Conserved Amino Acid Sequences Research Articles

In silico epitope prediction of Borrelia burgdorferi sensu lato antigens for the detection of specific antibodies

Despite many years of research, serodiagnosis of Lyme disease still faces many obstacles. Difficulties arise mainly due to the low degree of amino acid sequence conservation of the most immunogenic antigens among B. burgdorferi s.l. genospecies, as well as differences in protein production depending on the environment in which the spirochete is located. Mapping B-cell epitopes located on antigens allows for a better understanding of antibody-pathogen interactions which is essential for the development of new and more effective diagnostic tools. In this study, in silico B-cell epitope mapping was performed to determine the theoretical diagnostic potential of selected B. burgdorferi s.l. proteins (BB0108, BB0126, BB0298, BB0689, BB0323, FliL, PstS, SecD, EF-Tu). Bioinformatics software predicted 35 conserved linear and 31 conformational epitopes with the degree of identity among B. burgdorferi s.l. of at least 85%, which may prove to be useful in the development of a new tool for the diagnosis of Lyme disease.

Familial Thrombocytopenia-Associated Germline ETV6 P214L Mutation Results in XPO1-Mediated Nuclear Export

In recent years, a number of heterozygous germline mutations in ETV6 have been associated with familial thrombocytopenia and predisposition to hematologic malignancies. While most of these missense mutations occur in the DNA-binding-domain of ETV6, the recurrent P214L mutation is unique in that it occurs in the central disordered region of the protein. The ETV6 P214L mutation has previously been shown to disrupt the nuclear localization of ETV6 and the ability of ETV6 to act as a transcriptional repressor. Unexpectedly, we have found that the P214L missense mutation creates a recognition motif for the nuclear export protein XPO1. We have demonstrated in cellular and protein reconstitution assays that XPO1 directly engages the P214L but not the wild-type ETV6 construct. Consistent with this mechanism, we are able to fully rescue the nuclear localization of ETV6 P214L in mammalian cells with small molecule XPO1 inhibitors. We have also found that once localized to the nucleus, the P214L mutant construct exhibits functional consequences indistinguishable from wild-type ETV6 in Ba/F3 transformation assays. When looking at the evolutionary conservation of the ETV6 amino acid sequence involved in the XPO1 recognition motif, we observed that the synonymous P216L mutation in the mouse ETV6 ortholog does not create a nuclear export signal. In order to study XPO1-mediated export of ETV6 P214L and its impact on hematopoiesis, we have generated a mouse model in which two point mutations (P216L and S211I) have been introduced into a single endogenous ETV6 allele, which precisely matches the sequence of this region in human P214L carriers. Using CRISPR-Cas9, we generated 3 transgenic mouse lines: Etv6 P216L, Etv6 S211I, and Etv6 S211IP216L. Immunofluorescence of fixed bone marrow shows that ETV6 is only mislocalized in megakaryocytes from ETV6 S211I P216L mice. Complete blood counts revealed a significant decrease in platelet counts in ETV6 S211I P216L heterozygotes (485 x 10 3/µL) compared to ETV6 S211I heterozygotes (1140 x 10 3/µL, p<0.0001), ETV6 P216L heterozygotes (1065 x 10 3/µL, p<0.0006), and wild-type littermates (1254 x 10 3/µL, p<0.0001). Flow cytometry phenotyping of bone marrow from the three transgenic lines and wild-type littermates shows a significant decrease in the absolute numbers of hematopoietic stem cells (HSCs) and megakaryocyte precursors (MkPs) in ETV6 S211I P216L mice. In summary, we have determined that XPO1-mediated nuclear export is the mechanistic basis for ETV6 P214L mislocalization. We developed a mouse model that recapitulates this export signal in the endogenous ETV6 allele and found that this model demonstrates a thrombocytopenic phenotype as seen in human patients. Reductions in HSCs and MkPs suggest both a decreased fitness of HSCs, as well as dysregulated megakaryopoiesis in this model.

A molluscan IRF interacts with IKKα/β family protein and modulates NF-κB and MAPK activity

Interferon regulatory factor (IRF) family proteins are key transcription factors involved in vital physiological processes such as immune defense. However, the function of IRF in invertebrates, especially in marine shellfish is not clear. In this study, a new IRF gene (CfIRF2) was identified in the Zhikong scallop, Chlamys farreri, and its immune function was analyzed. CfIRF2 has an open reading frame of 1107 bp encoding 368 amino acids. The N-terminus of CfIRF2 consists of a typical IRF domain, with conserved amino acid sequences. Phylogenetic analysis suggested close evolutionary relationship with shellfish IRF1 subfamily proteins. Expression pattern analysis showed that CfIRF2 mRNA was expressed in all tissues, with the highest expression in the hepatopancreas and gills. CfIRF2 gene expression was substantially enhanced by a pathogenic virus (such as acute viral necrosis virus) and poly(I:C) challenge. Co-immunoprecipitation assay identified CfIRF2 interaction with the IKKα/β family protein CfIKK1 of C. farreri, demonstrating a unique signal transduction mechanism in marine mollusks. Moreover, CfIRF2 interacted with itself to form homologous dimers. Overexpression of CfIRF2 in HEK293T cells activated reporter genes containing interferon stimulated response elements and NF-κB genes in a dose-dependent manner and promoted the phosphorylation of protein kinases (JNK, Erk1/2, and P38). Our results provide insights into the functions of IRF in mollusks innate immunity and also provide valuable information for enriching comparative immunological theory for the prevention of diseases in scallop farming.

Role of Different Members of the AGPAT Gene Family in Milk Fat Synthesis in Bubalus bubalis.

During triacylglycerol synthesis, the acylglycerol-3-phosphate acyltransferase (AGPAT) family catalyzes the conversion of lysophosphatidic acid to phosphatidic acid and the acylation of sn-2 fatty acids. However, the catalytic activity of different AGPAT members is different. Therefore, this study aimed to investigate the mechanism through which different AGPATs affect the efficiency of TAG synthesis and fatty acid composition. The conservation of amino acid sequences and protein domains of the AGPAT family was analyzed, and the functions of AGPAT1, AGPAT3, and AGPAT4 genes in buffalo mammary epithelial cells (BMECs) were studied using RNA interference and gene overexpression. Prediction of the protein tertiary structure of the AGPAT family demonstrated that four conservative motifs (motif1, motif2, motif3, and motif6) formed a hydrophobic pocket in AGPAT proteins, except AGPAT6. According to cytological studies, AGPAT1, AGPAT3, and AGPAT4 were found to promote the synthesis and fatty acid compositions of triacylglycerol, especially UFA compositions of triacylglycerol, by regulating ACSL1, FASN, GPAM, DGAT2, and PPARG gene expression. This study provides new insights into the role of different AGPAT gene family members involved in TAG synthesis, and a reference for improving the fatty acid composition of milk.

Functional Roles of the Conserved Amino Acid Sequence Motif C, the Antiporter Motif, in Membrane Transporters of the Major Facilitator Superfamily.

The biological membrane surrounding all living cells forms a hydrophobic barrier to the passage of biologically important molecules. Integral membrane proteins called transporters circumvent the cellular barrier and transport molecules across the cell membrane. These molecular transporters enable the uptake and exit of molecules for cell growth and homeostasis. One important collection of related transporters is the major facilitator superfamily (MFS). This large group of proteins harbors passive and secondary active transporters. The transporters of the MFS consist of uniporters, symporters, and antiporters, which share similarities in structures, predicted mechanism of transport, and highly conserved amino acid sequence motifs. In particular, the antiporter motif, called motif C, is found primarily in antiporters of the MFS. The antiporter motif's molecular elements mediate conformational changes and other molecular physiological roles during substrate transport across the membrane. This review article traces the history of the antiporter motif. It summarizes the physiological evidence reported that supports these biological roles.

Alkyl Sulfatase of Cholera Vibrios

The aim of the work was to study the structure of the alkyl sulfatase (asu) gene in Vibrio cholerae strains of various serogroups, as well as to compare nucleotide and amino acid sequences of alkyl sulfatases using various methods of bioinformatic analysis.Materials and methods. 483 strains of V. cholerae O1, O139 and nonO1/nonO139 serogroups were employed in the work. The search for the gene, its recurrence, and localization was carried out applying the Blast software. The nucleotide and corresponding amino acid sequences of the gene, as well as its structure, were studied using bioinformatic analysis. Sequencing was performed on the MiSeq (Illumina) platform. The enzymatic activity was detected using a medium, confirming the presence/absence of the gene by PCR in vitro and in silico.Results and discussion. Bioinformatic analysis of the nucleotide and corresponding amino acid sequences of the asu gene has been carried out and its structure investigated. Four functional domains have been identified. In the beta-lactamase domain, a conservative amino acid sequence -HAHADH- has been found in all strains of cholera vibrios, which is part of the Zn2+ binding motif. It has been established that the alkyl sulfatase of cholera vibrios belongs to the family of Zn2+-dependent β-lactamases. Blast analysis has revealed the similarity of nucleotide and amino acid sequences of alkyl sulfatases in representatives of V. cholerae O1 and O139 serogroups (ctxAB+tcpA+) and representatives of the genera Aeromonas and Pseudomonas, which is in the line with the data of 3D modeling of the amino acid sequence structures of the alkyl sulfatase enzyme in these microorganisms. The bioinformatic analysis of nucleotide and amino acid sequences of alkyl sulfatases in cholera vibrios has showed the conservativeness of these sequences in toxigenic strains and the presence of a number of single mutations in the asu gene in atoxigenic ones. The presence or absence of the asu gene has been established by PCR in vitro and in silico and confirmed by the results obtained using the Blast program. It is demonstrated that the presence/absence of the asu gene correlates with the ability/inability of O139 strains to hydrolyze SDS on the medium. These results can be used in studying mechanisms of cholera vibrios adaptation, persistence and pathogenicity.

RNA viruses of Crithidia bombi, a parasite of bumblebees

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi – a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

Allosteric modulation of SHP2: Quest from known to unknown.

Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) is a key regulatory factor in the cell cycle and its activating mutations play an important role in the development of various cancers, making it an important target for antitumor drugs. Due to the highly conserved amino acid sequence and positively charged nature of the active site of SHP2, it is difficult to discover inhibitors with high affinity for the catalytic site of SHP2 and sufficient cell permeability, making it considered an "undruggable" target. However, the discovery of allosteric regulation mechanisms provides new opportunities for transforming undruggable targets into druggable ones. Given the limitations of orthosteric inhibitors, SHP2 allosteric inhibitors have become a more selective and safer research direction. In this review, we elucidate the oncogenic mechanism of SHP2 and summarize the discovery methods of SHP2 allosteric inhibitors, providing new strategies for the design and improvement of SHP2 allosteric inhibitors.

NOX family NADPH oxidases in mammals: Evolutionary conservation and isoform-defining sequences

NADPH oxidases are superoxide-producing enzymes that play a role in host defense, biosynthetic pathways, as well as cellular signaling. Humans have 7 NOX isoforms (NOX1-5, DUOX1,2), while mice and rats lack NOX5 and therefore have only 6 NOX isoforms. Whether all human NOX isoforms or their subunits (CYBA, NCF1, 2, 4, NOXO1, NOXA1, DUOXA1, 2) are present and conserved in other mammalian species is unknown. In this study, we have analyzed the conservation of the NOX family during mammalian evolution using an in-silico approach. Complete genomic sequences of 164 mammalian species were available. The possible absence of genes coding for NOX isoforms was investigated using the NCBI orthologs database followed by manual curation. Conservation of a given NOX isoform during mammalian evolution was evaluated by multiple alignment and identification of highly conserved sequences. There was no convincing evidence for the absence of NOX2, 3, 4, and DUOX1, 2 in all the available mammalian genome. However, NOX5 was absent in 27 of 31 rodent, in 2 of 3 lagomorph and in 2 out of 18 bat species. NOX1 was absent in all sequenced Afrotheria and Monotremata species, as well as in 3 of 18 bat species. NOXA1 was absent in all Afrotheria and in 3 out of 4 Eulipotyphla species. We also investigated amino acid sequence conservation among given NOX isoforms. Highly conserved sequences were observed for most isoforms except for NOX5. Interestingly, the highly conserved region of NOX2 sequence was relatively small (11 amino acids), as compared to NOX1, 3, 4.The highly conserved domains are different from one NOX isoform to the other, raising the possibility of distinct evolutionary conserved functional domains. Our results shed a new light on the essentiality of different NOX isoforms. We also identified isoform-defining sequences, i.e., hitherto undescribed conserved domains within specific NOX isoforms.

Advancing evolution: Bacteria break down gene silencer to express horizontally acquired genes.

Horizontal gene transfer advances bacterial evolution. To benefit from horizontally acquired genes, enteric bacteria must overcome silencing caused when the widespread heat-stable nucleoid structuring (H-NS) protein binds to AT-rich horizontally acquired genes. This ability had previously been ascribed to both anti-silencing proteins outcompeting H-NS for binding to AT-rich DNA and RNA polymerase initiating transcription from alternative promoters. However, we now know that pathogenic Salmonella enterica serovar Typhimurium and commensal Escherichia coli break down H-NS when this silencer is not bound to DNA. Curiously, both species use the same protease - Lon - to destroy H-NS in distinct environments. Anti-silencing proteins promote the expression of horizontally acquired genes without binding to them by displacing H-NS from AT-rich DNA, thus leaving H-NS susceptible to proteolysis and decreasing H-NS amounts overall. Conserved amino acid sequences in the Lon protease and H-NS cleavage site suggest that diverse bacteria degrade H-NS to exploit horizontally acquired genes.

Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50°C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50°C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells1

Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction. However, its expression and function in chickens are still unclear. In the present study, ovarian tissues from 30 w and 15 w laying chickens were analyzed using RNA-seq to identify the differentially expressed gene TFPI2. The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs. In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT–PCR) and Western blotting. Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at different developmental states, such as primordial follicles, the ovarian stroma, and the granulosa and theca layers of prehierarchical follicles (6-8 mm) and preovulatory follicles (F1). In vitro, Follicle stimulating hormone or Luteinizing hormone (FSH/LH) stimulated the expression of TFPI2 in chicken granulosa cells. FSH-/LH-induced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways. Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes (STAR, Cyp17a1, Cyp19a1 and 3B-HSD) as well as MMPs (MMP2, 7, 9 and 11). Mechanistically, TFPI2 inhibited the expression of MMP7 via the Wnt signaling pathway. These findings indicate that TFPI2 may play an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase calcineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.

Genome-wide identification of Alfin Like (AL) transcription factors and their regulatory role in abiotic stress responses in Poplar (Populus trichocarpa)

Poplar is a valuable natural resource that is extensively utilized to manufacture many types of paper and wood products. It has considerable economic and ecological importance. However, forest dieback caused by environmental stressors has been increasing in recent years. Abiotic stress is managed by the Alfin-like (AL) gene in plants. The highly conserved DUF3594 domain and PHD-finger motif, which belong to the family Alfin-like transcription factors (AL-TFs), are essential for the metabolic and physiological reactions of plants to abiotic stress. Although the environmental stress sensitivity of AL-TFs has not yet been elucidated, these TFs play a vital role in regulating plant growth. We performed a database search and discovered 9 AL TFs. Using the AL-protein sequences from a variety of plant species, gene structures as well as conserved amino acid sequences, cis-elements of promoter regions, and events in the evolution of genes were examined. A phylogenetic tree was constructed and divided into six distinct groups. The expression of PtAL genes was specific to certain organs, and their gene mapping reveled that these genes are situated on seven out of the nineteen chromosomes present in Populus trichocarpa. Moreover, the majority of PtAL genes were strongly expressed in response to abiotic conditions such as low temperature (4 °C), high temperature (39 °C), salt (100 mM NaCl solution), and drought (Water holding). Two PtAL genes (PtAL4 and PtAL6) were sensitive to all abiotic stressors evaluated. The highly expressed PtAL genes (PtAL5 and PtAL7) discovered in this study could be employed in the Populus breeding program to generate stress-resistant cultivars. Our research shows that PtAL genes are very important for plant growth and development and for their ability to cope with environmental stress. This discovery offers valuable insights for improving plant stress tolerance.

The transcription factor Ste12-like increases the mycelial abiotic stress tolerance and regulates the fruiting body development of Flammulina filiformis.

Flammulina filiformis is one of the most commercially important edible fungi worldwide, with its nutritional value and medicinal properties. It becomes a good model species to study the tolerance of abiotic stress during mycelia growth in edible mushroom cultivation. Transcription factor Ste12 has been reported to be involved in the regulation of stress tolerance and sexual reproduction in fungi. In this study, identification and phylogenetic analysis of ste12-like was performed by bioinformatics methods. Four ste12-like overexpression transformants of F. filiformis were constructed by Agrobacterium tumefaciens-mediated transformation. Phylogenetic analysis showed that Ste12-like contained conserved amino acid sequences. All the overexpression transformants were more tolerant to salt stress, cold stress and oxidative stress than wild-type strains. In the fruiting experiment, the number of fruiting bodies of overexpression transformants increased compared with wild-type strains, but the growth rate of stipes slowed down. It suggested that gene ste12-like was involved in the regulation of abiotic stress tolerance and fruiting body development in F. filiformis.

Combining In Vitro and In Silico New Approach Methods to Investigate Type 3 Iodothyronine Deiodinase Chemical Inhibition Across Species.

New approach methodologies (NAMs) are being developed to reduce and replace vertebrate animal testing in support of ecotoxicology and risk assessment. The US Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) bioinformatic tool was used to evaluate amino acid sequence conservation of the type 3 iodothyronine deiodinase (DIO3) enzyme across species to demonstrate NAM applications for understanding effects of chemical interactions with a specific protein target. Existing literature was used to identify critical amino acids for thyroid hormone binding and interaction with a reducing cofactor. The SeqAPASS tool identifies whether known critical amino acids involved in ligand binding are exact, partial, or not matches across species compared with a template species based on molecular weight and side chain classification. This evaluation guided the design of variant proteins representing critical amino acid substitutions found in various species. Site-directed mutagenesis of the wild-type (WT) human DIO3 gene sequence was used to create six variant proteins expressed in cell culture, which were then tested in vitro for chemical inhibition. Significant differences in in vitro median inhibitory concentration results were observed among variants for potential competitive inhibitors. A molecular model representing the WT human DIO3 was constructed using Molecular Operating Environment (MOE) software and mutated in silico to create the six variants. The MOE Site Finder tool identified the proposed catalytic and cofactor sites and potential alternative binding sites. Virtual docking did not provide affinity scores with sufficient resolution to rank the potency of the chemical inhibitors. Chemical characteristics, function and location of substituted amino acids, and complexities of the protein target are important considerations in developing NAMs to evaluate chemical susceptibility across species. Environ Toxicol Chem 2023;42:1032-1048. © 2023 University of Wisconsin-Madison. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Rotary properties of hybrid F1-ATPases consisting of subunits from different species

F1-ATPase (F1) is an ATP-driven rotary motor protein ubiquitously found in many species as the catalytic portion of FoF1-ATP synthase. Despite the highly conserved amino acid sequence of the catalytic core subunits: α and β, F1 shows diversity in the maximum catalytic turnover rate Vmax and the number of rotary steps per turn. To study the design principle of F1, we prepared eight hybrid F1s composed of subunits from two of three genuine F1s: thermophilic Bacillus PS3 (TF1), bovine mitochondria (bMF1), and Paracoccus denitrificans (PdF1), differing in the Vmax and the number of rotary steps. The Vmax of the hybrids can be well fitted by a quadratic model highlighting the dominant roles of β and the couplings between α-β. Although there exist no simple rules on which subunit dominantly determines the number of steps, our findings show that the stepping behavior is characterized by the combination of all subunits.

Fusarium mindanaoense sp. nov., a New Fusarium Wilt Pathogen of Cavendish Banana from the Philippines Belonging to the F. fujikuroi Species Complex.

The pathogen causing Fusarium wilt in banana is reported to be Fusarium oxysporum f. sp. cubense (FOC). In 2019, wilt symptoms in banana plants (cultivar: Cavendish) in the Philippines were detected, i.e., the yellowing of the leaves and discoloration of the pseudostem and vascular tissue. The fungus isolated from the vascular tissue was found to be pathogenic to Cavendish bananas and was identified as a new species, F. mindanaoense, belonging to the F. fujikuroi species complex (FFSC); species classification was assessed using molecular phylogenetic analyses based on the tef1, tub2, cmdA, rpb1, and rpb2 genes and morphological analyses. A reciprocal blast search using genomic data revealed that this fungus exclusively included the Secreted in Xylem 6 (SIX6) gene among the SIX homologs related to pathogenicity; it exhibited a highly conserved amino acid sequence compared with that of species in the FFSC, but not with that of FOC. This was the first report of Fusarium wilt in Cavendish bananas caused by a species of the genus Fusarium other than those in the F. oxysporum species complex.

Upstream open reading frame-mediated upregulation of ANAC082 expression in response to nucleolar stress in Arabidopsis.

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5'-untranslated region (5'-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 expression in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5'-UTR were treated with reagents that induced nucleolar stress, expression of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent repressive effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent regulation was lost in ANAC103 during evolution.

Evolutionary conservation of sequence motifs at sites of protein modification

Gene duplications are common in biology and are likely to be an important source of functional diversification and specialization. The yeast Saccharomyces cerevisiae underwent a whole-genome duplication event early in evolution, and a substantial number of duplicated genes have been retained. We identified more than 3500 instances where only one of two paralogous proteins undergoes posttranslational modification despite having retained the same amino acid residue in both. We also developed a web-based search algorithm (CoSMoS.c.) that scores conservation of amino acid sequences based on 1011 wild and domesticated yeast isolates and used it to compare differentially modified pairs of paralogous proteins. We found that the most common modifications-phosphorylation, ubiquitylation, and acylation but not N-glycosylation-occur in regions of high sequence conservation. Such conservation is evident even for ubiquitylation and succinylation, where there is no established 'consensus site' for modification. Differences in phosphorylation were not associated with predicted secondary structure or solvent accessibility but did mirror known differences in kinase-substrate interactions. Thus, differences in posttranslational modification likely result from differences in adjoining amino acids and their interactions with modifying enzymes. By integrating data from large-scale proteomics and genomics analysis, in a system with such substantial genetic diversity, we obtained a more comprehensive understanding of the functional basis for genetic redundancies that have persisted for 100 million years.