SEA domain autoproteolysis accelerated by conformational strain: energetic aspects

A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G^− 1S^+ 1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N → O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t_½) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by ∼ 10 kcal mol^− 1 compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to ∼ 11 and ∼ 15 kcal mol^− 1, respectively, but ∼ 7 kcal mol^− 1 of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of ∼ 7 kcal mol^− 1 was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G^− 1A^+ 1VVV motif) and the wild-type protein. The results imply that cleavage by N → O acyl shift alone would proceed with a t_½ of ∼ 2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.     

Structural basis for differential receptor binding of cholera and Escherichia coli heat-labile toxins: influence of heterologous amino acid substitutions in the cholera B-subunit

The closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1–25 region were combined with those in the 75–83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33→Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.     

VIP in cerebrospinal fluid of patients with multiple sclerosis

The concentration of VIP was measured radioimmunochemically in cerebrospinal fluid (CSF) from 14 healthy volunteers and from 22 patients with multiple sclerosis. Significantly lower levels of VIP was obtained in the patients (18 +/- 3 pmol/l) than in controls (37 +/- 4 pmol/l). There was no correlation between the level of VIP in CSF and other CSF parameters such as albumin. IgG or cell content; nor between VIP concentration and the physical handicap or neuropsychiatric symptoms. There was a trend towards lower values of VIP in patients with steadily progressing rather than intermittent course of the disease but the difference between the groups was not significant.     

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

Summary To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.     

Molecular cloning of chicken prepro-orexin cDNA and preferential expression in the chicken hypothalamus

Chicken prepro-orexin cDNA has been cloned, sequenced and characterized. The predicted amino acid sequence of chicken prepro-orexin cDNA revealed that orexin-A and -B are highly conserved among vertebrate species. In situ hybridization and immunohistochemistry localized orexin-positive cell bodies in the periventricular hypothalamic nucleus extending into the lateral hypothalamic area. Comparisons of orexin gene expression in the brains of 24-h-fasted and ad libitum-fed chickens were made using semi-quantitative RT-PCR. No significant differences in orexin mRNA expression were observed.     

Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer

Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C(14)H(29)) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12 degrees C in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase.     

A Battery of Cell- and Structure-specific Markers for the Adult Porcine Retina

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathological changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and reproducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina. (J Histochem Cytochem 58:377–389, 2010)     

Summer and Winter Marine Heatwaves Favor an Invasive Over Native Seaweeds

Marine heatwaves (MHWs) are emerging as forceful agents of ecosystem change and are increasing in frequency, duration, and intensity with climate change. During MHWs, physiological thresholds of native species may be exceeded while the performance of invasive species with warm affinities may be enhanced. As a consequence, MHWs could significantly alter an ecosystem's invasive dynamics, but such interactions are poorly understood. Following a 10-d acclimation period, we investigated the physiological resistance and resilience of an intertidal rock pool assemblage invaded by the seaweed Sargassum muticum to realistic 14-d marine heatwave scenarios (+1.5°C, +2.0°C, +3.5°C) followed by a 14-d recovery period. We conducted mesocosm experiments in both summer and winter to investigate temporal variability of MHWs. MHW treatments had clear negative impacts on native seaweeds (Fucus serratus and Chondrus crispus) while enhancing the performance of S. muticum. This pattern was consistent across season indicating that acclimation to cooler ambient temperatures results in winter MHWs having significant impacts on native species. As climate warming advances, this may ultimately lead to changes in competitive interactions and potentially exclusion of native species, while invasive species may proliferate and become more conspicuous within temperate rocky shore environments.