Transmembrane Structure Research Articles

Bioinformatics analysis and expression pattern of NAC transcription factor family of Angelica dahurica var. formosana from Sichuan province

NAC(NAM/ATAF/CUC) protein plays an important role in plant growth and development, secondary cell wall formation and stress response. In this study, based on the sequencing data of Angelica dahurica, the NAC family was systematically analyzed using bioinformatics methods and its expression pattern was analyzed. Studies showed that 75 candidate genes had been selected from the NAC transcription factor family of A. dahurica, with the protein size of 148-641, all of which were unstable hydrophilic proteins. Most NAC proteins were localized in the nucleus, and had complete NAC domain. Phylogenetic analysis of NAC family proteins of A.dahurica and Arabidopsis thaliana showed that among the 17 subfamilies, NAC members were unevenly distributed in each subfamily, indicating that the evolution of species is developing in multiple directions. Among them, ANAC063 subfamily contained no NAC sequence of A. dahurica, which might be due to the functional evolution of the species. Analysis of protein transmembrane structure and signal peptide showed that NAC transcription factor could carry out transmembrane transportation, but its signal peptide function had not been found. Expression analysis showed that most transcription factors responded to abiotic stress and hormones to varying degrees, and the effects of hormones were obvious, especially ABA and IAA. In different organs of A. dahurica, most members of the NAC family had higher expression in root phloem, followed by root xylem. This study lays a foundation for further research on the function of A. dahurica NAC transcription factor and for solving the biological problems of A. dahurica.

Molecular characterization and functional analysis of scavenger receptor class B from black tiger shrimp (Penaeus monodon)

Abstract Background Scavenger receptor class B (SRB) is a multifunctional protein in animals that participates in physiological processes, including recognition of a wide range of ligands. Astaxanthin is a major carotenoid found in shrimp. However, the molecular mechanism of astaxanthin and SRB protein binding has not been reported. Results In the present study, a member of the SRB subfamily, named PmSRB, was identified from the transcriptome of black tiger shrimp (Penaeus monodon). The open reading frame of PmSRB was 1557 bp in length and encoded 518 amino acids. The structure of PmSRB included a putative transmembrane structure at the N-terminal region and a CD36 domain. Multiple sequence alignment indicated that the CD36 domain were conserved. Phylogenetic analysis showed four separate branches (SRA, SRB, SRC, and croquemort) in the phylogenetic tree and that PmSRB was clustered with SRB of Eriocheir sinensis. Quantitative real-time polymerase chain reaction showed that the PmSRB gene was widely expressed in all tissues tested, with the highest expression level observed in the lymphoid organ and brain. Subcellular localization analysis revealed that PmSRB-GFP (green fluorescent protein) fusion proteins were predominantly localized in the cell membrane. The recombinant proteins of PmSRB showed binding activities against astaxanthin in vitro. Conclusions PmSRB was identified and characterized in this study. It is firstly reported that PmSRB may take as an important mediator of astaxanthin uptake in shrimp. How to cite: Fan S, Wang F, Xie Z, et al. Molecular characterization and functional analysis of scavenger receptor class B from black tiger shrimp (Penaeus monodon). Electron J Biotechnol 2021; 51. https://doi.org/10.1016/j.ejbt.2021.03.003

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H+-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H+-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H+-ATPase isoforms were identified and named SmPHA1–SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H+-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H+-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.

Prediction of ATPase cation transporting 13A2 molecule in Petromyzon marinus and pan-cancer analysis into human tumors from an evolutionary perspective.

The ATPase cation transporting 13A2 protein (ATP13A2), which maintains the homeostasis of mitochondria and lysosomes, plays a significant role in human neurodegenerative diseases and cancer. Through constructing a lamprey proteome database, employing multiple sequence alignment and phylogenetic analysis, 5 ATP13A2 proteins from Petromyzon marinus (Pm-ATP13A2) were identified based on the evolutionary perspective. The motif and domain analysis showed that the ATP13A2 protein was conserved. The multiple phosphorylation sites and transmembrane structures highlighted the characteristics of ATP13A2 as the P-ATPase-V cation transporting protein. Based on the information provided by the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, this study was conducted as a preliminary investigation of the carcinogenic effects of the ATP13A2 gene in a variety of tumors. The ATP13A2 was strongly expressed in most tumors, except in two types of nervous system tumors glioblastoma multiforme (GBM) and brain lower grade glioma (LGG). Moreover, the expression of ATP13A2 was strongly correlated with the prognosis of tumor patients. The high expression of ATP13A2 was obviously related to the poor prognosis of LGG. The poor prognosis of LGG patients may affect the ATP13A2 expression through the immune cells and radiotherapy. Also, cancer-related fibroblast infiltration was observed. All in all, this work offers more insights into the molecular evolution of the ATP13A2 protein and facilitates the understanding of the carcinogenic effects of the ATP13A2 in different tumors. Our discussion also promotes the study into the successful evolution of the vertebrate brain and the mechanism of clinical brain-related diseases.

Cloning, sequence analysis, and expression of tyrp1a and tyrp2 genes related to body colour in different developmental stages and tissues of rainbow trout Oncorhynchus mykiss

Tyrosinase-related protein 1 (tyrp1) and tyrosinase-related protein 2 (tyrp2) genes are important downstream regulatory factors for body colour formation in animals. The present study aimed to explore the relationship between tyrp1a and tyrp2 expression and body colour variation between wild-type (WT) and yellow mutant (YM) rainbow trout. The full-length cDNA sequences of trout tyrp1a and tyrp2 were obtained using rapid amplification of cDNA ends, and the bioinformatic analysis was performed. Quantitative real-time PCR (qRT-PCR) was used to investigate the different expression levels of tyrp1a and tyrp2 in different developmental stages and tissues. The full-length cDNAs of tyrp1a and tyrp2 were 2409 bp and 2219 bp encoding predicted proteins of 522 and 529 amino acid residues, respectively. Both proteins possess six conserved domains, including a signal peptide, an EGF-motif, a Pfam tyrosinase motif, a transmembrane structure, and two copper binding sites (CuA and CuB). Amino acid sequence comparisons showed higher conservation of Tyrp1a and Tyrp2 proteins amongst fishes than amongst other vertebrates, which was further confirmed by phylogenetic analysis. qRT-PCR analysis revealed that tyrp1a and tyrp2 expression levels in WT rainbow trout, and tyrpla in YM rainbow trout, were detected from the fertilised stage to 12 months post hatching (12 M), while tyrp1a did not expressed until the blastula in YM rainbow trout. In addition, the expression levels of both of the genes post hatching were significantly higher than those in the embryonic stages, which showed extremely low expression levels. Additionally, there were extremely significant differences (P < 0.01) in expression at the same stages between WT and YM rainbow trout, e.g., at the blastula, gastrula, 7 days post hatching (7 dph), 10 dph, 2 M, 3 M, 6 M, and 12 M stages for tyrp1a; and at the 4-cell, 16-cell, multicell, blastula, somites, heartbeating, 1 dph, 5 dph, 7 dph, 3 M, 6 M, and 12 M stages for tyrp2. Besides, variable expression levels of tyrp1a and tyrp2 were detected in all tissues; significantly high expression was detected in the dorsal skin and eye compared with that in the other tissues in WT and YM rainbow trout (P < 0.05). These results suggested that the expression level changes of tyrp1a and tyrp2 might be closely related to the variation of rainbow trout body colour, which will enrich our knowledge of the molecular mechanism of skin colour variation in rainbow trout and provide data for research into fish skin colour inheritance and improvement.

Transmembrane 163 (TMEM163) Protein: A New Member of the Zinc Efflux Transporter Family

A growing body of evidence continues to demonstrate the vital roles that zinc and its transporters play on human health. The mammalian solute carrier 30 (SLC30) family, with ten current members, controls zinc efflux transport in cells. TMEM163, a recently reported zinc transporter, has similar characteristics in both predicted transmembrane domain structure and function to the cation diffusion facilitator (CDF) protein superfamily. This review discusses past and present data indicating that TMEM163 is a zinc binding protein that transports zinc in cells. We provide a brief background on TMEM163’s discovery, transport feature, protein interactome, and similarities, as well as differences, with known SLC30 (ZnT) protein family. We also examine recent reports that implicate TMEM163 directly or indirectly in various human diseases such as Parkinson’s disease, Mucolipidosis type IV and diabetes. Overall, the role of TMEM163 protein in zinc metabolism is beginning to be realized, and based on current evidence, we propose that it is likely a new CDF member belonging to mammalian SLC30 (ZnT) zinc efflux transporter proteins.

Extracellular expression of natural cytosolic nitrilase from Rhodococcus zopfii through constructing a transmembrane tunnel structure in Escherichia coli cells

Nitrilases are a class of industrially important hydrolases that convert nitriles to carboxylic acids. As a natural cytosolic enzyme, production of nitrilase suffer from inclusion body and cumbersome purification procedure. Extracellular expression of recombinant proteins offers advantages including improved protein folding and simplified downstream bioprocesses, which is more suitable for industrial production. Herein, extracellular expression of nitrilase (RzNIT) from Rhodococcus zopfii in Escherichia coli cells was investigated using active and passive transport strategies. The active transport of RzNIT mediated by N-terminal fused signal peptides and secretory proteins had negative effects on both solubility and activity. For the passive transport, PhiX174 protein E was co-expressed to form a 40−70 nm transmembrane tunnel structure in cell membranes. RzNIT was spontaneously released from cytoplasm into extracellular medium through the constructed hole. Under the optimum conditions, the extracellular RzNIT activity reached 7.6 U/mL (86 % of its total activity) toward 3-cyanopyridine. The calculated concentration of extracellular RzNIT reached 0.13 mg/mL. The obtained results provide a novel and efficient strategy for extracellular production of recombinant nitrilases.

TMPSS: A Deep Learning-Based Predictor for Secondary Structure and Topology Structure Prediction of Alpha-Helical Transmembrane Proteins.

Alpha transmembrane proteins (αTMPs) profoundly affect many critical biological processes and are major drug targets due to their pivotal protein functions. At present, even though the non-transmembrane secondary structures are highly relevant to the biological functions of αTMPs along with their transmembrane structures, they have not been unified to be studied yet. In this study, we present a novel computational method, TMPSS, to predict the secondary structures in non-transmembrane parts and the topology structures in transmembrane parts of αTMPs. TMPSS applied a Convolutional Neural Network (CNN), combined with an attention-enhanced Bidirectional Long Short-Term Memory (BiLSTM) network, to extract the local contexts and long-distance interdependencies from primary sequences. In addition, a multi-task learning strategy was used to predict the secondary structures and the transmembrane helixes. TMPSS was thoroughly trained and tested against a non-redundant independent dataset, where the Q3 secondary structure prediction accuracy achieved 78% in the non-transmembrane region, and the accuracy of the transmembrane region prediction achieved 90%. In sum, our method showcased a unified model for predicting the secondary structure and topology structure of αTMPs by only utilizing features generated from primary sequences and provided a steady and fast prediction, which promisingly improves the structural studies on αTMPs.

A systematic review of the Uridine diphosphate-Galactose/Glucose-4-epimerase (UGE) in plants

Uridine diphosphate (UDP)-Galactose/Glucose-4-epimerase (UGE) is the third enzyme involved in the Leloir pathway. It catalyzes the conversion of UDP-Galactose to UDP-Glucose, which is a rate-limiting step for polysaccharides biosynthesis. As the main cell wall materials, polysaccharides play an irreplaceable role throughout the whole life history of plant. In this review, 1243 UGE mRNA sequences registered in NCBI were obtained and their evolutionary relationship was analyzed by constructing a neighbor-joining tree based on representative sequences. The physicochemical properties of 15 specific UGEs were analyzed, and four UGE sequences of Setaria italica, Zea mays, Ricinus Communis, and Oryza sativa were selected for further secondary structure, three-dimensional protein modeling, transmembrane structure, and signal peptide prediction analyses. The results demonstrate that UGEs are mostly conservative without signal peptides and exert activities in cytoplasm. Then, studies related to the catalytic reaction, characteristic structure, function, and regulation of UGE were summarized, which provide strong evidence that UGE plays a crucial role in cell growth, cell differentiation, cell-to-cell communication, primary metabolism, secondary metabolism, and defense responses. However, the molecular mechanisms whereby UGE regulates the plant stress resistance and useful secondary metabolites accumulation are far from clear. This paper will lay a foundation for further studies and applications of UGE.

Identification of AhATL1 interaction proteins participating in drought stress memory in peanut

Plants have evolved an adaptive mechanism named drought stress memory to improve resistance and increase survival chances, by means of coordination of physiological responses and changes of gene expression patterns. The ATP-binding cassette (ABC) superfamily G proteins are membrane proteins that are involved in abscisic acid (ABA) transport and the response to drought stress. The ABC protein AhATL1 could play an important role in drought resistance by affecting ABA importation and stomatal closure and could participate in drought stress memory in Arachis hypogaea L. Here, we screened the interacting proteins of AhATL1 by co-immunoprecipitation and mass spectrometry, in order to further explore the function of AhATL1 in response to repeated dehydration stress. Subsequently, we screened for proteins that may be related to abscisic acid transport and identified AhLBP, AhGCP, AhSHT and AhHSP. Furthermore, we predicted their signal peptides, transmembrane structures, functional domains, and analyzed their gene expression levels, demonstrating they may play a role in peanut drought stress memory. Yeast two-hybrid assay showed that AtSLAC1 but not AhLBP, AhGCP, AhSHT, AhHSP or AhSLAC1 interacted with AhATL1. However, confocal microscopic analysis demonstrated that AhATL1 was co-localized with AhHSP, AhSLAC1 and AtSALC1. And BiFC assays showed that AhATL1 interacted with AhSLAC1 and AtSALC1, indicating that a close relationship existed between them. The findings provide further insights into the role of AhATL1 in drought stress memory and a useful reference for exploring the role of the ABA signaling pathway in plant drought stress memory. Supplemental data for this article is available online at https://dx.doi.org/10.1080/13102818.2021.2013734 .

Genome-wide identification of Cellulose-like synthase D gene family in Dendrobium catenatum

Dendrobium catenatum, which grows on the semi humid rocks in the mountains, has been at the top of the ‘Nine Immortals of China’ since ancient times. It is a kind of yin tonic medicine and its main active component is polysaccharide. Cellulose synthase-like D(CslD) genes were predicted to catalyse the biosynthesis of 1,4-β-d-glycan backbone of hemicelluloses, which plays fundamental roles in plant development. To investigate the role of CslD in the development of D. catenatum, eight CslD genes (DcCslD1,2a,2b,3a,3b,4a,4b,5) were identified. The results of protein prediction and analysis showed that CslD2a/2b/4a/4b proteins were acidic proteins. All the proteins had obvious hydrophobic or hydrophilic regions, and had transmembrane structure. Phylogenetic analysis revealed that the DcCslD family could be divided into group I, II, III and IV. DcCslD proteins had a typical Cellulose synthase domain and similar protein structures to the CslDs of other plants. Their promoter regions contain cis regulatory elements related to stress and hormone response. The results of qRT-PCR showed that the identified DcCslDs were differentially expressed in roots, stems and leaves. Most of them were highly expressed in stems and leaves. The environmental stresses examination showed that the expression levels of DcCslD5 were closely associated with drought-recovery treatment; the expression levels of DcCslD1, DcCslD2a, DcCslD2b, DcCslD3a and DcCslD5 were significantly influenced by low temperature. This study systematically analyzed the sequence characteristics of CslD protein of D. catenatum, which can provide reference for further study on the function of CslD protein in the polysaccharide metabolism of D. catenatum. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1941252.

Identification, molecular structure and expression characteristics of Torso like gene in Anopheles dirus

To characterize Torso-like (tsl) gene and investigate its expression characteristics in Anopheles dirus, so as to provide a theoretical basis for subsequent functional studies of the tsl gene. According to the coding sequences of Drosophila melanogaster and An. gambiae tsl genes, the complete genome of An. dirus was retrieved and the An. dirus tsl gene was characterized. Specific primers were designed and the target gene was amplified using PCR and reverse-transcription PCR assays. The physicochemical properties, signal peptide, transmembrane structure, secondary structure and tertiary structure of the encoded protein TSL were analyzed using bioinformatics tools, and a phylogenetic analysis was performed. In addition, the specific expression of the tls gene was detected in various tissues of An. dirus using a quantitative real-time PCR assay. The An. dirus tsl gene was 16 751 bp in length with a CDS region of 1 134 bp, encoding 377 amino acids, and the encoded TSL protein was a stably hydrophilic protein. The TSL protein was predicted to be a secretory protein that was located in extra-membrane regions containing signal peptides. The secondary structure of the TSL protein contained α-helix (51.72%), extended strand (12.20%), β-bridge (4.78%) and random coil (31.30%) in the secondary structure, and a 3D homology model was generated using 5cj9.1.A as a template. Phylogenetic analysis revealed a close genetic relationship in the TSL protein between An. dirus and An. farauti. In addition, quantitative real-time PCR assay detected the tsl gene expression in the head, chest, abdomen and foot of An. dirus, with the highest expression in the head and low expression in the foot. The tsl gene is characterized in An. dirus at a genomic level, and the prediction of the TSL protein structure and the elucidation of the tissue-specific tsl gene expression in An. dirus provide a basis for the further studies on the gene functions.

Open-pFind Verified Four Missing Proteins from Multi-Tissues.

The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on. A higher resolution mass spectrometry (MS) interpretation engine might provide an opportunity to identify these buried MPs in complex samples by the combination with multi-tissue large-scale proteomics. In this study, open-pFind was used to dig MPs from 20 pairs of healthy human tissues by Wang et al. ( Mol. Syst. Biol. 2019, 15 (2), e8503) combined with our large-scale testis data set digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity for different amino acid residues ( J. Proteme Res. 2019, 18 (12), 4189-4196). A total of 1 535 536 peptides with 17 283 477 peptide-spectrum matches (PSMs) were mapped to 14 279 protein entries at a false discovery rate of <1% at the PSM, peptide, and protein levels. A total of 103 MP candidates were identified, among which 86 candidates had more unique peptide numbers compared with our single testis tissue. After rigorous screening, manual checks, peptide synthesis, and matching with documented peptides from PeptideAtlas, we validated four MPs, P0C7T8 (duodenum and small intestine), Q8WWZ4 (stomach and rectum), Q8IV35 (fallopian tube), and O14921 (tonsil), at the protein level. All MS raw files have been deposited to the ProteomeXchange with identifier PXD021391.

Isolation and Comparison of Eight SWEET17 Genes from Six Loquat Cultivars

The plant SWEET (Sugars Will Eventually be Exported Transporters) proteins, as newly discovered sugar transporters, play various important roles during plant depending on the transmembrane gradient of sugar concentration. SWEET17 protein is one of the SWEET family members transporting fructose in particular. In this study, we identified eight SWEET17s from six loquats (Eriobotrya japonica (Thunb.) Lindl.) cultivars and named them as Ej_NHB_SWEET17, Ej_BL_SWEET17a, Ej_ZH_SWEET17, Ej_MM-_SWEET17a, Ej_MM_SWEET17b, Ej_BL_SWEET17b, Ej_GY_SWEET17 and Ep_LY_SWEET17. All of the eight loquat SWEET17s have five to six transmembrane structures. Phylogenetic analysis indicated that Ej_NHB_SWEET17 had the farthest genetic relationship with other seven loquat SWEET17s, indicating that ‘Ninghaibai’ cultivar was far away from the other five loquat varieties possibly. The similarities of nucleotide and amino acid sequences between Ej_NHB_SWEET17 and the other loquat SWEET17s were only about 61 and 46%, respectively, while that among other seven SWEET17s were greater than 90%. The expression levels of loquat SWEET17s in leaves were positively correlated with the content of fructose, sorbitol and total sugar in various fruit from the six cultivars, in which the sugar contents in fruits of different cultivars varied significantly, that was probably related to expression levels of loquat SWEET17. Our results indicated that the expression level of loquat SWEET17s was probably related to the regulation of sugar contents of mature fruit, experimental confirmation needs to be performed. Together, these results indicated that EjSWEET17s possibly played important roles in the sugar accumulation of fruit, which could serve as the basis for further functional identification of such genes.

Cloning and expression analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase genes from Siraitia grosvenorii

3-Hydroxy-3-methylglutaryl coenzyme A reductase(HMGR) is the first rate-limiting enzyme in the mevalonic acid(MVA)pathway and it is an important regulatory site in the metabolism of terpenoids in the cytoplasm. In this study, Siraitia grosvenorii that had been pollinated 0 day,1 day,3 days,15 days and 30 days were used as experimental materials. Based on the transcriptome data, two HMGR genes were cloned from S. grosvenorii cDNA and named SgHMGR2(GenBank Accession Numbers MT270447) and SgHMGR3(GenBank Accession Number MT270448). The two genes contain open reading frames(ORFs) of 1 746 bp and 1 782 bp, encoding 582 and 594 amino acids, and their molecular masses are estimated to be 62.7,63.2 kDa, respectively. Isoelectric point are 8.34 and 7.47, both of which do not contain signal peptides, are non-secretory proteins, and have two transmembrane structures. Combining the conserved regions of the proteins and the analysis of the evolutionary tree, it was confirmed that the genes are indeed HMGR family genes. Real-time PCR was used to detect the expression pattern of SgHMGRs at different times after pollination, and the highest expression level was 15 days after pollination. Finally, two full-length SgHMGRs were cloned from S. grosvenorii for the first time, and the differential expression of SgHMGRs at different times after pollination was revealed, providing a research basis for the mining of key enzyme gene elements in the biosynthesis pathway of S. grosvenorii terpenoids.

Helical ensembles outperform ideal helices in molecular replacement.

The conventional approach in molecular replacement is the use of a related structure as a search model. However, this is not always possible as the availability of such structures can be scarce for poorly characterized families of proteins. In these cases, alternative approaches can be explored, such as the use of small ideal fragments that share high, albeit local, structural similarity with the unknown protein. Earlier versions of AMPLE enabled the trialling of a library of ideal helices, which worked well for largely helical proteins at suitable resolutions. Here, the performance of libraries of helical ensembles created by clustering helical segments is explored. The impacts of different B-factor treatments and different degrees of structural heterogeneity are explored. A 30% increase in the number of solutions obtained by AMPLE was observed when using this new set of ensembles compared with the performance with ideal helices. The boost in performance was notable across three different fold classes: transmembrane, globular and coiled-coil structures. Furthermore, the increased effectiveness of these ensembles was coupled to a reduction in the time required by AMPLE to reach a solution. AMPLE users can now take full advantage of this new library of search models by activating the `helical ensembles' mode.

DP7-C-modified liposomes enhance immune responses and the antitumor effect of a neoantigen-based mRNA vaccine

To date, many clinical trials have been carried out with neoantigen-specific mRNA vaccines, and positive results have been achieved. However, further improvements in the efficiency of the intracellular delivery of mRNA and the production of a stronger immune response are still worth studying. In this study, we used the cholesterol-modified cationic peptide DP7 (VQWRIRVAVIRK), which was developed in our previous study, with a transmembrane structure and immunoadjuvant function to modify DOTAP liposomes to create a common mRNA delivery system. This system was intended to improve the efficiency of the delivery of mRNA encoding individualized neoantigens to dendritic cells (DCs) and enhance the activation of DCs. The system serves dual functions as a carrier and as an immunoadjuvant. As a carrier of mRNA, DP7-C-modified DOTAP liposomes (DOTAP/DP7-C) could transfer mRNA efficiently into different type of DCs in vitro. As an immunoadjuvant, DOTAP/DP7-C liposomes were shown to be more efficacious in stimulating DC maturation, CD103+ DC (contributing to antigen presentation) production and proinflammatory cytokine secretion than DOTAP liposomes both in vitro and in vivo. In animal studies, the subcutaneous administration of DOTAP/DP7-C/LL2 neoantigen-encoding mRNA complexes significantly inhibited the growth of LL2 in situ and the growth of subcutaneous tumors and stimulated the production of antigen-specific lymphocyte reactions, which were superior to the DOTAP/LL2 neoantigen-encoding mRNA complex group. In conclusion, DOTAP/DP7-C liposomes may serve as a potential universal mRNA delivery system, providing a simple method to increase the efficiency of intracellular mRNA delivery and the immunostimulatory activity of DCs.

Protein-lipid architecture of a cholinergic postsynaptic membrane.

The cholinergic postsynaptic membrane is an acetyl-choline receptor-rich membrane mediating fast chemical communication at the nerve-muscle synapse. Here, cryo-EM is used to examine the protein-lipid architecture of this membrane in tubular vesicles obtained from the (muscle-derived) electric organ of the Torpedo ray. As reported earlier, the helical arrangement of the protein component of the vesicles facilitates image averaging and enables us to determine how cholesterol and phospho-lipid molecules are distributed in the surrounding matrix, using headgroup size as a means to discriminate between the two kinds of lipid. It is shown that cholesterol segregates preferentially around the receptors in both leaflets of the lipid bilayer, interacting robustly with specific transmembrane sites and creating a network of bridging microdomains. Cholesterol interactions with the receptor are apparently essential for stabilizing and maintaining its physiological architecture, since the transmembrane structure contracts, involving displacements of the helices at the outer membrane surface by ∼2 Å (1-3 Å), when this lipid is extracted. The microdomains may promote cooperativity between neighbouring receptors, leading to an enhanced postsynaptic response.

Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a “stealth” carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.

Unveiling the complexity of the litchi transcriptome and pericarp browning by single-molecule long-read sequencing

Litchi is a perennial fruit crop with a highly heterozygous genome and complex transcripts. Although fruit senescence and pericarp browning have been extensively studied, the underlying regulatory mechanisms are still poorly understood. In this study, we used long-read sequencing technology in combination with RNA-seq analysis to investigate the diversity and complexity of the litchi transcriptome, as well as differential express of transcripts during litchi fruit storage. We obtained a reference transcriptome with 50,808 unique full-length isoforms, including 41,290 coding sequences (CDS), 1658 transcription factors, 22,100 simple sequence repeats, 2434 long noncoding RNAs, and 151 alternative splicing (AS) events. In addition, 41,290 isoforms had transmembrane helical structure, 33,579 isoforms contained Pfam protein domains, 14,348 isoforms contained SMART protein domains, 4180 isoforms had signal-peptide structure, 19,183 isoforms contained glycosylation sites, and 30,436 isoforms contained furin protease cleavage sites. Using this transcriptome, 1272 isoforms were found to be differentially expressed in litchi fruit pericarp during postharvest storage. The postharvest storage could be divided into a ‘senescence onset’ stage and a subsequent ‘browning’ stage according to RNA-seq data. During the ‘senescence onset’ stage, the expression of isoforms related to senescence and stress response was significantly up-regulated. During the ‘browning’ stage, the expression of isoforms related to cell wall degradation, oxidation, and disease response was significantly up-regulated. In addition, qPCR analysis showed that the expression changes of 30 isoforms during the postharvest storage of both ‘Huaizhi’ and ‘Guiwei’ fruit were consistent with the RNA-seq results, indicating high reliability of our results and inferences.