Structural and functional comparison of proteolytic enzymes from plant latex and snake venoms

This work describes classification, functions, location, inhibition, activation, and therapeutic applications of proteases from snake venoms and vegetables. Snake venoms and vegetables can present toxins that unchain necrosis or proteolysis due to the direct cytotoxic action of venom proteases. These proteases are potential tools in the development of drugs for the prevention and treatment of several illnesses. We report herein mainly fibrinogenolytic metallo proteases and serine proteases (“thrombin-like”). These enzymes are extensively used in the treatment and prevention of thrombotic disorders, since they serve as defibrinogenating agents. The therapeutic uses of fibrin(ogen)olytic metallo proteases hold promise for clinical application due to potential in reversing the effects of thrombosis; this has been shown to be an alternative approach to the prevention and treatment of cardiovascular disorders, which are among the most prominent causes of mortality around the world. Plant proteases can be utilized for many cellular and molecular activities, in antibacterial and anticancer therapies, and in the treatment of snakebites, inhibiting snake venom activities such as blood-clotting, defibrinogenation, and fibrin(ogen)olytic and hemorrhagic actions. These toxins also display potential for clinical use in the treatment of hemostatic disorders.     

Molecular cloning of a mitogenic proteinase from Carica candamarcensis: its potential use in wound healing

Cysteine proteinases from the Caricaceae belong to the C1 family of the CA clan and display papain-like structured, the archetype enzyme for this group of proteins. Carica candamarcensis, also named Vasconcellea cundinamarcensis, a member of Caricaceae family common to many areas in South America, contains cysteine proteinases with proteolytic activity five to eight-fold higher than those from latex of Carica papaya. The cysteine protease CMS2MS2 from C. candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In this study, the cDNA cloning, expression and evaluation of biological activity of a CMS2MS2-like protein from C. candamarcensis is reported. The 650 bp fragment was cloned in bacteria and the DNA sequence confirmed a cysteine-proteinase similar to CMS2MS2. The recombinant protein is 30 kDa, induces a mitogenic response, and enhances ERK1/2 phosphorylation, like the non-recombinant enzyme, but lacks either amidase or caseinolytic activity. The mitogenic activity of this protein and its lack of proteolytic activity underscore a potential for use in wound healing treatment.     

Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva

A high-molecular-weight mucin-glycoprotein (MG1) was isolated from human submandibular-sublingual saliva and was comprised of 14.9% protein, 29.0% N-acetylglucosamine, 9.4% N-acetylgalactosamine, 10.5% fucose, 24.2% galactose, 0.9% mannose, 4.0% N-acetylneuraminic acid, and 7.0% sulfate. Carbohydrate units were O-glycosidically linked and ranged in size from 4 to 16 residues. The biophysical properties of MG1 were compared to those of a smaller mucin (MG2) also isolated from submandibular-sublingual saliva. Fluorescence spectroscopy demonstrated that MG1 bound both 1-anilino-8-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPNA) in stable hydrophobic binding sites (melting temperature, 47 +/- 2 degrees C), whereas MG2 did not bind these hydrophobic probes. These hydrophobic domains occurred on nonglycosylated or naked portions of MG1 since Pronase treatment eliminated ANS binding. Reduction of disulfide bridges in MG1 increased the number of available hydrophobic binding sites. High ionic strength (0 to 2 M NaCl) had no effect on ligand binding, whereas lowering pH (9 to 2) increased ANS binding without affecting NPNA complexation. Circular dichroism (CD) data suggested that MG1's carbohydrate chains dominated its spectrum. In contrast, the peptide backbone dominated the CD spectrum of MG2. Collectively, the results of this study indicate that human submandibular-sublingual saliva contains two structurally distinct mucins.     

Structural and functional comparison of HemN to other radical SAM enzymes

Radical SAM enzymes have only recently been recognized as an ancient family sharing an unusual radical-based reaction mechanism. This late appreciation is due to the extreme oxygen sensitivity of most radical SAM enzymes, making their characterization particularly arduous. Nevertheless, realization that the novel apposition of the established cofactors S-adenosylmethionine and [4Fe-4S] cluster creates an explosive source of catalytic radicals, the appreciation of the sheer size of this previously neglected family, and the rapid succession of three successfully solved crystal structures within a year have ensured that this family has belatedly been noted. In this review, we report the characterization of two enzymes: the established radical SAM enzyme, HemN or oxygen-independent coproporphyrinogen III oxidase from Escherichia coli, and littorine mutase, a presumed radical SAM enzyme, responsible for the conversion of littorine to hyoscyamine in plants. The enzymes are compared to other radical SAM enzymes and in particular the three reported crystal structures from this family, HemN, biotin synthase and MoaA, are discussed.     

Systemic blood pressure response to the inhibition of two hyperpolarizing pathways: a comparison to NO-synthase inhibition

The impact on blood pressure of two vasodilating mechanisms, underlied by vascular smooth muscle hyperpolarization, was studied and compared to that induced by nitric oxide NO mechanism. Systemic blood pressure, after inhibitory intervention in arachidonic acid metabolism cytochrome P-450 inhibition by miconazole 0.5 mg/100 g b.w. , one of the hyperpolarizing pathways, did not change. After the inhibition of the action voltage-dependent K(+) channels operator by 4-aminopyridine 0.1 mg/100 g b.w. , the other hyperpolarizing pathway, blood pressure declined slightly from 132.3+/-3.2 mm Hg to 116.5+/-5.0 mm Hg, P<0.05 . Inhibition of nitric oxide production L-NAME 5 mg/100 g b.w. increased blood pressure considerably 123.5+/-2.7 mm Hg to 155.4+/-3.1 mm Hg, P<0.001 . After inhibition of the hyperpolarizing pathway by miconazole, hypotension induced by acetylcholine (Ach, 10 microg represented 63.0+/-1.9 mm Hg vs control value 78.6+/-5.2 mm Hg P<0.001 , by bradykinin (BK) 100 microg 59.4+/-3.9 mm Hg vs control value 71.2+/-6.1 mm Hg P<0.05 . After inhibition of the hyperpolarizing pathway by 4-aminopyridine, hypotension induced by ACh 10 microg achieved 64.6+/-2.5 mm Hg vs control value 78.4+/-2.8 mm Hg P<0.001 and that induced by BK 100 microg 56.6+/-5.3 mm Hg vs control value 72.3+/-2.5 mm Hg P<0.001 . ACh or BK hypotension after the inhibition of the above hyperpolarizing pathways was significantly attenuated. On the contrary, after NO-synthase inhibition the hypotension to ACh was significantly enhanced. Blood pressure decrease after ACh 10 microg hypotension was 91.8+/-4.1 mm Hg vs control value 79.3+/-3.3 mm Hg P<0.01 , and after BK 100 microg it was 78.4+/-7.1 mm Hg vs control value 68.3+/-5.2 mm Hg. A different basal BP response, but equally attenuated hypotension to Ach and BK, was detected after the inhibition of two selected hyperpolarizing pathways. In cotrast, the inhibition of NO production elicited an increase in systemic BP and augmentation of ACh and BK hypotension. The effectiveness of further hyperpolarizing mechanisms in relation to systemic BP regulation and nitric oxide level remains open.     

Structural analysis and biochemical properties of laccase enzymes from two Pediococcus species

Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6-DMP, and non-conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well-studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.     

Cold adaption of enzymes: Structural comparison between salmon and bovine trypsins

The crystal structure of an anionic form of salmon trypsin has been determined at 1.82 Å resolution. We report the first structure of a trypsin from a phoikilothermic organism in a detailed comparison to mammalian trypsins in order to look for structural rationalizations for the cold-adaption features of salmon trypsin. This form of salmon trypsin (T II) comprises 222 residues, and is homologous to bovine trypsin (BT) in about 65% of the primary structure. The tertiary structures are similar, with an overall displacement in main chain atomic positions between salmon trypsin and various crystal structures of bovine trypsin of about 0.8 Å. Intramolecular hydrogen bonds and hydrophobic interactions are compared and discussed in order to estimate possible differences in molecular flexibility which might explain the higher catalytic efficiency and lower thermostability of salmon trypsin compared to bovine trypsin. No overall differences in intramolecular interactions are detected between the two structures, but there are differences in certain regions of the structures which may explain some of the observed differences in physical properties. The distribution of charged residues is different in the two trypsins, and the impact this might have on substrate affinity has been discussed. © 1994 Wiley-Liss, Inc.     

Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: a comparison to Homo sapiens B-ZIP motifs

Basic region-leucine zipper (B-ZIP) proteins are a class of dimeric sequence-specific DNA-binding proteins unique to eukaryotes. We have identified 67 B-ZIP proteins in the Arabidopsis thaliana genome. No A.thaliana B-ZIP domains are homologous with any Homo sapiens B-ZIP domains. Here, we predict the dimerization specificity properties of the 67 B-ZIP proteins in the A.thaliana genome based on three structural properties of the dimeric alpha-helical leucine zipper coiled coil structure: (i) length of the leucine zipper, (ii) placement of asparagine or a charged amino acid in the hydrophobic interface and (iii) presence of interhelical electrostatic interactions. Many A.thaliana B-ZIP leucine zippers are predicted to be eight or more heptads in length, in contrast to the four or five heptads typically found in H.sapiens, a prediction experimentally verified by circular dichroism analysis. Asparagine in the a position of the coiled coil is typically observed in the second heptad in H.sapiens. In A.thaliana, asparagine is abundant in the a position of both the second and fifth heptads. The particular placement of asparagine in the a position helps define 14 families of homodimerizing B-ZIP proteins in A.thaliana, in contrast to the six families found in H.sapiens. The repulsive interhelical electrostatic interactions that are used to specify heterodimerizing B-ZIP proteins in H.sapiens are not present in A.thaliana. Instead, we predict that plant leucine zippers rely on charged amino acids in the a position to drive heterodimerization. It appears that A.thaliana define many families of homodimerizing B-ZIP proteins by having long leucine zippers with asparagine judiciously placed in the a position of different heptads.     

Structural comparison of two esterases from Drosophila mojavensis isolated by immunoaffinity chromatography.

Antibodies raised against esterase-4 and esterase-5 from Drosophila mojavensis were coupled to Protein A-Sepharose CL-4B to prepare high-efficiency immunomatrices used for their purification. Final purification was achieved by anion-exchange h.p.l.c., in the case of esterase-5 followed by gel-filtration h.p.l.c. The resultant esterase preparations were homogeneous, as judged by gel-filtration h.p.l.c., SDS/polyacrylamide-gel electrophoresis and non-denaturing gel electrophoresis. Esterase-4 and esterase-5 are the products of a duplicated gene. They are differently localized in the insect's body and expressed in different periods during development. Although both enzymes exhibit little immunological cross-reactivity, their amino acid compositions show few significant differences and their N-terminal sequences are largely identical, which clearly show their common origin.     

Biochemical comparison of two glucose 6-phosphate dehydrogenase isozymes from a cold-adapted Pseudomonas mandelii

Cold-adapted bacteria primarily have two glucose 6-phosphate dehydrogenase isozymes (G6PD, also known as zwf), zwf-1 for the Entner–Doudoroff pathway and zwf-2 for the oxidative pentose phosphate pathway. Although the roles of zwfs in carbon metabolism and antioxidant defense have been reported, the biochemical properties of zwfs at low and moderate temperatures have not been fully described. In this study, we cloned and characterized zwf-1 (Pmzwf-1) and zwf-2 (Pmzwf-2) from a cold-adapted bacterium Pseudomonas mandelii JR-1. Pmzwf-1 and Pmzwf-2 were expressed in Escherichia coli BL21 (DE3) as soluble tetrameric proteins. Both Pmzwf proteins were active at 4 °C, but Pmzwf-1 exhibited overall better biochemical properties than those of Pmzwf-2, including 10–30% higher specific activity at 4–40 °C as well as consistent conformational flexibility and thermal stability in the 4–40 °C range. Pmzwf-2 showed reduced thermal stability at moderate temperatures. Furthermore, the mRNA expression of Pmzwf-1 was higher than that of Pmzwf-2 at both 4 °C and 25 °C. These results indicate that Pmzwfs are cold-adapted enzymes, but Pmzwf-1 can function at both low to moderate temperatures while Pmzwf-2 is primarily functional at low temperatures. Our results suggest distinct temperature adaptations of two G6PD isozymes in P. mandelii JR-1, adaptations that are metabolic pathway dependent.

     

Metal-ion-dependent catalysis and specificity of CCA-adding enzymes: a comparison of two classes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases] catalyze synthesis of the conserved and essential CCA sequence to the tRNA 3' end. These enzymes are divided into two classes of distinct structures that differ in the overall orientation of the head to tail domains. However, the catalytic core of the two classes is conserved and contains three carboxylates in a geometry commonly found in DNA and RNA polymerases that use the two-metal-ion mechanism for phosphoryl transfer. Two important aspects of the two-metal-ion mechanism are tested here for CCA enzymes: the dependence on metal ions for catalysis and for specificity of nucleotide addition. Using the archaeal Sulfolobus shibabae enzyme as an example of the class I, and the bacterial Escherichia coli enzyme as an example of the class II, we show that both enzymes depend on metal ions for catalysis, and that both use primarily Mg2+ and Mn2+ as the "productive" metal ions, but several other metal ions such as Ca2+ as the "nonproductive" metal ions. Of the two productive metal ions, Mg2+ specifically promotes synthesis of the correct CCA, whereas Mn2+ preferentially accelerates synthesis of the noncognate CCC and poly(C). Thus, despite evolution of structural diversity of two classes, both classes use metal ions to determine catalysis and specificity. These results provide critical insights into the catalytic mechanism of CCA synthesis to allow the two classes to be related to each other, and to members of the larger family of DNA and RNA polymerases.     

Mechanisms of insulin resistance in cultured fibroblasts from a patient with leprechaunism: resistance to proteolytic activation of glycogen synthase by trypsin

Summary Post-receptor or post-binding events in the action of insulin have been investigated in cultured skin fibroblasts from an infant with leprechaunism. Both diminished binding of insulin and multiplication-stimulating activity (MSA) to these cells as well as deficits distal to binding were described in a previous publication. Exposure of control fibroblasts to low concentrations (0.001 to 0.01%) of trypsin for one min without glucose in the medium activated the enzyme glycogen synthase; activation was less than that observed with a maximally effective concentration (10^–6 M) of insulin alone. In cells from the patient with leprechaunism, the effect of trypsin was much smaller than in the control fibroblasts. Exposing the control cells to soybean trypsin inhibitor before addition of trypsin prevented activation of glycogen synthase and demonstrated the specificity of the proteolytic action of trypsin. The rates of activation and inactivation of glycogen synthase in vitro were similar in extracts of the control subject's and the patient's fibroblasts and indicated that the enzymes regulating the phosphorylation/ dephosphorylation of glycogen synthase were intact in the patient's cells. Total glycogen synthase activity and glycogen content were also indistinguishable in control and leprechaun fibroblasts. These results are compatible with the presence of an abnormality in the structure or availability of the protease substrate from which chemical mediators of insulin action are formed in the patient's cells. Two possible models for a receptor-coupling complex are proposed. Either a mutation in a regulator-substrate unit of the receptor-coupling complexes for insulin and certain insulin-like growth factors or an alteration in the environment of the unit are postulated to explain the findings.Established Investigator of the American Diabetes Association.Abbreviations MSa multiplication-stimulating activity - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - EMEM growth medium, described in text - DPBS Dulbecco's phosphate-buffered saline - IM incubation medium, described in text - EDTA ethylenediaminetetraacetic acid - DTT dithiothreitol - ATP adenosine 5-triphosphate - UDPG uridine-5-diphosphoglucose - Tris tris (hydroxymethyl) aminomethane An abstract of this work was submitted for the Forty-Second Annual Meeting of the American Diabetes Association (Diabetes 31: 124A, 1982).     

Adaptation of enzymes to temperature: myofibrillar adenosine triphosphatase from two terrestrial isopod species

A comparison of the temperature profiles of the myofibrillar ATPase in Porcellio spinicornis and Metoponorthus pruinosus showed that Porcellio enzyme had a maximum activity between pH 6.8-7.2 at 9 degrees C, and that from Metoponorthus between pH 7.0-7.4 at 45 degrees C. The energy of activation for Porcellio enzyme was estimated to be 2.964 kcal/mol and that for Metoponorthus enzyme 9.91 X 10(-1) kcal/mol. The significance of these findings in relation to the natural habitats of these two species is discussed.     

How to obtain NNT from Cohen's d: comparison of two methods

Background

In the literature we find many indices of size of treatment effect (effect size: ES). The preferred index of treatment effect in evidence-based medicine is the number needed to treat (NNT), while the most common one in the medical literature is Cohen''s d when the outcome is continuous. There is confusion about how to convert Cohen''s d into NNT.

Methods

We conducted meta-analyses of individual patient data from 10 randomized controlled trials of second generation antipsychotics for schizophrenia (n = 4278) to produce Cohen''s d and NNTs for various definitions of response, using cutoffs of 10% through 90% reduction on the symptom severity scale. These actual NNTs were compared with NNTs calculated from Cohen''s d according to two proposed methods in the literature (Kraemer, et al., Biological Psychiatry, 2006; Furukawa, Lancet, 1999).

Results

NNTs from Kraemer''s method overlapped with the actual NNTs in 56%, while those based on Furukawa''s method fell within the observed ranges of NNTs in 97% of the examined instances. For various definitions of response corresponding with 10% through 70% symptom reduction where we observed a non-small number of responders, the degree of agreement for the former method was at a chance level (ANOVA ICC of 0.12, p = 0.22) but that for the latter method was ANOVA ICC of 0.86 (95%CI: 0.55 to 0.95, p<0.01).

Conclusions

Furukawa''s method allows more accurate prediction of NNTs from Cohen''s d. Kraemer''s method gives a wrong impression that NNT is constant for a given d even when the event rate differs.