scholarly journals Five Silkworm 30K Proteins Are Involved in the Cellular Immunity against Fungi

Insects ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 107
Author(s):  
Lin Ye ◽  
Yan Zhang ◽  
Zhaoming Dong ◽  
Pengchao Guo ◽  
Dongchao Zhao ◽  
...  
Keyword(s):  
Cellular Immunity ◽  
Storage Proteins ◽  
Polyclonal Antibodies ◽  
Expression System ◽  
Transcriptional Expression ◽  
Binding Assays ◽  
Agarose Beads ◽  
Escherichia Coli Expression ◽  
Molecular Patterns

Background: 30K proteins are a major group of nutrient storage proteins in the silkworm hemolymph. Previous studies have shown that 30K proteins are involved in the anti-fungal immunity; however, the molecular mechanism involved in this immunity remains unclear. Methods: We investigated the transcriptional expression of five 30K proteins, including BmLP1, BmLP2, BmLP3, BmLP4, and BmLP7. The five recombinant 30K proteins were expressed in an Escherichia coli expression system, and used for binding assays with fungal cells and hemocytes. Results: The transcriptional expression showed that the five 30K proteins were significantly upregulated after injection of pathogen-associated molecular patterns to the fifth instar larvae, indicating the possibility of their involvement in immune response. The binding assay showed that only BmLP1 and BmLP4 can bind to both fungal cells and silkworm hemocytes. Furthermore, we found that BmLP1-coated and BmLP4-coated agarose beads promote encapsulation of hemocytes in vitro. The hemocyte encapsulation was blocked when the BmLP1-coated beads were preincubated with BmLP1 specific polyclonal antibodies. Conclusions: These results demonstrate that 30K proteins are involved in the cellular immunity of silkworms by acting as pattern recognition molecules to directly recruit hemocytes to the fungal surface.

scholarly journals SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhaoming Dong ◽  
Lingna An ◽  
Mengyao Lu ◽  
Muya Tang ◽  
Haiqin Chen ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Fungal Spores ◽  
Immune Defense ◽  
Proteinase K ◽  
Binding Assays ◽  
Activity Inhibition ◽  
Agarose Beads ◽  
Molecular Patterns

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to β-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Expression of adenylate kinase fused mouse ubiquitin active enzyme in Escherichia coli and its application in ubiquitination

2021 ◽  
Author(s):  
Xiaoliang Liu ◽  
Ling Hu ◽  
Yuan Zhang ◽  
Hongtao Li
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Adenylate Kinase ◽  
Energy Supply ◽  
Expression System ◽  
Synthesis Process ◽  
Cellular Functions ◽  
Escherichia Coli Expression

Abstract Ubiquitination, is involved in the regulation of numerous cellular functions. Researches in the ubiquitin realm rely heavily on ubiquitination assays in vitro and require large amounts of ubiquitin-activating enzyme (UBA1) and keep ATP supplies. But UBA1 is hard to be obtained with large quantities using reported methods. We fused Escherichia coli adenylate kinase (adk) and mouse UBA1 obtained fusion protein adk-mUBA1. The expression level of adk-mUBA1 increased about 8-fold than that of mUBA1 in Escherichia coli expression system, and adk-mUBA1 was easily purified to 90% purity via two purification steps. The purified adk-mUBA1 protein was functional for ubiquitination and could use ATP in addition to ADP as energy supply and had a higher catalytic activity than mUBA1 in cell lysis. Adk-mUBA1 can be applied to preparing ubiquitin modified substrates and kinds of ubiquitin chains in chemical synthesis process and is preferable application than mUBA1 in vitro ubiquitination.

scholarly journals Polypeptidic Taxol-Tropins: Targeting paclitaxel to the tumor microenvironment

2021 ◽  
Vol 5 (3) ◽  
pp. 01-11
Author(s):  
Erlinda M. Gordon ◽  
Seiya Liu ◽  
Sant P. Chawla ◽  
Frederick L. Hall
Keyword(s):  
Tumor Microenvironment ◽  
Targeted Delivery ◽  
Tumor Targeting ◽  
Drug Binding ◽  
Peptide Sequence ◽  
Human Pancreatic Cancer ◽  
Binding Assays ◽  
Agarose Beads

Background and Rationale: Although PTX is widely used as a single chemotherapeutic agent and in various combination regimens, its clinical utility is hindered by acquired drug resistance and serious dose-limiting side effects that result from the ungoverned biodistribution of the taxane. Hypothesis: Conceptually, the precision, validity, and efficiency of paclitaxel delivery to tumor compartments might be substantially improved by “actively targeting” the exposed collagenous (XC-) proteins presented within the tumor microenvironment (TME)—XC-proteins physically exposed by the pathologic biochemical processes of tumor invasion, reactive stroma formation, and neo-angiogenesis. Objective: An adaptive bioengineering approach aims to apply pathotropic tumor-targeting functionality to paclitaxel (PTX), a powerful cytotoxic taxane which exhibits anti-tubulin / anti-mitotic / anti-cancer activities against a broad range of solid tumors. Materials and Methods: Synthetic peptide XC-targeting probes (< 40 aa) and polypeptide aptamers (40 to 53 aa), 85 - 99% purity, were prepared by 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis, purified by high performance liquid chromatography (HPLC), and verified by mass spectrometry and amino acid analysis, and the XC-targeting probes were FITC-labeled. Analysis of fluorescence in XC-binding assays was visualized with an Ultra Bright Blue Light trans-illuminator equipped with an amber filter; photo-documentation was provided by a Leica V-Lux 1 digital camera; and comparative fluorescence was quantified using a Quantus benchtop fluorimeter (Promega). The tumor-targeting properties of Taxol-Tropins were tested in vitro by Taxol-aptamer binding assays and collagen-agarose binding assays and the bioactivities of PTX bound non-covalently toTaxol-Tropin aptamers were tested on XC-agarose beads. Further, the tumor targeting property of the Taxol-Tropin aptamers was tested in vivo in a murine model of metastatic cancer. Results: Here we report on the first actively targeted delivery of paclitaxel utilizing bifunctional polypeptide targeting onco-aptamers, called Taxol-Tropins, which: (i) bind PTX upon simple mixing with suitably high affinities and; (ii) bind exposed XC-proteins, thereby promoting enhanced partitioning and drug delivery into the TME. The bifunctional peptide sequence-optimized Taxol-Tropins bound tightly non-covalently to PTX and also exhibited high affinity and selectivity for XC-agarose beads in vitro. Importantly, the cytotoxic bioactivity of the Taxol-Tropin-bound-PTX molecule was well preserved in cellulo, as was demonstrated by cytocidal activity observed in MDA-MB-231 breast cancer cell cultures. Tumor-targeted PTX delivery by Taxol-Tropin onco-aptamers in vivo was modeled by subcutaneous xenografts of human pancreatic cancer in nude mice: where intense fluorescence of the PTX probe was observed in tumors of mice injected with the Taxol-Tropin-bound-PTX within minutes after intravenous injection, but not in untreated mice or mice treated with non-targeted PTX probe. Conclusions: These results demonstrate the feasibility of pro-actively targeting PTX, a clinically important small molecule, using Taxol-Tropins: synthetic polypeptide onco-aptamers, revealing optimized drug binding sequences and structural modifications pertinent to further clinical development of the tumor-targeting platform which may indeed shift the Therapeutic Index of PTX to one of greater clinical efficacy at lower drug doses.

scholarly journals Protein degradation in a TX-TL cell-free expression system using ClpXP protease

2015 ◽  
Author(s):  
Zachary Sun ◽  
Jongmin Kim ◽  
Vipul Singhal ◽  
Richard M Murray
Keyword(s):  
Protein Degradation ◽  
Expression System ◽  
Free Expression ◽  
Batch Mode ◽  
Protein Targets ◽  
Resource Limited ◽  
Escherichia Coli Expression ◽  
Cell Free Expression System

An in vitro S30-based Escherichia coli expression system (“Transcription-Translation”, or “TX-TL”) has been developed as an alternative prototyping environment to the cell for synthetic circuits [1-5]. Basic circuit elements, such as switches and cascades, have been shown to function in TX-TL, as well as bacteriophage assembly [2, 6]. Circuits can also be prototyped from basic parts within 8 hours, avoiding cloning and transformation steps [7]. However, most published results have been obtained in a “batch mode” reaction, where factors that play an important role for in vivo circuit dynamics – namely protein degradation and protein dilution – are severely hindered or are not present. This limits the complexity of circuits built in TX-TL without steady-state or continuous-flow solutions [8-10]. However, alternate methods that enable dilution either require extra equipment and expertise or demand lower reaction throughput. We explored the possibility of supplementing TX-TL with ClpXP, an AAA+ protease pair that selectively degrades tagged proteins [11], to provide finely-tuned degradation. The mechanism of ClpXP degradation has been extensively studied both in vitro and in vivo [12-15]. However, it has not been characterized for use in synthetic circuits – metrics such as toxicity, ATP usage, degradation variation over time, and cellular loading need to be determined. In particular, TX-TL in batch mode is known to be resource limited [16], and ClpXP is known to require significant amounts of ATP to unfold different protein targets [17, 18]. We find that ClpXP’s protein degradation dynamics is dependent on protein identity, but can be determined experimentally. Degradation follows Michaels-Menten kinetics, and can be fine tuned by ClpX or ClpP concentration. Added purified ClpX is also not toxic to TX-TL reactions. Therefore, ClpXP provides a controllable way to introduce protein degradation and dynamics into synthetic circuits in TX-TL.

scholarly journals Mapping residues of SUMO precursors essential in differential maturation by SUMO-specific protease, SENP1

2005 ◽  
Vol 386 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Zheng XU ◽  
Shannon W. N. AU
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Tissue Distribution ◽  
Catalytic Domain ◽  
Expression System ◽  
Proteolytic Processing ◽  
Maturation Process ◽  
Specific Protease ◽  
Escherichia Coli Expression

SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that regulates cellular function of a variety of target proteins. SUMO proteins are expressed as their precursor forms. Cleavage of the residues after the ‘GG’ region of these precursors by SUMO-specific proteases in maturation is a prerequisite for subsequent sumoylation. To understand further this proteolytic processing, we expressed and purified SENP1 (sentrin-specific protease 1), one of the SUMO-specific proteases, using an Escherichia coli expression system. We show that SENP1 is capable of processing all SUMO-1, -2 and -3 in vitro; however, the proteolytic efficiency of SUMO-1 is the highest followed by SUMO-2 and -3. We demonstrate further that the catalytic domain of SENP1 (SENP1C) alone can determine the substrate specificity towards SUMO-1, -2 and -3. Replacement of the C-terminal fragments after the ‘GG’ region of SUMO-1 and -2 precursors with that of the SUMO-3, indicates that the C-terminal fragment is essential for efficient maturation. In mutagenesis analysis, we further map two residues immediately after the ‘GG’ region, which determine the differential maturation. Distinct patterns of tissue distribution of SENP1, SUMO-1, -2 and -3 are characterized. Taken together, we suggest that the observed differential maturation process has its physiological significance in the regulation of the sumoylation pathway.

scholarly journals Biochemical characterization of the human placental transcription factor GCMa/1

2005 ◽  
Vol 83 (2) ◽  
pp. 188-195 ◽  
Author(s):  
Chenchen Lin ◽  
Meiyao Lin ◽  
Hungwen Chen
Keyword(s):  
Transcription Factor ◽  
Biochemical Characterization ◽  
Expression System ◽  
In Vitro Transcription ◽  
Tata Box ◽  
Transcription System ◽  
Multiple Copies ◽  
Escherichia Coli Expression ◽  
Set Up

Glial cells missing (GCM) proteins are a novel family of zinc-containing transcription factors. Human GCMa/1 is primarily expressed in placental trophoblast cells and regulates SYNCYTIN gene expression, which mediates fusion of cytotrophoblasts to form the syncytiotrophoblast layer of the human placenta. To biochemically characterize the transcriptional activity of GCMa/1, we set up an in vitro transcription system for human GCMa/1 (hGCMa/1). Using G-free reporter constructs carrying multiple copies of wild-type or mutant GCMa-binding site (GBS) in front of a synthetic TATA box, we observed specific transcriptional activities of recombinant hGCMa/1 proteins prepared from a baculovirus – insect cell or Escherichia coli expression system. We further characterized GCMa/1-mediated tran scriptional activation on the native syncytin promoter. Using G-free reporter constructs containing the native syncytin promoter, a TATA box downstream of the proximal GBS in the syncytin promoter was shown to be essential for the transcription activation directed by hGCMa/1. Therefore, our results demonstrate positive transcriptional activities of GCMa/1 in vitro and provide a better understanding of GCMa/1-mediated SYNCYTIN gene expression.Key words: syncytin, transcription factor, GCMa/1, placenta.

scholarly journals Identification and Characterization of the LysR-Type Transcriptional Regulator HsdR for Steroid-Inducible Expression of the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni

2011 ◽  
Vol 78 (4) ◽  
pp. 941-950 ◽  
Author(s):  
Wenjie Gong ◽  
Guangming Xiong ◽  
Edmund Maser
Keyword(s):  
Polyclonal Antibodies ◽  
Hydroxysteroid Dehydrogenase ◽  
Carbonyl Reductase ◽  
Comamonas Testosteroni ◽  
Binding Assays ◽  
Knockout Mutant ◽  
Mobility Shift ◽  
Content Type ◽  
Key Enzyme

ABSTRACT3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) fromComamonas testosteroniis a key enzyme in steroid degradation in soil and water. 3α-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Previously, we have shown that the induction ofhsdAexpression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking ofhsdAtranscription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem.276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem.278:47400–47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3α-HSD/CR expression inC. testosteronihas been identified. ThehsdRgene is located 2.58 kb downstream fromhsdAon theC. testosteroniATCC 11996 chromosome with an orientation opposite that ofhsdA. ThehsdRgene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of thehsdAgene, electrophoresis mobility shift assays showed that HsdR specifically binds to thehsdApromoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore,in vitrobinding assays indicated that HsdR can come into contact with RNA polymerase. As expected, anhsdRknockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-typeC. testosteroniafter testosterone induction. In conclusion, HsdR is a positive transcription factor for thehsdAgene and promotes the induction of 3α-HSD/CR expression inC. testosteroni.

scholarly journals Epitope-Mapping Studies Define Two Major Neutralization Sites on the Vaccinia Virus Extracellular Enveloped Virus Glycoprotein B5R

2005 ◽  
Vol 79 (10) ◽  
pp. 6260-6271 ◽  
Author(s):  
Lydia Aldaz-Carroll ◽  
J. Charles Whitbeck ◽  
Manuel Ponce de Leon ◽  
Huan Lou ◽  
Lauren Hirao ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Expression System ◽  
Antigenic Structure ◽  
Functional Domain ◽  
Type I ◽  
Enveloped Virus

ABSTRACT Vaccinia extracellular enveloped virus (EEV) is critical for cell-to-cell and long-range virus spread both in vitro and in vivo. The B5R gene encodes an EEV-specific type I membrane protein that is essential for efficient EEV formation. The majority of the B5R ectodomain consists of four domains with homology to short consensus repeat domains followed by a stalk. Previous studies have shown that polyclonal antibodies raised against the B5R ectodomain inhibit EEV infection. In this study, our goal was to elucidate the antigenic structure of B5R and relate this to its function. To do this, we produced multimilligram quantities of vaccinia virus B5R as a soluble protein [B5R(275t)] using a baculovirus expression system. We then selected and characterized a panel of 26 monoclonal antibodies (MAbs) that recognize B5R(275t). Five of these MAbs neutralized EEV and inhibited comet formation. Two other MAbs were able only to neutralize EEV, while five others were able only to inhibit comet formation. This suggests that the EEV neutralization and comet inhibition assays measure different viral functions and that at least two different antigenic sites on B5R are important for these activities. We further characterized the MAbs and the antigenic structure of B5R(275t) by peptide mapping and by reciprocal MAb blocking studies using biosensor analysis. The epitopes recognized by neutralizing MAbs were localized to SCR1-SCR2 and/or the stalk of B5R(275t). Furthermore, the peptide and blocking data support the concept that SCR1 and the stalk may be in juxtaposition and may be part of the same functional domain.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

2015 ◽  
Vol 197 (14) ◽  
pp. 2383-2391 ◽  
Author(s):  
Semen A. Leyn ◽  
Irina A. Rodionova ◽  
Xiaoqing Li ◽  
Dmitry A. Rodionov
Keyword(s):  
Carbon Dioxide ◽  
Transcriptional Regulation ◽  
Comparative Genomics ◽  
Dna Binding ◽  
Transcriptional Control ◽  
Binding Assays ◽  
Promoter Regions ◽  
Content Type ◽  
Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

Sign in / Sign up

Export Citation Format

Share Document