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PubMed2002

Loop folds in proteins and evolutionary conservation of folding nuclei.

We show that loops of close contacts involving hydrophobic residues are important in protein folding. Contrary to Berezovsky Berezovsky and Trifonov (J Biomol Struct Dyn 20, 5-6, 2002) the loops important in protein folding usually are much larger in size than 23-31 residues, being instead comparable to the size of the protein for single domain proteins. Additionally what is important are not single loop contacts, but a highly interconnected network of such loop contacts, which provides extra stability to a protein fold and which leads to their conservation in evolution.

Amino Acid Sequence↗
PubMedDate not supplied

Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (review).

All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.

Bacterial Proteins↗
PubMed2004

Genomic organization of mouse desmocollin genes reveals evolutionary conservation.

Desmosomal cadherins are a family of calcium regulated proteins involved in the formation of desmosomes, a type of cell junction important in maintaining cell adhesion and tissue stability. The desmosomal plaque consists of members of the desmosomal cadherin, plakin and armadillo family of proteins. Desmosomal cadherins are transmembrane glycoproteins that interact with desmosomal cadherins of the adjacent cells via their extracellular repeat domains and are divided in two subfamilies, the desmogleins (Dsg) and the desmocollins (Dsc). On the cytoplasmic side, the cadherins connect to the intermediate filament (IF) network indirectly by interacting with plakin and armadillo proteins. Here, we report the elucidation of the genomic structure of two mouse desmocollin genes, Dsc2 and Dsc3. Interestingly, at the genomic level, desmocollins show a higher degree of similarity to the classical cadherins, such as E-cadherin, than to the desmogleins.

Animals↗
PubMed1997

Functional analysis of the evolutionary conserved arginine 182 residue in human glutathione S-transferase P1-1.

The mutational replacement of Arg182 with threonine markedly decreased the specific activities for GSH-conjugation reaction. The Kcat of R182T for GSH-[1-chloro-2,4-dinitrobenzene] conjugation reaction was about 100-fold smaller than that of the wild type. On the other hand, the affinity for GSH of R182T was not significantly affected. The pKa of the thiol group of GSH bound in R182T was approximately 0.9 pK units higher than those in the wild type, but the Kcat/Km1-chloro-2,4-dinitrobenzene values at high pH were not so much lower than those of the wild type. The thermostability of R182T was significantly lower than that of the wild type. Therefore, Arg182 seems to be important for the construction of the active enzyme structure.

Amino Acid Sequence↗
PubMed1999

Evolutionary conservation of the membrane fusion machine.

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

Amino Acid Sequence↗
PubMed1991

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast.

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.

Amino Acid Sequence↗
PubMed1981

Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man.

We describe the biochemical properties and cell surface distributions of three human T cell antigens (Leu-1, Leu-2a, and Leu-2b) which we postulate to be the homologues of the Lyt-1, Lyt-2, and Lyt-3 antigens that distinguish functional T cell subsets in the mouse. Leu-l, like Lyt-1, is on all thymocytes and peripheral T cells and is present in greater amounts on the helper/inducer subset than on the cytotoxic/suppressor subset. Both antigens increase in parallel fashion during T cell maturation in the thymus and each antigen is carried on a single 67,000-molecular weight (relative) (M(r)) polypeptide chain. Surprisingly, Leu-1 and Lyt-1 each are also expressed in readily detectable amounts on some B celI Ieukemias but not detectably so on normal B cells. Leu-2a and Leu-2b are antigens found only on suppressor/cytotoxic cells in the human and are very similar to the murine Lyt-2 and Lyt-3 antigens. In both species, the two antigens are on the same disulfide- linked multimeric molecules. Disulfide-bond reduction in both species yields subunits of similar size and charge. Lyt-3 and Leu-2b are extremely sensitive to trypsin digestion on viable cells whereas Lyt-2 and Leu-2a are much less so. A different membrane antigen, Leu-3, is an exclusive marker of the helper/inducer subset in man. No mouse homologue for this 55,000-M(r) protein is known. The maintenance of the homologous molecules on functionally distinct T cell subpopulations in two evolutionarily distant species suggests that the Lyt and Leu antigens perform essential functions for the cells on which they are found.

Animals↗
PubMed1993

Evolutionary conservation of major histocompatibility complex-DR/peptide/T cell interactions in primates.

Many major histocompatibility complex (MHC) polymorphisms originate from ancient structures that predate speciation. As a consequence, members of the Mhc-DRB1*03 allelic lineage are not only present in humans but in chimpanzees and rhesus macaques as well. This emphasizes that Mhc-DRB1*03 members must have been present in a common ancestor of these primate species that lived about 30 million years ago. Due to the accumulation of genetic variation, however, alleles of the Mhc-DRB1*03 lineage exhibit species-unique sequences. To investigate the biological importance of such conservation and variation, we have studied both the binding and antigen presentation capacity of various trans-species Mhc-DRB1*03 lineage members. Here we show that p3-13 of the 65-kD heat-shock protein (hsp65) of Mycobacterium leprae and M. tuberculosis binds not only to HLA-DR17(3) but also to some chimpanzee and rhesus macaque class II-positive cells. Comparison of the corresponding human, chimpanzee, and rhesus macaque Mhc-DRB1*03 lineage members revealed the presence of uniquely shared amino acid residues, at positions 9-13 and 26-31, of the antigen-binding site that are critical for p3-13 binding. In addition it is shown that several nonhuman primate antigen-presenting cells that bind p3-13 can activate HLA-DR17-restricted T cells. Certain amino acid replacements, however, in Mhc-DRB1*03 lineage members did not influence peptide binding or T cell recognition. Therefore, these studies demonstrate that some polymorphic amino acid residues (motifs) within the antigen-binding site of MHC class II molecules that are crucial for peptide binding and recognition by the T cell receptor have been conserved for over 30 million years.

Amino Acid Sequence↗
PubMed1994

Evolutionary conservation of function among mammalian, avian, and viral homologs of the Bcl-2 oncoprotein.

The bcl-2 gene was originally cloned because of its involvement in B-cell lymphomas and encodes a 25-kD integral membrane protein that has been shown to inhibit programmed cell death (also termed apoptosis) in a wide variety of circumstances. The Epstein-Barr Virus (EBV) also has been implicated in B-cell malignancies and interestingly contains an open reading frame (BHRF-1) predicting a 19-kD protein with 22% homology to Bcl-2. To compare the functions of p26-Bcl-2 and p19-BHRF-1, we stably introduced expression plasmids encoding these proteins into a murine interleukin-3 (IL-3)-dependent hemopoietic cell line, 32D. Removal of IL-3 from cultures of control-transfected 32D cells resulted in internucleosomal DNA cleavage (a hallmark of programmed cell death) and loss of cell survival. In contrast, 32D cells containing high levels of p26-Bcl-2 or p19-BHRF-2 proteins exhibited prolonged survival and markedly delayed DNA degradation under the same conditions of IL-3 deprivation. As a first attempt to determine the functional importance of amino acid sequences that are conserved between the Bcl-2 and BHRF-1 proteins, we used site-specific mutagenesis to replace two conserved cysteine residues with alanines (positions 158 and 219) in the human Bcl-2 protein. Comparisons of the wild-type and cysteine-minus human Bcl-2 proteins in S49 lymphoma cells revealed equivalent ability to block glucocorticoid-induced cell death and DNA fragmentation, indicating that these two conserved cysteines are not critical for Bcl-2 oncoprotein function. Investigations in 32D cells of an avian homolog of Bcl-2 cloned from the chicken also revealed conservation of function with the human Bcl-2 protein, despite the presence of a 48-amino-acid region of divergent sequence. Taken together, these data demonstrate that despite marked differences in their predicted amino-acid sequences, the human, chicken, and EBV versions of Bcl-2 have retained the structural characteristics necessary to interface with pathways involved in the regulation of programmed cell death in murine cells. The findings thus contribute to the mapping of functional domains in Bcl-2 proteins, and raise the possibility that the EBV-encoded p19-BHRF-1 protein may be able to substitute for p26-Bcl-2 in the development of some types of cancer.

Amino Acid Sequence↗
PubMed1996

The tilapia prolactin I gene: evolutionary conservation of the regulatory elements directing pituitary-specific expression.

To study the elements involved in the pituitary specific transcriptional regulation of the tilapia prolactin I gene (tiPRL I), we have cloned and entirely sequenced a 3.4-kb genomic fragment immediately upstream from the first exon. In footprinting experiments, three tilapia sequences are protected from DNase I digestion by rat pituitary extracts (base pair coordinates -643 to -593, -160 to -111, and -73 to -46). Computer analysis of the nucleotide sequence reveals significant homology to mammalian binding sites for Pit-1, a transcription factor that is known to mediate pituitary-specific expression of the PRL genes in mammals. The tiPRL I 5'-flanking sequences can direct transient expression of a linked luciferase reporter gene in transfected rat pituitary cell lines and tilapia pituitary primary cell cultures. Transient expression experiments with 5'-deletion mutants reveal three regulatory regions. Two have a stimulatory effect on transcription and one an inhibitory effect. Electrophoretic mobility-shift assays (EMSA) demonstrate that the rat Pit-1 factor specifically binds to tilapia DNA sequences. Several such tilapia Pit-1 binding sites mediate activation of a linked heterologous promoter in transfected rat and tilapia pituitary cells. As evidenced by EMSA, a Pit-1-like protein is present in tilapia pituitary extracts. All these data point to a high conservation of the molecular mechanisms involved in pituitary-specific expression of the PRL genes in vertebrates.

Animals↗
PubMed1993

Analysis of chicken CD4 by monoclonal antibodies indicates evolutionary conservation between avian and mammalian species.

We have created a panel of mouse monoclonal antibodies detecting different epitopes on avian CD4 molecule. Two-color immunofluorescence analysis shows that chicken peripheral alpha beta T cells are either CD4 or CD8 single positive whereas most gamma delta T cells are CD4-negative both in the thymus and peripheral tissues. Unlabeled antibody competition analysis by flow cytometry demonstrates that several different epitopes on chicken CD4 are recognized by these antibodies. Antibodies precipitate a monomeric glycoprotein from surface-labeled chicken thymocytes and T cells with relative molecular mass (M(r) of 64 kd as analyzed by SDS gel electrophoresis. Removal of N-linked carbohydrates by endoglycosidase-F increases the electrophoretic mobility and reveals the core protein size with M(r) of 45 kd. The anti-CD4 antibodies inhibit antigen-induced cellular proliferation of a keyhole limpet hemocyanin (KLH) -specific T cell line. They synergize in the blocking of T cell proliferation with anti-class II major histocompatibility complex (MHC)-specific antibodies indicating that chicken CD4 is involved in antigen recognition process by CD4+ T cells. We also show that chicken CD4 is down-modulated in a similar manner as its mammalian equivalent when thymocytes are stimulated in vitro with phorbol esters. Altogether these findings suggest functional and biochemical conservation of the CD4 molecule in evolution.

Animals↗
PubMed2002

Cytoplasmic localization and evolutionary conservation of MEI-218, a protein required for meiotic crossing-over in Drosophila.

During Drosophila oogenesis, the oocyte is formed within a 16-cell cyst immediately after four incomplete cell divisions. One of the primary events in oocyte development is meiotic recombination. Here, we report the intracellular localization of the MEI-218 protein that is specifically required for meiotic crossing-over. To understand the role of mei-218 in meiosis and to study the regulation of genes required for meiotic recombination, we characterized the expression pattern of its RNA and protein. Furthermore, we cloned and sequenced mei-218 from two other Drosophila species. The mei-218 RNA and protein have a similar expression pattern, appearing first in early meiotic prophase and then rapidly disappearing as prophase is completed. This pattern corresponds to a specific appearance of the mei-218 gene product in the region of the ovary where meiotic prophase occurs. Although mei-218 is required for 95% of all crossovers, the protein is found exclusively in the cytoplasm. Based on these results, we suggest that mei-218 does not have a direct role in recombination but rather regulates other factors required for the production of crossovers. We propose that mei-218 is a molecular link between oocyte differentiation and meiosis.

3' Untranslated Regions↗
PubMed1998

Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway.

In normal and transformed cells, the F-box protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2. Here we identify human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo. CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2. The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.

Amino Acid Sequence↗
PubMed1999

Structure-function analysis of an evolutionary conserved protein, DAP3, which mediates TNF-alpha- and Fas-induced cell death.

A novel approach to the isolation of positive mediators of programmed cell death, based on random inactivation of genes by expression of anti sense RNAs, was employed to identify mediators of interferon-gamma-induced apoptosis. One of the several genes identified is DAP3, which codes for a 46 kDa protein with a potential nucleotide-binding motif. Structure-function studies of the protein indicate that the intact full-length protein is required for its ability to induce apoptosis when overexpressed. The N-terminal 230 amino acids, on the other hand, act in a dominant-negative fashion. Both of these functions are dependent on the integrity of the nucleotide binding motif. Expression of anti-sense DAP3 RNA and of the dominant interfering form of DAP3 both protected cells from apoptosis induced by activation of Fas and tumor necrosis factor alpha (TNF-alpha) receptors. Thus, DAP3 is implicated as a positive mediator of these death-inducing stimuli. It functions downstream of the receptor signaling complex and its death promoting effects depend on caspase activity. In the nematode Caenorhabditis elegans, a potential homolog of DAP3 showing 35% identity and 64% similarity to the human protein was isolated. Overexpression of the nematode DAP3 cDNA in mammalian cells induced cell death, indicating that the protein is conserved at the functional level as well as the structural level.

Amino Acid Sequence↗
PubMed1994

Evolutionary conservation of the structure and expression of alternatively spliced Ultrabithorax isoforms from Drosophila.

In Drosophila melanogaster, alternatively spliced mRNAs from the homeotic gene Ultrabithorax (Ubx) encode a family of structurally distinct homeoprotein isoforms. The developmentally regulated expression patterns of these isoforms suggest that they have specialized stage- and tissue-specific functions. To evaluate the functional importance of UBX isoform diversity and gain clues to the mechanism that regulates processing of Ubx RNAs, we have investigated whether the Ubx RNAs of other insects undergo similar alternative splicing. We have isolated and characterized Ubx cDNA fragments from D. melanogaster, Drosophila pseudoobscura, Drosophila hydei and Drosophila virilis, species separated by as much as 60 million years of evolution, and have found that three aspects of Ubx RNA processing have been conserved. (1) These four species exhibit identical patterns of optional exon use in a region adjacent to the homeodomain. (2) These four species produce the same family of UBX protein isoforms with identical amino acid sequences in the optional exons, even though the common amino-proximal region has undergone substantial divergence. The nucleotide sequences of the optional exons, including third positions of rare codons, have also been conserved strongly, suggesting functional constraints that are not limited to coding potential. (3) The tissue- and stage-specific patterns of expression of different UBX isoforms are identical among these Drosophila species, indicating that the developmental regulation of the alternative splicing events has also been conserved. These findings argue for an important role of alternative splicing in Ubx function. We discuss the implications of these results for models of UBX protein function and the mechanism of alternative splicing.

Alternative Splicing↗
PubMed1997

Linkage groups of protein-coding genes in western palearctic water frogs reveal extensive evolutionary conservation.

Among progeny of a hybrid (Rana shqiperica x R. lessonae) x R. lessonae, 14 of 22 loci form four linkage groups (LGs): (1) mitochondrial aspartate aminotransferase, carbonate dehydratase-2, esterase 4, peptidase D; (2) mannosephosphate isomerase, lactate dehydrogenase-B, sex, hexokinase-1, peptidase B; (3) albumin, fructose-biphosphatase-1, guanine deaminase; (4) mitochondrial superoxide dismutase, cytosolic malic enzyme, xanthine oxidase. Fructose-biphosphate aldolase-2 and cytosolic aspartate aminotransferase possibly form a fifth LG. Mitochondrial aconitate hydratase, alpha-glucosidase, glyceraldehyde-3-phosphate dehydrogenase, phosphogluconate dehydrogenase, and phosphoglucomutase-2 are unlinked to other loci. All testable linkages (among eight loci of LGs 1, 2, 3, and 4) are shared with eastern palearctic water frogs. Including published data, 44 protein loci can be assigned to 10 of the 13 chromosomes in Holarctic Rana. Of testable pairs among 18 protein loci, agreement between Palearctic and Nearctic Rana is complete (125 unlinked, 14 linked pairs among 14 loci of five syntenies), and Holarctic Rana and Xenopus laevis are highly concordant (125 shared nonlinkages, 13 shared linkages, three differences). Several Rana syntenies occur in mammals and fish. Many syntenies apparently have persisted for 60-140 x 10(6) years (frogs), some even for 350-400 x 10(6) years (mammals and teleosts).

Animals↗
PubMed2000

RNA editing of the Drosophila para Na(+) channel transcript. Evolutionary conservation and developmental regulation.

Post-transcriptional editing of pre-mRNAs through the action of dsRNA adenosine deaminases results in the modification of particular adenosine (A) residues to inosine (I), which can alter the coding potential of the modified transcripts. We describe here three sites in the para transcript, which encodes the major voltage-activated Na(+) channel polypeptide in Drosophila, where RNA editing occurs. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites was also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures were evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na(+) channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs.

Alternative Splicing↗