Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A–binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein–associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome–ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB—and thus peroxisome–ER contact sites—differently. Moreover, we demonstrate that GSK3β (glycogen synthase kinase-3 β) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome–ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.

In silico construction of a multiepitope Zika virus vaccine using immunoinformatics tools

Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine’s average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation.

GFAP: ultra-fast and accurate gene functional annotation software for plants

Sequence alignment is the basis of gene functional annotation for unknow sequences. Selecting closely related species as the reference species should be an effective way to improve the accuracy of gene annotation for plants, compared with only based on one or some model plants. Therefore, limited species number in previous software or website is disadvantageous for plant gene annotation. Here, we collected the protein sequences of 236 plant species with known genomic information from 63 families. After that, these sequences were annotated by pfam, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to construct our databases. Furthermore, we developed the software, Gene Annotation Software for Plants (GFAP), to perform gene annotation using our databases. GFAP, an open-source software running on Windows and MacOS systems, is an efficient and network independent tool. GFAP can search the protein domain, GO and KEGG information for 43000 genes within 4 minutes. In addition, GFAP can also perform the sequence alignment, statistical analysis and drawing. The website of https://gitee.com/simon198912167815/gfap-database provides the software, databases, testing data and video tutorials for users. GFAP contained large amount of plant-species information. We believe that it will become a powerful tool in gene annotation using closely related species for phytologists.

Pregnancy-specific glycoproteins: evolution, expression, functions, and disease associations.

Pregnancy-specific glycoproteins (PSGs) are members of the immunoglobulin superfamily and are closely related to the predominantly membrane-bound CEACAM proteins. PSGs are produced by placental trophoblasts and secreted into the maternal bloodstream at high levels where they may regulate maternal immune and vascular functions through receptor binding and modulation of cytokine and chemokine expression and activity. PSGs may have autocrine and paracrine functions in the placental bed, and PSGs can activate soluble and extracellular matrix bound TGF-β, with potentially diverse effects on multiple cell types. PSGs are also found at high levels in the maternal circulation, at least in human, where they may have endocrine functions. In a non-reproductive context, PSGs are expressed in the gastrointestinal tract and their deregulation may be associated with colorectal cancer and other diseases. Like many placental hormones, PSGs are encoded by multigene families and they have an unusual phylogenetic distribution, being found predominantly in species with hemochorial placentation, with the notable exception of the horse in which PSG-like proteins are expressed in the endometrial cups of the epitheliochorial placenta. The evolution and expansion of PSG gene families appears to be a highly active process, with significant changes in gene numbers and protein domain structures in different mammalian lineages, and reports of extensive copy number variation at the human locus. Against this apparent diversification, the available evidence indicates extensive conservation of PSG functions in multiple species. These observations are consistent with maternal-fetal conflict underpinning the evolution of PSGs.

DARTS: An Algorithm for Domain-Associated Retrotransposon Search in Genome Assemblies

Retrotransposons comprise a substantial fraction of eukaryotic genomes, reaching the highest proportions in plants. Therefore, identification and annotation of retrotransposons is an important task in studying the regulation and evolution of plant genomes. The majority of computational tools for mining transposable elements (TEs) are designed for subsequent genome repeat masking, often leaving aside the element lineage classification and its protein domain composition. Additionally, studies focused on the diversity and evolution of a particular group of retrotransposons often require substantial customization efforts from researchers to adapt existing software to their needs. Here, we developed a computational pipeline to mine sequences of protein-coding retrotransposons based on the sequences of their conserved protein domains—DARTS (Domain-Associated Retrotransposon Search). Using the most abundant group of TEs in plants—long terminal repeat (LTR) retrotransposons (LTR-RTs)—we show that DARTS has radically higher sensitivity for LTR-RT identification compared to the widely accepted tool LTRharvest. DARTS can be easily customized for specific user needs. As a result, DARTS returns a set of structurally annotated nucleotide and amino acid sequences which can be readily used in subsequent comparative and phylogenetic analyses. DARTS may facilitate researchers interested in the discovery and detailed analysis of the diversity and evolution of retrotransposons, LTR-RTs, and other protein-coding TEs.

Transcriptome-wide identification of coding and noncoding RNA-binding proteins defines the comprehensive RNA interactome of Leishmania mexicana

Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labelling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of twenty Leishmania deubiquitinases including the recently characterised L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterise the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 145 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterise the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology.

The genetic origin and architecture of sex determination

Here, I demonstrate that sex determination and sexual dimorphism across tree of life are deeply related to polyamine biochemistry in cells, especially to the synteny of genes: [SAT1-NR0B1], [SAT2-SHBG] and DMRT1. This synteny was found to be most distinct in mammals. Further, the common protein domain of SAT1 and SAT2 - PF00583 was shown to be present in the genome of the last universal common ancestor (LUCA). Protein domain-domain interaction analysis of LUCA’s genes suggests the possibility that LUCA had developed an immune defence against viruses. This domain-domain interaction analysis is the first scientific evidence indicating that viruses existed at least 3.5 billions years ago and probably co-existed with LUCA on early Hadean Earth.

The genetic origin and architecture of sex determination

Here, I demonstrate that sex determination and sexual dimorphism across tree of life are deeply related to polyamine biochemistry in cells, especially to the synteny of genes: [SAT1-NR0B1], [SAT2-SHBG] and DMRT1. This synteny was found to be most distinct in mammals. Further, the common protein domain of SAT1 and SAT2 - PF00583 was shown to be present in the genome of the last universal common ancestor (LUCA). Protein domain-domain interaction analysis of LUCAs genes suggests the possibility that LUCA had developed an immune defence against viruses. This domain-domain interaction analysis is the first scientific evidence indicating that viruses existed at least 3.5 billions years ago and probably co-existed with LUCA on early Hadean Earth.

DARTS: an Algorithm for Domain-Associated RetroTransposon Search in Genome Assemblies

Retrotransposons comprise a substantial fraction of eukaryotic genomes reaching the highest proportions in plants. Therefore, identification and annotation of retrotransposons is an important task in studying regulation and evolution of plant genomes. A majority of computational tools for mining transposable elements (TEs) are designed for subsequent genome repeat masking, often leaving aside the element lineage classification and its protein domain composition. Additionally, studies focused on diversity and evolution of a particular group of retrotransposons often require substantial customization efforts from researchers to adapt existing software to their needs. Here, we developed a computational pipeline to mine sequences of protein-coding retrotransposons based on the sequences of their conserved protein domains - DARTS. Using the most abundant group of TEs in plants - long terminal repeat (LTR) retrotransposons (LTR-RTs), we show that DARTS has radically higher sensitivity of LTR-RTs identification compared to a widely accepted LTRharvest tool. DARTS can be easily customized for specific user needs. As a result, DARTS returns a set of structurally annotated nucleotide and amino acid sequences which can be readily used in subsequent comparative and phylogenetic analyses. DARTS should facilitate researchers interested in discovery and in-detail analysis of diversity and evolution of retrotransposons, LTR-RTs, and other protein-coding TEs.