Protein Sequence Length Research Articles

Generation of a high confidence set of domain-domain interface types to guide protein complex structure predictions by AlphaFold.

While the release of AlphaFold (AF) represented a breakthrough for the prediction of protein complex structures, its sensitivity, especially when using full length protein sequences, still remains limited. Modeling success rates might increase if AF predictions were guided by likely interacting protein fragments. This approach requires available sets of highly confident protein-protein interface types. Computational resources, such as 3did, infer interacting globular domain types from observed contacts in protein structures. Assessing the accuracy of these predicted interface types is difficult because we lack hand-curated reference sets of verified domain-domain interface (DDI) types. To improve protein complex modeling of DDIs by AF, we manually inspected 80 randomly selected DDI types from the 3did resource to generate a first reference set of DDI types. Identified cases of DDI type nonapproval (40%) primarily resulted from inaccurate Pfam domain matches, crystal contacts, and synthetic protein constructs. Using logistic regression, we predicted a subset of 2411 out of 5724 considered DDI types in 3did to be of high confidence, which we subsequently applied to 53000 human-protein interactions to predict DDIs followed by AF modeling. We obtained highly confident AF models for 604 out of 1129 predicted DDIs. Of note, for 47% of them no confident AF structural model could be obtained using full length protein sequences. Code is available at https://github.com/KatjaLuckLab/DDI_manuscript.

Characterization of the Heat Shock Transcription Factor Family in Lycoris radiata and Its Potential Roles in Response to Abiotic Stresses.

Heat shock transcription factors (HSFs) are an essential plant-specific transcription factor family that regulates the developmental and growth stages of plants, their signal transduction, and their response to different abiotic and biotic stresses. The HSF gene family has been characterized and systematically observed in various species; however, research on its association with Lycoris radiata is limited. This study identified 22 HSF genes (LrHSFs) in the transcriptome-sequencing data of L. radiata and categorized them into three classes including HSFA, HSFB, and HSFC, comprising 10, 8, and 4 genes, respectively. This research comprises basic bioinformatics analyses, such as protein sequence length, molecular weight, and the identification of its conserved motifs. According to the subcellular localization assessment, most LrHSFs were present in the nucleus. Furthermore, the LrHSF gene expression in various tissues, flower developmental stages, two hormones stress, and under four different abiotic stresses were characterized. The data indicated that LrHSF genes, especially LrHSF5, were essentially involved in L. radiata development and its response to different abiotic and hormone stresses. The gene-gene interaction network analysis revealed the presence of synergistic effects between various LrHSF genes' responses against abiotic stresses. In conclusion, these results provided crucial data for further functional analyses of LrHSF genes, which could help successful molecular breeding in L. radiata.

Pan-Genome-Wide Identification and Transcriptome-Wide Analysis of ZIP Genes in Cucumber

The ZRT/IRT-like proteins (ZIPs) play critical roles in the absorption, transport, and intracellular balance of metal ions essential for various physiological processes in plants. However, little is known about the pan-genomic characteristics and properties of ZIP genes in cucumber (Cucumis sativus L.). In this study, we identified 10 CsZIP genes from the pan-genome of 13 C. sativus accessions. Among them, only CsZIP10 showed no variation in protein sequence length. We analyzed the gene structure, conserved domains, promoter cis-elements, and phylogenetic relationships of these 10 CsZIP genes derived from “9930”. Based on phylogenetic analysis, the CsZIP genes were classified into three branches. Amino acid sequence comparison revealed the presence of conserved histidine residues in the ZIP proteins. Analysis of promoter cis-elements showed that most promoters contained elements responsive to plant hormones. Expression profiling in different tissues showed that most CsZIP genes were expressed at relatively low levels in C. sativus leaves, stems, and tendrils, and CsZIP7 and CsZIP10 were specifically expressed in roots, indicating their potential involvement in the absorption and transport of metal ions. Transcriptomic data indicated that these 10 ZIP genes displayed responses to both downy mildew and powdery mildew, and CsZIP1 was significantly downregulated after both salt and heat treatments. In conclusion, this study deepens our understanding of the ZIP gene family and enhances our knowledge of the biological functions of CsZIP genes in C. sativus.

Use of 2D FFT and DTW in Protein Sequence Comparison.

Protein sequence comparison remains a challenging work for the researchers owing to the computational complexity due to the presence of 20 amino acids compared with only four nucleotides in Genome sequences. Further, protein sequences of different species are of different lengths; it throws additional changes to the researchers to develop methods, specially alignment-free methods, to compare protein sequences. In this work, an efficient technique to compare protein sequences is developed by a graphical representation. First, the classified grouping of 20 amino acids with a cardinality of 4 based on polar class is considered to narrow down the representational range from 20 to 4. Then a unit vector technique based on a two-quadrant Cartesian system is proposed to provide a new two-dimensional graphical representation of the protein sequence. Now, two approaches are proposed to cope with the varying lengths of protein sequences from various species: one uses Dynamic Time Warping (DTW), while the other one uses a two-dimensional Fast Fourier Transform (2D FFT). Next, the effectiveness of these two techniques is analyzed using two evaluation criteria-quantitative measures based on symmetric distance (SD) and computational speed. An analysis is performed on five data sets of 9 ND4, 9 ND5, 9 ND6, 12 Baculovirus, and 24 TF proteins under the two methods. It is found that the FFT-based method produces the same results as DTW but in less computational time. It is found that the result of the proposed method agrees with the known biological reference. Further, the present method produces better clustering than the existing ones.

Protein-DNA Binding Residues Prediction Using a Deep Learning Model With Hierarchical Feature Extraction.

Biologically important effects occur when proteins bind to other substances, of which binding to DNA is a crucial one. Therefore, accurate identification of protein-DNA binding residues is important for further understanding of the protein-DNA interaction mechanism. Although wet-lab methods can accurately obtain the location of bound residues, it requires significant human, financial and time costs. There is thus an urgent need to develop efficient computational-based methods. Most current state-of-the-art methods are two-step approaches: the first step uses a sliding window technique to extract residue features; the second step uses each residue as an input to the model for prediction. This has a negative impact on the efficiency of prediction and ease of use. In this study, we propose a sequence-to-sequence (seq2seq) model that can input the entire protein sequence of variable length and use two modules, Transformer Encoder Block and Feature Extracting Block, for hierarchical feature extraction, where Transformer Encoder Block is used to extract global features, and then Feature Extracting Block is used to extract local features to further improve the recognition capability of the model. The comparison results on two benchmark datasets, namely PDNA-543 and PDNA-41, prove the effectiveness of our method in identifying protein-DNA binding residues.

Novel and improved Caenorhabditis briggsae gene models generated by community curation

BackgroundThe nematode Caenorhabditis briggsae has been used as a model in comparative genomics studies with Caenorhabditis elegans because of their striking morphological and behavioral similarities. However, the potential of C. briggsae for comparative studies is limited by the quality of its genome resources. The genome resources for the C. briggsae laboratory strain AF16 have not been developed to the same extent as C. elegans. The recent publication of a new chromosome-level reference genome for QX1410, a C. briggsae wild strain closely related to AF16, has provided the first step to bridge the gap between C. elegans and C. briggsae genome resources. Currently, the QX1410 gene models consist of software-derived gene predictions that contain numerous errors in their structure and coding sequences. In this study, a team of researchers manually inspected over 21,000 gene models and underlying transcriptomic data to repair software-derived errors.ResultsWe designed a detailed workflow to train a team of nine students to manually curate gene models using RNA read alignments. We manually inspected the gene models, proposed corrections to the coding sequences of over 8,000 genes, and modeled thousands of putative isoforms and untranslated regions. We exploited the conservation of protein sequence length between C. briggsae and C. elegans to quantify the improvement in protein-coding gene model quality and showed that manual curation led to substantial improvements in the protein sequence length accuracy of QX1410 genes. Additionally, collinear alignment analysis between the QX1410 and AF16 genomes revealed over 1,800 genes affected by spurious duplications and inversions in the AF16 genome that are now resolved in the QX1410 genome.ConclusionsCommunity-based, manual curation using transcriptome data is an effective approach to improve the quality of software-derived protein-coding genes. The detailed protocols provided in this work can be useful for future large-scale manual curation projects in other species. Our manual curation efforts have brought the QX1410 gene models to a comparable level of quality as the extensively curated AF16 gene models. The improved genome resources for C. briggsae provide reliable tools for the study of Caenorhabditis biology and other related nematodes.

Characterization of the Heat Shock Transcription Factor Family in Medicago sativa L. and Its Potential Roles in Response to Abiotic Stresses.

Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The HSF gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 HSF genes (MsHSFs) in the tetraploid alfalfa genome ("Xinjiangdaye" reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 MsHSFs and tissue-wide expression for 28 MsHSFs. Based on transcriptomic data analysis, 21, 11 and 27 MsHSFs responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT-PCR, MsHSF27/33 expression gradually increased with cold, salt and drought stress condition duration; MsHSF6 expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of MsHSFs and for genetic improvement of stress resistance in alfalfa.

PROVAL: A framework for comparison of protein sequence embeddings

High throughput sequencing technology leads to a significant increase in the number of generated protein sequences and the anchor database UniProt doubles approximately every two years. This large set of annotated data is used by many bioinformatics algorithms. Searching within these databases, typically without using any annotations, is challenging due to the variable lengths of the entries and the used non-standard comparison measures. A promising strategy to address these issues is to find fixed-length, information-preserving representations of the variable length protein sequences. A systematic algorithmic evaluation of the proposals is however surprisingly missing. In this work, we analyze how different algorithms perform in generating general protein sequence representations and provide a thorough evaluation framework PROVAL. The strategies range from a proximity representation using classical Smith–Waterman algorithm to state-of-the-art embedding techniques by means of transformer networks. The methods are evaluated by, e.g., the molecular function classification, embedding space visualization, computational complexity and the carbon footprint.

Evolution is not uniform along protein sequence length

Evolution is not uniform along protein sequence length

Evolution is not uniform along protein sequence length

Evolution is not uniform along protein sequence length

Evolution is not uniform along protein sequence length

Evolution is not uniform along protein sequence length

Cross-attention PHV: Prediction of human and virus protein-protein interactions using cross-attention–based neural networks

Viral infections represent a major health concern worldwide. The alarming rate at which SARS-CoV-2 spreads, for example, led to a worldwide pandemic. Viruses incorporate genetic material into the host genome to hijack host cell functions such as the cell cycle and apoptosis. In these viral processes, protein–protein interactions (PPIs) play critical roles. Therefore, the identification of PPIs between humans and viruses is crucial for understanding the infection mechanism and host immune responses to viral infections and for discovering effective drugs. Experimental methods including mass spectrometry-based proteomics and yeast two-hybrid assays are widely used to identify human-virus PPIs, but these experimental methods are time-consuming, expensive, and laborious. To overcome this problem, we developed a novel computational predictor, named cross-attention PHV, by implementing two key technologies of the cross-attention mechanism and a one-dimensional convolutional neural network (1D-CNN). The cross-attention mechanisms were very effective in enhancing prediction and generalization abilities. Application of 1D-CNN to the word2vec-generated feature matrices reduced computational costs, thus extending the allowable length of protein sequences to 9000 amino acid residues. Cross-attention PHV outperformed existing state-of-the-art models using a benchmark dataset and accurately predicted PPIs for unknown viruses. Cross-attention PHV also predicted human–SARS-CoV-2 PPIs with area under the curve values >0.95. The Cross-attention PHV web server and source codes are freely available at https://kurata35.bio.kyutech.ac.jp/Cross-attention_PHV/ and https://github.com/kuratahiroyuki/Cross-Attention_PHV, respectively.

The Design and Implementation of an Improved Lightweight BLASTP on CUDA GPU

In the field of computational biology, sequence alignment is a very important methodology. BLAST is a very common tool for performing sequence alignment in bioinformatics provided by National Center for Biotechnology Information (NCBI) in the USA. The BLAST server receives tens of thousands of queries every day on average. Among the procedures of BLAST, the hit detection process whose core architecture is a lookup table is the most time-consuming. In the latest work, a lightweight BLASTP on CUDA GPU with a hybrid query-index table was proposed for servicing the sequence query length shorter than 512, which effectively improved the query efficiency. According to the reported protein sequence length distribution, about 90% of sequences are equal to or smaller than 1024. In this paper, we propose an improved lightweight BLASTP to speed up the hit detection time for longer query sequences. The largest sequence is enlarged from 512 to 1024. As a result, one more bit is required to encode each sequence position. To meet the requirement, an extended hybrid query-index table (EHQIT) is proposed to accommodate three sequence positions in a four-byte table entry, making only one memory access sufficient to retrieve all the position information as long as the number of hits is equal to or smaller than three. Moreover, if there are more than three hits for a possible word, all the position information will be stored in contiguous table entries, which eliminates branch divergence and reduces memory space for pointers to overflow buffer. A square symmetric scoring matrix, Blosum62, is used to determine the relative score made by matching two characters in a sequence alignment. The experimental results show that for queries shorter than 512 our improved lightweight BLASTP outperforms the original lightweight BLASTP with speedups of 1.2 on average. When the number of hit overflows increases, the speedup can be as high as two. For queries shorter than 1024, our improved lightweight BLASTP can provide speedups ranging from 1.56 to 3.08 over the CUDA-BLAST. In short, the improved lightweight BLASTP can replace the original one because it can support a longer query sequence and provide better performance.

Species-Specific Duplication and Adaptive Evolution of a Candidate Sex Pheromone Receptor Gene in Weather Loach.

The release and sensation of sex pheromone play a role in the reproductive success of vertebrates including fish. Previous studies have shown that the weather loach Misgurnus anguillicaudatus perceives sex pheromones by olfaction to stimulate courtship behavior. It was speculated that weather loaches use smell to recognize intraspecific mates. However, the identification of loach pheromone receptor has not been reported. By comparative transcriptomic approach, we found that the olfactory receptor gene or114-1 was male-biasedly expressed in the olfactory epithelium of M. anguillicaudatus, M. bipartitus and the closely related species Paramisgurnus dabryanus. This sex-biased expression pattern implicated that or114-1 presumably encoded a sex pheromone receptor in loaches. M. bipartitus and P. dabryanus, like zebrafish, possess one or114-1 only. However, in M. anguillicaudatus, or114-1 has two members: Ma_or114-1a and Ma_or114-1b. Ma_or114-1a, not Ma_or114-1b, showed sex-differential expression in olfactory epithelium. Ma_or114-1b has base insertions that delayed the stop codon, causing the protein sequence length to be extended by 8 amino acids. Ma_or114-1a was subject to positive selection resulting in adaptive amino acid substitutions, which indicated that its ligand binding specificity has probably changed. This adaptive evolution might be driven by the combined effects of sexual selection and reinforcement of premating isolation between the sympatric loach species.

Correction to: Key genes in the phenylpropanoids biosynthesis pathway have different expression patterns under various abiotic stresses in the Iranian red and green cultivars of sweet basil (Ocimum basilicum L.)

Phenylpropanoids are a large and important family of plant secondary metabolites. The biosynthetic pathway of phenylpropanoids is induced in plants under environmental stresses to cope with these harmful conditions. In the present study, for the first time, we identified and characterized one reference gene (ACTIN) and three key biosynthetic genes (4CL, C4H and CVOMT) in the Iranian red and green cultivars of sweet basil. Also, the expression patterns of 4CL, C4H and CVOMT biosynthetic genes were determined for the first time in the Iranian red and green cultivars of sweet basil under cold, drought, heat, light and salt stresses. The results showed that the ACTIN, 4CL, C4H and CVOMT genes identified in the Iranian cultivars are identical to other cultivars in terms of all characteristics such as ORF length, protein sequence length, molecular weight, functional domains, lack of signal peptide, subcellular localization site, and secondary structures. Our results also revealed that the 4CL, C4H and CVOMT biosynthetic genes have different expression in the Iranian red and green cultivars of sweet basil under abiotic stresses and their expression patterns are cultivar dependent. The findings of this study can advance our knowledge of phenylpropanoids biosynthesis in plants under environmental stresses. These findings also can be used in plant breeding programs for stress tolerance in sweet basil and other plants.

Key genes in the phenylpropanoids biosynthesis pathway have different expression patterns under various abiotic stresses in the Iranian red and green cultivars of sweet basil (Ocimum basilicum L.)

Phenylpropanoids are a large and important family of plant secondary metabolites. The biosynthetic pathway of phenylpropanoids is induced in plants under environmental stresses to cope with these harmful conditions. In the present study, for the first time, we identified and characterized one reference gene (ACTIN) and three key biosynthetic genes (4CL, C4H and CVOMT) in the Iranian red and green cultivars of sweet basil. Also, the expression patterns of 4CL, C4H and CVOMT biosynthetic genes were determined for the first time in the Iranian red and green cultivars of sweet basil under cold, drought, heat, light and salt stresses. The results showed that the ACTIN, 4CL, C4H and CVOMT genes identified in the Iranian cultivars are identical to other cultivars in terms of all characteristics such as ORF length, protein sequence length, molecular weight, functional domains, lack of signal peptide, subcellular localization site, and secondary structures. Our results also revealed that the 4CL, C4H and CVOMT biosynthetic genes have different expression in the Iranian red and green cultivars of sweet basil under abiotic stresses and their expression patterns are cultivar dependent. The findings of this study can advance our knowledge of phenylpropanoids biosynthesis in plants under environmental stresses. These findings also can be used in plant breeding programs for stress tolerance in sweet basil and other plants.

Transcriptome-wide and expression analysis of the NAC gene family in pepino (Solanum muricatum) during drought stress.

Solanum muricatum (Pepino) is an increasingly popular solanaceous crop and is tolerant of drought conditions. In this study, 71 NAC transcription factor family genes of S. muricatum were selected to provide a theoretical basis for subsequent in-depth study of their regulatory roles in the response to biological and abiotic stresses, and were subjected to whole-genome analysis. The NAC sequences obtained by transcriptome sequencing were subjected to bioinformatics prediction and analysis. Three concentration gradient drought stresses were applied to the plants, and the target gene sequences were analyzed by qPCR to determine their expression under drought stress. The results showed that the S. muricatum NAC family contains 71 genes, 47 of which have conserved domains. The protein sequence length, molecular weight, hydrophilicity, aliphatic index and isoelectric point of these transcription factors were predicted and analyzed. Phylogenetic analysis showed that the S. muricatum NAC gene family is divided into seven subfamilies. Some NAC genes of S. muricatum are closely related to the NAC genes of Solanaceae crops such as tomato, pepper and potato. The seedlings of S. muricatum were grown under different gradients of drought stress conditions and qPCR was used to analyze the NAC expression in roots, stems, leaves and flowers. The results showed that 13 genes did not respond to drought stress while 58 NAC genes of S. muricatum that responded to drought stress had obvious tissue expression specificity. The overall expression levels in the root were found to be high. The number of genes at extremely significant expression levels was very large, with significant polarization. Seven NAC genes with significant responses were selected to analyze their expression trend in the different drought stress gradients. It was found that genes with the same expression trend also had the same or part of the same conserved domain. Seven SmNACs that may play an important role in drought stress were selected for NAC amino acid sequence alignment of Solanaceae crops. Four had strong similarity to other Solanaceae NAC amino acid sequences, and SmNAC has high homology with the Solanum pennellii. The NAC transcription factor family genes of S. muricatum showed strong structural conservation. Under drought stress, the expression of NAC transcription factor family genes of S. muricatum changed significantly, which actively responded to and participated in the regulation process of drought stress, thereby laying foundations for subsequent in-depth research of the specific functions of NAC transcription factor family genes of S. muricatum.

Villin Family Members Associated with Multiple Stress Responses in Cotton

Villin (VLN) is considered to be one of the most important actin-binding proteins, participates in modulating the actin cytoskeleton dynamics, plays essential role in plant development and resisting adverse environments. However, systematic studies of the VLN gene family have not been reported in cotton (Gossypium). In this study, 14 GhVLNs were identified in G. hirsutum. These GhVLN genes were distributed in 6 A-subgenome chromosomes and 6 D-subgenome chromosomes of the allotetraploid upland cotton and classified into three phylogenetical groups based on the classification model of AtVLNs. In addition, the 14 GhVLN genes have highly conserved gene structure and motif architecture. The number of introns was ranged from 18 to 22 and the length of protein sequences was varied from 901 to 1077. Six gelsolin homology domains, G1–G6, and villin headpiece domain, VHP, were existed in all GhVLNs with the exception of two VLNs (GhVLN6 and GhVLN13) which lacked VHP. Cis-elements analysis revealed that the promoter regions of GhVLNs contained various light related components and also elements responsible for phytohormones and stresses response, indicating that, when subjected to those adverse environments, cotton plants may activate the response system by targeting VLN genes to survive the crisis. Heatmaps showed that the GhVLN genes exhibited various expression patterns, some were accumulated in certain tissues, root, petal, stamen or elongating fibers, and some were obviously induced by environmental changes. Especially GhVLN3 and GhVLN10 were highly and preferentially expressed in elongating fibers and distinctly upregulated by abiotic (salt, PEG, cold and heat) and biotic (Verticillium dahliae V991) stresses. This study may provide useful information for biological function identification of GhVLN genes and gene resources for creating high-quality and various resistant cotton germplasms.

New insights on the function of plant acyl carrier proteins from comparative and evolutionary analysis

Acyl carrier proteins (ACPs) play a central role in both plastidial and mitochondrial Type II fatty acid synthesis in plant cells. However, a large proportion of plant ACPs remain functionally uncharacterized, and their evolutionary history remains elusive. In present study, 97 putative ACPs were identified from ten angiosperm species examined. Based on phylogenetic analysis, ACP genes were grouped into plastidial (cpACP: ACP1/2/3/4/5) and mitochondrial (mtACP: mtACP1/mtACP2/mtACP3) ACPs. Protein sequence (motifs and length), tertiary structure, and gene structure (exon number, average intron length, and intron phase) were highly conserved in different ACP subclades. The differentiation of ACPs into distinct types occurred 85–98 and 45–57 million years ago. A limited proportion of ACP genes experience tandem or segmental duplication, corresponding to two rounds of whole genome duplication. Ka/Ks ratios revealed that duplicated ACP genes underwent a purifying selection. Regarding expression patterns, most ACPs were expressed constitutively and tissue-specifically. Notably, the average expression levels of ACP1, mtACP3, and mtACP1 were positively correlated with those of ACP3, ACP4, and mtACP2, respectively. Analysis of cis-elements showed that seven motifs (CACTFTPPCA1, DOFCOREZM, GT1CONSENSUS, CAATBOX1, ARR1AT, POLLEN1LELAT52, and GATABOX) related to tissue-specific, ABA, and light-mediated gene regulation were ubiquitous in all ACPs investigated, which shed new light on the regulation patterns of these central enzymatic partners of the FAS system. This study presents a thorough overview of angiosperm ACP gene families and provides informative clues for the functional characterization of plant ACPs in the future.

Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence.

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the Begomovirus genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.