, we cloned a cDNA corresponding to a COX protein from the R variety of P. homomalla. The deduced peptide sequence shows 80% identity with the 15S-specific coral COX from the Arctic soft coral Gersemia fruticosa and ≈50% identity to mammalian COX-1 and COX-2. The predicted tertiary structure shows high homology with mammalian COX isozymes having all of the characteristic structural units and the amino acid residues important in catalysis. Some structural differences are apparent around the peroxidase active site, in the membrane-binding domain, and in the pattern of glycosylation. When expressed in Sf9 cells, the P. homomalla enzyme forms a 15R-prostaglandin endoperoxide together with 11R-hydroxyeicosatetraenoic acid and 15R-hydroxyeicosatetraenoic acid as by-products. The endoperoxide gives rise to 15R-prostaglandins and 12R-hydroxyheptadecatrienoic acid, identified by comparison to authentic standards. Evaluation of the structural differences of this 15R-COX isozyme should provide new insights into the substrate binding and stereospecificity of the dioxygenation reaction of arachidonic acid in the cyclooxygenase active site.

Characterization of a Novel Human SMC Heterodimer Homologous to the Schizosaccharomyces pombe Rad18/Spr18 Complex

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27–28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.