Morpholinone mediated oxazolone‐free C‐terminus amide coupling permitting a convergent strategy for peptide synthesis

3‐Substituted‐5‐phenylmorpholinones have been demonstrated to act as N‐protected C‐terminus activated α‐amino acids capable of undergoing solution phase N‐terminus peptide extension following standard coupling procedures. The N‐acylated morpholinones do not undergo epimerisation of the stereocentre of the C‐terminus amino acid residue as oxazolone formation is sterically prevented, although C‐terminus peptide coupling is still possible. This convergent approach to peptide synthesis is exemplified by the preparation of L‐ala‐L‐ala‐L‐ala and L‐ala‐D‐ala‐L‐ala. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

C5 conserved region of hydrophilic C‐terminal part of Saccharomyces cerevisiae Nha1 antiporter determines its requirement of Erv14 COPII cargo receptor for plasma‐membrane targeting

Erv14, a conserved cargo receptor of COPII vesicles, helps the proper trafficking of many but not all transporters to the yeast plasma membrane, for example, three out of five alkali‐metal‐cation transporters in Saccharomyces cerevisiae. Among them, the Nha1 cation/proton antiporter, which participates in cell cation and pH homeostasis, is a large membrane protein (985 aa) possessing a long hydrophilic C‐terminus (552 aa) containing six conserved regions (C1–C6) with unknown function. A short Nha1 version, lacking almost the entire C‐terminus, still binds to Erv14 but does not need it to be targeted to the plasma membrane. Comparing the localization and function of ScNha1 variants shortened at its C‐terminus in cells with or without Erv14 reveals that only ScNha1 versions possessing the complete C5 region are dependent on Erv14. In addition, our broad evolutionary conservation analysis of fungal Na⁺/H⁺ antiporters identified new conserved regions in their C‐termini, and our experiments newly show C5 and other, so far unknown, regions of the C‐terminus, to be involved in the functionality and substrate specificity of ScNha1. Taken together, our results reveal that also relatively small hydrophilic parts of some yeast membrane proteins underlie their need to interact with the Erv14 cargo receptor.     

A novel KRT71 variant in curly‐coated dogs

Curly fur is a common phenotype in many dog breeds, known to result from a missense variant (c.451C>T) in exon 2 of the keratin 71 (KRT71) gene. During screening for this variant across various breeds, we found that Curly Coated Retrievers (CCRs) fixed with the trait did not carry the known variant. By analysis of whole‐genome sequencing data of one CCR we identified a novel genetic cause for curly fur. We found a novel structural variant in exon 7 of the KRT71 gene (c.1266_1273delinsACA) that was predicted to result in a frameshift and stop loss, therefore significantly affecting the structure of the protein, if translated. The variant was also found at lower frequencies in five other breeds, including Lagotto Romagnolo, Bichon Frise, Spanish Water Dog, Chesapeake Bay Retriever and Irish Terrier. One curly‐coated Lagotto carried neither of the two KRT71 variants. These results identify a second variant for curly coat in KRT71 and suggest the existence of additional alleles. This study enables the development of an additional KRT71 gene test for breeders to understand and manage coat types.     

Tagging of Weakly Expressed Toxoplasma gondii Calcium‐Related Genes with High‐Affinity Tags

Calcium ions regulate a diversity of cellular functions in all eukaryotes. The cytosolic Ca²⁺ concentration is tightly regulated at the physiological cytosolic concentration of 50–100 nm . The Toxoplasma gondii genome predicts the presence of several genes encoding potential Ca²⁺ channels, pumps, and transporters. Many of these genes are weakly expressed and likely tightly regulated due to their potential impact to the physiology of the cell. Endogenous tagging has been widely used to localize proteins in T. gondii but low level of expression of many of them makes visualization of tags difficult and sometimes impossible. The use of high‐performance tags for labeling proteins expressed at low level is ideal for investigating the localization of these gene products. We designed a Carboxy‐terminus tagging plasmid containing the previously characterized “spaghetti monster‐HA” (smHA) or “spaghetti monster‐MYC” (smMYC) tags. These tags consist of 10 copies of a single epitope (HA or MYC) inserted into a darkened green fluorescence protein scaffold. We localized six proteins of various levels of expression. Clonal lines were isolated and validated by PCR, western blot, and immunofluorescence analyses. Some gene products were only visible when tagged with smHA and in one case the smHA revealed a novel localization previously undetected.     

Specific Interactions Between gC1qR and α1‐Adrenoceptor Subtypes

Abstract

The multi‐functional protein gC1qR has been reported to interact with an arginine‐rich motif in the C‐tail of hamster α_1B‐adrenoceptors (ARs), controlling their expression and subcellular localization. Since a similar motif is present in α_1D‐, but not α_1A‐ARs, we studied the specificity of this interaction. Human α₁‐ARs, tagged at their amino termini with Flag epitopes, were coexpressed in HEK293 cells with gC1qR containing a hemaglutinin (HA) tag at its carboxy terminus. Immunoprecipitation studies showed that Flag‐α_1B‐ or α_1D‐, but not α_1A‐ARs, caused coimmunoprecipitation of HA‐gC1qR, while immunoprecipitation of HA‐gC1qR caused coimmunoprecipitation of Flag‐α_1B‐ or α_1D‐, but not α_1A‐ARs, supporting specific interactions between subtypes. C‐terminal truncation of Flag‐α₁‐ARs prevented interaction with HA‐gC1qR, supporting previous conclusions about the role of the C‐terminal arginine‐rich motif. These studies suggest that gC1qR interacts specifically with α_1B‐ and α_1D‐, but not α_1A‐ARs, and this interaction depends on the presence of an intact C‐tail.     

Structural insights into the stabilization of active,tetrameric DszC by its C‐terminus

Dibenzothiophene (DBT) is a typical sulfur‐containing compound found in fossil fuels. This compound and its derivatives are resistant to the hydrodesulfurization method often used in industry, but they are susceptible to enzymatic desulfurization via the 4S pathway, which is a well‐studied biochemical pathway consisting of four enzymes. DBT monooxygenase (DszC) from Rhodococcus erythropolis is involved in the first step of the 4S pathway. We determined the crystal structure of DszC, which reveals that, in contrast to several homologous proteins, the C‐terminus (410–417) of DszC participates in the stabilization of the substrate‐binding pocket. Analytical ultracentrifugation analysis and enzymatic assays confirmed that the C‐terminus is important for the stabilization of the active conformation of the substrate‐binding pocket and the tetrameric state. Therefore, the C‐terminus of DszC plays a significant role in the catalytic activity of this enzyme. Proteins 2014; 82:2733–2743. © 2014 Wiley Periodicals, Inc.     

An Approach to Incorporate Multi‐Enzyme Digestion into C‐TAILS for C‐Terminomics Studies

Protein C‐termini study is still a challenging task and far behind its counterpart, N‐termini study. MS based C‐terminomics study is often hampered by the low ionization efficiency of C‐terminal peptides and the lack of efficient enrichment methods. We previously optimized the C‐terminal amine‐based isotope labeling of substrates (C‐TAILS) method and identified 369 genuine protein C‐termini in Escherichia coli. A key limitation of C‐TAILS is that the prior protection of amines and carboxylic groups at protein level makes Arg‐C as the only specific enzyme in practice. Herein, we report an approach combining multi‐enzyme digestion and C‐TAILS, which significantly increases the identification rate of C‐terminal peptides and consequently improves the applicability of C‐TAILS in biological studies. We carry out a systematic study and confirm that the omission of the prior amine protection at protein level has a negligible influence and allows the application of multi‐enzyme digestion. We successfully apply five different enzyme digestions to C‐TAILS, including trypsin, Arg‐C, Lys‐C, Lys‐N, and Lysarginase. As a result, we identify a total of 722 protein C‐termini in E. coli, which is at least 66% more than the results using any single enzyme. Moreover, the favored enzyme and enzyme combination are discovered. Data are available via ProteomeXchange with identifier PXD004275.     

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self‐assembling peptides and a C‐terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self‐assembling peptide. Upon simple separation, the target protein or peptide with an authentic N‐terminus is then released in the solution by intein‐mediated cleavage. Different combinations of four self‐assembling peptides (ELK16, L₆KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI‐CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI‐CM, which has pH‐shift inducible cleavage, was found to work well with three self‐assembling peptides (L₆KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx‐strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N‐terminus, which is especially effective for peptides of 30‐100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.     

A second KRT71 allele in curly coated dogs

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant ¹⁵¹Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein‐changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C‐terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs.     

Ichthyosis with confetti: clinics,molecular genetics and management

Ichthyosis with confetti (IWC) is an autosomal dominant congenital ichthyosis also known as ichthyosis variegata or congenital reticular ichthyosiform erythroderma. It manifests at birth with generalized ichthyosiform erythroderma or with a collodion baby picture. The erythrodermic and ichthyotic phenotype persists during life and its severity may modify. However, the hallmark of the disease is the appearance, in childhood or later in life, of healthy skin confetti-like spots, which increase in number and size with time. IWC is a very rare genodermatosis, with a prevalence <1/1,000,000 and only 40 cases reported worldwide. The most important associated clinical features include ear deformities, mammillae hypoplasia, palmoplantar keratoderma, hypertrichosis and ectropion. IWC is due to dominant negative mutations in the KRT10 and KRT1 genes, encoding for keratins 10 and keratin 1, respectively. In this context, healthy skin confetti-like spots represent “repaired” skin due to independent events of reversion of keratin gene mutations via mitotic recombination. In most cases, IWC clinical suspicion is delayed until the detection of white skin spots. Clinical features, which may represent hint to the diagnosis of IWC even before appearance of confetti-like spots, include ear and mammillae hypoplasia, the progressive development of hypertrichosis and, in some patients, of adherent verrucous plaques of hyperkeratosis. Altogether the histopathological finding of keratinocyte vacuolization and the nuclear staining for keratin 10 and keratin 1 by immunofluorescence are pathognomonic. Nevertheless, mutational analysis of KRT10 or KRT1 genes is at present the gold standard to confirm the diagnosis. IWC has to be differentiated mainly from congenital ichthyosiform erythroderma. Differential diagnosis also includes syndromic ichthyoses, in particular Netherton syndrome, and the keratinopathic ichthyoses. Most of reported IWC cases are sporadic, but familial cases with autosomal dominant mode of inheritance have been also described. Therefore, knowledge of the mutation is the only way to properly counsel the couples. No specific and satisfactory therapy is currently available for IWC. Like for other congenital ichthyoses, topical treatments (mainly emollients and keratolytics) are symptomatic and offer only temporary relief. Among systemic treatments, retinoids, in particular acitretin, improve disease symptoms in most patients. Although at present there is no curative therapy for ichthyoses, treatments have improved considerably over the years and the best therapy for each patient is always the result of both physician and patient efforts.     

Total synthesis and membrane modifying properties of the lipopeptaibol trikoningin KB II and its analogues with acyl chains of different length at the N‐ and C‐termini

Trikoningin KB II, a ten‐amino acid residue lipopeptaibol blocked at the N‐terminus by the n‐octanoyl group and at the C‐terminus by the 1,2‐amino alcohol l ‐leucinol, and extracted from the fungus Trichoderma koningii, exhibits membrane‐modifying properties. We have synthesized by solution‐phase methods trikoningin KB II and several analogues with acyl chains of different length at the N‐ and C‐termini. Permeability measurements showed that an appropriate length of the linear acyl chain is a more important characteristic for the onset of significant membrane‐modifying activity than its position in the peptide chain. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Immunolocalization of keratin‐associated beta‐proteins in developing epidermis of lizard suggests that adhesive setae contain glycine–cysteine‐rich proteins

The localization of specific keratin‐associated beta‐proteins (formerly referred to as beta‐keratins) in the embryonic epidermis of lizards is not known. Two specific keratin‐associated beta‐proteins of the epidermis, one representing the glycine‐rich subfamily (HgG5) and the other the glycine‐cysteine medium‐rich subfamily (HgGC10), have been immunolocalized at the ultrastructural level in the lizard Anolis lineatopus. The periderm and granulated subperiderm are most immunonegative for these proteins. HgG5 is low to absent in theOberhäutchen layer while is present in the forming beta‐layer, and disappears in mesos‐ and alpha‐layers. Instead, HgGC10 is present in the Oberhäutchen, beta‐, and also in the following alpha‐layers, and specifically accumulates in the developing adhesive setae but not in the surrounding cells of the clear layer. Therefore, setae and their terminal spatulae that adhere to surfaces allowing these lizards to walk vertically contain cysteine–glycine rich proteins. The study suggests that, like in adult and regenerating epidermis, the HgGC10 protein is not only accumulated in cells of the beta‐layer but also in those forming the alpha‐layer. This small protein therefore is implicated in resistance, flexibility, and stretching of the epidermal layers. It is also hypothesized that the charges of these proteins may influence adhesion of the setae of pad lamellae. Conversely, glycine‐rich beta‐proteins like HgG5 give rise to the dense, hydrophobic, and chromophobic corneous material of the resistant beta‐layer. This result suggests that the differential accumulation of keratin‐associated beta‐proteins over the alpha‐keratin network determines differences in properties of the stratified layers of the epidermis of lizards. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Design,synthesis, and comparative evaluation of 99mTc(CO)3‐labeled N‐terminal and C‐terminal modified asparagine–glycine–arginine peptide constructs

The present study describes modification of asparagine–glycine–arginine (NGR) peptide at N‐terminally and C‐terminally by introduction of a tridentate chelating scaffold via click chemistry reaction. The N‐terminal and C‐terminal modified peptides were radiometalated with [^99mTc(CO)₃]⁺ precursor. The influence of these moieties at the two termini on the targeting properties of NGR peptide was determined by in vitro cell uptake studies and in vivo biodistribution studies. The two radiolabeled constructs did not exhibit any significant variation in uptake in murine melanoma B16F10 cells during in vitro studies. In vivo studies revealed nearly similar tumor uptake of N‐terminally modified peptide construct 5 and C‐terminally construct 6 at 2 h p.i. (1.9 ± 0.1 vs 2.4 ± 0.2% ID/g, respectively). The tumor‐to‐blood (T/B) and tumor‐to‐liver (T/L) ratios of the two radiometalated peptides were also quite similar. The two constructs cleared from all the major organs (heart, lungs, spleen, stomach, and blood) at 4 h p.i. (<1% ID/g). Blocking studies carried out by coinjection of cCNGRC peptide led to approximately 50% reduction in the tumor uptake at 2 h p.i. This work thus illustrates the possibility of convenient modification/radiometalation of NGR peptide at either N‐ or C‐terminus without hampering tumor targeting and pharmacokinetics.     

Controlled Atomic Solubility in Mn‐Rich Composite Material to Achieve Superior Electrochemical Performance for Li‐Ion Batteries

The quest for high energy density and high power density electrode materials for lithium‐ion batteries has been intensified to meet strongly growing demand for powering electric vehicles. Conventional layered oxides such as Co‐rich LiCoO₂ and Ni‐rich Li(Ni_xMn_yCo_z)O₂ that rely on only transition metal redox reaction have been faced with growing constraints due to soaring price on cobalt. Therefore, Mn‐rich electrode materials excluding cobalt would be desirable with respect to available resources and low cost. Here, the strategy of achieving both high energy density and high power density in Mn‐rich electrode materials by controlling the solubility of atoms between phases in a composite is reported. The resulting Mn‐rich material that is composed of defective spinel phase and partially cation‐disordered layered phase can achieve the highest energy density, ≈1100 W h kg^?1 with superior power capability up to 10C rate (3 A g^?1) among other reported Mn‐rich materials. This approach provides new opportunities to design Mn‐rich electrode materials that can achieve high energy density and high power density for Li‐ion batteries.     

Inhibition of chlamydial class Ic ribonucleotide reductase by C‐terminal peptides from protein R2

Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese–iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C‐terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical‐containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese–iron cofactor is similar to that of the tyrosyl‐radical‐containing RNRs, involving reversible long‐range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2‐derived oligopeptide blocks the transfer pathway. We have constructed three‐dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20‐mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C‐terminus of protein R2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Engineering hyperthermostability into a mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N‐terminus substitution

A mesophilic xylanase from Aspergillus oryzae CICC40186 (abbreviated to AoXyn11A) belongs to glycoside hydrolase family 11. The thermostability of AoXyn11A was significantly improved by substituting its N‐terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11^TS. The suitable N‐terminus of AoXyn11A to be replaced was selected by the comparison of B‐factors between AoXyn11A and EvXyn11^TS, which were generated and calculated after a 15 ns molecular dynamic (MD) simulation process. Then, the predicted hybrid xylanase (designated AEx11A) was modeled, and subjected to a 2 ns MD simulation process for calculating its total energy value. The N‐terminus substitution was confirmed by comparing the total energy value of AEx11A with that of AoXyn11A. Based on the in silico design, the AEx11A was constructed and expressed in Pichia pastoris GS115. After 72 h of methanol induction, the recombinant AEx11A (reAEx11A) activity reached 82.2 U/mL. The apparent temperature optimum of reAEx11A was 80°C, much higher than that of reAoXyn11A. Its half‐life was 197‐fold longer than that of reAoXyn11A at 70°C. Compared with reAoXyn11A, the reAEx11A displayed a slight alteration in K_m but a decrease in V_max. Biotechnol. Bioeng. 2013; 110: 1028–1038. © 2012 Wiley Periodicals, Inc.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Recent research has implicated the C‐terminus of G‐protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C‐tail has proven a formidable task. Here, a peptide corresponding to the full‐length C‐tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α‐helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self‐association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (¹⁵N‐ and ¹³C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐helical domains. The first domain, S401‐F412, corresponds to the helix 8 common to G protein‐coupled receptors while the second domain, A440‐M461, is a newly identified structural motif in the distal region of the carboxyl‐terminus of the receptor. Molecular modeling of the C‐tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 565–573, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com