A Second Class of Acetylcholinesterase-Deficient Mutants of the Nematode CAENORHABDITIS ELEGANS

In Johnson et al. (1981), the Caenorhabditis elegans mutant strain PR1000, homozygous for the ace-1 mutation p1000, is shown to be deficient in the class A subset of acetylcholinesterases, which comprises approximately one-half of the total C. elegans acetylcholinesterase activity. Beginning with this strain, we have isolated 487 new behavioral and morphological mutant strains. Two of these, independently derived, lack approximately 98% of the wild-type acetylcholinesterase activity and share the same specific uncoordinated phenotype; both move forward in a slow and uncoordinated manner, and when mechanically stimulated to induce reversal, both hypercontract and become temporarily paralyzed. In addition to the ace-1 mutation, both strains also harbor recessive mutations in the same newly identified gene, ace-2, which maps to chromosome I and is therefore not linked to ace-1. Gene dosage experiments suggest that ace-2 is a structural gene for the remaining class B acetylcholinesterases, which are not affected by ace-1.—The uncoordinated phenotype of the newly isolated, doubly mutant strains depends on both the ace-1 and ace-2 mutations; homozygosity for either mutation alone produces normally coordinated animals. This result implies functional overlap of the acetylcholinesterases controlled by ace-1 and ace-2, perhaps at common synapses. Consistent with this, light microscopic histochemical staining of permeabilized whole mounts indicates some areas of possible spatial overlap of these acetylcholinesterases (nerve ring, longitudinal nerve cords). In addition, there is at least one area where only ace-2-controlled acetylcholinesterase activity appears (pharyngeo-intestinal valve).     

The carboxylesterase/cholinesterase gene family in invertebrate deuterostomes

Carboxylesterase/cholinesterase family members are responsible for controlling the nerve impulse, detoxification and various developmental functions, and are a major target of pesticides and chemical warfare agents. Comparative structural analysis of these enzymes is thus important. The invertebrate deuterostomes (phyla Echinodermata and Hemichordata and subphyla Urochordata and Cephalochordata) lie in the transition zone between invertebrates and vertebrates, and are thus of interest to the study of evolution. Here we have investigated the carboxylesterase/cholinesterase gene family in the sequenced genomes of Strongylocentrotus purpuratus (Echinodermata), Saccoglossus kowalevskii (Hemichordata), Ciona intestinalis (Urochordata) and Branchiostoma floridae (Cephalochordata), using sequence analysis of the catalytic apparatus and oligomerisation domains, and phylogenetic analysis. All four genomes show blurring of structural boundaries between cholinesterases and carboxylesterases, with many intermediate enzymes. Non-enzymatic proteins are well represented. The Saccoglossus and Branchiostoma genomes show evidence of extensive gene duplication and retention. There is also evidence of domain shuffling, resulting in multidomain proteins consisting either of multiple carboxylesterase domains, or of carboxylesterase/cholinesterase domains linked to other domains, including RING finger, chitin-binding, immunoglobulin, fibronectin type 3, CUB, cysteine-rich-Frizzled, caspase activation and 7tm-1, amongst others. Such gene duplication and domain shuffling in the carboxylesterase/cholinesterase family appears to be unique to the invertebrate deuterostomes, and we hypothesise that these factors may have contributed to the evolution of the morphological complexity, particularly of the nervous system and neural crest, of the vertebrates.     

Analysis of structural motifs of proteins using sets of codes, describing local structures]

An amino acid sequence pattern conserved among a family of proteins is called motif. It is usually related to the specific function of the family. On the other hand, functions of proteins are achieved by their 3D structures. Specific local structures, called structural motifs, are considered related to their functions. However, searching for common structural motifs in different proteins is much more difficult than for common sequence motifs. We are attempting in this study to convert the information about the structural motifs into a set of one-dimensional digital strings, i.e., a set of codes, to compare them more easily by computer and to investigate their relationship to functions more quantitatively. By applying the Delaunay tessellation to a 3D structure of a protein, we can assign each local structure to a unique code that is defined so as to reflect its structural feature. Since a structural motif is defined as a set of the local structures in this paper, the structural motif is represented by a set of the codes. In order to examine the ability of the set of the codes to distinguish differences among the sets of local structures with a given PROSITE pattern that contain both true and false positives, we clustered them by introducing a similarity measure among the set of the codes. The obtained clustering shows a good agreement with other results by direct structural comparison methods such as a superposition method. The structural motifs in homologous proteins are also properly clustered according to their sources. These results suggest that the structural motifs can be well characterized by these sets of the codes, and that the method can be utilized in comparing structural motifs and relating them with function.     

Structural homology discloses a bifunctional structural motif at the N-termini of G alpha proteins

In a family of proteins, often the three-dimensional structure has been experimentally determined only for one member or a few members of the family. Homology modeling can be used to model the structures of all other members of the family and thus allow comparison of these structures. This approach was applied to heterotrimeric G proteins that require anchorage to the plasma membrane to properly interact with membrane-bound receptors and downstream effectors. Lipid modification by palmitoylation is a fundamental contributor to this localization, but the signals leading to this modification are still unknown. In this work, homology models of all the different human G(alpha) paralogs were generated using automated homology modeling, and the electrostatic potential of these proteins was calculated and visualized. This approach identifies a basic, positively charged, structural motif in the N-termini of heterotrimeric G proteins, which is not readily discernible from sequence alone. The basic motif is much reduced in those G(alpha) subunits that also undergo myristoylation, suggesting that the basic patches and myristoylation play overlapping roles. These motifs can affect both membrane affinity and orientation and determine the palmitoylation of G(alpha) subunits in cooperation with the G(betagamma) subunits, as has been corroborated by previous experimental studies. Furthermore, other palmitoylated proteins such as GAP-43 and RGS proteins share this alpha-helical basic motif in their N-terminus. It therefore appears that this structural motif is more widely applicable as a membrane-targeting and palmitoylation-determining signal. The work presented here highlights the possibilities available for experimentalists to discover structural motifs that are not readily observed by analysis of the linear sequence.     

An analysis of acetylcholinesterase sequence for predicting mechanisms of its non-catalytic actions

The enzyme acetylcholinesterase (AChE) which belongs to the family of alpha/beta hydrolases is well known for hydrolyzing the neurotransmitter acetylcholine (ACh). In addition to its catalytic function, AChE appears to play a significant non-catalytic role in development, regeneration and modulation of properties of neurons. However the mechanisms underlying these important actions of AChE are unknown. It prompted the analysis of the sequence of fetal bovine serum AChE to seek clue(s) for the mechanisms of AChE non-catalytic actions. The searches for motifs, finger prints and domains demonstrated the presence of a highly conserved carboxylesterase type B signature in AChE from slime molds to human. Interestingly, the presence of finger prints for a metabotropic glutamate receptor, gap junction protein connexin, a calcium binding motif, several phosphorylation sites, and a motif similar to nicotinic acetylcholine receptor were also found in the sequence of AChE suggesting a role for AChE in cell signalling. The similarity search using gapped BLAST retrieved previously known similar proteins, namely neurotactin, neuroligin and thyroglobulin. When aligned neurotactin, neuroligin and bovine AChE showed the presence of three stretches of consensus, one of which is carboxylesterase B signature. The identity of the other two are not known. Amino acids in these stretches in AChE may be involved in possible growth factor like action and awaits experimental verification. Thus sequence analysis can be very useful for biologists while searching for a possible mechanism of action of a protein. Further, these results underscore the significance of performing both local and global searches to identify possible functional domains as well as consensus sequence.     

Identifying DNA-binding proteins using structural motifs and the electrostatic potential

Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif.     

Analysis of Protein Structural Motifs in Terms of Sets of Codes Representing Local Structures

An amino acid sequence pattern conserved among a family of proteins is called motif. It is usually related to the specific function of the family. On the other hand, functions of proteins are realized through their 3D structures. Specific local structures, called structural motifs, are considered as related to their functions. However, searching for common structural motifs in different proteins is much more difficult than for common sequence motifs. We are attempting in this study to convert the information about the structural motifs into a set of one-dimensional digital strings, i.e., a set of codes, to compare them more easily by computer and to investigate their relationship to functions more quantitatively. By applying the Delaunay tessellation to a 3D structure of a protein, we can assign each local structure to a unique code that is defined so as to reflect its structural feature. Since a structural motif is defined as a set of the local structures in this paper, the structural motif is represented by a set of the codes. In order to examine the ability of the set of the codes to distinguish differences among the sets of local structures with a given PROSITE pattern that contain both true and false positives, we clustered them by introducing a similarity measure among the set of the codes. The obtained clustering shows a good agreement with other results by direct structural comparison methods such as a superposition method. The structural motifs in homologous proteins are also properly clustered according to their sources. These results suggest that the structural motifs can be well characterized by these sets of the codes, and that the method can be utilized in comparing structural motifs and relating them with function.     

Common fold in helix-hairpin-helix proteins

Helix–hairpin–helix (HhH) is a widespread motif involved in non-sequence-specific DNA binding. The majority of HhH motifs function as DNA-binding modules, however, some of them are used to mediate protein–protein interactions or have acquired enzymatic activity by incorporating catalytic residues (DNA glycosylases). From sequence and structural analysis of HhH-containing proteins we conclude that most HhH motifs are integrated as a part of a five-helical domain, termed (HhH)₂ domain here. It typically consists of two consecutive HhH motifs that are linked by a connector helix and displays pseudo-2-fold symmetry. (HhH)₂ domains show clear structural integrity and a conserved hydrophobic core composed of seven residues, one residue from each α-helix and each hairpin, and deserves recognition as a distinct protein fold. In addition to known HhH in the structures of RuvA, RadA, MutY and DNA-polymerases, we have detected new HhH motifs in sterile alpha motif and barrier-to-autointegration factor domains, the α-subunit of Escherichia coli RNA-polymerase, DNA-helicase PcrA and DNA glyco­s­y­lases. Statistically significant sequence similarity of HhH motifs and pronounced structural conservation argue for homology between (HhH)₂ domains in different protein families. Our analysis helps to clarify how non-symmetric protein motifs bind to the double helix of DNA through the formation of a pseudo-2-fold symmetric (HhH)₂ functional unit.     

Conservation of context-dependent splicing activity in distant Muscleblind homologs

The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species.     

Comparative analysis of editosome proteins in trypanosomatids

Detailed comparisons of 16 editosome proteins from Trypanosoma brucei, Trypanosoma cruzi and Leishmania major identified protein motifs associated with catalysis and protein or nucleic acid interactions that suggest their functions in RNA editing. Five related proteins with RNase III-like motifs also contain a U1-like zinc finger and either dsRBM or Pumilio motifs. These proteins may provide the endoribonuclease function in editing. Two other related proteins, at least one of which is associated with U-specific 3′ exonuclease activity, contain two putative nuclease motifs. Thus, editosomes contain a plethora of nucleases or proteins presumably derived from nucleases. Five additional related proteins, three of which have zinc fingers, each contain a motif associated with an OB fold; the TUTases have C-terminal folds reminiscent of RNA binding motifs, thus indicating the presence of numerous nucleic acid and/or protein binding domains, as do the two RNA ligases and a RNA helicase, which provide for additional catalytic steps in editing. These data indicate that trypanosomatid RNA editing is orchestrated by a variety of domains for catalysis, molecular interaction and structure. These domains are generally conserved within other protein families, but some are found in novel combinations in the editosome proteins.     

Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors.     

An Ancient Autoproteolytic Domain Found in GAIN,ZU5 and Nucleoporin98

A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain.     

Cyst germination proteins of the potato pathogen Phytophthora infestans share homology with human mucins

We have cloned genes of Phytophthora infestans, the causal agent of potato late blight, that are activated shortly before the onset of invasion of the host tissue. The three genes isolated appear to be arranged in a genomic cluster and belong to a small polymorphic gene family. A conspicuous feature of the deduced proteins is an internal octapeptide repeat with the consensus sequence TTYAP TEE. Because of this structural motif, these novel P. infestans proteins were named Car (Cyst-germination-specific acidic repeat) proteins. One of the genes, car90, codes for 1,489 amino acids including 120 octapeptide tandem repeats. Car proteins are transiently expressed during germination of cysts and formation of appressoria and are localized at the surface of germlings. The structural motif of tandemly repeated oligopeptides also occurs in a prominent class of proteins, the mucins, from mammals. The P. infestans Car proteins share 51% sequence homology with the tandem repeat region of a subfamily of human mucins. According to the physiological functions ascribed to mucins, we suggest that Car proteins may serve as a mucous cover protecting the germling from desiccation, physical damage, and adverse effects of the plant defense response and may assist in adhesion to the leaf surface.     

The interaction of Thrombospondins with extracellular matrix proteins

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias.     

The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

Background

Arthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families.

Methodology/Principal Findings

Two short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread.

Conclusions

This survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly.     

PE-only/PE_PGRS proteins of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> contain a conserved tetra-peptide sequence DEVS/DXXS that is a potential caspase-3 cleavage motif

Mycobacterium tuberculosis H37Rv is an intracellular pathogen responsible for causing tuberculosis in humans. The M. tuberculosis genome has been shown to contain a very large and unique family of PE proteins made of two sub-families: PE-only and PE_PGRS proteins. These two subtypes of proteins play a crucial role in the pathogenesis of the microbe. However, despite numerous investigations, the role of these proteins in disease development remains obscure. In this study, sequence analysis with a search for short conserved motifs revealed a conserved tetra-peptide motif DEVS/DXXS at the PE domain of almost every PE-only and PE_PGRS protein. The motif was found at a distance of 43–46 amino acids from the N-terminal of PE_PGRS proteins, and at a distance of between 35 and 82 amino acids of the PE-only proteins. As phosphorylation of the serine residue of this tetra-peptide could yield a motif similar to the caspase-3 binding recognition sequence DEVD/E, the region from a representative PE_PGRS protein (PE_PGRS45) was docked to human caspase-3. Strong interactions of only the protein containing the phosphorylated motif (DEVpS/DXXpS) to caspase-3 were observed. This suggested that the conserved DEVS/DXXS motif could have evolved for phosphorylation and subsequent recognition by caspase-3. These findings have important implications in unravelling the role of these PE proteins in mycobacterial infection.     

Conserved pore-forming regions in polypeptide-transporting proteins

Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the beta-barrel-shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organelles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the beta-barrel-shaped polypeptide-transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C-terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed.     

Characteristics of SNARE proteins are defined by distinctive properties of SNARE motifs

BackgroundSyntaxin-1A and Sso1 are syntaxin family SNARE proteins engaged in synaptic vesicle fusion and yeast exocytosis. The syntaxin-1A SNARE motif can form a fusogenic SNARE complex with Sso1 partners. However, a chimera in which the SNARE motif in syntaxin-1A is introduced into Sso1 was not functional in yeast because the chimera is retained in the ER. Through the analysis of the transport defect of Sso1/syntaxin-1A chimeric SNAREs, we found that their SNARE motifs have distinctive properties.MethodsSso1, syntaxin-1A, and Sso1/syntaxin-1A chimeric SNAREs were expressed in yeast cells and their localization and interaction with other SNAREs are analyzed.ResultsSNARE proteins containing the syntaxin-1A SNARE motif exhibit a transport defect because they form a cis-SNARE complex in the ER. Ectopic SNARE complex formation can be prevented in syntaxin-1A by binding to a Sec1/Munc-18-like (SM) protein. In contrast, the SNARE motif of Sso1 does not form an ectopic SNARE complex. Additionally, we found that the SNARE motif in syntaxin-1A, but not that in Sso1, self-interacts, even when it is in the inactive form and bound to the SM protein.ConclusionsThe SNARE motif in syntaxin-1A, but not in Sso1, likely forms ectopic SNARE complex. Because of this property, the SM protein is necessary for syntaxin-1A to prevent its promiscuous assembly and to promote its export from the ER.General significanceProperties of SNARE motifs affect characteristics of SNARE proteins. The regulatory mechanisms of SNARE proteins are, in part, designed to handle such properties.