Extra Cysteine Residue Research Articles

First transcriptome analysis of the venom glands of the scorpion Hottentotta zagrosensis (Scorpions: Buthidae) with focus on venom lipolysis activating peptides

IntroductionScorpion venom is a rich source of biological active peptides and proteins. Transcriptome analysis of the venom gland provides detailed insights about peptide and protein venom components. Following the transcriptome analysis of different species in our previous studies, our research team has focused on the Hottentotta zagrosensis as one of the endemic scorpions of Iran to obtain information about its venom proteins, in order to develop biological research focusing on medicinal applications of scorpion venom components and antivenom production. To gain insights into the protein composition of this scorpion venom, we performed transcriptomic analysis.MethodsTranscriptomic analysis of the venom gland of H. zagrosensis, prepared from the Khuzestan province, was performed through Illumina paired-end sequencing (RNA-Seq), Trinity de novo assembly, CD-Hit-EST clustering, and annotation of identified primary structures using bioinformatics approaches.ResultsTranscriptome analysis showed the presence of 96.4% of complete arthropod BUSCOs, indicating a high-quality assembly. From total of 45,795,108 paired-end 150 bp trimmed reads, the clustering step resulted in the generation of 101,180 de novo assembled transcripts with N50 size of 1,149 bp. 96,071 Unigenes and 131,235 transcripts had a significant similarity (E-value 1e-3) with known proteins from UniProt, Swissprot, Animal toxin annotation project, and the Pfam database. The results were validated using InterProScan. These mainly correspond to ion channel inhibitors, metalloproteinases, neurotoxins, protease inhibitors, protease activators, Cysteine-rich secretory proteins, phospholipase A enzymes, antimicrobial peptides, growth factors, lipolysis-activating peptides, hyaluronidase, and, phospholipase D. Our venom gland transcriptomic approach identified several biologically active peptides including five LVP1-alpha and LVP1-beta isoforms, which we named HzLVP1_alpha1, HzLVP1_alpha2, HzLVP1_alpha3, HzLVP1_beta1, and HzLVP1_beta and have extremely characterized here.DiscussionExcept for HzLVP1_beta1, all other identified LVP1s are predicted to be stable proteins (instability index <40). Moreover, all isoform of LVP1s alpha and beta subunits are thermostable, with the most stability for HzLVP1_alpha2 (aliphatic index = 71.38). HzLVP1_alpha2 has also the highest half-life. Three-dimensional structure of all identified proteins compacts with three disulfide bridges. The extra cysteine residue may allow the proteins to form a hetero- or homodimer. LVP1 subunits of H. zagrosensis potentially interact with adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), two key enzymes in regulation of lipolysis in adipocytes, suggesting pharmacological properties of these identified proteins.

Genotype-phenotype spectrum and prognosis of early-onset Marfan syndrome

BackgroundMarfan syndrome is a genetic connective tissue disorder affecting skeletal, ocular, and cardiovascular organ systems. Previous research found that pathogenic variants clustered in exons 24–32 of fibrillin-1 (FBN1) gene result in more severe clinical phenotypes. Furthermore, genotype-phenotype correlation studies suggested that more severe cardiovascular phenotypes were related to variants held responsible for haploinsufficiency. Our objective was to analyze the differences in clinical manifestations and genotypes of individuals with early-onset Marfan syndrome and to assess their impact on management strategies.MethodsWe analyzed clinical and genetic data of a new patient with early-onset Marfan syndrome together with 51 previously reported ones in the PubMed database between 1991 and 2022.ResultsAnalysis showed 94% (49/52) of pathogenic variants clustered in exons 24–32 of the FBN1. The most common skeletal features were arachnodactyly (98%), reduced elbow extension (48%), pectus deformity (40%), and scoliosis (39%). Haploinsufficiency variants were reported as having poor outcome in 87.5% of the cases. Among patients carrying variants that substitute a cysteine for another amino acid and those that do not change cysteine content, cardiac intervention was found to be associated with a better outcome (p = 0.035 vs. p = 0.002). Variants that create an extra cysteine residue were found to be associated with a higher risk of ectopia lentis. Additionally, children up to 36-months-old were more often reported as still alive at the time of publication compared to newborns (p < 0.01).ConclusionsOur findings have implications for prognosis, because different genotype groups and their resulting phenotype may require personalized care and management.

Genomic, transcriptomic, and epigenomic analysis of a medicinal snake, Bungarus multicinctus, to provides insights into the origin of Elapidae neurotoxins.

The many-banded krait, Bungarus multicinctus, has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia. Characterization of its venoms classified chief phyla of modern animal neurotoxins. However, the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome. Here, we present the 1.58Gbp genome of B. multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8Mbp. Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications. The truncation of glycosylphosphatidylinositol anchor in the 3'-terminal of a LY6E paralog released modern three-finger toxins (3FTxs) from membrane tethering before the Colubroidea divergence. Subsequent expansion and mutations diversified and recruited these 3FTxs. After the cobra/krait divergence, the modern unit-B of β-bungarotoxin emerged with an extra cysteine residue. A subsequent point substitution in unit-A enabled the β-bungarotoxin covalent linkage. The B. multicinctus gene expression, chromatin topological organization, and histone modification characteristics were featured by transcriptome, proteome, chromatin conformation capture sequencing, and ChIP-seq. The results highlighted that venom production was under a sophisticated regulation. Our findings provide new insights into snake neurotoxin research, meanwhile will facilitate antivenom development, toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.

Characterization of Glutenin Genes in Bread Wheat by Third-Generation RNA Sequencing and the Development of a Glu-1Dx5 Marker Specific for the Extra Cysteine Residue.

High-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) in a mature grain play important roles in the formation of a glutenin macropolymer and gluten quality. To characterize the expressed glutenin genes of the bread wheat variety Xinmai 26 during seed development, a total of 18 full-length transcripts were obtained by the newly emerged third-generation RNA sequencing of the PacBio Sequel II platform, including 5 transcripts of HMW-GS genes and 13 transcripts of LMW-GS genes (8 intact genes and 5 pseudogenes). Combined with the patterns of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), allelic types of the obtained glutenin genes were, respectively, determined, wherein molecular characterization deduced by transcript1528 (1Dx5) and transcript907 (Glu-A3c) indicated their great influence on dough quality. In addition, a specific functional marker dCAPS5 was developed for the single-nucleotide substitution at position 353 of the 1Dx5 subunit, which was further intensively compared with the other proposed markers to efficiently utilize the 1Dx5 subunit with the extra cysteine residue. This study provides an efficient method to accurately identify and utilize glutenin genes in bread wheat, which is helpful in understanding the contributions of glutenin genes to wheat quality.

Characterization of novel low-molecular-weight glutenin subunit genes from the diploid wild wheat relative Aegilops umbellulata

AbstractLow molecular weight glutenin subunits (LWM-GSs) play a crucial role in determining wheat flour processing quality. In this work, 35 novel LMW-GS genes (32 active and three pseudogenes) from three Aegilops umbellulata (2n = 2x = 14, UU) accessions were amplified by allelic-specific PCR. We found that all LMW-GS genes had the same primary structure shared by other known LMW-GSs. Thirty-two active genes encode 31 typical LMW-m-type subunits. The MZ424050 possessed nine cysteine residues with an extra cysteine residue located in the last amino acid residue of the conserved C-terminal III, which could benefit the formation of larger glutenin polymers, and therefore may have positive effects on dough properties. We have found extensive variations which were mainly resulted from single-nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) among the LMW-GS genes in Ae. umbellulata. Our results demonstrated that Ae. umbellulata is an important source of LMW-GS variants and the potential value of the novel LMW-GS alleles for wheat quality improvement.

A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG controlConclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. DisclosuresRoss: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

A Redox-Based Autoinduction Strategy to Facilitate Expression of 5xCys-Tagged Proteins for Electrobiofabrication.

Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices.

Low-molecular-weight glutenin subunit LMW-N13 improves dough quality of transgenic wheat

In our previous study, a novel LMW-GS designated as LMW-N13 with a unique molecular structure was identified from Aegilops uniaristata. LMW-N13 has been characterized as the largest LMW-GS, so far, and possesses an extra cysteine residue compared with typical LMW-GS. In order to analyze the contribution of LMW-N13 to dough quality, in this work, three transgenic wheat lines overexpressing LMW-N13 were generated. Compared with non-transformation (NT) lines, transgenic (TG) lines demonstrated superior dough properties. These superior dough properties were accompanied by the higher contents of glutenin macropolymer (GMP) and total protein. The microstructure of the dough was further investigated by scanning electron microscopy; starch granules in NT lines were smaller than those in transgenic lines. The protein matrix in NT lines was relatively loose and discontinuous. Conversely, the protein matrix in transgenic lines was more continuous and tight. The application of LMW-N13 in wheat breeding is also discussed.

1Mx2.1, a novel high-molecular-weight glutenin subunit from Aegilops comosa and its relationship to high-quality dough

AbstractHigh-molecular-weight glutenin subunit (HMW-GS) of endosperm is mainly correlated with dough quality of bread wheat. In wheat cultivars, the HMW-GS genes with good processing quality are limited. However, there are an amount of excellent HMW-GS genes presenting in wheat-related species. In this work, two novel HMW-GS genes located on 1 M chromosome from Aegilops comosa have been cloned, designated as 1Mx2.1 and 1My12.1, respectively. The molecular structure of 1Mx2.1 and 1My12.1 showed high similarity with the published HMW-GS, but containing unique structures. 1Mx2.1 contained an extra cysteine residue in the repetitive domain, and 1My12.1 lost the conservative cysteine residue in the C-terminal domain. In vitro mixing test has indicated that 1Mx2.1 contributes excellent dough quality. The Ae. comosa can be used as an important genetic resource for wheat quality improvement.

Expression and characterization of Flavikunin: A Kunitz-type serine protease inhibitor identified in the venom gland cDNA library of Bungarus flaviceps.

Trancriptomic analysis of the venom gland cDNA library of Bungarus flaviceps revealed Kunitz-type serine protease inhibitor as one of the major venom protein families with three groups A, B, C. One of the group B isoforms named Flavikunin, which lacked an extra cysteine residue involved in disulfide bond formation in β-bungarotoxin, was synthesized, cloned, and overexpressed in Escherichia coli. To decipher the structure-function relationship, the P1 residue of Flavikunin, histidine, was mutated to alanine and arginine. Purified wild-type and mutant Flavikunins were screened against serine proteases-thrombin, factor Xa, trypsin, chymotrypsin, plasmin, and elastase. The wild-type and mutant Flavikunin (H∆R) inhibited plasmin with an IC 50 of 0.48 and 0.35 µM, respectively. The in-silico study showed that P1 residue of wild-type and mutant (H∆R) Flavikunin interacted with S1' and S1 site of plasmin, respectively. Thus, histidine at the P1 position was found to be involved in plasmin inhibition with mild anticoagulant activity.

Characterization of a Novel 4.0-kb Y-type HMW-GS from Eremopyrum distans

A novel 4.0-kb Fy was sequenced and bacterially expressed. This gene, the largest y-type HMW-GS currently reported, is 4,032-bp long and encodes a mature protein with 1,321 amino acid (AA) residues. The 4.0-kb Fy shows novel modifications in all domains. In the N-terminal, it contains only 67 AA residues, as three short peptides are absent. In the repetitive domain, the undecapeptide RYYPSVTSPQQ is completely lost and the dodecapeptide GSYYPGQTSPQQ is partially absent. A novel motif unit, PGQQ, is present in addition to the two standard motif units PGQGQQ and GYYPTSPQQ. Besides, an extra cysteine residue also occurs in the middle of this domain. The large molecular mass of the 4.0-kb Fy is mainly due to the presence of an extra-long repetitive domain with 1,279 AA residues. The novel 4.0-kb Fy gene is of interest in HMW-GS gene evolution as well as to wheat quality improvement with regard to its longest repetitive domain length and extra cysteines residues.

Characterization of novel LMW glutenin subunit genes at the Glu-M3 locus from Aegilops comosa.

We report the characterization of seven novel low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops comosa (2n = 2x = 14, MM). We found that all seven LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Three genes (comosa-M1, comosa-M2, and comosa-M3) encode LMW-m-type subunits, two (comosa-I1 and comosa-I2) encode LWM-i-type subunits, and two (comosa-L1 and comosa-L2) encode LWM-l-type subunits. The comosa-M1 possessed seven cysteine residues, which resulted from a single-nucleotide polymorphism (SNP) of the G-A transition in the fifth position of the N-terminal sequence. Two l-type subunits, comosa-L1 and comosa-L2, contained nine cysteine residues with an extra cysteine residue located in the signal peptide and repetitive domain. In addition, a long insertion of 13 residues (LGQQPQ8/LVQQPQ8) was present in the end of the C-terminal II. Phylogenetic analysis implied that the comosa-L2 may be the intermediate type during the evolution of LMW-l and LMW-i-type genes. Our results demonstrated that the novel LMW-GSs, such as comosa-M1, comosa-L1, and comosa-L2, may have positive effects on dough properties.

Strong and oriented conjugation of nanobodies onto magnetosomes for the development of a rapid immunomagnetic assay for the environmental detection of tetrabromobisphenol-A.

Variable domain of heavy chain antibody (nanobody, Nb) derived from camelids is an efficient reagent in monitoring environmental contaminants. Oriented conjugates of Nbs and bacterial magnetic particles (BMPs) provide new tools for the high-throughput immunoassay techniques. An anti-tetrabromobisphenol-A (TBBPA) Nb genetically integrated with an extra cysteine residue at the C terminus was immobilized onto BMPs enclosed within the protein membrane, using a heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithiol) propionate, to form a solid BMP-Nb complex. A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) based on the combination of BMP-Nb and T5-horseradish peroxidase was developed for the analysis of TBBPA, with a total assay time of 30min and a half-maximum signal inhibition concentration (IC50) of 1.04ng/mL in PBS (pH10, 10% methanol and 0.137 moL/L NaCl). This assay can even be performed in 100% methanol, with an IC50 value of 44.3ng/mL. This assay showed quantitative recoveries of TBBPA from spiked canal water (114-124%) and sediment (109-113%) samples at 1.0-10ng/mL (or ng/g (dw)). TBBPA residues determined by this assay in real canal water samples were below the limit of detection (LOD) and in real sediments were between <LOD and 23.4ng/g (dw). The BMP-Nb-based ELISA shows promising application in environmental monitoring. Graphical abstract ᅟ.

Characterization of novel D-hordeins from Psathyrostachys juncea

Three genes encoding novel D-hordeins, Ns 1.3, Ns 2.6, and Ns 2.9 were isolated from Psathyrostachys juncea. The Ns 1.3 differed from Ns 2.6 and Ns 2.9 by having a shorter open reading frame ( 2.5 kb), and was probably not expressed as a normal protein, while the activities for Ns 2.6 and Ns 2.9 were verified by bacterial expression. Though highly similar primary structure to wheat high molecular mass glutenin subunits (HMM-GSs) and barley D-hordeins, Ns 2.6 and Ns 2.9 had more cysteine residues (nine in total) and a larger molecular mass than HMMGSs, and a longer N-terminal length than D-hordeins. Phylogenetic analysis revealed that the Ps. juncea D-hordeins were divided into Ns 1.3 type and Ns 2.6/Ns 2.9 type. Divergence times indicated that Ns 1.3 diverged the earliest from the orthologous Triticeae locus, while Ns 2.6 and Ns 2.9 and the D-hordeins from two Hordeum species diverged nearly at the same time from those loci, and the divergence between the D-hordeins of H. chilense and Ns 2.6/Ns 2.9 was more recent than between the two Hordeum species. The novel Ps. juncea D-hordeins have the potential to be very important for improving the end-use quality of wheat flours because of the presence of extra cysteine residues and longer repetitive domain, in addition they can contribute to the understanding of the evolution of Triticeae prolamins.

Analysis of novel high-molecular-weight prolamins from Leymus multicaulis (Kar. et Kir.) Tzvelev and L. chinensis (Trin. ex Bunge) Tzvelev.

Nine novel high-molecular-weight prolamins (HMW-prolamins) were isolated from Leymus multicaulis and L. chinensis. Based on the structure of the repetitive domains, all nine genes were classified as D-hordeins but not high-molecular-weight glutenin subunits (HMW-GSs) that have been previously isolated in Leymus spp. Four genes, Lmul 1.2, 2.4, 2.7, and Lchi 2.5 were verified by bacterial expression, whereas the other five sequences (1.3 types) were classified as pseudogenes. The four Leymus D-hordein proteins had longer N-termini than those of Hordeum spp. [116/118 vs. 110 amino acid (AA) residues], whereas three (Lmul 1.2, 2.4, and 2.7) contained shorter N-termini than those of the Ps. juncea (116 vs. 118 AA residues). Furthermore, Lmul 1.2 was identified as the smallest D-hordein, and Lmul 1.2 and 2.7 had an additional cysteines. Phylogenetic analysis supported that the nine D-hordeins of Leymus formed two independent clades, with all the 1.3 types clustered with Ps. juncea Ns 1.3, whereas the others were clustered together with the D-hordeins from Hordeum and Ps. juncea and the HMW-GSs from Leymus. Within the clade of four D-hordein genes and HMW-GSs, the HMW-GSs of Leymus formed a separated branch that served as an intermediate between the D-hordeins of Ps. juncea and Leymus. These novel D-hordeins may be potentially utilized in the improvement of food processing properties particularly those relating to extra cysteine residues. The findings of the present study also provide basic information for understanding the HMW-prolamins among Triticeae species, as well as expand the sources of D-hordeins from Hordeum to Leymus.

Gold nanoparticles functionalized with angiogenin-mimicking peptides modulate cell membrane interactions.

Angiogenin is a protein crucial in angiogenesis, and it is overexpressed in many cancers and downregulated in neurodegenerative diseases, respectively. The protein interaction with actin, through the loop encompassing the 60-68 residues, is an essential step in the cellular cytoskeleton reorganization. This, in turn, influences the cell proliferation and migration processes. In this work, hybrid nanoassemblies of gold nanoparticles with angiogenin fragments containing the 60-68 sequence were prepared and characterized in their interaction with both model membranes of supported lipid bilayers (SLBs) and cellular membranes of cancer (neuroblastoma) and normal (fibroblasts) cell lines. The comparison between physisorption and chemisorption mechanisms was performed by the parallel investigation of the 60-68 sequence and the peptide analogous containing an extra cysteine residue. Moreover, steric hindrance and charge effects were considered with a third analogous peptide sequence, conjugated with a fluorescent carboxyfluorescein (Fam) moiety. The hybrid nanobiointerface was characterized by means of ultraviolet-visible, atomic force microscopy and circular dichroism, to scrutinize plasmonic changes, nanoparticles coverage and conformational features, respectively. Lateral diffusion measurements on SLBs "perturbed" by the interaction with the gold nanoparticles-peptides point to a stronger membrane interaction in comparison with the uncoated nanoparticles. Cell viability and proliferation assays indicate a slight nanotoxicity in neuroblastoma cells and a proliferative activity in fibroblasts. The actin staining confirms different levels of interaction between the hybrid assemblies and the cell membranes.

Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics

BackgroundPreclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1–1 versus Hp 2–2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200–900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions.ResultsIn the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies.ConclusionsWe present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

DNA Modulates the Interaction of Genetically Engineered DNA-Binding Proteins and Gold Nanoparticles: Diagnosis of High-Risk HPV Infection.

Gene detection has an important role in diagnosing several serious diseases and genetic defects in modern clinical medicine. Herein, we report a fast and convenient gene detection method based on the modulation of the interaction between a heat-resistant double-stranded DNA (dsDNA)-binding protein (Sso7d) and gold nanoparticles (Au NPs). We prepared a recombinant Cys-Sso7d, which is Sso7d with an extra cysteine (Cys) residue in the N-terminus, through protein engineering to control the interaction between Sso7d and Au NPs. Cys-Sso7d exhibited a stronger affinity for Au NPs and more easily induced the aggregation of Au NPs than Sso7d. In addition, Cys-Sso7d retained its ability to bind with dsDNA. The aggregation of Au NPs induced by Cys-Sso7d was diminished in the presence of dsDNA, which could be utilized as a transduction mechanism for the detection of the polymerase chain reaction (PCR) products of human papillomavirus (HPV) gene fragments (HPV types 16 and 18). The Cys-Sso7d/Au NP probe could detect as few as 1 copy of the HPV gene. The sensitivity and specificity of the Cys-Sso7d/Au NP probe for Pap smear clinical specimens (n = 52) for HPV 16 and HPV 18 detection were 85.7%/100.0% and 85.7%/91.7%, respectively. Our results demonstrate that the Cys-Sso7d/Au NP probe can be used to diagnose high-risk HPV types in Pap smear samples with high sensitivity, specificity, and accuracy.