Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

Purifying selection and birth-and-death evolution in the ubiquitin gene family

Ubiquitin is a highly conserved protein that is encoded by a multigene family. It is generally believed that this gene family is subject to concerted evolution, which homogenizes the member genes of the family. However, protein homogeneity can be attained also by strong purifying selection. We therefore studied the proportion (pS) of synonymous nucleotide differences between members of the ubiquitin gene family from 28 species of fungi, plants, and animals. The results have shown that pS is generally very high and is often close to the saturation level, although the protein sequence is virtually identical for all ubiquitins from fungi, plants, and animals. A small proportion of species showed a low level of pS values, but these values appeared to be caused by recent gene duplication. It was also found that the number of repeat copies of the gene family varies considerably with species, and some species harbor pseudogenes. These observations suggest that the members of this gene family evolve almost independently by silent nucleotide substitution and are subjected to birth-and-death evolution at the DNA level.

Identification of a renal-specific oxido-reductase in newborn diabetic mice

Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is widely distributed in mammalian tissues and has been implicated in complications of diabetes, including diabetic nephropathy. To identify a renal-specific reductase belonging to the AKR family, representational difference analyses of cDNA from diabetic mouse kidney were performed. A full-length cDNA with an ORF of 855 nt and yielding a ≈1.5-kb mRNA transcript was isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had ≈91% and ≈97% amino acid identity with mouse protein. In vitro translation of the cDNA yielded a protein product of ≈33 kDa. Northern and Western blot analyses, using the cDNA and antirecombinant protein antibody, revealed its expression exclusively confined to the kidney. Like ALR2, the expression was up-regulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm–Horsfall protein nor aquaporin-2. The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members where it is confined to the C terminus. Fluorescence quenching and reactive blue agarose chromatography studies revealed that it binds to NADPH with high affinity (KdNADPH = 66.9 ± 2.3 nM). This binding domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the other AKR members. The identified protein is designated as RSOR because it is renal-specific with properties of an oxido-reductase, and like ALR2 it may be relevant in the renal complications of diabetes mellitus.

Proposed mechanism for stability of proteins to evolutionary mutations

It is shown that the sequence-ordering tendencies induced by design into different fast-folding, thermally stable native structures interfere. This interference results in a type of quasiorthogonality between optimal native structures, which divides sequence space into fast-folding, thermally stable families surrounded by slow-folding, low stability shells. A concrete example of this effect is provided by using a simple α carbon type model in which a complete correspondence is established between sequence and structure. It is speculated that gaps can occur in the space of protein-like sequences separating the sequence families and resulting in a mechanism for stability and diversity of protein sequence information.

Interactions of the chaperone Hsp104 with yeast Sup35 and mammalian PrP

[PSI+] is a genetic element in yeast for which a heritable change in phenotype appears to be caused by a heritable change in the conformational state of the Sup35 protein. The inheritance of [PSI+] and the physical state of Sup35 in vivo depend on the protein chaperone Hsp104 (heat shock protein 104). Although these observations provide a strong genetic argument in support of the “protein-only” or “prion” hypothesis for [PSI+], there is, as yet, no direct evidence of an interaction between the two proteins. We report that when purified Sup35 and Hsp104 are mixed, the circular dichroism (CD) spectrum differs from that predicted by the addition of the proteins’ individual spectra, and the ATPase activity of Hsp104 is inhibited. Similar results are obtained with two other amyloidogenic substrates, mammalian PrP and β-amyloid 1-42 peptide, but not with several control proteins. With a group of peptides that span the PrP protein sequence, those that produced the largest changes in CD spectra also caused the strongest inhibition of ATPase activity in Hsp104. Our observations suggest that (i) previously described genetic interactions between Hsp104 and [PSI+] are caused by direct interaction between Hsp104 and Sup35; (ii) Sup35 and PrP, the determinants of the yeast and mammalian prions, respectively, share structural features that lead to a specific interaction with Hsp104; and (iii) these interactions couple a change in structure to the ATPase activity of Hsp104.

Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair

ERCC1–XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5′ to 3′ away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1–XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1–XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.

GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources

GlycoSuiteDB is a relational database that curates information from the scientific literature on glyco­protein derived glycan structures, their biological sources, the references in which the glycan was described and the methods used to determine the glycan structure. To date, the database includes most published O-linked oligosaccharides from the last 50 years and most N-linked oligosaccharides that were published in the 1990s. For each structure, information is available concerning the glycan type, linkage and anomeric configuration, mass and composition. Detailed information is also provided on native and recombinant sources, including tissue and/or cell type, cell line, strain and disease state. Where known, the proteins to which the glycan structures are attached are reported, and cross-references to the SWISS-PROT/TrEMBL protein sequence databases are given if applicable. The GlycoSuiteDB annotations include literature references which are linked to PubMed, and detailed information on the methods used to determine each glycan structure are noted to help the user assess the quality of the structural assignment. GlycoSuiteDB has a user-friendly web interface which allows the researcher to query the database using mono­isotopic or average mass, monosaccharide composition, glycosylation linkages (e.g. N- or O-linked), reducing terminal sugar, attached protein, taxonomy, tissue or cell type and GlycoSuiteDB accession number. Advanced queries using combinations of these parameters are also possible. GlycoSuiteDB can be accessed on the web at http://www.glycosuite.com.

The emotif database

The emotif database is a collection of more than 170 000 highly specific and sensitive protein sequence motifs representing conserved biochemical properties and biological functions. These protein motifs are derived from 7697 sequence alignments in the BLOCKS+ database (released on June 23, 2000) and all 8244 protein sequence alignments in the PRINTS database (version 27.0) using the emotif-maker algorithm developed by Nevill-Manning et al. (Nevill-Manning,C.G., Wu,T.D. and Brutlag,D.L. (1998) Proc. Natl Acad. Sci. USA, 95, 5865–5871; Nevill-Manning,C.G., Sethi,K.S., Wu,T.D. and Brutlag,D.L. (1997) ISMB-97, 5, 202–209). Since the amino acids and the groups of amino acids in these sequence motifs represent critical positions conserved in evolution, search algorithms employing the emotif patterns can identify and classify more widely divergent sequences than methods based on global sequence similarity. The emotif protein pattern database is available at http://motif.stanford.edu/emotif/.

Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas’ disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of ∼1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

A Selaginella lepidophylla Trehalose-6-Phosphate Synthase Complements Growth and Stress-Tolerance Defects in a Yeast tps1 Mutant1

The accumulation of the disaccharide trehalose in anhydrobiotic organisms allows them to survive severe environmental stress. A plant cDNA, SlTPS1, encoding a 109-kD protein, was isolated from the resurrection plant Selaginella lepidophylla, which accumulates high levels of trehalose. Protein-sequence comparison showed that SlTPS1 shares high similarity to trehalose-6-phosphate synthase genes from prokaryotes and eukaryotes. SlTPS1 mRNA was constitutively expressed in S. lepidophylla. DNA gel-blot analysis indicated that SlTPS1 is present as a single-copy gene. Transformation of a Saccharomyces cerevisiae tps1Δ mutant disrupted in the ScTPS1 gene with S. lepidophylla SlTPS1 restored growth on fermentable sugars and the synthesis of trehalose at high levels. Moreover, the SlTPS1 gene introduced into the tps1Δ mutant was able to complement both deficiencies: sensitivity to sublethal heat treatment at 39°C and induced thermotolerance at 50°C. The osmosensitive phenotype of the yeast tps1Δ mutant grown in NaCl and sorbitol was also restored by the SlTPS1 gene. Thus, SlTPS1 protein is a functional plant homolog capable of sustaining trehalose biosynthesis and could play a major role in stress tolerance in S. lepidophylla.

Measuring genome evolution

The determination of complete genome sequences provides us with an opportunity to describe and analyze evolution at the comprehensive level of genomes. Here we compare nine genomes with respect to their protein coding genes at two levels: (i) we compare genomes as “bags of genes” and measure the fraction of orthologs shared between genomes and (ii) we quantify correlations between genes with respect to their relative positions in genomes. Distances between the genomes are related to their divergence times, measured as the number of amino acid substitutions per site in a set of 34 orthologous genes that are shared among all the genomes compared. We establish a hierarchy of rates at which genomes have changed during evolution. Protein sequence identity is the most conserved, followed by the complement of genes within the genome. Next is the degree of conservation of the order of genes, whereas gene regulation appears to evolve at the highest rate. Finally, we show that some genomes are more highly organized than others: they show a higher degree of the clustering of genes that have orthologs in other genomes.

Identification and characterization of a melanin-concentrating hormone receptor

Melanin-concentrating hormone (MCH), a neuropeptide expressed in central and peripheral nervous systems, plays an important role in the control of feeding behaviors and energy metabolism. An orphan G protein-coupled receptor (SLC-1/GPR24) has recently been identified as a receptor for MCH (MCHR1). We report here the identification and characterization of a G protein-coupled receptor as the MCH receptor subtype 2 (MCHR2). MCHR2 has higher protein sequence homology to MCHR1 than any other G protein-coupled receptor. The expression of MCHR2 has been detected in many regions of the brain. In contrast to MCHR1, which is intronless in the coding region and is located at the chromosomal locus 22q13.3, the MCHR2 gene has multiple exons and is mapped to locus 6q21. MCHR2 is specifically activated by nanomolar concentrations of MCH, binds to MCH with high affinity, and signals through Gq protein. This discovery is important for a full understanding of MCH biology and the development of potential therapeutics for diseases involving MCH, including obesity.

Characterization of Euphorbia characias Latex Amine Oxidase1

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 105 m−1 cm−1 and 6000 m−1 cm−1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.

Molecular Characterization of an Arabidopsis Gene Encoding Hydroperoxide Lyase, a Cytochrome P-450 That Is Wound Inducible1

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.

Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase.

Recent studies on proteins whose N and C termini are in close proximity have demonstrated that folding of polypeptide chains and assembly of oligomers can be accomplished with circularly permuted chains. As yet no methodical study has been conducted to determine how extensively new termini can be introduced and where such termini cannot be tolerated. We have devised a procedure to generate random circular permutations of the catalytic chains of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and to select clones that produce active or stable holoenzyme containing permuted chains. A tandem gene construct was made, based on the desired linkage between amino acid residues in the C- and N-terminal regions of the polypeptide chain, and this DNA was treated with a suitable restriction enzyme to yield a fragment containing the rearranged coding sequence for the chain. Circularization achieved with DNA ligase, followed by linearization at random with DNase I, and incorporation of the linearized, repaired, blunt-ended, rearranged genes into a suitable plasmid permitted the expression of randomly permuted polypeptide chains. The plasmid with appropriate stop codons also contained pyrI, the gene encoding the regulatory chain of ATCase. Colonies expressing detectable amounts of ATCase-like molecules containing permuted catalytic chains were identified by an immunoblot technique or by their ability to grow in the absence of pyrimidines in the growth medium. Sequencing of positive clones revealed a variety of novel circular permutations. Some had N and C termini within helices of the wild-type enzyme as well as deletions and insertions. Permutations were concentrated in the C-terminal domain and only few were detected in the N-terminal domain. The technique, which is adaptable generally to proteins whose N and C termini are near each other, can be of value in relating in vivo folding of nascent, growing polypeptide chains to in vitro renaturation of complete chains and determining the role of protein sequence in folding kinetics.