scholarly journals Different Roles of DosS and DosT in the Hypoxic Adaptation of Mycobacteria

2010 ◽  
Vol 192 (19) ◽  
pp. 4868-4875 ◽  
Author(s):  
Min-Ju Kim ◽  
Kwang-Jin Park ◽  
In-Jeong Ko ◽  
Young Min Kim ◽  
Jeong-Il Oh
Keyword(s):  
Oxygen Tension ◽  
Response Regulator ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
High Sequence Similarity ◽  
Hypoxic Adaptation ◽  
The Difference ◽  
A Domains

ABSTRACT The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.

scholarly journals Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification of the guanylyltransferase-encoding segment

2007 ◽  
Vol 88 (1) ◽  
pp. 342-350 ◽  
Author(s):  
S. Supyani ◽  
Bradley I. Hillman ◽  
Nobuhiro Suzuki
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Expression System ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Sequence Alignments ◽  
High Sequence Similarity ◽  
Chestnut Blight Fungus ◽  
A Genome ◽  
Hypovirulent Strain

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1–S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1–S11 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [α-32P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133–667. Within this region, a sequence stretch (aa 170–250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltiviruses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of ‘turreted’ reoviruses, these results supported the a/vxxHx8Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.

scholarly journals The TFIID Components Human TAFII140 andDrosophila BIP2 (TAFII155) Are Novel Metazoan Homologues of Yeast TAFII47 Containing a Histone Fold and a PHD Finger

2001 ◽  
Vol 21 (15) ◽  
pp. 5109-5121 ◽  
Author(s):  
Yann-Gaël Gangloff ◽  
Jean-Christophe Pointud ◽  
Sylvie Thuault ◽  
Lucie Carré ◽  
Christophe Romier ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Protein A ◽  
Amino Acid Sequences ◽  
Molecular Organization ◽  
Phd Finger ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
High Degree

ABSTRACT The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAFIIs). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAFIIs and overall molecular organization. In recent years, it has been assumed that all the metazoan TAFIIs have been identified, yet no metazoan homologues of yeast TAFII47 (yTAFII47) and yTAFII65 are known. Both of these yTAFIIs contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAFII25. We have cloned a novel mouse protein, TAFII140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAFII140 shows extensive sequence similarity toDrosophila BIP2 (dBIP2) (dTAFII155), which we also show to be a component of DrosophilaTFIID. These proteins are metazoan homologues of yTAFII47 as their HFDs selectively heterodimerize with dTAFII24 and human TAFII30, metazoan homologues of yTAFII25. We further show that yTAFII65 shares two domains with theDrosophila Prodos protein, a recently described potential dTAFII. These conserved domains are critical for yTAFII65 function in vivo. Our results therefore identify metazoan homologues of yTAFII47 and yTAFII65.

scholarly journals N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert

Plants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 312 ◽  
Author(s):  
Vanessa Vieira ◽  
Bruno Peixoto ◽  
Mónica Costa ◽  
Susana Pereira ◽  
José Pissarra ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Research Gaps ◽  
Glycosylation Pattern ◽  
High Sequence Similarity ◽  
Post Translational Modifications ◽  
Vacuolar Sorting ◽  
Prevacuolar Compartment ◽  
Cardosin A ◽  
Comprehensive Study

In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features

1999 ◽  
Vol 181 (18) ◽  
pp. 5734-5741 ◽  
Author(s):  
Inessa Lysnyansky ◽  
Konrad Sachse ◽  
Ricardo Rosenbusch ◽  
Sharon Levisohn ◽  
David Yogev
Keyword(s):  
High Frequency ◽  
Sequence Similarity ◽  
Membrane Surface ◽  
Amino Acid Sequences ◽  
High Rate ◽  
Phenotypic Switching ◽  
Upstream Region ◽  
Mycoplasma Bovis ◽  
High Sequence Similarity ◽  
Polypeptide Structure

ABSTRACT Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogenMycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copyvsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of eachvsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the variousvsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.

scholarly journals Identification and Functional and Spectral Characterization of a Globin-coupled Histidine Kinase from Anaeromyxobacter sp. Fw109-5

2011 ◽  
Vol 286 (41) ◽  
pp. 35522-35534 ◽  
Author(s):  
Kenichi Kitanishi ◽  
Kazuo Kobayashi ◽  
Takeshi Uchida ◽  
Koichiro Ishimori ◽  
Jotaro Igarashi ◽  
...  
Keyword(s):  
Histidine Kinase ◽  
Oxygen Sensor ◽  
Response Regulator ◽  
Dissociation Rate ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Significant Activity ◽  
Distal Side ◽  
Two Component Signal Transduction ◽  
First Time

Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme:protein, 1:1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O2, and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O2 binding site at the heme distal side. The O2 dissociation rate constant, 0.10 s−1, was substantially increased up to 8.0 s−1 upon Y45L mutation. The resonance Raman frequencies representing νFe-O2 (559 cm−1) and νO-O (1149 cm−1) of the Fe(II)-O2 complex of Y45F mutant AfGcHK were distinct from those of the wild-type protein (νFe-O2, 557 cm−1; νO-O, 1141 cm−1), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.

scholarly journals Crystal structure of an alleged mannosylphosphate transferase Ktr6p at 3.06 Å

2014 ◽  
Vol 70 (a1) ◽  
pp. C1671-C1671
Author(s):  
Dmitry Rodionov ◽  
Daniella Marks ◽  
Pedro Romero ◽  
Albert Berghuis
Keyword(s):  
Room Temperature ◽  
Sequence Similarity ◽  
Transferase Activity ◽  
Molecular Replacement ◽  
Sequence Identity ◽  
Mannose Binding ◽  
The Difference ◽  
Resolution Dataset ◽  
Close Homolog

Ktr6p is an alleged mannosylphosphate transferase from yeast Golgi. It has been implicated in decorating both O-linked and N-lined glycans with mannosylphosphate in vivo. However, based on sequence similarity, Ktr6p belongs to GT15 family of α-1,2-mannosyltransferases. To address this disagreement, the soluble portion of Ktr6p was expressed in P. pastoris and purified by liquid chromatography. The purified protein, GDP-mannose and various acceptors were used in a number of direct and indirect activity assays, however, neither manosyltransferase nor mannosylphosphate transferase activity was detected. Ktr6p was crystallized in a number of PEG- containing conditions, but the crystals resisted all attempts at cryoprotection. Three crystals were used to collect a 3.06 Å resolution dataset on a home source at room temperature. The crystals belong to P 21 21 21 spacegroup with 2 molecules per asymmetric unit. The structure was solved by molecular replacement using a structure of Kre2p, a close homolog from GT15 family (40% sequence identity). The structure was refined to R/Rfree 16.1%/21.2% The overall structure of Ktr6p is very similar to the structure of Kre2p having less than 2 Å overall backbone RMSD. However even at 3 Å resolution the difference in the active site is striking. The guanine moiety binding pocked is occluded by a well-ordered loop making GDP-mannose binding impossible in this conformation. Several aminoacid substitutions in the Mn2+ coordinating environment suggest that Ktr6 does not depend on manganese for its postulated activity. These observations indicate that Ktr6p functions quite differently from Kre2p.

scholarly journals Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe

2020 ◽  
Vol 21 (23) ◽  
pp. 9062
Author(s):  
Barbara Kędzierska ◽  
Katarzyna Potrykus ◽  
Agnieszka Szalewska-Pałasz ◽  
Beata Wodzikowska
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Sequence Similarity ◽  
In Vitro Transcription ◽  
High Sequence Similarity ◽  
Sequence Composition ◽  
Repressor Complex ◽  
Transcriptional Fusions

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

scholarly journals NDE1 and NDEL1: twin neurodevelopmental proteins with similar ‘nature’ but different ‘nurture’

2013 ◽  
Vol 4 (5) ◽  
pp. 447-464 ◽  
Author(s):  
Nicholas J. Bradshaw ◽  
William Hennah ◽  
Dinesh C. Soares
Keyword(s):  
Neurodevelopmental Disorders ◽  
Sequence Similarity ◽  
Review Of The Literature ◽  
Post Translational Modification ◽  
Similar Nature ◽  
High Sequence Similarity ◽  
Basic Functions ◽  
Key Aspects ◽  
Paralogous Proteins

AbstractNuclear distribution element 1 (NDE1, also known as NudE) and NDE-like 1 (NDEL1, also known as Nudel) are paralogous proteins essential for mitosis and neurodevelopment that have been implicated in psychiatric and neurodevelopmental disorders. The two proteins possess high sequence similarity and have been shown to physically interact with one another. Numerous lines of experimental evidence in vivo and in cell culture have demonstrated that these proteins share common functions, although instances of differing functions between the two have recently emerged. We review the key aspects of NDE1 and NDEL1 in terms of recent advances in structure elucidation and cellular function, with an emphasis on their differing mechanisms of post-translational modification. Based on a review of the literature and bioinformatics assessment, we advance the concept that the twin proteins NDE1 and NDEL1, while sharing a similar ‘nature’ in terms of their structure and basic functions, appear to be different in their ‘nurture’, the manner in which they are regulated both in terms of expression and of post-translational modification within the cell. These differences are likely to be of significant importance in understanding the specific roles of NDE1 and NDEL1 in neurodevelopment and disease.

scholarly journals Isolation of Cysteine-Rich Peptides from Citrullus colocynthis

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1326
Author(s):  
Behzad Shahin-Kaleybar ◽  
Ali Niazi ◽  
Alireza Afsharifar ◽  
Ghorbanali Nematzadeh ◽  
Reza Yousefi ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
High Performance ◽  
De Novo ◽  
Sequence Similarity ◽  
In Silico Analysis ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Peptide Fragments ◽  
Citrullus Colocynthis ◽  
High Sequence Similarity

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

scholarly journals A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx.

1989 ◽  
Vol 9 (7) ◽  
pp. 3117-3121 ◽  
Author(s):  
P Staeheli ◽  
Y X Yu ◽  
R Grob ◽  
O Haller
Keyword(s):  
Influenza Virus ◽  
Resistance Gene ◽  
Virus Resistance ◽  
Genomic Dna ◽  
Sequence Similarity ◽  
Perca Fluviatilis ◽  
High Sequence Similarity ◽  
Double Stranded Rna ◽  
Virus Resistance Gene

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.

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