Acid Polypeptide Research Articles

Molecular characterization and functional analysis of duck CCCH-type zinc finger antiviral protein (ZAP)

This is the first study to clone duck CCCH-type zinc finger antiviral protein (duZAP) from Jingjiang duck (Anas platyrhynchos). Full-length duZAP cDNA was 2154 bp and encoded a 717-amino acid polypeptide containing four highly conserved CCCH-type finger motifs, a WWE domain and a poly (ADP-ribose) polymerase (PARP) domain. duZAP was expressed in multiple duck tissues, with the highest mRNA expression in the spleen. Overexpression of duZAP in duck embryo fibroblast cells (DEFs) led to activation of the transcription factors IRF1 and NF-κB, and induction of IFN-β. Analysis of deletion mutants revealed that both the WWE and PARP domains of duZAP were essential for activating the IFN-β promoter. Knockdown of duZAP in DEFs significantly reduced poly (I:C)- and duck Tembusu virus (DTMUV)-induced IFN-β activation. Our findings further the understanding of the role of duZAP in the duck innate immune response.

Antioxidant activity of linear and star-shaped polypeptides modified with dopamine and glutathione

The aim of this study was to investigate the effect of topology on the antioxidative activities of antioxidant-grafted polypeptides. The linear and star-shaped poly(L-glutamic acid) (PLG) polypeptides were synthesized by N-carboxyanhydrides (NCAs) ring opening polymerization (ROP) using primary amine and polyols as initiators, followed by deprotection. Graft copolypeptides (PLG-Dopa, PLG-GSH and PLG-Dopa-GSH) were obtained by EDC/NHS coupling chemistry using dopamine (Dopa) and glutathione (GSH). Successful synthesis of these polypeptides was confirmed by 1H NMR, MALDI TOF, GPC, UV–vis and IR spectra analyses. The conformation of polypeptides was investigated by circular dichroism (CD) spectroscopy, demonstrating the grafting of Dopa and GSH onto PLG was accompanied by a conformational change from a random coil to α-helical structure. The results showed that the antioxidant activities of PLG-Dopa and PLG-Dopa-GSH were mainly ascribed to Dopa. It was found that the conjugation of Dopa resulted in the decrease in its antioxidant activity and the antioxidant-grafted polypeptides exhibited comparable antioxidant activity regardless the polypeptide arm number. The conjugation of GSH could prolong polypeptide antioxidant activity by preventing Dopa from oxidation. It was found that both PLG-Dopa and PLG-Dopa-GSH exhibited good hemocompatibility and biocompatibility.

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review.

Hirudin, an acidic polypeptide secreted by the salivary glands of Hirudo medicinalis (also known as “Shuizhi” in traditional Chinese medicine), is the strongest natural specific inhibitor of thrombin found so far. Hirudin has been demonstrated to possess potent anti-thrombotic effect in previous studies. Recently, increasing researches have focused on the anti-thrombotic activity of the derivatives of hirudin, mainly because these derivatives have stronger antithrombotic activity and lower bleeding risk. Additionally, various bioactivities of hirudin have been reported as well, including wound repair effect, anti-fibrosis effect, effect on diabetic complications, anti-tumor effect, anti-hyperuricemia effect, effect on cerebral hemorrhage, and others. Therefore, by collecting and summarizing publications from the recent two decades, the pharmacological activities, pharmacokinetics, novel preparations and derivatives, as well as toxicity of hirudin were systematically reviewed in this paper. In addition, the clinical application, the underlying mechanisms of pharmacological effects, the dose-effect relationship, and the development potential in new drug research of hirudin were discussed on the purpose of providing new ideas for application of hirudin in treating related diseases.

ANGPTL8 roles in proliferation, metabolic diseases, hypothyroidism, polycystic ovary syndrome, and signaling pathways.

A new and atypical member of the ANGPTL family is angiopoietin-like protein 8 (ANGPTL8). This newly discovered hormone is a drug target that can be used to treat diabetes and dyslipidemia. The protein, as a hepatocyte-derived circulating factor, can control the triglyceride level of plasma. ANGPTL8 is significantly associated with inflammation and metabolic syndrome consequences such as obesity, diabetes, hypothyroidism, and PCOS. ANGPTL8 gene has four exons encoding a 22/5kDa weight of 198 amino acid polypeptides. A highly preserved ANGPTL8 gene among mammals exhibits the essential hormone functions of ANGPTL8. Nevertheless, the physiological function of this hormone in the body is poorly understood. Studies published in PubMed (2008-2020), Google Scholar (2004-2020), and Scopus (2004-2020) databases of clinical trials were reviewed. This analysis is aimed at collecting information on ANGPTL8. The emphasis of this review was on gathering information about the role of ANGPTL8 in the metabolism of glucose and lipids and cell proliferation. It addition to the different roles of ANGPTL8 in diabetes and lipid metabolism, this review emphasized on the protein role in signaling pathways. The study also proposes the signaling pathways that may be considered as a new target for treatment.

Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features.

Thymosin β4 (Tβ4) is a small, 44-amino acid polypeptide. It has been implicated in multiple processes, including cell movement, angiogenesis, and stemness. Previously, we reported that melanoma cell lines differ in Tβ4 levels. Studies on stable clones with silenced TMSB4X expression showed that Tβ4 impacted adhesion and epithelial-mesenchymal transition progression. Here, we show that the cells with silenced TMSB4X expression exhibited altered actin cytoskeleton’s organization and subcellular relocalization of two intermediate filament proteins: Nestin and Vimentin. The rearrangement of the cell cytoskeleton resulted in changes in the cells’ topology, height, and stiffness defined by Young’s modulus. Simultaneously, only for some A375 clones with a lowered Tβ4 level, we observed a decreased ability to initiate colony formation in soft agar, tumor formation in vivo, and alterations in Nanog’s expression level transcription factor regulating stemness. Thus, we show for the first time that in A375 cells, biomechanical properties are not directly coupled to stemness features, and this cell line is phenotypically heterogeneous.

CPSF30-L: A direct connection between mRNA polyadenylation and m6A RNA modification in plants

CPSF30-L: A direct connection between mRNA polyadenylation and m6A RNA modification in plants

Characterization and expression analysis of ascorbate peroxidase from the moss Dicranum scoparium during abiotic stresses

Ascorbate peroxidase (APX) is an important antioxidant enzyme responsible for the conversion of H2O2 to H2O and O2. In this study, APX was studied in the widespread boreal cushion moss Dicranum scoparium. Native PAGE of crude extracts of moss thalli revealed the presence of APX isoforms in D. scoparium with a range of molecular masses. An APX complementary DNA (cDNA) gene of 771-bp length was cloned and designated as DsAPX. The cloned coding domain sequence (CDS) encoded a 256 amino acid polypeptide, and the predicted protein product was calculated to have a molecular mass of 28.4 kDa with an isoelectric point of 5.7. Several highly conserved sites important for enzyme activity were predicted using bioinformatic tools. The DsAPX protein has similarities of 91%, 78%, 67% and 66% with APX homologs from Grimmia pilifera, Physcomitrella patens, Zea mays and Nicotiana tomentosiformis, respectively. The high homology with cytosolic APX from G. pilifera strongly suggests that the cloned DsAPX gene encodes a cytosolic APX. We studied the role of APX in the tolerance of this moss to abiotic stresses. Changes in both APX activity and DsAPX gene expression, estimated using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), showed that the gene was up-regulated in response to desiccation/rehydration, and expression was maintained during a heat stress of +50°C. By contrast, DsAPX was down-regulated by a freezing (–20°C) treatment. Results obtained extend our knowledge of the diversity of APX isoforms that occur in bryophytes and suggest that APX may be involved in the tolerance of D. scoparium to abiotic stresses.

Molecular characterization and expression analysis of a novel C-type lectin (CTL) gene in yellow catfish Pelteobagrus fulvidraco

C-type lectin (CTL) proteins contain special carbohydrate-recognition domains (CRDs) and play vital roles in activating the innate immune system. In this study, a CTL gene from freshwater yellow catfish (Pelteobagrus fulvidraco) was identified and named as PfCTL. Analysis revealed that the complete PfCTL cDNA was 718 bp in size, including a 42-bp 5′-untranslated region (UTR), 61-bp 3′-UTR, and 615-bp open reading frame (ORF) encoding a 204 amino acid polypeptide, which contained a signal peptide and CRD. The PfCTL protein contained two casein kinase II phosphorylation sites, four protein kinase C phosphorylation sites, and two N-myristoylation sites. The deduced PfCTL protein sequence has 37–83 % in comparison to CTL proteins from other fish. Further phylogenetic analysis revealed that PfCTL was closely related to the CTL of Ictalurus punctatus. Based on quantitative real-time polymerase chain reaction, the PfCTL gene was expressed in all tested tissues at different levels, with highest distribution in the spleen, followed by the head kidney, and lowest distribution in blood. After challenge with lipopolysaccharide and polyriboinosinic polyribocytidylic acid, the expression of PfCTL was up-regulated in the liver, spleen, head kidney, and blood at different time points. These results imply that this CTL gene may be an important pathogen pattern recognition molecule in the innate immune system of yellow catfish under pathogen stimulation.

Effect of surface charge conditions of carriers on the immobilization of β-d-glucosidase

In this study, a series of acidic or alkaline polypeptide chains were designed and grafted onto DEG-AM resin using Fmoc solid-phase synthesis to study the relationship between enzyme conformation and carrier surface charge. β-d-glucosidase (βGase) was then immobilized onto these modified carriers by adsorption. Each form of immobilized βGase showed decreasing specific activity compared to that of the free. It could be attributed to both the changes in the enzyme conformation and the decrease in mass transfer efficiency. The optimum temperature of free βGase, DEG@B3-βGase is 55 °C, which of DEG@A3-βGase is 65 °C and they all have the highest activity at pH 5. The Ea values ​​of free βGase, DEG@A3-βGase, and DEG@B3-βGase are 0.546 kJ/mol, 0.224 kJ/mol, and 0.446 kJ/mol, and the Km values were 1.30 mmol/L, 1.44 mmol/L and 2.63 mmol/L, respectively. It shows that free βGase and DEG@A3-βGase are more similar. Meanwhile, the free βGase (1.0 g/L, pH 5.0) stored at 4 °C has a shorter half-life (t1/2), which is only 9 days. However, the half-life of DEG@B3-βGase and DEG@A3-βGase is 20 days and over 60 days, indicating that the negative charged surface was conducive to maintenance of the structure and catalytic property of βGase.

A C-type lectin (OmCTL) in Onychostoma macrolepis: Binding ability to LPS, PGN and agglutinating activity against bacteria

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila, suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila, in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

The Ubiquitin Switch in Plant Stress Response.

Ubiquitin is a 76 amino acid polypeptide common to all eukaryotic organisms. It functions as a post-translationally modifying mark covalently linked to a large cohort of yet poorly defined protein substrates. The resulting ubiquitylated proteins can rapidly change their activities, cellular localization, or turnover through the 26S proteasome if they are no longer needed or are abnormal. Such a selective modification is essential to many signal transduction pathways particularly in those related to stress responses by rapidly enhancing or quenching output. Hence, this modification system, the so-called ubiquitin-26S proteasome system (UPS), has caught the attention in the plant research community over the last two decades for its roles in plant abiotic and biotic stress responses. Through direct or indirect mediation of plant hormones, the UPS selectively degrades key components in stress signaling to either negatively or positively regulate plant response to a given stimulus. As a result, a tightly regulated signaling network has become of much interest over the years. The ever-increasing changes of the global climate require both the development of new crops to cope with rapid changing environment and new knowledge to survey the dynamics of ecosystem. This review examines how the ubiquitin can switch and tune plant stress response and poses potential avenues to further explore this system.

Molecular characterization and expression analysis of double‐stranded RNA‐dependent protein kinase (PKR) in Dabry’s sturgeon ( Acipenser dabryanus )

Protein kinase RNA-activated (PKR) is an important component of the innate immune system induced by interferon. This antiviral protein senses a variety of stresses and pathogen-driven double-stranded RNAs (dsRNAs). In the present study, we cloned and characterized the PKR gene from Dabry's sturgeon (Acipenser dabryanus). Bioinformatics analysis revealed that the AdPKR gene comprises a coding sequence (CDS) of 1848 bp, encoding a 616 amino acids (aa) polypeptide. AdPKR shares high aa identity/similarity with proteins from other fish, ranging from 33.4%/51.8% to 87.6%/91.2%, and relatively low aa identity (13.9%) and similarity (26.8%) with human PKR. The AdPKR polypeptide has two dsRNA-binding motifs at the N-terminus and a conserved catalytic kinase domain at the C-terminus. The sturgeon PKR gene contains 17 exons split by 16 introns. Phylogenetic analysis clustered AdPKR together with its counterparts from other teleost fishes. Real-time fluorescene quantitative PCR (RT-qPCR) transcription profiles revealed highest AdPKR expression levels in brain, followed by middle kidney, skin, muscle and head kidney. AdPKR mRNA transcripts were significantly increased at 6 hr post-stimulation (hps) with polyinosinic:polycytidylic acid (poly I:C), remained high at 24 hps, then decreased to normal levels at 48 hps. These results indicate that AdPKR might play a vital role in antiviral immune responses.

Controlling the self-assembly of human calcitonin: a theoretical approach using molecular dynamics simulations.

Human calcitonin (hCT) is a 32-residue amino acid poly-peptide hormone which is secreted by the C-cells (also known as parafollicular cells) of thyroid glands. It acts to inhibit osteoclast cell hormones by reducing the cell function and regulating calcium and phosphate in blood. hCT has a high tendency to assemble into protofilaments with β-sheet conformations. Amyloid fibril formation of hCT reduces its bio-activity and limits its application as a therapeutic drug. Salmon calcitonin (sCT), which also carries the same disulfide bridge at the N and C-terminus, but differs at the 16 residue position from hCT, has less propensity to aggregate than hCT. Human calcitonin has much higher bio-activity than sCT if its aggregation propensity is reduced. Substituting the key residues which are responsible for the aggregation of hCT, is one of the ways to reduce its aggregation and fibril formation. hCT analogues with less aggregation tendency can be exploited as therapeutic drugs. In this work, we study the amyloidogenic behavior of hCT and its peptide based derivatives i.e., sCT, phCT, N17H hCT, Y12L hCT and DM hCT, through classical molecular dynamics (MD) simulations. Our study reveals that sCT is the least aggregation prone derivative, and the double mutation at position 12 and 17 can reduce the aggregation propensity of this peptide. Also, we have applied these mutant variants of hCT as peptide inhibitors in the self-aggregation of hCT. This study could help in understanding and preparing peptide-based inhibitors for hCT fibrillation and their applications as therapeutic drugs.

Cloning and characterization of a phosphomevalonate kinase gene from Sanghuangporus baumii

Sanghuangporus baumii, a macrofungus belonging to the Hymenochaetaceae family, has been widely used as a traditional medicine in Asia for centuries. Triterpenoids in S. baumii contribute to the pharmacological activities, however, their content is low in the fruiting body. Phosphomevalonate kinase (PMK) is an important rate-limiting enzyme in the triterpenoids synthesis pathway. In this study, a PMK gene (SbPMK) was cloned and analyzed. Sequence analysis showed that SbPMK has six exons and five introns. The 1,488 bp open reading frame encodes a 489 amino acid polypeptide with a predicted protein molecular weight of 52.47 kDa. The amino acid sequence of SbPMK shares highly similarity with that of PMK sequences from other species, especially in three functional motifs (MI, MII and MIII). The promoter of SbPMK contains typical cis-acting regulatory elements involved in MeJA responsiveness, light responsiveness, anaerobic induction, etc. Furthermore, recombinant SbPMK was overexpressed in Escherichia coli BL21, with a satisfactory solubility. During the fermentation culture of S. baumii, the biomass increased with fermentation and peaked on day 18 (4.80 g/L). The SbPMK expression and the triterpenoids content was peaked on day 6, and the triterpenoids content stabilized at 20.8 mg/g on day 18. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1938678 .

Cloning, Spatiotemporal Expression and Prokaryotic Expression of ACC Oxidase Gene (<I>BnACO2</i>) in Ramie

ACC oxidase (ACO) is a key enzyme in ethylene synthesis pathway. This study by rt-pcr with RACE technology get a ACO gene cloning, named  BnACO2 , total length of the gene cDNA sequence is 1 377 bp, the open reading frame of 957 bp, coded 318 amino acid polypeptide, predict its molecular weight and isoelectric point (pl) 36.145 kD and 5.60, respectively, bioinformatics analysis showed that  BnACO2  no encoding protein signal peptide and transmembrane domain structure, subcellular localization in the cytoplasm. The similarity of ACO gene nucleotide sequence and amino acid sequence was more than 82% and 84% respectively. Phylogenetic analysis showed that the ACO gene was closely related to jute and hemp. Real-time fluorescence quantitative PCR analysis showed that  BnACO2  gene was expressed in all parts of ramie, especially in female flower buds. The prokaryotic expression vector PQe- BnACO2  was successfully constructed by double enzyme digestion. At 37°C, 1.0mmol/L IPTG induced PQe- BnACO2  to express the target protein, and the molecular weight of the induced protein was about 36.15kda, which was consistent with the predicted protein size. This lays the foundation for further study on the function of  BnACO2  gene.

Development and radiosynthesis of a novel bifunctional tri-γ-glutamic acid polypeptide for gallium-68 labeling

Development and radiosynthesis of a novel bifunctional tri-γ-glutamic acid polypeptide for gallium-68 labeling

Resolving the Mechanisms of Soy Glycinin Self-Coacervation and Hollow-Condensate Formation.

Self-coacervation of animal-derived proteins has been extensively investigated while that of plant proteins remains largely unexplored. Here, we study the process of soy glycinin self-coacervation and transformation into hollow condensates. The protein hexameric structure composed of hydrophilic and hydrophobic polypeptides is crucial for coacervation. The process is driven by charge screening of the intrinsically disordered region of acidic polypeptides, allowing for weak hydrophobic interactions between exposed hydrophobic polypeptides. We find that the coacervate surface exhibits order, which stabilizes the coacervate shape during hollow-condensate formation. The latter process occurs via nucleation and growth of protein-poor phase in the coacervate interior, during which another ordered layer at the inner surface is formed. Aging enhances the stability of both coacervates and hollow condensates. Understanding plant protein coacervation holds promises for fabricating novel functional materials.

Cloning, purification, and characterization of recombinant endo- β-1,4-D-xylanase of Bacillus sp. From soil termite abdomen

Abstract A novel endo-β-1,4- d -xylanase (xynBT) was identified from Bacillus sp. in soil termite abdomen and successfully cloned in Escherichia coli TOP10 and expressed in Escherichia coli BL21 (DE3) via pET-30a (+) as an expression vector. The full length gene consist of 801 bp ORF encoding a 267 amino acid polypeptide. The deduced amino acid sequence of xynBT displayed homology with glycoside hydrolase (GH) family 11 xylanase. Recombinant XynBT (r-XynBT), which was purified, showed an optimal pH and temperature of 5.5 and 40 °C, respectively. This enzyme was purified by the Immobilized Metal Affinity Chromatography (IMAC) method and has a molecular mass of 30 kDa, which was observed via sodium dodecyl polyacrylamide sodium electrophoresis (SDS-PAGE). Purified r-XynBT was the most stable at pH 5 for up to 120 min pre-incubation time and had a residual activity of 83%. Purified r-XynBT was also stable between 30 and 40 °C for 80 min of pre-incubation and had a residual activity of more than 50%. The presence of metal cations K+ and Na+ on r-XynBT increased its activity, while metal cations Mg2+, Cu2+, Zn2+, and Fe3+ were inhibitors.

Design and Construction of pH-Selective Self-Lytic Liposome System

Liposomes are well-investigated drug or gene delivery vehicles for chemotherapy, used by taking advantage of their biocompatibility and biodegradability. A central question on the construction of intracellular liposomal delivery systems is to entrap the liposomes of interest in the highly acidic and proteolytic endosomal environment. In the other words, it is essential that the liposomes release a therapeutic drug into the cytosol before they are degraded in the endosome. As a strategy to enhance the endosome escape, the self-lytic liposomes with acidic pH-selective membrane active polypeptide are considered highly effective. Here, an acidic pH-selective membrane-lytic polypeptide (LPE) and its retro isomer (rLPE) were designed, and then their membrane-lytic activities against EggPC liposomes were determined. It was noticed that the rLPE polypeptide showed an increase in activity compared with the LPE polypeptide. Furthermore, the rLPE polypeptide was conjugated to liposomes via a flexible Gly-Gly-Gly-Gly linker to facilitate the pH-selective content release. The rLPE anchoring liposomes exhibited distinctly different contents release behavior at physiological and endosomal pHs, namely typical contents release from liposomes was positively observed at acidic pH range. The overarching goal of this paper is to develop efficient pH-selective therapeutic delivery systems by using our findings.

A Novel Surface-Exposed Polypeptide Is Successfully Employed as a Target for Developing a Prototype One-Step Immunochromatographic Strip for Specific and Sensitive Direct Detection of Staphylococcus aureus Causing Neonatal Sepsis.

Neonatal sepsis is a life-threatening condition and Staphylococcus aureus is one of its major causes. However, to date, no rapid and sensitive diagnostic tool has been developed for its direct detection. Bioinformatics analyses identified a surface-exposed 112-amino acid polypeptide of the cell wall protein NWMN_1649, a surface protein involved in cell aggregation and biofilm formation, as being a species-specific and highly conserved moiety. The polypeptide was cloned, purified, and used to immunize mice to raise specific immunoglobulins. The purified antibodies were conjugated to gold nano-particles and used to assemble an immunochromatographic strip (ICS). The developed prototype ICS detected as low as 5 µg purified polypeptide and 102 CFU/mL S. aureus within 15 min. The strip showed superior ability to directly detect S. aureus in neonatal sepsis blood specimens without prior sample processing. Moreover, it showed no cross-reaction in specimens infected with two other major causes of neonatal sepsis; coagulase-negative staphylococci and Klebsiella pneumoniae. The selected NWMN_1649-derived polypeptide demonstrates success as a promising biomolecule upon which a prototype ICS has been developed. This ICS provides a rapid, direct, sensitive, and specific option for the detection of S. aureus causing neonatal sepsis. Such a tool is urgently needed especially in resources-limited countries.