Evolution of the alpha-crystallin/small heat-shock protein family

The common characteristic of the alpha-crystallin/small heat-shock protein family is the presence of a conserved homologous sequence of 90-100 residues. Apart from the vertebrate lens proteins--alpha A- and alpha B-crystallin--and the ubiquitous group of 15-30-kDa heat-shock proteins, this family also includes two mycobacterial surface antigens and a major egg antigen of Schistosoma mansoni. Multiple small heat-shock proteins are especially present in higher plants, where they can be distinguished in at least two classes of cytoplasmic proteins and a chloroplast-located class. The alpha-crystallins have recently been found in many tissues outside the lens, and alpha B-crystallin, in particular, behaves in many respects like a small heat-shock protein. The homologous sequences constitute the C-terminal halves of the proteins and probably represent a structural domain with a more variable C-terminal extension. These domains must be responsible for the common structural and functional properties of this protein family. Analysis of the phylogenetic tree and comparison of the biological properties of the various proteins in this family suggest the following scenario for its evolution: The primordial role of the small heat-shock protein family must have been to cope with the destabilizing effects of stressful conditions on cellular integrity. The alpha-crystallin-like domain appears to be very stable, which makes it suitable both as a surface antigen in parasitic organisms and as a long-living lens protein in vertebrates. It has recently been demonstrated that, like the other heat-shock proteins, the alpha-crystallins and small heat-shock proteins function as molecular chaperones, preventing undesired protein-protein interactions and assisting in refolding of denatured proteins. Many of the small heat-shock proteins are differentially expressed during normal development, and there is good evidence that they are involved in cytomorphological reorganizations and in degenerative diseases. In conjunction with the stabilizing, thermoprotective role of alpha-crystallins and small heat-shock proteins, they may also be involved in signal transduction. The reversible phosphorylation of these proteins appears to be important in this respect.     

Evolution of the trappin multigene family in the Suidae

The purpose of the study was to examine the effects of manipulating lung volume (LV) on phonatory and articulatory kinematic behavior during sentence production in healthy adults. Five men and five women repeated the sentence "I sell a sapapple again" under five LV conditions. These included (1) speaking normally, (2) speaking after exhaling most of the air from the lungs, (3) speaking at end expiratory level (EEL), (4) speaking after a maximal inhalation, and (5) speaking after a maximal inhalation while attempting to maintain as normal a mode of speech as possible. From a multichannel recording, measures were made of LV, sound pressure level (SPL), fundamental frequency (F0) and semitone standard deviation (STSD), and upper and lower lip displacements and peak velocities. When compared with the reference condition, the sentence was spoken significantly more quickly at the lowest LV. SPL increased significantly for the high LV condition, as did the women's F0 and STSD. Upper lip displacements and peak velocities generally decreased for LVs other than the reference condition. Lower lip movements showed inconsistent changes as a function of LV. Adjustments to the LV for speech led to SPL and F0 changes consistent with a coordinated control of the respiratory system and the larynx. However, less consistent effects were observed in the articulatory kinematic measures, possibly because of a less direct biomechanical and neural control linkage between respiratory and articulatory structures.     

Sequence analysis of the AAA protein family

The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases.     

Identification and cloning of a glucan- and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade

Coelomic fluid of Eisenia foetida earthworms contains a 42-kDa protein named coelomic cytolytic factor 1 (CCF-1) that was described previously to be involved in cytolytic, opsonizing, and hemolytic properties of the coelomic fluid. Cloning and sequencing of CCF-1 reveal significant homology with the putative catalytic region of beta-1,3- and beta-1,3-1,4-glucanases. CCF-1 also displays homology with coagulation factor G from Limulus polyphemus and with Gram-negative bacteria-binding protein of Bombyx mori silkworm, two proteins involved in invertebrate defense mechanisms. We show that CCF-1 efficiently binds both beta-1,3-glucan and lipopolysaccharide. Moreover, CCF-1 participates in the activation of prophenoloxidase cascade via recognition of yeast and Gram-negative bacteria cell wall components. These results suggest that the 42-kDa CCF-1 protein of E. foetida coelomic fluid likely plays a role in the protection of earthworms against microbes.     

Complement-inhibiting peptides identified by proximity to indels in the C3/4/5 protein family

To find protein-protein interactive sites in complement component C3, we examined regions of C3 that are proximal to sites of length polymorphism or indels in the C345 protein family. We reasoned that indels probably mark protein interactive sites because they usually involve residues at protein surfaces. To test for the involvement of individual indels, we examined the effects on complement function of synthetic peptides corresponding to indel-proximal segments of C3. We inferred that if such a segment made direct contact with a C3 binding protein, then the corresponding peptide might also bind to that protein and inhibit binding to C3. Twenty-one peptides were tested; four of these inhibited complement-mediated erythrocyte lysis at < or =100 microM and complement-mediated killing of Escherichia coli at about threefold higher levels. These results indicate that the four peptides act as specific inhibitors of complement. They also suggest that indels can be effective guides for locating interactive sites in C3 and in any protein that is a member of a protein family. Because only a linear sequence is required, a focus on indels may be particularly useful for identifying interactive sites in proteins for which a three-dimensional structure is unavailable.     

Phylogeny of the cholecystokinin/gastrin family

The neuroendocrine peptides cholecystokinin (CCK) and gastrin, originally identified in mammals, are characterized by a common amidated C-terminal tetrapeptide sequence, Trp-Met-Asp-Phe.NH2, which also constitutes the minimal structure necessary for biological activity of both. Hence, it has been proposed that CCK and gastrin have evolved from a common ancestor. Although the occurrence of CCK/gastrin-related peptides has been suggested in representatives of several invertebrate phyla, the evidence, mostly based on immunoreactivity, has not been substantiated by peptide identification. Instead, CCK/gastrin-specific antibodies might be cross-reacting with Asp-Phe-amides, like the lymnaDFamides, isolated from the freshwater snail Lymnaea stagnalis. Cionin, isolated from Ciona intestinalis, a representative of the protochordates that occupy a key position at the transition to vertebrates, so far represents the oldest genuine member of the CCK/gastrin family, dating the emergence of these peptides back to at least 500 million years ago. The CCK/gastrin family appears to be represented in the whole chordate phylum, and in addition to mammals, CCK and gastrin have recently been identified in a number of nonmammalian species representing the major vertebrate classes, including fishes, amphibians, reptiles, and birds. This now makes it possible to consider the CCK/gastrin phylogeny based on structural information. A duplication of the ancestral gene appears to have already occurred before or during the appearance of cartilaginous fish, giving rise to two peptides most likely homologous to mammalian CCK and gastrin. Indicative of a function of gastrin, the acid secretory system appears to have developed concomitantly in sharks. The segregation of CCK and gastrin early in vertebrate evolution resembles the situation in other peptide families, in accordance with a suggested widespread pattern of multiplication within vertebrate peptide and protein families around 400 million years ago. At the amphibian level, two separate peptide systems, resembling mammalian CCK and gastrin, have been characterized by identification of the mature bioactive peptides, cDNAs, gene structures, primary and secondary sites of gene expression, and their physiological actions. The overall gene structure, including exon/intron organization, is similar in all mammalian and nonmammalian CCK/gastrin genes. CCK is well conserved in all vertebrate species investigated, while the mammalian gastrins at first sight appear as a distinct group with little similarity to the nonmammalian gastrins outside the invariant C-terminal tetrapeptide and the C-terminal flanking peptide of the prohormone. However, evidence indicates that the transition from nonmammalian to mammalian gastrin may not be as dramatic as first anticipated. In conclusion, the CCK/gastrin family appears to be represented in most, if not all, chordates, to which group it may also be limited. The two major classes, CCK and gastrin, probably arose as distinct peptide systems early in vertebrate history. While CCK is well conserved in all vertebrates, a major structural change of gastrin accompanied the transition to mammals.     

Evolution of the "classical" cadherin family of cell adhesion molecules in vertebrates

The cadherins are major mediators of calcium-dependent cell-cell adhesion and are also involved in cell signaling pathways during development. The classical cadherins, which are the definitive group of the cadherin superfamily, are transmembrane proteins that consist of an extracellular domain of five cadherin repeats, including an HAV tripeptide conserved in one binding surface within the first domain, and a highly conserved cytoplasmic domain that interacts with the actin cytoskeleton via the catenin proteins. These cadherins play major roles in vertebrate morphogenesis; they are expressed widely throughout development, antibodies to specific cadherins perturb a variety of developmental processes, and many gene knockouts are lethal at early stages of development. Phylogenetic analysis of the "classical" cadherins shows that in the vertebrates there are four paralog families. The rate of evolutionary change is radically different between the different paralogs, indicating that there are significantly different selection pressures on the functions of the various cadherins, both between the different paralogs in a single organism lineage and between different organism lineages within a single paralog family. There is also evidence for gene conversion between the E-cadherin and P-cadherin paralogs in Gallus gallus and possibly Xenopus laevis, but not between the same paralogs in the mammalian lineages. A scheme for the origin of the paralogs within the vertebrate lineage based on these analyses indicates that the presence of the four paralog families is a characteristic of vertebrates and that variation of cadherin structure and function is a significant factor in morphological evolution of vertebrates.     

Characterization of two patched receptors for the vertebrate hedgehog protein family

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, we have isolated the human PTCH2 gene. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates its activity in vivo. Chromosomal localization of PTCH2 places it on chromosome 1p33-34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells.     

Actin cytoskeleton organization regulated by the PAK family of protein kinases

Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton.     

The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait

Inspection of the genomes for the bacteria Bacillus subtilis 168, Borrelia burgdorferi B31, Escherichia coli K-12, Haemophilus influenzae KW20, Helicobacter pylori 26695, Mycoplasma genitalium G-37, and Synechocystis sp PCC 6803 and for the archaeons Archaeoglobus fulgidus VC-16 DSM4304, Methanobacterium thermoautotrophicum delta H, and Methanococcus jannaschii DSM2661 revealed that each contains at least one ORF whose predicted product displays sequence features characteristic of eukaryote-like protein-serine/threonine/tyrosine kinases and protein-serine/threonine/tyrosine phosphatases. Orthologs for all four major protein phosphatase families (PPP, PPM, conventional PTP, and low molecular weight PTP) were present in the bacteria surveyed, but not all strains contained all types. The three archaeons surveyed lacked recognizable homologs of the PPM family of eukaryotic protein-serine/threonine phosphatases; and only two prokaryotes were found to contain ORFs for potential phosphatases from all four major families. Intriguingly, our searches revealed a potential ancestral link between the catalytic subunits of microbial arsenate reductases and the protein-tyrosine phosphatases; they share similar ligands (arsenate versus phosphate) and features of their catalytic mechanism (formation of arseno-versus phospho-cysteinyl intermediates). It appears that all prokaryotic organisms, at one time, contained the genetic information necessary to construct protein phosphorylation-dephosphorylation networks that target serine, threonine, and/or tyrosine residues on proteins. However, the potential for functional redundancy among the four protein phosphatase families has led many prokaryotic organisms to discard one, two, or three of the four.     

Evolution of the serum amyloid A (SAA) protein superfamily

The serum amyloid A (SAA) superfamily comprises a number of genes and proteins characterized from a range of mammalian species. The majority of members described to date are dramatically induced during the acute-phase response, suggesting an important short-term beneficial role in the response to tissue injury and inflammation. However, important disease associations have also been proposed for certain SAAs during chronic inflammation. The nomenclature of many of the superfamily members has been the result of comparisons with previously reported sequences implying disease association and/or functional relatedness between such members. The evolutionary relationships of the SAA superfamily members have been investigated by comparisons at both the amino acid and the nucleotide level. The results indicate that all members of the superfamily within a species have been undergoing concerted evolution. This has important implications in ascribing functions and disease associations to individual SAA superfamily members and indicates that designations should not be based on the extent of amino acid identity alone but should be made only following direct experimental observation of the proteins themselves.     

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.     

gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COP I and II coatomer

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.