Conserved Amino Acid Sequences Research Articles

Structural Insights into the Iodothyronine Deiodinase 2 Catalytic Core and Deiodinase Catalysis and Dimerization

Iodothyronine deiodinases (Dio) are selenocysteine-containing membrane enzymes that activate and inactivate the thyroid hormones (TH) through reductive iodide eliminations. The three deiodinase isoforms are homodimers sharing highly conserved amino acid sequences, but they differ in their regioselectivities for the deiodination reaction and regulatory features. We have now solved a crystal structure of the mouse deiodinase 2 (Dio2) catalytic domain. It reveals a high overall similarity to the deiodinase 3 structure, supporting the proposed common mechanism, but also Dio2-specific features, likely mediating its unique properties. Activity studies with an artificially enforced Dio dimer further confirm that dimerization is required for activity and requires both the catalytic core and the enzyme’s N-terminus. Cross-linking studies reveal the catalytic core’s dimerization interface, providing insights into the architecture of the complete, active Dio homodimer.

ZNF512B binds RBBP4 via a variant NuRD interaction motif and aggregates chromatin in a NuRD complex-independent manner.

The evolutionarily conserved histone variant H2A.Z plays a crucial role in various DNA-based processes, but the mechanisms underlying its activity are not completely understood. Recently, we identified the zinc finger (ZF) protein ZNF512B as a protein associated with H2A.Z, HMG20A and PWWP2A. Here, we report that high levels of ZNF512B expression lead to nuclear protein and chromatin aggregation foci that form in a manner that is dependent on the ZF domains of ZNF512B. Notably, we demonstrate ZNF512B binding to the nucleosome remodeling and deacetylase (NuRD) complex. We discover a conserved amino acid sequence within ZNF512B that resembles the NuRD-interaction motif (NIM) previously identified in FOG-1 and other transcriptional regulators. By solving the crystal structure of this motif bound to the NuRD component RBBP4 and by applying several biochemical and biophysical assays, we demonstrate that this internal NIM is both necessary and sufficient for robust and high-affinity NuRD binding. Transcriptome analyses and reporter assays identify ZNF512B as a repressor of gene expression that can act in both NuRD-dependent and -independent ways. Our study might have implications for diseases in which ZNF512B expression is deregulated, such as cancer and neurodegenerative diseases, and hints at the existence of more proteins as potential NuRD interactors.

Immune History Modifies Disease Severity to HPAI H5N1 Clade 2.3.4.4b Viral Challenge.

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity-either from LAIV vaccination and/or from Cal09 infection-showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

M protein ectodomain-specific immunity restrains SARS-CoV-2 variants replication.

The frequent occurrence of mutations in the SARS-CoV-2 Spike (S) protein, with up to dozens of mutations, poses a severe threat to the current efficacy of authorized COVID-19 vaccines. Membrane (M) protein, which is the most abundant viral structural protein, exhibits a high level of amino acid sequence conservation. M protein ectodomain could be recognized by specific antibodies; however, the extent to which it is immunogenic and provides protection remains unclear. We designed and synthesized multiple peptides derived from coronavirus M protein ectodomains, and determined the secondary structure of specific peptides using circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect IgG responses against the synthesized peptides in clinical samples. To evaluate the immunogenicity of peptide vaccines, BALB/c mice were intraperitoneally immunized with peptide-keyhole limpet hemocyanin (KLH) conjugates adjuvanted with incomplete Freund's adjuvant (IFA). The humoral and T-cell immune responses induced by peptide-KLH conjugates were assessed using ELISA and ELISpot assays, respectively. The efficacy of the S2M2-30-KLH vaccine against SARS-CoV-2 variants was evaluated in vivo using the K18-hACE2 transgenic mouse model. The inhibitory effect of mouse immune serum on SARS-CoV-2 virus replication in vitro was evaluated using microneutralization assays. The subcellular localization of the M protein was evaluated using an immunofluorescent staining method, and the Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) activity of the S2M2-30-specific monoclonal antibody (mAb) was measured using an ADCC reporter assay. Seroconversion rates for ectodomain-specific IgG were observed to be high in both SARS-CoV-2 convalescent patients and individuals immunized with inactivated vaccines. To assess the protective efficacy of the M protein ectodomain-based vaccine, we initially identified a highly immunogenic peptide derived from this ectodomain, named S2M2-30. The mouse serum specific to S2M2-30 showed inhibitory effects on the replication of SARS-CoV-2 variants in vitro. Immunizations of K18-hACE2-transgenic mice with the S2M2-30-keyhole limpet hemocyanin (KLH) vaccine significantly reduced the lung viral load caused by B.1.1.7/Alpha (UK) infection. Further mechanism investigations reveal that serum neutralizing activity, specific T-cell response and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) correlate with the specific immuno-protection conferred by S2M2-30. The findings of this study suggest that the antibody responses against M protein ectodomain in the population most likely exert a beneficial effect on preventing various SARS-CoV-2 infections.

MotifQuest: An Automated Pipeline for Motif Database Creation to Improve Peptidomics Database Searching Programs.

Endogenous peptides are an abundant and versatile class of biomolecules with vital roles pertinent to the functionality of the nervous, endocrine, and immune systems and others. Mass spectrometry stands as a premier technique for identifying endogenous peptides, yet the field still faces challenges due to the lack of optimized computational resources for reliable raw mass spectra analysis and interpretation. Current database searching programs can exhibit discrepancies due to the unique properties of endogenous peptides, which typically require specialized search considerations. Herein, we present a high throughput, novel scoring algorithm for the extraction and ranking of conserved amino acid sequence motifs within any endogenous peptide database. Motifs are conserved patterns across organisms, representing sequence moieties crucial for biological functions, including maintenance of homeostasis. MotifQuest, our novel motif database generation algorithm, is designed to work in partnership with EndoGenius, a program optimized for database searching of endogenous peptides and that is powered by a motif database to capitalize on biological context to produce identifications. MotifQuest aims to quickly develop motif databases without any prior knowledge, a laborious task not possible with traditional sequence alignment resources. In this work we illustrate the utility of MotifQuest to expand EndoGenius' identification utility to other endogenous peptides by showcasing its ability to identify antimicrobial peptides. Additionally, we discuss the potential utility of MotifQuest to parse out motifs from a FASTA database file that can be further validated as new peptide drug candidates.

Immuno-informatics study identifies conserved T cell epitopes in non-structural proteins of Bluetongue virus serotypes: formulation of a computationally optimized next-generation broad-spectrum multi-epitope vaccine.

Bluetongue (BT) poses a significant threat to the livestock industry, affecting various animal species and resulting in substantial economic losses. The existence of numerous BT virus (BTV) serotypes has hindered control efforts, highlighting the need for broad-spectrum vaccines. In this study, we evaluated the conserved amino acid sequences within key non-structural (NS) proteins of BTV and identified numerous highly conserved murine- and bovine-specific MHC class I-restricted (MHC-I) CD8+ and MHC-II-restricted CD4+ epitopes. We then screened these conserved epitopes for antigenicity, allergenicity, toxicity, and solubility. Using these epitopes, we developed in silico-based broad-spectrum multiepitope vaccines with Toll-like receptor (TLR-4) agonists. The predicted proinflammatory cytokine response was assessed in silico using the C-IMMSIM server. Structural modeling and refinement were achieved using Robetta and GalaxyWEB servers. Finally, we assessed the stability of the docking complexes through extensive 100-nanosecond molecular dynamics simulations before considering the vaccines for codon optimization and in silico cloning. We found many epitopes that meet these criteria within NS1 and NS2 proteins and developed in silico broad-spectrum vaccines. The immune simulation studies revealed that these vaccines induce high levels of IFN-γ and IL-2 in the vaccinated groups. Protein-protein docking analysis demonstrated promising epitopes with strong binding affinities to TLR-4. The docked complexes were stable, with minimal Root Mean Square Deviation and Root Mean Square Fluctuation values. Finally, the in silico-cloned plasmids have high % of GC content with > 0.8 codon adaptation index, suggesting they are suitable for expressing the protein vaccines in prokaryotic system. These next-generation vaccine designs are promising and warrant further investigation in wet lab experiments to assess their immunogenicity, safety, and efficacy for practical application in livestock. Our findings offer a robust framework for developing a comprehensive, broad-spectrum vaccine, potentially revolutionizing BT control and prevention strategies in the livestock industry.

Functional characterization of a cystatin A from the bat Myotis davidii

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73–79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

Comparing Prion Proteins Across Species: Is Zebrafish a Useful Model?

Despite the considerable body of research dedicated to the field of neurodegeneration, the gap in knowledge on the prion protein and its intricate involvement in brain diseases remains substantial. However, in the past decades, many steps forward have been taken toward a better understanding of the molecular mechanisms underlying both the physiological role of the prion protein and the misfolding event converting it into its pathological counterpart, the prion. This review aims to provide an overview of the main findings regarding this protein, highlighting the advantages of many different animal models that share a conserved amino acid sequence and/or structure with the human prion protein. A particular focus will be given to the species Danio rerio, a compelling research organism for the investigation of prion biology, thanks to its conserved orthologs, ease of genetic manipulation, and cost-effectiveness of high-throughput experimentation. We will explore its potential in filling some of the gaps on physiological and pathological aspects of the prion protein, with the aim of directing the future development of therapeutic interventions.

Structural and functional investigation of the DHH/DHHA1 family proteins in Deinococcus radiodurans.

DHH/DHHA1 family proteins have been proposed to play critical roles in bacterial resistance to environmental stresses. Members of the most radioresistant bacteria genus, Deinococcus, possess two DHH/DHHA1 family proteins, RecJ and RecJ-like. While the functions of Deinococcus radiodurans RecJ (DrRecJ) in DNA damage resistance have been well characterized, the role and biochemical activities of D. radiodurans RecJ-like (DrRecJ-like) remain unclear. Phenotypic and transcriptomic analyses suggest that, beyond DNA repair, DrRecJ is implicated in cell growth and division. Additionally, DrRecJ-like not only affects stress response, cell growth, and division but also correlates with the folding/stability of intracellular proteins, as well as the formation and stability of cell membranes/walls. DrRecJ-like exhibits a preferred catalytic activity towards short single-stranded RNA/DNA oligos and c-di-AMP. In contrast, DrRecJ shows no activity against RNA and c-di-AMP. Moreover, a crystal structure of DrRecJ-like, with Mg2+ bound in an open conformation at a resolution of 1.97 Å, has been resolved. Subsequent mutational analysis was conducted to pinpoint the crucial residues essential for metal cation and substrate binding, along with the dimerization state, necessary for DrRecJ-like's function. This finding could potentially extend to all NrnA-like proteins, considering their conserved amino acid sequence and comparable dimerization forms.

Genomic Analysis of Brassinosteroid Biosynthesis Gene Family Reveals Its Roles in Cotton Development across Gossypium Species.

Cotton is a globally significant economic crop. Brassinosteroids (BRs) are crucial to cotton development. This study systematically analyzed the BR synthase gene family in four cotton species and identified 60 BR genes: 20 in Gossypium hirsutum (GhBRs), 20 in G. barbadense (GbBRs), 10 in G. arboreum (GaBRs), and 10 in G. raimondii (GrBRs). The analysis was extended to chromosomal localization, evolutionary relationships, domain features, and cis-regulatory elements in the promoter regions of BR synthase genes. The results showed that the BR synthase genes were evenly distributed across different subgenomes and chromosomes. Bioinformatics analyses revealed high conservation of amino acid sequences, secondary structures, and conserved domains among the subfamily members, which is closely linked to their pivotal roles in the BR biosynthesis pathway. Cis-element distribution analysis of the BR synthase genes further underscored the complexity of BR gene expression regulation, which is influenced by multiple factors, including plant hormones, abiotic stress, and transcription factors. Expression profiling of GhBRs genes in various cotton tissues and developmental stages highlighted the key roles of GhROT3-1 and GhDET2-1 in fiber elongation and initiation, respectively. Protein-protein interactions and transcription factor analyses further elucidated the regulatory mechanisms of GhROT3-1 and GhDET2-1 in cotton growth and development. This study lays a theoretical foundation for understanding the role of the BR signaling pathway in cotton development, facilitating molecular breeding.

An Inner Mitochondrial Membrane Microprotein from the SLC35A4 Upstream ORF Regulates Cellular Metabolism

Upstream open reading frames (uORFs) are cis-acting elements that can dynamically regulate the translation of downstream ORFs by suppressing downstream translation under basal conditions and, in some cases, increasing downstream translation under stress conditions. Computational and empirical methods have identified uORFs in the 5′-UTRs of approximately half of all mouse and human transcripts, making uORFs one of the largest regulatory elements known. Because the prevailing dogma was that eukaryotic mRNAs produce a single functional protein, the peptides and small proteins, or microproteins, encoded by uORFs were rarely studied. We hypothesized that a uORF in the SLC35A4 mRNA is producing a functional microprotein (SLC35A4-MP) because of its conserved amino acid sequence. Through a series of biochemical and cellular experiments, we find that the 103-amino acid SLC35A4-MP is a single-pass transmembrane inner mitochondrial membrane (IMM) microprotein. The IMM contains the protein machinery crucial for cellular respiration and ATP generation, and loss of function studies with SLC35A4-MP significantly diminish maximal cellular respiration, indicating a vital role for this microprotein in cellular metabolism. The findings add SLC35A4-MP to the growing list of functional microproteins and, more generally, indicate that uORFs that encode conserved microproteins are an untapped reservoir of functional microproteins.

Comprehensive identification and analysis of DUF640 genes associated with rice growth

Protein domains with conserved amino acid sequences and uncharacterized functions are called domains of unknown function (DUF). The DUF640 gene family plays a crucial role in plant growth, particularly in light regulation, floral organ development, and fruit development. However, there exists a lack of systematic understanding of the evolutionary relationships and functional differentiation of DUF640 within the Oryza genus. In this study, 61 DUF640 genes were identified in the Oryza genus. The expression of DUF640s is induced by multiple hormonal stressors including abscisic acid (ABA), cytokinin (CK), ethylene (ETH), and indole-3-acetic acid (IAA). Specifically, OiDUF640-10 expression significantly increased after ETH treatment. Transgenic experiments showed that overexpressing OiDUF640-10 lines were sensitive to ETH, and seedling length was obstructed. Evolutionary analysis revealed differentiation of the OiDUF640-10 gene in O. sativa ssp. indica and japonica varieties, likely driven by natural selection during the domestication of cultivated rice. These results indicate that OiDUF640-10 plays a vital role in the regulation of rice seedling length.

A loss of function mutation in CLDN25 causing Pelizaeus-Merzbacher-like leukodystrophy.

Claudin-25 (CLDN-25), also known as Claudin containing domain 1, is an uncharacterized claudin family member. It has less conserved amino acid sequences when compared to other claudins. It also has a very broad tissue expression profile and there is currently a lack of functional information from murine knockout models. Here, we report a de novo missense heterozygous variant in CLDN25 (c. 745G>C, p. A249P) found in a patient diagnosed with Pelizaeus-Merzbacher-like leukodystrophy and presenting with symptoms such as delayed motor development, several episodes of tonic absent seizures and generalized dystonia. The variant protein does not localize to the cell-cell borders where it would normally be expected to be expressed. Amino acid position 249 is located 4 amino acids from the C-terminal end of the protein where most claudin family members have a conserved binding motif for the key scaffolding protein ZO-1. However, CLDN-25 does not contain this motif. Here, we show that the C-terminal end of CLDN-25 is required for its junctional localization in a ZO-1 independent manner. The A249P mutant protein as well as a deletion mutant lacking its last 5 C-terminal amino acids also failed to localize to the cell-cell border in vitro. Intriguingly, cellular knockout of CLDN25, in vitro, appeared to increase the integrity of the tight junction between 2 contacting cells, while driving highly unusual increased movement of solutes between cells. We propose that the barrier function of CLDN-25 is akin to a decoy claudin, whereby decreasing its expression in "leaky" epithelial cells and endothelial cells will drive dynamic changes in the adhesion and interaction capacity of cell-cell contact points. While it remains unclear how this de novo CLDN-25 mutant induces leukodystrophy, our findings strongly suggest that this mutation induces haploinsufficiency of CLDN-25. Elucidating the function of this uncharacterized claudin protein will lead to a better understanding of the role of claudin proteins in health and disease.

Burkholderia pseudomallei Complex Subunit and Glycoconjugate Vaccines and Their Potential to Elicit Cross-Protection to Burkholderia cepacia Complex.

Burkholderia are a group of Gram-negative bacteria that can cause a variety of diseases in at-risk populations. B. pseudomallei and B. mallei, the etiological agents of melioidosis and glanders, respectively, are the two clinically relevant members of the B. pseudomallei complex (Bpc). The development of vaccines against Bpc species has been accelerated in recent years, resulting in numerous promising subunits and glycoconjugate vaccines incorporating a variety of antigens. However, a second group of pathogenic Burkholderia species exists known as the Burkholderia cepacia complex (Bcc), a group of opportunistic bacteria which tend to affect individuals with weakened immunity or cystic fibrosis. To date, there have been few attempts to develop vaccines to Bcc species. Therefore, the primary goal of this review is to provide a broad overview of the various subunit antigens that have been tested in Bpc species, their protective efficacy, study limitations, and known or suspected mechanisms of protection. Then, we assess the reviewed Bpc antigens for their amino acid sequence conservation to homologous proteins found in Bcc species. We propose that protective Bpc antigens with a high degree of Bpc-to-Bcc sequence conservation could serve as components of a pan-Burkholderia vaccine capable of protecting against both disease-causing groups.

Comparative analysis of the organelle genomes of Aconitum carmichaelii revealed structural and sequence differences and phylogenetic relationships

In this study, we conducted an assembly and analysis of the organelle genomes of Aconitum carmichaelii. Our investigation encompassed the examination of organelle genome structures, gene transfer events, and the environmental selection pressures affecting A. carmichaelii. The results revealed distinct evolutionary patterns in the organelle genomes of A. carmichaelii. Especially, the plastome exhibited a more conserved structure but a higher nucleotide substitution rate (NSR), while the mitogenome displayed a more complex structure with a slower NSR. Through homology analysis, we identified several instances of unidirectional protein-coding genes (PCGs) transferring from the plastome to the mitogenome. However, we did not observe any events which genes moved from the mitogenome to the plastome. Additionally, we observed multiple transposable element (TE) fragments in the organelle genomes, with both organelles showing different preferences for the type of nuclear TE insertion. Divergence time estimation suggested that rapid differentiation occurred in Aconitum species approximately 7.96 million years ago (Mya). This divergence might be associated with the reduction in CO2 levels and the significant uplift of the Qinghai-Tibet Plateau (QTP) during the late Miocene. Selection pressure analysis indicated that the dN/dS values of both organelles were less than 1, suggested that organelle PCGs were subject to purification selection. However, we did not detect any positively selected genes (PSGs) in Subg. Aconitum and Subg. Lycoctonum. This observation further supports the idea that stronger negative selection pressure on organelle genes in Aconitum results in a more conserved amino acid sequence. In conclusion, this study contributes to a deeper understanding of organelle evolution in Aconitum species and provides a foundation for future research on the genetic mechanisms underlying the structure and function of the Aconitum plastome and mitogenome.

DIVERSITY OF H2A HISTONES AND THEIR IMPLICATIONS FOR NUCLEOSOME STRUCTURAL PROPERTIES

Histone proteins are key epigenetic factors, which play an important role in chromatin dynamics and gene activity regulation. They are divided into two broad classes: canonical histones and their variants. Canonical histones are expressed mainly during the S-phase of the cell cycle, as they are involved in DNA packaging during cell division. Histone variants are histone genes that are expressed and regulate chromatin dynamics throughout the cell cycle. Due to the functional and species diversity, various families of histone variants are distinguished. Some proteins may diff er slightly from canonical histones, while others, on the contrary, may have many important structural and functional features that aff ect nucleosome stability and chromatin dynamics. In order to assess the variability of the H2A histone family and their role in nucleosome structure, we performed a bioinformatic analysis of the amino acid sequences of the H2A histone family. The clustering performed by the UPGMA method made it possible to reveal two main subfamilies of H2A proteins: short H2A and other H2A variants demonstrating highly conserved amino acid sequences. We also constructed and analyzed multiple alignments for various H2A histone subfamilies. It is important to note that the proteins of the short H2A subfamily are not only the least conserved within the H2A family, but also have features that signifi cantly aff ect the structural properties of the nucleosome. In addition, we performed a phylogenetic analysis of short H2A, which resulted in the identifi cation and characterization of individual clades on the phylogenetic tree for the variants H2A.B, H2A.P, H2A.Q, H2A.L.

Evidence That Aquaporin 11 (AQP11) in the Spiny Dogfish (Squalus acanthias) May Represent a Pseudogene.

Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (Squalus acanthias) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5' and 3' RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5' and 3' RACE amplifications depend on the presence of a 5' cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene.

Genome-Wide Identification of DUF668 Gene Family and Expression Analysis under F. solani, Chilling, and Waterlogging Stresses in Zingiber officinale.

The domains of unknown function (DUF) superfamilies contain proteins with conserved amino acid sequences without known functions. Among them, DUF668 was indicated widely involving the stress response of plants. However, understanding ZoDUF668 is still lacking. Here, 12 ZoDUF668 genes were identified in ginger by the bioinformatics method and unevenly distributed on six chromosomes. Conserved domain analysis showed that members of the same subfamily had similar conserved motifs and gene structures. The promoter region of ZoDUF668s contained the light, plant hormone and stress-responsive elements. The prediction of miRNA targeting relationship showed that nine ginger miRNAs targeted four ZoDUF668 genes through cleavage. The expression patterns of 12 ZoDUF668 genes under biotic and abiotic stress were analyzed using RT-qPCR. The results showed that the expression of seven ZoDUF668 genes was significantly downregulated under Fusarium solani infection, six ZoDUF668 genes were upregulated under cold stress, and five ZoDUF668 genes were upregulated under waterlogging stress. These results indicate that the ZoDUF668 gene has different expression patterns under different stress conditions. This study provides excellent candidate genes and provides a reference for stress-resistance research in ginger.

Domain of unknown function (DUF) proteins in plants: function and perspective.

Domains of unknown function (DUFs), which are deposited in the protein family database (Pfam), are protein domains with conserved amino acid sequences and uncharacterized functions. Proteins with the same DUF were classified as DUF families. Although DUF families are generally not essential for the survival of plants, they play roles in plant development and adaptation. Characterizing the functions of DUFs is important for deciphering biological puzzles. DUFs were generally studied through forward and reverse genetics. Some novelty approaches, especially the determination of crystal structures and interaction partners of the DUFs, should attract more attention. This review described the identification of DUF genes by genome-wide and transcriptome-wide analyses, summarized the function of DUF-containing proteins, and addressed the prospects for future studies in DUFs in plants.

Crystal structure of the Arabidopsis SPIRAL2 C-terminal domain reveals a p80-Katanin-like domain.

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.