Ergothioneine and mitochondria: An important protective mechanism?

Mitochondrial dysfunction is implicated in a wide range of human disorders including many neurodegenerative and cardiovascular diseases, metabolic diseases, cancers, and respiratory disorders. Studies have suggested the potential of l-ergothioneine (ET), a unique dietary thione, to prevent mitochondrial damage and improve disease outcome. Despite this, no studies have definitively demonstrated uptake of ET into mitochondria. Moreover, the expression of the known ET transporter, OCTN1, on the mitochondria remains controversial. In this study, we utilise mass spectrometry to demonstrate direct ET uptake in isolated mitochondria as well as its presence in mitochondria isolated from ET-treated cells and animals. Mitochondria isolated from OCTN1 knockout mice tissues, have impaired but still detectable ET uptake, raising the possibility of alternative transporter(s) which may facilitate ET uptake into the mitochondria. Our data confirm that ET can enter mitochondria, providing a basis for further work on ET in the prevention of mitochondrial dysfunction in human disease.

Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress-related proteins.

Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such gene targeting/gene tagging (GT) can alter expression of the target gene. The enzyme encoded by Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localisation have been difficult to assay. We used GT to insert YFP in frame with the 5' or 3' ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3' end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localisation and tissue-specific expression. In contrast, insertion of YFP at the 5' end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene.

Transient Expression Vector Construction, Subcellular Localisation, and Evaluation of Antiviral Potential of Flagellin BP8-2.

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture.

Isolation, characterisation, and expression profiling of DXS and DXR genes in Atractylodes lancea.

1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2-C-methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea.

Phylogenetic analysis and stress response of the plant U2 small nuclear ribonucleoprotein B″ gene family.

BACKGROUND: Alternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein B″ (U2B″), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2B″, and its mechanism remains unclear. RESULT: Phylogenetic analysis, including gene and protein structures, revealed that U2B″ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2B″ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein-protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins. CONCLUSION: U2B″ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.

CropPAL for discovering divergence in protein subcellular location in crops to support strategies for molecular crop breeding.

Agriculture faces increasing demand for yield, higher plant-derived protein content and diversity while facing pressure to achieve sustainability. Although the genomes of many of the important crops have been sequenced, the subcellular locations of most of the encoded proteins remain unknown or are only predicted. Protein subcellular location is crucial in determining protein function and accumulation patterns in plants, and is critical for targeted improvements in yield and resilience. Integrating location data from over 800 studies for 12 major crop species into the cropPAL2020 data collection showed that while >80% of proteins in most species are not localised by experimental data, combining species data or integrating predictions can help bridge gaps at similar accuracy. The collation and integration of over 61 505 experimental localisations and more than 6 million predictions showed that the relative sizes of the protein catalogues located in different subcellular compartments are comparable between crops and Arabidopsis. A comprehensive cross-species comparison showed that between 50% and 80% of the subcellulomes are conserved across species and that conservation only depends to some degree on the phylogenetic relationship of the species. Protein subcellular locations in major biosynthesis pathways are more often conserved than in metabolic pathways. Underlying this conservation is a clear potential for subcellular diversity in protein location between species by means of gene duplication and alternative splicing. Our cropPAL data set and search platform (https://crop-pal.org) provide a comprehensive subcellular proteomics resource to drive compartmentation-based approaches for improving yield, protein composition and resilience in future crop varieties.

New fluorescent auxin probes visualise tissue-specific and subcellular distributions of auxin in Arabidopsis.

In a world that will rely increasingly on efficient plant growth for sufficient food, it is important to learn about natural mechanisms of phytohormone action. In this work, the introduction of a fluorophore to an auxin molecule represents a sensitive and non-invasive method to directly visualise auxin localisation with high spatiotemporal resolution. The state-of-the-art multidisciplinary approaches of genetic and chemical biology analysis together with live cell imaging, liquid chromatography-mass spectrometry (LC-MS) and surface plasmon resonance (SPR) methods were employed for the characterisation of auxin-related biological activity, distribution and stability of the presented compounds in Arabidopsis thaliana. Despite partial metabolisation in vivo, these fluorescent auxins display an uneven and dynamic distribution leading to the formation of fluorescence maxima in tissues known to concentrate natural auxin, such as the concave side of the apical hook. Importantly, their distribution is altered in response to different exogenous stimuli in both roots and shoots. Moreover, we characterised the subcellular localisation of the fluorescent auxin analogues as being present in the endoplasmic reticulum and endosomes. Our work provides powerful tools to visualise auxin distribution within different plant tissues at cellular or subcellular levels and in response to internal and environmental stimuli during plant development.

Guided Antitumoural Drugs: (Imidazol-2-ylidene)(L)gold(I) Complexes Seeking Cellular Targets Controlled by the Nature of Ligand L.

Three [1,3-diethyl-4-(p-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazol-2-ylidene](L)gold(I) complexes, 4 a (L=Cl), 5 a (L=PPh3 ), and 6 a (L=same N-heterocyclic carbene (NHC)), and their fluorescent [4-(anthracen-9-yl)-1,3-diethyl-5-phenylimidazol-2-ylidene](L)gold(I) analogues, 4 b, 5 b, and 6 b, respectively, were studied for their localisation and effects in cancer cells. Despite their identical NHC ligands, the last three accumulated in different compartments of melanoma cells, namely, the nucleus (4 b), mitochondria (5 b), or lysosomes (6 b). Ligand L was also more decisive for the site of accumulation than the NHC ligand because the couples 4 a/4 b, 5 a/5 b, and 6 a/6 b, carrying different NHC ligands, afforded similar results in cytotoxicity tests, and tests on targets typically found at their sites of accumulation, such as DNA in nuclei, reactive oxygen species and thioredoxin reductase in mitochondria, and lysosomal membranes. Regardless of the site of accumulation, cancer cell apoptosis was eventually induced. The concept of guiding a bioactive complex fragment to a particular subcellular target by secondary ligand L could reduce unwanted side effects.

In-Pero: Exploiting Deep Learning Embeddings of Protein Sequences to Predict the Localisation of Peroxisomal Proteins.

Peroxisomes are ubiquitous membrane-bound organelles, and aberrant localisation of peroxisomal proteins contributes to the pathogenesis of several disorders. Many computational methods focus on assigning protein sequences to subcellular compartments, but there are no specific tools tailored for the sub-localisation (matrix vs. membrane) of peroxisome proteins. We present here In-Pero, a new method for predicting protein sub-peroxisomal cellular localisation. In-Pero combines standard machine learning approaches with recently proposed multi-dimensional deep-learning representations of the protein amino-acid sequence. It showed a classification accuracy above 0.9 in predicting peroxisomal matrix and membrane proteins. The method is trained and tested using a double cross-validation approach on a curated data set comprising 160 peroxisomal proteins with experimental evidence for sub-peroxisomal localisation. We further show that the proposed approach can be easily adapted (In-Mito) to the prediction of mitochondrial protein localisation obtaining performances for certain classes of proteins (matrix and inner-membrane) superior to existing tools.

Cdc6 as a novel target in cancer: Oncogenic potential, senescence and subcellular localisation.

Cdc6 is a key replication licencing factor with a pivotal role in regulating the process of DNA replication, rendering it an important investigatory focus in tumourigenesis. Indeed, Cdc6 overexpression has been found to be a feature in certain tumours and has been associated as an early event in malignancies. With a focus on pancreatic cancer, there are evidence of its convergence in downstream pathways implicated in major genetic alterations found in pancreatic cancer, primarily KRAS. There is also data of its direct influence on protumourigenic processes as a transcriptional regulator, repressing the key tumour suppressor loci CDH1 (E-Cadherin) and influencing epithelial to mesenchymal transition (EMT). Moreover, gene amplification of Cdc6 as well as of E2F (an upstream regulator of Cdc6) have also been found to be a key feature in tumours overexpressing Cdc6, further highlighting this event as a potential driver of tumourigenesis. In this review, we summarise the evidence for the role of Cdc6 overexpression in cancer, specifically that of pancreatic cancer. More importantly, we recapitulate the role of Cdc6 as part of the DNA damage response and on senescence-an important antitumour barrier-in the context of pancreatic cancer. Finally, recent emerging observations suggest that the potential of the subcellular localisation of Cdc6 in inducing senescence. In this regard, we speculate and hypothesise potentially exploitable mechanisms in the context of inducing senescence via a novel pathway involving cytoplasmic retention of Cdc6 and Cyclin E.

The stability of CREB3/Luman is regulated by protein kinase CK2 phosphorylation.

CREB3 (Luman) is a family member of ER resident transcription factors, which are cleaved upon the induction of ER stress. Their N-terminal fragments shuttle into the nucleus where they regulate the transcription of target genes. Here, we found that human CREB3 is phosphorylated within its transcription activation domain on serine 46 by protein kinase CK2. Further analyses revealed that the phosphorylation of this site does neither affect the cleavage by S1P/S2P proteases, nor the nuclear localisation nor the transcriptional activity of CREB3. However, phosphorylation at serine 46 reduced the stability of CREB3.

The complete functional characterisation of the terpene synthase family in tomato.

Analysis of the updated reference tomato genome found 34 full-length TPS genes and 18 TPS pseudogenes. Biochemical analysis has now identified the catalytic activities of all enzymes encoded by the 34 TPS genes: one isoprene synthase, 10 exclusively or predominantly monoterpene synthases, 17 sesquiterpene synthases and six diterpene synthases. Among the monoterpene and sesquiterpene and diterpene synthases, some use trans-prenyl diphosphates, some use cis-prenyl diphosphates and some use both. The isoprene synthase is cytosolic; six monoterpene synthases are plastidic, and four are cytosolic; the sesquiterpene synthases are almost all cytosolic, with the exception of one found in the mitochondria; and three diterpene synthases are found in the plastids, one in the cytosol and two in the mitochondria. New trans-prenyltransferases (TPTs) were characterised; together with previously characterised TPTs and cis-prenyltransferases (CPTs), tomato plants can make all cis and trans C10 , C15 and C20 prenyl diphosphates. Every type of plant tissue examined expresses some TPS genes and some TPTs and CPTs. Phylogenetic comparison of the TPS genes from tomato and Arabidopsis shows expansions in each clade of the TPS gene family in each lineage (and inferred losses), accompanied by changes in subcellular localisations and substrate specificities.

Identification, characterisation and preliminary functional analysis of IRAK-M in grass carp (Ctenopharyngodon idella).

Interleukin-1 receptor-associated kinase (IRAK) family members play important roles in myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) signaling, the crucial innate immune pathway in vertebrates. In the present study, the IRAK family gene IRAK-M (also called IRAK3) from grass carp (Ctenopharyngodon idella) was cloned and characterised. IRAK-M was mainly enriched in the spleen, and the significantly altered expression was observed after grass carp reovirus (GCRV) infection. Subcellular localisation showed that IRAK-M protein distributed uniformly in the entire cell and co-localised with MyD88 in the cytoplasm of transfected cells. Additionally, the interaction between IRAK-M and MyD88 was confirmed by bimolecular fluorescence complementation (BiFC) system. Moreover, deficient of IRAK-M in C. idella kidney cell line (CIK) with small interference RNA (siRNA) upregulated polyinosinic:polycytidylic acid (poly(I:C))-induced inflammatory cytokines production, including interleukin 8 (IL-8), IL-6, and tumour necrosis factor α (TNF-α), which reveals that IRAK-M functions as a negative regulator of inflammatory cytokines. Taken together, our results demonstrate that IRAK-M gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the IRAK-M in teleosts.

Characterisation and function of TRIM23 in grass carp (Ctenopharyngodon idella).

Tripartite motif (TRIM) proteins are key components of the innate immune system, functioning as antiviral restriction factors or modulating signaling cascades that lead to proinflammatory cytokine induction. In the present study, the TRIM family gene TRIM23 from grass carp (Ctenopharyngodon idella) was cloned and characterised. TRIM23 was moderately expressed in the examined tissues, and the significantly altered expression was observed after grass carp reovirus (GCRV) and poly(I:C) infection. Dual-luciferase activity assay showed that TRIM23, especially its C-terminal domain ARF, depressed the promoter activity of IRF3 and IRF7. The subcellular localisation showed that TRIM23 protein was located in the cytoplasm and could be recruited by both TRAF6 and MyD88. Furthermore, TRIM23 was confirmed to interact with either TRAF6 or MyD88 by the bimolecular fluorescence complementation (BiFC) system in CIK cells. Additionally, autophagy was enhanced by over-expressed TRIM23 in 293T cells. Taken together, our results demonstrate that TRIM23 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the TRIM23 in teleosts.

Identification, expression and functional characterisation of CYP1A in grass carp (Ctenopharyngodon idella).

In mammal, CYP1A has attracted special attention due to its important roles in the oxidative metabolism. In fish, the researches on CYP1A are more focus on its roles in pollution in water environments, but the immune function is unclear. In the study, CiCYP1A gene was cloned from grass carp (Ctenopharyngodon idella). Tissue distribution exhibited an overwhelmingly high basal expression levels in the liver. After GCRV infection, CiCYP1A showed a potent response, indicating CiCYP1A was involved in GCRV-induced immunity. Subcellular localisation showed CiCYP1A was distributed in the cytoplasm. Besides, dual-luciferase activity assays revealed CYP1A was relevant for IFN-I signaling pathway modulation, furthermore, overexpressed CYP1A potently suppressed the mRNA expression of IRF3 and IFN-I but not IRF7. The results provide new sights into exploring immune function of CiCYP1A in teleosts.

Ectopic expression of VpSTS29, a stilbene synthase gene from Vitis pseudoreticulata, indicates STS presence in cytosolic oil bodies.

MAIN CONCLUSION: Stilbene synthase (STS) and its metabolic products are accumulated in senescing grapevine leaves. Ectopic expression of VpSTS29 in Arabidopsis shows the presence of VpSTS29 in oil bodies and increases trans-piceid in developing leaves. Stilbenes are the natural antimicrobial phytoalexins that are synthesised via the phenylpropanoid pathway. STS is the key enzyme catalysing the production of stilbenes. We have previously reported that the VpSTS29 gene plays an important role in powdery mildew resistance in Vitis pseudoreticulata. However, the synthesis and accumulation of these stilbene products in plant cells remain unclear. Here, we demonstrate that VpSTS29 is present in cytosolic oil bodies and can be transported into the vacuole at particular plant-developmental stages. Western blot and high-performance liquid chromatography showed that STS and trans-piceid accumulated in senescent grape leaves and in pVpSTS29::VpSTS29-expressing Arabidopsis during age-dependent leaf senescence. Subcellular localisation analyses indicated VpSTS29-GFP was present in the cytoplasm and in STS-containing bodies in Arabidopsis. Nile red staining, co-localisation and immunohistochemistry analyses of leaves confirmed that the STS-containing bodies were oil bodies and that these moved randomly in the cytoplasm and vacuole. Detection of protein profiles revealed that no free GFP was detected in the pVpSTS29::VpSTS29-GFP-expressing protoplasts or in Arabidopsis during the dark-light cycle, demonstrating that GFP fluorescence distributed in the STS-containing bodies and vacuole was the VpSTS29-GFP fusion protein. Intriguingly, in comparison to the controls, over-expression of VpSTS29 in Arabidopsis resulted in relatively high levels of trans-piceid, chlorophyll content and of photochemical efficiency accompanied by delayed leaf senescence. These results provide exciting new insights into the subcellular localisation of STS in plant cells and information about stilbene synthesis and storage.

Molecular cloning and functional characterisation of NLRX1 in grass carp (Ctenopharyngodon idella).

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. In the present study, the NLR family gene NLRX1 from grass carp (Ctenopharyngodon idella) was cloned and characterised. NLRX1 was widely expressed in all tissues examined, albeit at varying levels. After exposure to the grass carp reovirus (GCRV), NLRX1 mRNA expression levels were altered in immune organs, and dramatically altered in liver. Subcellular localisation indicated that NLRX1 protein co-localised with the mitochondria in the transfected cells. Additionally, the bimolecular fluorescence complementation (BiFC) system was introduced to detect the interaction between tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) and NLRX1. Moreover, deficient of NLRX1 in CIK cells with small interference RNA (siRNA) promoted polyinosinic:polycytidylic acid (poly (I:C))-induced IFN-related genes production, including IRF3, IRF7, and IFN-I, which reveals that NLRX1 is a negative regulator of IFN. Taken together, our results demonstrate that NLRX1 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the NLRX1 in teleosts.

Functional characterisation of the YIPF protein family in mammalian cells.

In this study, we carry out a systematic characterisation of the YIPF family of proteins with respect to their subcellular localisation profile, membrane topology and functional effects on the endomembrane system. YIPF proteins primarily localise to the Golgi complex and can be grouped into trans-Golgi-localising YIPFs (YIPF1 and YIPF2) and cis-Golgi-localising YIPFs (YIPF3, YIPF4 and YIPF5), with YIPF6 and YIPF7 showing a broader profile being distributed throughout the Golgi stack. YIPF proteins have a long soluble N-terminal region, which is orientated towards the cytosol, followed by 5 closely stacked transmembrane domains, and a C terminus, orientated towards the lumen of the Golgi. The significance of YIPF proteins for the maintenance of the morphology of the Golgi was tested by RNA interference, revealing a number of specific morphological changes to this organelle on their depletion. We propose a role for this family of proteins in regulating membrane dynamics in the endomembrane system.

A Na+ channel receptor of FMRFamide in the cephalopod Sepiella japonica: Identification, characterisation, and expression profiling during different stages of gonadal development.

FMRFamide, a member of the neuropeptide family, is involved in numerous physiological processes. FMRFamide-activated sodium channels (FaNaCs) are a family of non-voltage-gated, amiloride-sensitive, Na+-selective channels triggered by the neuropeptide FMRFamide. In the present study, the full-length cDNA of the FaNaC receptor of Sepiella japonica (SjFaNaC) was cloned. The cDNA of SjFaNaC was 3004 bp long with an open reading frame (ORF) of 1812 bp, encoding 603 amino acid residues with no signal peptide at the N-terminus. Sequence analysis indicated that SjFaNaC shared a high identity with other cephalopods FaNaCs and formed a sister clade with bivalves. The protein structure was predicted using SWISS-MODEL with AcFaNaC as the template. Quantitative real-time PCR (qRT-PCR) revealed that SjFaNaC transcripts were highly expressed in both female and male reproductive organs, as well as in the optic lobe and brain of the central nervous system (CNS). Results of in situ hybridisation (ISH) showed that SjFaNaC mRNA was mainly distributed in the medulla and deep retina of the optic lobe and in both the supraesophageal and subesophageal masses of the brain. Subcellular localisation indicated that the SjFaNaC protein was localised intracellularly and on the cell surface of HEK293T cells. In summary, these findings may lay the foundation for future exploration of the functions of SjFaNaC in cephalopods.