Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.     

Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys14?Cys39 bond into the ovomucoid third domain from silver pheasant

P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹⁴? Cys³⁹ bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹⁴? Cys³⁹ bond into the flexible N‐terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH‐independent hydrolysis constant (K_hyd°) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K_hyd°, but has no effect on the thermal stability. The site‐specific disulfide introduction into the flexible N‐terminal loop of natural Kazal‐type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The functional role of Cys3–Cys4 loop in hydrophobin HGFI

Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic–hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3–Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3–Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water–air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3–Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3–Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic–hydrophilic interfaces.     

Two Cys or Not Two Cys? That Is the Question; Alternative Oxidase in the Thermogenic Plant Sacred Lotus

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32°C to 35°C across ambient temperatures of 8°C to 40°C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys₁ and Cys₂, are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys₁ with α-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys₁. Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys₁ had been replaced by serine; however, Cys₂ was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys₁ and Cys₂. An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

Thermogenesis in Sacred Lotus

Sacred lotus (Nelumbo nucifera) is a thermogenic plant that regulates the temperature of its floral chamber between 32°C and 35°C for up to 4 d (Seymour and Schultze-Motel, 1996). Heating of plant tissues has been described as an adaptation to attract insect pollinators either by volatilization of scent compounds (Meeuse, 1975) or by providing a heat reward (Seymour et al., 1983), protect floral parts from low temperatures (Knutson, 1974), or provide the optimum temperature for floral development (Ervik and Barfod, 1999; Seymour et al., 2009). In sacred lotus, heat is produced by high rates of alternative pathway respiration (Watling et al., 2006; Grant et al., 2008); however, the mechanisms of heat regulation, which likely occur at a cellular level, remain unclear.

Alternative Oxidase

Alternative pathway respiration is catalyzed by the alternative oxidase protein (AOX), which acts as a terminal oxidase in the electron transport chain but, unlike the energy conserving cytochrome pathway (COX), complexes III and IV are bypassed and energy is released as heat. Traditionally, AOX activity was measured using oxygen consumption of tissue, cells, or isolated mitochondria in the presence or absence of AOX and COX inhibitors. However, this method does not accurately measure activity in vivo but does indicate the capacity of the alternative pathway (Ribas-Carbo et al., 1995; Day et al., 1996). The only method to date to accurately determine AOX activity, that is, flux of electrons through the AOX pathway in vivo, is to use oxygen isotope discrimination techniques (for review, see Robinson et al., 1995). Determining AOX activity in vivo is important because heat production in plants could be due to activity of either the AOX and/or plant uncoupling proteins. Using oxygen fractionation techniques, we have shown that flux through the AOX pathway is responsible for heating in sacred lotus (Watling et al., 2006; Grant et al., 2008). Furthermore, we were unable to detect any uncoupling protein in these tissues (Grant et al., 2008). AOX protein content within the sacred lotus receptacle increases markedly prior to thermogenesis, but it remains constant during heating (Grant et al., 2008), suggesting that regulation of heating occurs through posttranslational modification of the protein.

Posttranslational Regulation of AOX Protein

The plant AOX is a cyanide-insensitive dimeric protein located in the inner mitochondrial membrane (Day and Wiskich, 1995). The dimer subunits (monomers) can be linked via a noncovalent association (reduced protein) or covalently through the formation of a disulfide bridge (oxidized protein; Umbach and Siedow, 1993). The reduced protein when run on SDS-PAGE has a molecular mass of approximately 30 to 35 kD and the oxidized protein 60 to 71 kD; this holds true for AOX from a number of species, including soybean (Glycine max) roots and cotyledons (Umbach and Siedow, 1993), tobacco (Nicotiana tabacum) leaf (Day and Wiskich, 1995), and the thermogenic spadix of Arum maculatum (Hoefnagel and Wiskich, 1998).Regulation of AOX has been well studied in nonthermogenic plant species, and two mechanisms have been identified. Most AOX isoforms have two highly conserved Cys residues, Cys₁ and Cys₂ (defined in Berthold et al., 2000 and Holtzapffel et al., 2003), located near the N-terminal hydrophilic domain of the protein. In these isoforms, Cys₁ can either be reduced on both subunits of the AOX dimer, or the Cys₁ sulfhydryl groups can be oxidized to form a disulfide bridge (Rhoads et al., 1998). Reduction/oxidation modulation of AOX in vitro can be achieved using the sulfhydryl reductant dithiothreitol (DTT) to reduce the protein or diamide to oxidize the Cys residues. The reduced dimer can be further activated via the interaction of Cys₁ with α-keto acids, principally pyruvate (Rhoads et al., 1998; see McDonald [2008] for a model of posttranslational regulation of AOX). In addition, Cys₂ may also be involved in regulating AOX activity through interaction with the α-keto acid glyoxylate (which can also stimulate activity at Cys₁; Umbach et al., 2002).Recently, however, AOX proteins with different regulatory properties have been reported. Naturally occurring AOX proteins without the two regulatory Cys residues have been identified and, along with site-directed mutagenesis studies, used to further elucidate the specific roles of Cys₁ and Cys₂. The LeAOX1b isoform from tomato (Lycopersicon esculentum), which has a Ser residue at the position of Cys₁ and thus does not form disulfide linked dimers, is also activated by succinate rather than pyruvate when expressed in Saccharomyces cerevisiae (Holtzapffel et al., 2003). In Arabidopsis (Arabidopsis thaliana), uncharged or hydrophobic amino acid substitutions of either Cys result in an inactive enzyme, while positively charged substitutions produce an enzyme with higher than wild type basal activity but that is insensitive to pyruvate or succinate (Umbach et al., 2002). Single substitutions at Cys₁ or Cys₂ have revealed that glyoxylate can activate AOX via both Cys residues, but only one is needed for glyoxylate stimulation (Umbach et al., 2002, 2006). Double substitution mutants were not stimulated by either pyruvate or glyoxylate (Umbach et al., 2006).Previously, we determined that thermogenesis via the AOX pathway in the sacred lotus receptacle is precisely regulated through changes in AOX flux rather than changes to protein content (Grant et al., 2008). In this study, we investigated the nature of this regulation in mitochondria isolated from heating receptacles. Our aim was to elucidate the reduction/oxidation behavior of the AOX protein and the mechanisms of activation of cyanide-resistant respiration in sacred lotus receptacles to provide insights into the mechanism(s) of heat regulation in this species. We further investigated AOX regulation by determining the amino acid sequence of two novel AOX genes isolated from thermogenic receptacle tissue of sacred lotus.     

Contribution of HOGG1 Ser³²⁶Cys polymorphism to the development of prostate cancer in smokers: meta-analysis of 2779 cases and 3484 controls

The HOGG1 gene catalyzes the excision of modified bases and removal of DNA damage adducts. It may play an important role in the prevention of carcinogenesis. Ser³²⁶Cys polymorphism localizes in exon 7 of the hOGG1 gene. It takes the form of an amino acid substitution, from serine to cysteine, in codon 326. Several epidemiological association studies have been conducted on this polymorphism and its relationship with the risk of prostate cancer. However, results have been conflicting. To resolve this conflict, we conducted a meta-analysis on the association between this polymorphism and prostate cancer, taking into account race, country, sources of controls, and smoking status. A total of nine studies covering 2779 cases and 3484 controls were included in the current meta-analysis. Although no significant association was found between hOGG1 Ser³²⁶Cys polymorphism and prostate cancer susceptibility in the pooled analysis, individuals with Ser/Cys+Cys/Cys genotypes were found to have greater risk of prostate cancer if they were also smokers (OR = 2.66, 95% CI = 1.58−4.47) rather than non-smokers (OR = 2.18, 95% CI = 1.13−4.19), compared with those with Ser/Ser genotype. In conclusion, our meta-analysis demonstrates that hOGG1 Ser³²⁶Cys polymorphism is a risk factor for prostate cancer in smokers. Further studies are needed to confirm this relationship.     

Critical Role of Cys168 in Noggin Protein＇s Biological Function

Previous studies have indicated that noggin exerts its neural inducing effect by binding and antagonizing bone morphogenetic protein 4 (BMP4). In order to further clarify the relationship between the structure and the function of noggin, and elucidate the possible mechanism responsible for noggin-BMP4 interaction, we generated three noggin mutants, C168S, C174S and C197S, by using a site-directed mutagenesis method. Ectopic expression of wild-type (WT) noggin, C174S or C197S, in Xenopus animal caps (ACs) by mRNA injection converted the explants (prospective ectoderm) into neural tissue, as indicated by the neural-like morphology and expression of the neural cell adhesion molecule (NCAM) in the ACs. In contrast, ACs expressing C168S suffered an epidermal fate similar to the control caps. Similarly, among the three mutants, only C168S lost the dorsalizing function. These studies highlight the critical role played by Cys168 in noggin‘s biological activities. It probably participates in the formation of an intermolecular disulfide bridge.     

Effect of cortisone on the developmental pattern of the neutral and the acid β-galactosidases of the small intestine of the rat

1. The developmental pattern and effect of cortisone on acid beta-galactosidase and neutral beta-galactosidase were studied in postnatal rats by a recently proposed method for their independent determination. 2. After birth the acid beta-galactosidase activity increases in the ileum, whereas it decreases slightly in the jejunum. On day 16 after birth the activity in the ileum decreases and in 20-day-old rats activity in both parts of the intestine decreases to adult values. In suckling animals the activity in the ileum exceeds the jejunal activity severalfold and in adult animals the activity in the jejunum is slightly higher than that in the ileum. 3. Neutral beta-galactosidase activity is high after birth and decreases in both jejunum and ileum after day 20 after birth. In 12-20-day-old rats activity in both parts is essentially the same, but in adult animals jejunal activity exceeds ileal activity four-to five-fold. 4. Cortisone (0.5, 2.0 or 5.0mg/100g body wt. daily for 4 days) does not influence the activity of either enzyme in 60-day-old rats. Acid beta-galactosidase activity is decreased after cortisone treatment in 8-, 12-, 16-and 18-day-old rats, with sensitivity to cortisone increasing with the approach of weaning. No effect of cortisone on acid beta-galactosidase is seen in 8-day-old rats. Neutral beta-galactosidase activity is increased in the ileum of 8-, 12-, 16- and 18-day old rats, but only in the jejunum of 8-and 12-day-old rats.     

胰蛋白酶分子中二硫键Cys129—Cys232的定位改造研究

对胰蛋白酶所特有的二硫键（１２９,２３２）进行了定位改造,将Ｃｙｓ突变为Ｓｅｒ,以观察其对胰蛋白酶稳定性及活性的影响,采用蛋白工程的方法,构建了三个突变体Ｃ１２９Ｓ,Ｃ２３２Ｓ和Ｃ１２９Ｓ／Ｃ２３２Ｓ,在Ｅ．ｃｏｌｉＸ－９０菌体中进行表达,表达产物用含胰蛋酶特异性底物ＴＡＭＥ的活性胶检测活性,发现Ｃ２３２Ｓ失胰蛋白酶活性,而Ｃ１２９Ｓ和Ｃ１２９Ｓ／Ｃ２３２Ｓ保留了胰蛋白酶活性,在盐酸胍作用了比较双     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

胰蛋白酶分子中二硫键Cys129－Cys232的定位改造研究

对胰蛋白酶所特有的二硫键［１２９,２３２］进行了定位改造,将Ｃｙｓ突变为Ｓｅｒ,以观察其对胰蛋白酶稳定性及活性的影响。采用蛋白质工程的方法,构建了三个突变体Ｃ１２９Ｓ、Ｃ２３２Ｓ和Ｃ１２９Ｓ／Ｃ２３２Ｓ．在Ｅ．ｃｏｌｉＸ－９０菌体中进行表达,表达产物用含胰蛋白酶特异性底物ＴＡＭＥ的活性胶检测活性,发现Ｃ２３２Ｓ丧失胰蛋白酶活性,而Ｃ１２９Ｓ和Ｃ１２９Ｓ／Ｃ２３２Ｓ保留了胰蛋白酶活性。在盐酸胍作用下比较双突变体和野生型胰蛋白酶活性,发现突变体Ｃ１２９Ｓ／Ｃ２３２Ｓ的稳定性有所降低。结果表明二硫键Ｃｙｓ１２９－Ｃｙｓ２３２对于胰蛋白酶的活性是非必需的,可能在稳定蛋白质的结构上发挥着重要作用。     

Phylogeography of the harlequin beetle-riding pseudoscorpion and the rise of the Isthmus of Panamá

Molecular and geological evidence indicates that the emergence of the Isthmus of Panamá influenced the historical biogeography of the Neotropics in a complex, staggered manner dating back at least 9 Myr bp. To assess the influence of Isthmus formation on the biogeography of the harlequin beetle-riding pseudoscorpion, Cordylochernes scorpioides, we analysed mitochondrial COI sequence data from 71 individuals from 13 locations in Panamá and northern South America. Parsimony and likelihood-based phylogenies identified deep divergence between South American and Panamanian clades. In contrast to low haplotype diversity in South America, the Panamanian Cordylochernes clade is comprised of three highly divergent lineages: one clade consisting predominantly of individuals from central Panamá (PAN A), and two sister clades (PAN B1 and PAN B2) of western Panamanian pseudoscorpions. Breeding experiments demonstrated a strictly maternal mode of inheritance, indicating that our analyses were not confounded by nuclear-mitochondrial pseudogenes. Haplotype diversity is striking in western Atlantic Panamá, where all three Panamanian clades can occur in a single host tree. This sympatry points to the existence of a cryptic species hybrid zone in western Panamá, a conclusion supported by interclade crosses and coalescence-based migration rates. Molecular clock estimates yield a divergence time of approximately 3 Myr between the central and western Panamanian clades. Taken together, these results are consistent with a recent model in which a transitory proto-Isthmus enabled an early wave of colonization out of South America at the close of the Miocene, followed by sea level rise, inundation of the terrestrial corridor and then a second wave of colonization that occurred when the Isthmus was completed approximately 3 Myr bp.     

Did the evolution of the phytoplankton fuel the diversification of the marine biosphere?

We discuss the possible links between the fossil record of marine biodiversity, nutrient availability and primary productivity. The parallelism of the fossil records of marine phytoplankton and faunal biodiversity implicates the quantity (primary productivity) and quality (stoichiometry) of phytoplankton as being critical to the diversification of the marine biosphere through the Phanerozoic. The relatively subdued marine biodiversity of the Palaeozoic corresponds to a time of relatively low macronutrient availability and poor food quality of the phytoplankton as opposed to the diversification of the Modern Fauna through the Mesozoic–Cenozoic. Increasing nutrient runoff to the oceans through the Phanerozoic resulted from orogeny, the emplacement of Large Igneous Provinces (LIPs), the evolution of deep-rooting forests and the appearance of more easily decomposable terrestrial organic matter that enhanced weathering. Positive feedback by bioturbation of an expanding benthos played a critical role in evolving biogeochemical cycles by linking the oxidation of dead organic matter and the recycling of nutrients back to the water column where they could be re-utilized. We assess our conclusions against a recently published biogeochemical model for geological time-scales. Major peaks of marine diversity often occur near rising or peak fluxes of silica, phosphorus and dissolved reactive oceanic phosphorus; either major or minor ⁸⁷Sr/⁸⁶Sr peaks; and frequently in the vicinity of major (Circum-Atlantic Magmatic Province) and minor volcanic events, some of which are associated with Oceanic Anoxic Events. These processes appear to be scale-dependent in that they lie on a continuum between biodiversification on macroevolutionary scales of geological time and mass extinction.     

Imperfect Batesian mimicry—the effects of the frequency and the distastefulness of the model

Batesian mimicry is the resemblance between unpalatable models and palatable mimics. The widely accepted idea is that the frequency and the unprofitability of the model are crucial for the introduction of a Batesian mimic into the prey population. However, experimental evidence is limited and furthermore, previous studies have considered mainly perfect mimicry (automimicry). We investigated imperfect Batesian mimicry by varying the frequency of an aposematic model at two levels of distastefulness. The predator encountered prey in a random order, one prey item at a time. The prey were thus presented realistically in a sequential way. Great tits (Parus major) were used as predators. This experiment, with a novel signal, supports the idea that Batesian mimics gain most when the models outnumber them. The mortalities of the mimics as well as the models were significantly dependent on the frequency of the model. Both prey types survived better the fewer mimics there were confusing the predator. There were also indications that the degree of distastefulness of the model had an effect on the survival of the Batesian mimic: the models survived significantly better the more distasteful they were. The experiment supports the most classical predictions in the theories of the origin and maintenance of Batesian mimicry.     

Microspectrophotometry of the photoreceptors of palaeognathous birds — the emu and the tinamou

Summary With the aid of a microspectrophotometer the visual pigments and oil globules in the retina of the emu (Dromiceius novae-hollandiae), the brushland tinamou (Nothoprocta c. cinerascens) and the Chilean tinamou (Nothoprocta perdicaria sanborni) were characterized. All three of these palaeognathous birds contain in their rods a typical rhodopsin with _max near 500 nm. Each of these birds has cones containing iodopsin-like visual pigments with _max in the 560–570 nm spectral region. No unequivocal evidence was obtained for the presence of cone pigments other than this iodopsin-like pigment, although one cell thought to be a cone, and containing a visual pigment with _max near 498 nm, was observed in the retina of the brushland tinamou. The oil globule systems of the three palaeognathous species are identical to each other and are much simpler than is typical for neognathous birds in that only two different types of globule are present, one with _T50 at 508 nm and another with _T50 at 568 nm. Comparison of the data with observations made on neognathous species indicates (1) that palaeognathous birds probably have poorer color discrimination capabilities than neognathous birds and (2) that the tinamou is more closely related to the ratites than to the galliform species.This study was supported, in part, by NIH Grant No. EY01839 (A.J. Sillman), NIH Grant No. EY00323 (W.N. McFarland) and NSF Grant No. 78-07657 (E.R. Loew). The authors thank E. Clinite, R. Dunford, C. Murphy, R. Riis and D. Weathers for their valuable assistance. Thanks also go to R.E. Burger for his gift of the emus.