Biochemical evidences for M1-, M17- and M18-like aminopeptidases in marine invertebrates from Cuban coastline.

Metallo-aminopeptidases (mAPs) control many physiological processes. They are classified in different families according to structural similarities. Neutral mAPs catalyze the cleavage of neutral amino acids from the N-terminus of proteins or peptide substrates; they need one or two metallic cofactors in their active site. Information about marine invertebrate's neutral mAPs properties is scarce; available data are mainly derived from genomics and cDNA studies. The goal of this work was to characterize the biochemical properties of the neutral APs activities in eight Cuban marine invertebrate species from the Phyla Mollusca, Porifera, Echinodermata, and Cnidaria. Determination of substrate specificity, optimal pH and effects of inhibitors (1,10-phenanthroline, amastatin, and bestatin) and cobalt on activity led to the identification of distinct neutral AP-like activities, whose biochemical behaviors were similar to those of the M1 and M17 families of mAPs. Additionally, M18-like glutamyl AP activities were detected. Thus, marine invertebrates express biochemical activities likely belonging to various families of metallo-aminopeptidases.

Matrix Metalloproteinases in Invertebrates.

Matrix Metalloproteinases (MMPs) belong to a family of metal-dependent endopeptidases which contain a series of conserved pro-peptide domains and catalytic domains. MMPs have been widely found in plants, animals, and microorganisms. MMPs are involved in regulating numerous physiological processes, pathological processes, and immune responses. In addition, MMPs play a key role in disease occurrence, including tumors, cardiovascular diseases, and other diseases. Compared with invertebrate MMPs, vertebrate MMPs have diverse subtypes and complex functions. Therefore, it is difficult to study the function of MMPs in vertebrates. However, it is relatively easy to study invertebrate MMPs because there are fewer subtypes of MMPs in invertebrates. In the present review, the structure and function of MMPs in invertebrates were summarized, which will provide a theoretical basis for investigating the regulatory mechanism of MMPs in invertebrates.

AID/APOBEC-like cytidine deaminases are ancient innate immune mediators in invertebrates.

In the course of both innate and adaptive immunity, cytidine deaminases within the activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) family modulate immune responses by mutating specific nucleic acid sequences of hosts and pathogens. The evolutionary emergence of these mediators, however, seems to coincide precisely with the emergence of adaptive immunity in vertebrates. Here, we show a family of genes in species within two divergent invertebrate phyla-the echinoderm Strongylocentrotus purpuratus and the brachiopod Lingula anatina-that encode proteins with similarities in amino acid sequence and enzymatic activities to the vertebrate AID/APOBECs. The expression of these invertebrate factors is enriched in tissues undergoing constant, direct interactions with microbes and can be induced upon pathogen challenge. Our findings suggest that AID/APOBEC proteins, and their function in immunity, emerged far earlier than previously thought. Thus, cytidine deamination is probably an ancient innate immune mechanism that predates the protostome/deuterostome divergence.

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa.

BACKGROUND: The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. RESULTS: Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. CONCLUSION: Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future effort to design logical, potent, and durable anti-SIAH-based anticancer strategies against oncogenic K-RAS-driven metastatic human cancers. Thus, this method of evolutionary study should be of interest in cancer biology.

Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, ß and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates.

A novel C-type lysozyme from Mytilus galloprovincialis: insight into innate immunity and molecular evolution of invertebrate C-type lysozymes.

A c-type lysozyme (named as MgCLYZ) gene was cloned from the mussel Mytilus galloprovincialis. Blast analysis indicated that MgCLYZ was a salivary c-type lysozyme which was mainly found in insects. The nucleotide sequence of MgCLYZ was predicted to encode a polypeptide of 154 amino acid residues with the signal peptide comprising the first 24 residues. The deduced mature peptide of MgCLYZ was of a calculated molecular weight of 14.4 kD and a theoretical isoelectric point (pI) of 8.08. Evolution analysis suggested that bivalve branch of the invertebrate c-type lysozymes phylogeny tree underwent positive selection during evolution. By quantitative real-time RT-PCR (qRT-PCR) analysis, MgCLYZ transcript was widely detected in all examined tissues and responded sensitively to bacterial challenge in hemocytes and hepatopancreas. The optimal temperature and pH of recombinant MgCLYZ (rMgCLYZ) were 20°C and 4, respectively. The rMgCLYZ displayed lytic activities against Gram-positive bacteria including Micrococcus luteus and Staphyloccocus aureus, and Gram-negative bacteria including Vibrio anguillarum, Enterobacter cloacae, Pseudomonas putida, Proteus mirabilis and Bacillus aquimaris. These results suggest that MgCLYZ perhaps play an important role in innate immunity of M. galloprovincialis, and invertebrate c-type lysozymes might be under positive selection in a species-specific manner during evolution for undergoing adaptation to different environment and diverse pathogens.

A bifunctional invertebrate-type lysozyme from the disk abalone, Haliotis discus discus: genome organization, transcriptional profiling and biological activities of recombinant protein.

Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545 bp with an open reading frame of 393 bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3 kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu(30) and Asp(41)), isopeptidase activity (His(107)), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60 °C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.

The evolutionary portrait of metazoan NAD salvage.

Nicotinamide Adenine Dinucleotide (NAD) levels are essential for cellular homeostasis and survival. Main sources of intracellular NAD are the salvage pathways from nicotinamide, where Nicotinamide phosphoribosyltransferases (NAMPTs) and Nicotinamidases (PNCs) have a key role. NAMPTs and PNCs are important in aging, infection and disease conditions such as diabetes and cancer. These enzymes have been considered redundant since either one or the other exists in each individual genome. The co-occurrence of NAMPT and PNC was only recently detected in invertebrates though no structural or functional characterization exists for them. Here, using expression and evolutionary analysis combined with homology modeling and protein-ligand docking, we show that both genes are expressed simultaneously in key species of major invertebrate branches and emphasize sequence and structural conservation patterns in metazoan NAMPT and PNC homologues. The results anticipate that NAMPTs and PNCs are simultaneously active, raising the possibility that NAD salvage pathways are not redundant as both are maintained to fulfill the requirement for NAD production in some species.

Identification of novel phospholipase A2 group IX members in metazoans.

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA2 pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA2 sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA2s while metazoan representatives are similar with GIX PLA2 of the marine snail Conus magus. Members of GXIV and GIX PLA2s show modest overall sequence similarity (21-35%) but considerable motif conservation within the putative Ca(2+)-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA2s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA2 homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA2 deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA2s, two of which correspond to those localized in GXIV PLA2s. Phylogenetic analysis demonstrates that metazoan GIX PLA2s cluster separate from the bacterial and fungal GXIV PLA2s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA2 pfam06951. Duplicate PLA2 pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA2s and support the idea of the common evolutionary origin of GXIV and GIX PLA2 pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.

Pharmacological analyses of protein kinases regulating egg maturation in marine nemertean worms: a review and comparison with Mammalian eggs.

For development to proceed normally, animal eggs must undergo a maturation process that ultimately depends on phosphorylations of key regulatory proteins. To analyze the kinases that mediate these phosphorylations, eggs of marine nemertean worms have been treated with pharmacological modulators of intracellular signaling pathways and subsequently probed with immunoblots employing phospho-specific antibodies. This article both reviews such analyses and compares them with those conducted on mammals, while focusing on how egg maturation in nemerteans is affected by signaling pathways involving cAMP, mitogen-activated protein kinases, Src-family kinases, protein kinase C isotypes, AMP-activated kinase, and the Cdc2 kinase of maturation-promoting factor.

Structure-based evolutionary relationship of glycosyltransferases: a case study of vertebrate ß1,4-galactosyltransferase, invertebrate ß1,4-N-acetylgalactosaminyltransferase and α-polypeptidyl-N-acetylgalactosaminyltransferase.

Cell surface glycans play important cellular functions and are synthesized by glycosyltransferases. Structure and function studies show that the donor sugar specificity of the invertebrate ß1,4-N-acetyl-glactosaminyltransferase (ß4GalNAc-T) and the vertebrate ß1,4-galactosyltransferase I (ß4Gal-T1) are related by a single amino acid residue change. Comparison of the catalytic domain crystal structures of the ß4Gal-T1 and the α-polypeptidyl-GalNAc-T (αppGalNAc-T) shows that their protein structure and sequences are similar. Therefore, it seems that the invertebrate ß4GalNAc-T and the catalytic domain of αppGalNAc-T might have emerged from a common primordial gene. When vertebrates emerged from invertebrates, the amino acid that determines the donor sugar specificity of the invertebrate ß4GalNAc-T might have mutated, thus converting the enzyme to a ß4Gal-T1 in vertebrates.

Alternative oxidase in animals: unique characteristics and taxonomic distribution.

Alternative oxidase (AOX), a ubiquinol oxidase, introduces a branch point into the respiratory electron transport chain, bypassing complexes III and IV and resulting in cyanide-resistant respiration. Previously, AOX was thought to be limited to plants and some fungi and protists but recent work has demonstrated the presence of AOX in most kingdoms of life, including animals. In the present study we identified AOX in 28 animal species representing nine phyla. This expands the known taxonomic distribution of AOX in animals by 10 species and two phyla. Using bioinformatics we found AOX gene sequences in members of the animal phyla Porifera, Placozoa, Cnidaria, Mollusca, Annelida, Nematoda, Echinodermata, Hemichordata and Chordata. Using reverse-transcriptase polymerase chain reaction (RT-PCR) with degenerate primers designed to recognize conserved regions of animal AOX, we demonstrated that AOX genes are transcribed in several animals from different phyla. An analysis of full-length AOX sequences revealed an amino acid motif in the C-terminal region of the protein that is unique to animal AOXs. Animal AOX also lacks an N-terminal cysteine residue that is known to be important for AOX enzyme regulation in plants. We conclude that the presence of AOX is the ancestral state in animals and hypothesize that its absence in some lineages, including vertebrates, is due to gene loss events.

Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: a reconsideration of the implications for insecticide design.

Previously we used site-directed mutagenesis, in vitro expression, and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase, cholinesterase 2 from amphioxus, by the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). We created the mutants C310A, C466A, C310A/C466A and C310A/F312I to assess the roles of the two cysteines and a proposal that the increased rate of inactivation previously found in an F312I mutant was due to increased access of sulfhydryl reagents to Cys310. Our results indicated that both of the cysteines could be involved in inactivation by sulfhydryl reagents, but that the cysteine near the acyl pocket was more accessible. We speculated that the inactivation of aphid AChEs by sulfhydryl reagents was due to the presence of a cysteine homologous to Cys310 and proposed that this residue could be a target for a specific insecticide. Here we reconsider this proposal.

Remodeling the model organism: matrix metalloproteinase functions in invertebrates.

The matrix metalloproteinase (MMP) family of extracellular proteases is conserved throughout the animal kingdom. Studies of invertebrate MMPs have demonstrated they are involved in tissue remodeling. In Drosophila, MMPs are required for histolysis, tracheal growth, tissue invasion, axon guidance, and dendritic remodeling. Recent work demonstrates that MMPs also participate in Drosophila tumor invasion. In Caenorhabditis elegans an MMP is involved in anchor cell invasion; a Hydra MMP is important for regeneration and maintaining cell identity; and a sea urchin MMP degrades matrix to allow hatching. In worms and in flies, MMPs are regulated by the JNK pathway.

Marine glutathione S-transferases.

The aquatic environment is generally affected by the presence of environmental xenobiotic compounds. One of the major xenobiotic detoxifying enzymes is glutathione S-transferase (GST), which belongs to a family of multifunctional enzymes involved in catalyzing nucleophilic attack of the sulfur atom of glutathione (gamma-glutamyl-cysteinylglycine) to an electrophilic group on metabolic products or xenobiotic compounds. Because of the unique nature of the aquatic environment and the possible pollution therein, the biochemical evolution in terms of the nature of GSTs could by uniquely expressed. The full complement of GSTs has not been studied in marine organisms, as very few aquatic GSTs have been fully characterized. The focus of this article is to present an overview of the GST superfamily and their critical role in the survival of organisms in the marine environment, emphasizing the critical roles of GSTs in the detoxification of marine organisms and the unique characteristics of their GSTs compared to those from non-marine organisms.

The prophenoloxidase-activating system in invertebrates.

A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed. The proPO-activating system (proPO system) is triggered by the presence of minute amounts of compounds of microbial origins, such as beta-1,3-glucans, lipopolysaccharides, and peptidoglycans, which ensures that the system will become active in the presence of potential pathogens. The presence of specific proteinase inhibitors prevents superfluous activation. Concomitant with proPO activation, many other immune reactions will be produced, such as the generation of factors with anti-microbial, cytotoxic, opsonic, or encapsulation-promoting activities.

The role of Ca2+/calmodulin-stimulable adenylyl cyclases as molecular coincidence detectors in memory formation.

Evidence from systems as diverse as mollusks, insects and mammals has revealed that adenylyl cyclase, cyclic adenosine 3',5'-monophosphate (cAMP) cascade, cAMP-dependent protein kinases and their substrates are required for the cellular events underlying the short-term and long-term forms of memory. In Aplysia and Drosophila models, the coincident activation of independent paths converge to produce a synergistic activation of Ca2+/calmodulin-stimulable adenylyl cyclase, thereby enhancing the cAMP level that appears as the primary mediator of downstream events that strengthen enduring memory. In mammals, in which long-term memories require hippocampal function, our understanding of the role of adenylyl cyclases is still fragmentary. Of the differently regulated isoforms present in the hippocampus, the susceptibility of type 1 and type 8 to stimulation by the complex Ca2+/calmodulin and their expression in the hippocampus suggest a role for these two isoforms as a molecular coincidence device for hippocampus-related memory function. Here, we review the key features of Ca2+/calmodulin stimulable adenylyl cyclases, as well as the involvement of cAMP-regulated signaling pathway in the processes of learning and memory.

Intracellular proteinases of invertebrates: calcium-dependent and proteasome/ubiquitin-dependent systems.

Cytosolic proteinases carry out a variety of regulatory functions by controlling protein levels and/or activities within cells. Calcium-dependent and ubiquitin/proteasome-dependent pathways are common to all eukaryotes. The former pathway consists of a diverse group of Ca(2+)-dependent cysteine proteinases (CDPs; calpains in vertebrate tissues). The latter pathway is highly conserved and consists of ubiquitin, ubiquitin-conjugating enzymes, deubiquitinases, and the proteasome. This review summarizes the biochemical properties and genetics of invertebrate CDPs and proteasomes and their roles in programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, gametogenesis and fertilization, development and pattern formation, cell-cell recognition, signal transduction and learning, and photoreceptor light adaptation. These pathways carry out bulk protein degradation in the programmed death of the intersegmental and flight muscles of insects and of individuals in a colonial ascidian; molt-induced atrophy of crustacean claw muscle; and responses of brine shrimp, mussels, and insects to environmental stress. Selective proteolysis occurs in response to specific signals, such as in modulating protein kinase A activity in sea hare and fruit fly associated with learning; gametogenesis, differentiation, and development in sponge, echinoderms, nematode, ascidian, and insects; and in light adaptation of photoreceptors in the eyes of squid, insects, and crustaceans. Proteolytic activities and specificities are regulated through proteinase gene expression (CDP isozymes and proteasomal subunits), allosteric regulators, and posttranslational modifications, as well as through specific targeting of protein substrates by a diverse assemblage of ubiquitin-conjugases and deubiquitinases. Thus, the regulation of intracellular proteolysis approaches the complexity and versatility of transcriptional and translational mechanisms.

Glutatión S-transferasas en no-vertebrados y en mamíferos: su importancia en la detoxificación de insecticidas / The Glutathione S-transferases (GST) of non-vertebrate organisms and mammals

Las Glutatión S-transferasas (GST) de organismos no-vertebrados no han sido estudiadas con la misma intensidad que las de mamíferos. El interés en las GST en no-vertebrados radica en su importancia como protección bioquímica de los organismos expuestos a compuestos químicos ambientales. En efecto, se ha observado que niveles elevados de GST podrían estar asociados con la tolerancia a pesticidas. La intención de esta actualización es revisar el nivel de conocimiento actual sobre estas enzimas en no-vertebrados, comparándolas con las de mamíferos. Evaluar la contribución de estos estudios al conocimiento del rol de las glutinatión transferasas en general, e intentar discernir la dirección de las futuras investigaciones en este campo