scholarly journals The Rib43a Protein Is Associated with Forming the Specialized Protofilament Ribbons of Flagellar Microtubules inChlamydomonas

2000 ◽  
Vol 11 (1) ◽  
pp. 201-215 ◽  
Author(s):  
Jan M. Norrander ◽  
Aimee M. deCathelineau ◽  
Jennifer A. Brown ◽  
Mary E. Porter ◽  
Richard W. Linck
Keyword(s):  
Homo Sapiens ◽  
Sequence Similarity ◽  
Germ Cell Tumors ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Dimensional Structure ◽  
Polymorphism Analysis ◽  
Basal Bodies ◽  
Coding Region ◽  
Expression Library

Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of tubulin and several associated proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon proteins astektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of tektins and other ribbon proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon protein rib43a. Using antibodies against rib43a to screen an expression library, we recovered a full-length cDNA clone that encodes a 42,657-Da polypeptide. On Northern blots, the rib43a cDNA hybridized to a 1.7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The cDNA was used to isolate RIB43a, an ∼4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43agene on linkage group III. Sequence analysis of theRIB43a gene indicates that the substantially coiled-coil rib43a protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens(genomic, normal fetal kidney, and endometrial and germ cell tumors) but little sequence similarity to other proteins including tektins. Affinity-purified antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure.

scholarly journals BLASTing away preconceptions in crystallization trials

2019 ◽  
Vol 75 (3) ◽  
pp. 184-192 ◽  
Author(s):  
Gabriel Jan Abrahams ◽  
Janet Newman
Keyword(s):  
Protein Data Bank ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Protein Sequences ◽  
Protein Molecule ◽  
Data Bank ◽  
Dimensional Structure ◽  
Critical Step ◽  
Quantitative Verification ◽  
Better Than

Crystallization is in many cases a critical step for solving the three-dimensional structure of a protein molecule. Determining which set of chemicals to use in the initial screen is typically agnostic of the protein under investigation; however, crystallization efficiency could potentially be improved if this were not the case. Previous work has assumed that sequence similarity may provide useful information about appropriate crystallization cocktails; however, the authors are not aware of any quantitative verification of this assumption. This research investigates whether, given current information, one can detect any correlation between sequence similarity and crystallization cocktails. BLAST was used to quantitate the similarity between protein sequences in the Protein Data Bank, and this was compared with three estimations of the chemical similarities of the respective crystallization cocktails. No correlation was detected between proteins of similar (but not identical) sequence and their crystallization cocktails, suggesting that methods of determining screens based on this assumption are unlikely to result in screens that are better than those currently in use.

Hemoglobin Biosynthesis in Vitreoscilla stercoraria DW: Cloning, Expression, and Characterization of a New Homolog of a Bacterial Globin Gene

1998 ◽  
Vol 64 (6) ◽  
pp. 2220-2228 ◽  
Author(s):  
Meenal Joshi ◽  
Shekhar Mande ◽  
Kanak L. Dikshit
Keyword(s):  
Globin Gene ◽  
Three Dimensional ◽  
Fold Increase ◽  
Binding Pocket ◽  
Growth Conditions ◽  
Dimensional Structure ◽  
Strictly Aerobic ◽  
Coding Region ◽  
Oxygen Control ◽  
Constitutive Production

ABSTRACT In the strictly aerobic, gram-negative bacteriumVitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure ofVitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla,V. stercoraria DW. Several silent changes were observed within the coding region of the V. stercoraria vgb gene. Apart from that, V. stercoraria Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation ofV. stercoraria Hb displays a slower autooxidation rate. The differences between Vitreoscilla Hb and V. stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within theV. stercoraria Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V. stercoraria Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscillastrain C1, production of Hb in V. stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V. stercoraria vgb promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species ofVitreoscilla. The relatively slower autooxidation rate ofV. stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V. stercorariaDW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.

scholarly journals Genome Wide Characterization & Phylogenetic Analysis of TNF Genes in Homo Sapiens

2021 ◽  
Vol 9 (2) ◽  
Author(s):  
Saif S ◽  
◽  
Mazhar MW ◽  
Sikandar M ◽  
Waqas N ◽  
...  
Keyword(s):  
Tumor Necrosis ◽  
Homo Sapiens ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Inflammatory Signaling ◽  
Genome Wide ◽  
Intron Structure ◽  
Tnf Gene ◽  
Necrosis Factor ◽  
Stimulation Of

Tumor Necrosis Factor is very important inflammatory signaling unit that do an important role in immune system. It functions by attachment and stimulation of different receptor’s Cysteine Rich Domains (CDRs). A number of TNF receptors mediated factors have been identified having a major role in signal transduction pathways of TNF gene family. There are about 18 TNF homologues that are identified as a major cause of many disorders like cancer, Diabetes, AIDS and many other lethal inflammations. In this study the genome wide identification of TNF gene was done. Different tools and databases were used. Identification of conserved domains was done by using pfam and homology analysis showed that the TNF might be a member of TRAFs superfamily. Structural analysis of gene showed the number of introns and exons by a three-dimensional structure of TNF gene. The TNF gene family's exon-intron structure was found to be very similar in this study. The distribution of genes across chromosomes, on the other hand, was extremely varied. Collectively, the newly discovered genes can provide a wealth of information for manipulating the TNF genome to develop drugs and strategies to treat a variety of diseases.

scholarly journals Molecular cloning of the rat analogue of human CD59: structural comparison with human CD59 and identification of a putative active site

1994 ◽  
Vol 304 (2) ◽  
pp. 595-601 ◽  
Author(s):  
N K Rushmere ◽  
R A Harrison ◽  
C W van den Berg ◽  
B P Morgan
Keyword(s):  
Molecular Cloning ◽  
Rat Kidney ◽  
Amino Acid Level ◽  
Three Dimensional ◽  
Glycosylation Site ◽  
Dimensional Structure ◽  
Flanking Sequence ◽  
Coding Region ◽  
Amino Terminal ◽  
Human Cd59

We have previously described the purification and partial characterization of the rat analogue of the human complement regulatory molecule CD59 [Hughes, Piddlesden, Williams, Harrison and Morgan (1992) Biochem. J. 284, 169-176]. We present here the molecular cloning and full sequence analysis of this molecule. A PCR-based approach utilizing primers designed from the amino-terminal protein sequence was used to isolate a full-length cDNA clone from a rat kidney cDNA library. This clone encoded a 92 bp 5′-flanking sequence, a 66 bp signal peptide and a 315 bp coding region containing putative glycosylation and GPI-anchor signals. The 3′ untranslated flanking region was approximately 1.1 kbp long and included the poly-A tail and a CATA repeating sequence. The coding region was 58% identical with the human cDNA at the nucleotide level and 44% identical at the amino acid level. Despite this relatively low overall sequence conservation, several highly conserved stretches were apparent, particularly in the N-terminal portion of the molecule, in the cysteine-rich region immediately preceding the site of glycolipid attachment and in the C-terminal peptide removed during glycolipid attachment. An N-glycosylation site was identified at Asn-16 and a putative glycosylphosphatidylinositol anchor addition site at Asn-79, indicating that the mature processed protein was two residues longer than human CD59. Comparison of the sequences of rat and human CD59, together with consideration of the published three-dimensional structure of human CD59 and functional data, implicates specific regions of the protein in interactions with C-8 and/or C-9.

scholarly journals BAR Domain-Containing FAM92 Proteins Interact with Chibby1 To Facilitate Ciliogenesis

2016 ◽  
Vol 36 (21) ◽  
pp. 2668-2680 ◽  
Author(s):  
Feng-Qian Li ◽  
Xingwang Chen ◽  
Cody Fisher ◽  
Saul S. Siller ◽  
Klara Zelikman ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Cultured Cells ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Basal Bodies ◽  
Membrane Remodeling ◽  
Bar Domains

Chibby1 (Cby1) is a small, conserved coiled-coil protein that localizes to centrioles/basal bodies and plays a crucial role in the formation and function of cilia. During early stages of ciliogenesis, Cby1 is required for the efficient recruitment of small vesicles at the distal end of centrioles to facilitate basal body docking to the plasma membrane. Here, we identified family with sequence similarity 92, member A (FAM92A) and FAM92B, which harbor predicted lipid-binding BAR domains, as novel Cby1-interacting partners using tandem affinity purification and mass spectrometry. We found that in cultured cell lines, FAM92A colocalizes with Cby1 at the centrioles/basal bodies of primary cilia, while FAM92B is undetectable. In airway multiciliated cells, both FAM92A and -92B colocalize with Cby1 at the base of cilia. Notably, the centriolar localization of FAM92A and -92B depends largely on Cby1. Knockdown of FAM92A in RPE1 cells impairs ciliogenesis. Consistent with the membrane-remodeling properties of BAR domains, FAM92A and -92B in cooperation with Cby1 induce deformed membrane-like structures containing the small GTPase Rab8 in cultured cells. Our results therefore suggest that FAM92 proteins interact with Cby1 to promote ciliogenesis via regulation of membrane-remodeling processes.

scholarly journals Hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi as a target for structure-based inhibitor design: crystallization and inhibition studies with purine analogs.

1997 ◽  
Vol 41 (8) ◽  
pp. 1686-1692 ◽  
Author(s):  
A E Eakin ◽  
A Guerra ◽  
P J Focia ◽  
J Torres-Martinez ◽  
S P Craig
Keyword(s):  
Trypanosoma Cruzi ◽  
De Novo ◽  
Homo Sapiens ◽  
Three Dimensional ◽  
New Drugs ◽  
Dimensional Structure ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Inhibitor Design ◽  
Purine Nucleotides ◽  
Purine Analogs

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi is a potential target for enzyme structure-based inhibitor design, based on previous studies which indicate that these parasites lack the metabolic enzymes required for de novo synthesis of purine nucleotides. By using a bacterial complement selection system, 59 purine analogs were assayed for their interaction with the HPRTs from T. cruzi and Homo sapiens. Eight compounds were identified from the bacterial assay to have an affinity for the trypanosomal enzyme. Inhibition constants for four of these compounds against purified recombinant trypanosomal and human HPRTs were determined and compared. The results confirm that the recombinant system can be used to identify compounds which have affinity for the trypanosomal HPRT. Furthermore, the results provide evidence for the importance of chemical modifications at positions 6 and 8 of the purine ring in the binding of these compounds to the HPRTs. An accurate three-dimensional structure of the trypanosomal enzyme will greatly enhance our understanding of the interactions between HPRTs and these compounds. Toward this end, crystallization conditions for the trypanosomal HPRT and preliminary analysis of X-ray diffraction data to a resolution of 2 A is reported. These results represent significant progress toward a structure-based approach to the design of inhibitors of the HPRT of trypanosomes with the long-range goal of developing new drugs for the treatment of Chagas' disease.

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

2014 ◽  
Vol 70 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Sonia Fieulaine ◽  
Michel Desmadril ◽  
Thierry Meinnel ◽  
Carmela Giglione
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Human Counterpart ◽  
Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

scholarly journals In-silico analysis of non-synonymous-SNPs of STEAP2: To provoke the progression of prostate cancer

2016 ◽  
Vol 11 (1) ◽  
pp. 402-416 ◽  
Author(s):  
Muhammad Naveed ◽  
Sana Tehreem ◽  
Shamsa Mubeen ◽  
Fareeha Nadeem ◽  
Fatima Zafar ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Three Dimensional ◽  
In Silico Analysis ◽  
Genome Project ◽  
Dimensional Structure ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Computational Tools ◽  
Epithelial Antigens ◽  
Novel Drug

AbstractAs a novel biomarker from the STEAP family, STEAP2 encodes six transmembrane epithelial antigens to prostate cancer. The overexpression of STEAP2 is predicted as the second most common cancer in the world that is responsible for male cancer-related deaths. Nonsynonymous SNPs are important group of SNPs which lead to alternations in encoded polypeptides. Changes in the amino acid sequence of gene products can lead to abnormal tissue function. The present study firstly sorted out those SNPs which exist in the coding region of STEAP2 and evaluated their impact through computational tools. Secondly, the three-dimensional structure of STEAP2 was formed through I-TASSER and validated by different software. Genomic data has been retrieved from the 1000 Genome project and Ensembl and subsequently analysed using computational tools. Out of 177 non-synonymous single nucleotide polymorphisms (nsSNPs) within the coding region, 42 mis-sense SNPs have been predicted as deleterious by all analyses. Our research shows a welldesigned computational methodology to inspect the prostate cancer associated nsSNPs. It can be concluded that these nsSNPs can play their role in the up-regulation of STEAP2 which further leads to progression of prostate cancer. It can benefit scientists in the handling of cancerassociated diseases related to STEAP2 through developing novel drug therapies.

scholarly journals NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

2007 ◽  
Vol 18 (2) ◽  
pp. 348-361 ◽  
Author(s):  
Patricia C. Abad ◽  
Jason Lewis ◽  
I. Saira Mian ◽  
David W. Knowles ◽  
Jennifer Sturgis ◽  
...  
Keyword(s):  
Mammary Epithelial Cells ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Mammary Epithelium ◽  
Chromatin Organization ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Deoxyribonuclease I ◽  
C Terminus

The coiled-coil protein NuMA is an important contributor to mitotic spindle formation and stabilization. A potential role for NuMA in nuclear organization or gene regulation is suggested by the observations that its pattern of nuclear distribution depends upon cell phenotype and that it interacts and/or colocalizes with transcription factors. To date, the precise contribution of NuMA to nuclear function remains unclear. Previously, we observed that antibody-induced alteration of NuMA distribution in growth-arrested and differentiated mammary epithelial structures (acini) in three-dimensional culture triggers the loss of acinar differentiation. Here, we show that in mammary epithelial cells, NuMA is present in both the nuclear matrix and chromatin compartments. Expression of a portion of the C terminus of NuMA that shares sequence similarity with the chromatin regulator HPC2 is sufficient to inhibit acinar differentiation and results in the redistribution of NuMA, chromatin markers acetyl-H4 and H4K20m, and regions of deoxyribonuclease I-sensitive chromatin compared with control cells. Short-term alteration of NuMA distribution with anti-NuMA C-terminus antibodies in live acinar cells indicates that changes in NuMA and chromatin organization precede loss of acinar differentiation. These findings suggest that NuMA has a role in mammary epithelial differentiation by influencing the organization of chromatin.

scholarly journals Pkg2, a Novel Transmembrane Protein Ser/Thr Kinase of Streptomyces granaticolor

1999 ◽  
Vol 181 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Richard Nádvorník ◽  
Tomáš Vomastek ◽  
Jiří Janeček ◽  
Zuzana Techniková ◽  
Pavel Branny
Keyword(s):  
Amino Acids ◽  
Tandem Repeats ◽  
Transmembrane Protein ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Chromosomal Dna ◽  
Serine Threonine Kinase ◽  
Significant Sequence Similarity ◽  
Threonine Kinase

ABSTRACT A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed inEscherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.

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