Sequence analysis and phylogenetic study of some toxin proteins of snakes and related non-toxin proteins of chordates

Snakes are equipped with their venomic armory to tackle different prey and predators in adverse natural world. The venomic composition of snakes is a mix of biologically active proteins and polypeptides. Among different components snake venom cytotoxins and short neurotoxin are non-enzymatic polypeptide candidates with in the venom. These two components structurally resembled to three-finger protein superfamily specific scaffold. Different non-toxin family members of three-finger protein superfamily are involved in different biological roles. In the present study we analyzed the snake venom cytotoxins, short neurotoxins and related non-toxin proteins of different chordates in terms of amino acid sequence level diversification profile, polarity profile of amino acid sequences, conserved pattern of amino acids and phylogenetic relationship of these toxin and nontoxin protein sequences. Sequence alignment analysis demonstrates the polarity specific molecular enrichment strategy for better system adaptivity. Occurrence of amino acid substitution is high in number in toxin sequences. In non-toxin body proteins there are less amino acid substitutions. With the help of conserved residues these proteins maintain the three-finger protein scaffold. Due to system specific adaptation toxin and non-toxin proteins exhibit a varied type of amino acid residue distribution in sequence stretch. Understanding of Natural invention scheme (recruitment of venom proteins from normal body proteins) may help us to develop futuristic engineered bio-molecules with remedial properties.     

Physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates

Snake venom contains a diverse array of proteins and polypeptides. Cytotoxins and short neurotoxins are non-enzymatic polypeptide components of snake venom. The three-dimensional structure of cytotoxin and short neurotoxin resembles a three finger appearance of three-finger protein super family. Different family members of three-finger protein super family are employed in diverse biological functions. In this work we analyzed the cytotoxin, short neurotoxin and related non-toxin proteins of other chordates in terms of functional analysis, amino acid compositional (%) profile, number of amino acids, molecular weight, theoretical isoelectric point (pI), number of positively charged and negatively charged amino acid residues, instability index and grand average of hydropathy with the help of different bioinformatical tools. Among all interesting results, profile of amino acid composition (%) depicts that all sequences contain a conserved cysteine amount but differential amount of different amino acid residues which have a family specific pattern. Involvement in different biological functions is one of the driving forces which contribute the vivid amino acid composition profile of these proteins. Different biological system dependent adaptation gives the birth of enriched bio-molecules. Understanding of physicochemical properties of these proteins will help to generate medicinally important therapeutic molecules for betterment of human lives.     

The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing proteins was confirmed by their binding to pyruvate carboxylase, a biotin-containing enzyme, as well as to biotinylated ovalbumin and biotinylated bovine serum albumin but not to their nonbiotinylated counterparts. Activated HD-73 toxin also inhibited the enzymatic activity of pyruvate carboxylase. The biotin binding site is likely contained in domain III of the toxin. Two highly conserved regions within domain III are similar in sequence to the biotin binding sites of avidin, streptavidin, and a biotin-specific monoclonal antibody. In particular, block 4 of the B. thuringiensis toxin contains the YAS biotin-specific motif. On the basis of its N-terminal amino acid sequence, the 120-kDa biotin-containing protein is totally distinct from the 120-kDa aminopeptidase N reported to be a receptor for Cry1Ac toxin.     

Adrenomedullin and related peptides: receptors and accessory proteins.

Adrenomedullin (AM), alpha- and beta-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes between CRLR or CTR and RAMPs are required for ligand recognition.     

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.     

Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

Cholera and the related Escherichia coli-associated diarrheal disease are important problems confronting Third World nations and any area where water supplies can become contaminated. The disease is extremely debilitating and may be fatal in the absence of treatment. Symptoms are caused by the action of cholera toxin, secreted by the bacterium Vibrio cholerae, or by a closely related heat-labile enterotoxin, produced by Escherichia coli, that causes a milder, more common traveler's diarrhea. Both toxins bind receptors in intestinal epithelial cells and insert an enzymatic subunit that modifies a G protein associated with the adenylate cyclase complex. The consequent stimulated production of cyclic AMP, or other factors such as increased synthesis of prostaglandins by intoxicated cells, initiates a metabolic cascade that results in the excessive secretion of fluid and electrolytes characteristic of the disease. The toxins have a very high degree of structural and functional homology and may be evolutionarily related. Several effective new vaccine formulations have been developed and tested, and a growing family of endogenous cofactors is being discovered in eukaryotic cells. The recent elucidation of the three-dimensional structure of the heat-labile enterotoxin has provided an opportunity to examine and compare the correlations between structure and function of the two toxins. This information may improve our understanding of the disease process itself, as well as illuminate the role of the toxin in studies of signal transduction and G-protein function.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

Membrane interactions of G proteins and other related proteins

Guanine nucleotide-binding proteins, G proteins, propagate incoming messages from receptors to effector proteins. They switch from an inactive to active state by exchanging a GDP molecule for GTP, and they return to the inactive form by hydrolyzing GTP to GDP. Small monomeric G proteins, such as Ras, are involved in controlling cell proliferation, differentiation and apoptosis, and they interact with membranes through isoprenyl moieties, fatty acyl moieties, and electrostatic interactions. This protein-lipid binding facilitates productive encounters of Ras and Raf proteins in defined membrane regions, so that signals can subsequently proceed through MEK and ERK kinases, which constitute the canonical MAP kinase signaling cassette. On the other hand, heterotrimeric G proteins undergo co/post-translational modifications in the alpha (myristic and/or palmitic acid) and the gamma (farnesol or geranylgeraniol) subunits. These modifications not only assist the G protein to localize to the membrane but they also help distribute the heterotrimer (Galphabetagamma) and the subunits generated upon activation (Galpha and Gbetagamma) to appropriate membrane microdomains. These proteins transduce messages from ubiquitous serpentine receptors, which control important functions such as taste, vision, blood pressure, body weight, cell proliferation, mood, etc. Moreover, the exchange of GDP by GTP is triggered by nucleotide exchange factors. Membrane receptors that activate G proteins can be considered as such, but other cytosolic, membranal or amphitropic proteins can accelerate the rate of G protein exchange or even activate this process in the absence of receptor-mediated activation. These and other protein-protein interactions of G proteins with other signaling proteins are regulated by their lipid preferences. Thus, G protein-lipid interactions control the features of messages and cell physiology.     

Cholera toxin modifies diverse GTP-modulated regulatory proteins

Using thin layer chromatography (TLC) and various colorimetric procedures, the exometabolite of $\text{Leishmania}\text{donovani}$ was shown to be a novel glycopeptidophosphosphingolipid. In aqueous medium the exometabolite aggregated to form micellar structures of high molecular weight. Purity of the various preparations and the novel nature of the micellar structures was demonstrated by TLC. These micelles are unique because they do not break up upon solvation in organic solvents. This indicates that once the supramolecular structure is established, its integrity is maintained by forces other than the apolar ones involved in its formation.     

Growth of Aeromonas spp. at 4°C and related toxin production

Over the last few years Aeromonas spp. have been considered to be potential intestinal pathogens (Altwegg & Geiss 1989). Recently, detailed evidence has linked a strain from contaminated shrimp with a clinical isolate from a patient with diarrhoea and found them to be the same (Altwegg et al. 1991). It is now known that Aeromonas spp. are present in many foods, especially those of animal origin (Palumbo et al. 1989). Furthermore, previous studies have shown that these organisms are quite capable of growing at refrigeration temperatures. For example, Palumbo et al. (1985) found that seven out of 14 strains showed significant growth within 14 d at 5°C. However, most studies have only examined a relatively small number of strains, which were not speciated and were simply called A. hydrophila.
In this paper we examine the growth of 36 strains of aeromonas (divided into A. hydrophila, A. sobria and A. caviae ) at 4°C, and their ability to produce cyotoxins at 4°C, 30°C and 37°C.     

Cellular ageing related proteins secreted by human fibroblasts.

Fibroblast secreted proteins participate in the formation of extracellular matrix. Extracellular matrix affects growth factor action, mediates cell adhesion and supports cell growth. Structural and quantitative characteristics of secreted proteins are modified in a similar manner, during both in vivo and in vitro cellular ageing. Such ageing related modifications may either be directly controlled by primary ageing causes, or evolve from a reformation of the extracellular matrix induced by a few ageing defects in key proteins such as fibronectin. They may result in the further inhibition of cell adhesion, cell stimulation by growth factors and, eventually, of cell proliferative ability.     

Characterization of proteins structurally related to human N-acetyl-beta-D-glucosaminidase.

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000--200000) were present both in the unadsorbed fraction and in the 0.05--0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000--70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.     

Prediction of functional residues in water channels and related proteins.

In this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity. The protein sequences were also analyzed using statistical tools (comparisons of means and correspondence analysis). Five key positions were clearly identified where the residues are specific for each functional subgroup and exhibit high dissimilar physico-chemical properties. Moreover, we have found that the putative channels for small neutral solutes clearly differ from the aquaporins by the amino acid content and the length of predicted loop regions, suggesting a substrate filter function for these loops. From these results, we propose a signature pattern for water transport.     

Plant TPX2 and related proteins

At the onset of mitosis, microtubules form a bipolar spindle around the prophase nucleus. TPX2 is phosphorylated during mitosis and acts as a spindle assembly factor that nucleates microtubules in the close vicinity of chromosomes, independent of the centrosomes. Furthermore, it activates the kinase Aurora A and targets the Xenopus kinesin-like protein 2 to spindle poles. We have characterized the plant orthologue of TPX2 that possesses all identified functional domains of its animal counterpart. Moreover, we have demonstrated that it is exported before nuclear envelope breakdown and that its activity around the nuclear envelope is essential for prospindle assembly. Here, we compare the sequences of several characterized TPX2 domains, allowing us to define TPX2. We propose that true TPX2 orthologues share simultaneously all these conserved domains and that other proteins possessing only some of these functional blocks may be considered as TPX2-related proteins.Key words: mitosis, microtubules, spindle assembly, TPX2 signature, targeting domains, Prosite motifs, evolution