scholarly journals Ca 2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C

2015 ◽  
Vol 14 (5) ◽  
pp. 486-494 ◽  
Author(s):  
Sharon King-Keller ◽  
Christina A. Moore ◽  
Roberto Docampo ◽  
Silvia N. J. Moreno
Keyword(s):  
Enzymatic Activity ◽  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Calcium Binding ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Content Type ◽  
Ef Hand

ABSTRACT We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei . The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca 2+ concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ), with a higher affinity for PIP 2 . We found that modification of a single amino acid in the EF-hand motif greatly affected the protein's Ca 2+ sensitivity and substrate preference, demonstrating the role of this motif in Ca 2+ regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP 2 . Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP 2 and a contribution to the inositol 1,4,5-trisphosphate (IP 3 )/diacylglycerol (DAG) pathway.

scholarly journals The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton

2010 ◽  
Vol 9 (10) ◽  
pp. 1466-1475 ◽  
Author(s):  
Edith Luginbuehl ◽  
Damaris Ryter ◽  
Judith Schranz-Zumkehr ◽  
Michael Oberholzer ◽  
Stefan Kunz ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Trypanosoma Brucei ◽  
Cell Body ◽  
Intracellular Signaling ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Single Amino Acid ◽  
Content Type ◽  
Gfp Reporter ◽  
Camp Levels

ABSTRACT The precise subcellular localization of the components of the cyclic AMP (cAMP) signaling pathways is a crucial aspect of eukaryotic intracellular signaling. In the human pathogen Trypanosoma brucei, the strict control of cAMP levels by cAMP-specific phosphodiesterases is essential for parasite survival, both in cell culture and in the infected host. Among the five cyclic nucleotide phosphodiesterases identified in this organism, two closely related isoenzymes, T. brucei PDEB1 (TbrPDEB1) (PDEB1) and TbrPDEB2 (PDEB2) are predominantly responsible for the maintenance of cAMP levels. Despite their close sequence similarity, they are distinctly localized in the cell. PDEB1 is mostly located in the flagellum, where it forms an integral part of the flagellar skeleton. PDEB2 is mainly located in the cell body, and only a minor part of the protein localizes to the flagellum. The current study, using transfection of procyclic trypanosomes with green fluorescent protein (GFP) reporters, demonstrates that the N termini of the two enzymes are essential for determining their final subcellular localization. The first 70 amino acids of PDEB1 are sufficient to specifically direct a GFP reporter to the flagellum and to lead to its detergent-resistant integration into the flagellar skeleton. In contrast, the analogous region of PDEB2 causes the GFP reporter to reside predominantly in the cell body. Mutagenesis of selected residues in the N-terminal region of PDEB2 demonstrated that single amino acid changes are sufficient to redirect the reporter from a cell body location to stable integration into the flagellar skeleton.

scholarly journals The gene family of EF-hand calcium-binding proteins from the flagellum of Trypanosoma brucei

1994 ◽  
Vol 304 (3) ◽  
pp. 833-841 ◽  
Author(s):  
Y Wu ◽  
J Deford ◽  
R Benjamin ◽  
M G Lee ◽  
L Ruben
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Direct Repeat ◽  
Calcium Binding Proteins ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Genomic Clones ◽  
Ef Hand

The flagellum of Trypanosoma brucei contains calmodulin, and a separate family of antigenically related EF-hand calcium-binding proteins which we call calflagins. The following study evaluates the structure and genomic organization of the calflagin family. Genomic Southern blots indicated that multiple copies of calflagin genes occurred in T. brucei, and that all of these copies were contained in a single 23 kb XhoI-XhoI fragment on chromosomes 15 and 16 mRNAs of 1.2 and 1.6 kb were identified in bloodstream and procyclic life-cycle stages. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. The calflagin genes were arranged tandemly along the genomic fragments. Three new members of the calflagin family were sequenced from a cDNA clone and the two genomic clones. Two unrelated families of 3′ flanking sequences were downstream from the calflagin genes. An open reading frame that was unrelated to any calflagin sequence was at the 5′ end of the 2.5 kb genomic fragment. The deduced amino acid sequences of the genomic clones (called Tb-24 and Tb-1.7g) were similar to the previously described Tb-17. Each encoded an approximately 24 kDa protein which contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. The cDNA encoded a protein (called Tb-44A) which was approximately twice as large as the other calflagins. The large size resulted from a nearly direct repeat of 186 amino acids. In general, variability among the T. brucei calflagins was greater than observed for related proteins from Trypanosoma cruzi. We demonstrate that this variability resulted from amino acid substitutions at the N-terminus, C-terminal extensions, and duplication of internal segments.

scholarly journals An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

2015 ◽  
Vol 197 (16) ◽  
pp. 2622-2630 ◽  
Author(s):  
Neus Sanchez-Alberola ◽  
Susana Campoy ◽  
David Emerson ◽  
Jordi Barbé ◽  
Ivan Erill
Keyword(s):  
In Silico ◽  
Transcriptional Regulatory Network ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Binding Motifs ◽  
Content Type ◽  
Lexa Protein ◽  
Lexa Binding

ABSTRACTThe SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In theBetaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacteriumSideroxydans lithotrophicus.In silicoandin vitroanalyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of theBacillus subtilisLexA-binding motif. We confirm that the closely relatedGallionella capsiferriformansshares the same LexA-binding motif, andin silicoanalyses indicate that this motif is also conserved in theNitrosomonadalesand theMethylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within theBetaproteobacteria. However, theirlexAgene is unrelated to the standardBetaproteobacterialexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon ofS. lithotrophicusis comparable in size and function to that of many otherBetaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain ofS. lithotrophicusLexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make theB. subtilis-type motif an optimal interface for multiple LexA sequences.IMPORTANCEUnderstanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

Tuning the Equilibrium Ion Affinity and Selectivity of the EF-Hand Calcium Binding Motif:  Substitutions at the Gateway Position†

Biochemistry ◽  
1996 ◽  
Vol 35 (21) ◽  
pp. 6697-6705 ◽  
Author(s):  
Steven K. Drake ◽  
Keith L. Lee ◽  
Joseph J. Falke
Keyword(s):  
Calcium Binding ◽  
Binding Motif ◽  
Ef Hand

scholarly journals Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Yuki Honda ◽  
Qian Zang ◽  
Yasuhiro Shimizu ◽  
Mohammad Dadashipour ◽  
Zilian Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Thermostable Enzyme ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Content Type ◽  
Mutant Proteins ◽  
Sulfolobus Tokodaii ◽  
Site Saturation ◽  
Thermophilic Archaeon

ABSTRACT The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii. Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. IMPORTANCE It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.

scholarly journals CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

2011 ◽  
Vol 193 (22) ◽  
pp. 6276-6287 ◽  
Author(s):  
Boris R. Belitsky ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Binding Motif ◽  
Downstream Site ◽  
Upstream Site ◽  
Binding Motifs ◽  
Content Type ◽  
A Minor

CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons inBacillus subtilis. Some of the most strongly repressed targets of CodY, thenupNOPQ(formerly,yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of thenupNgene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6- to 8-foldnupNrepression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site tonupNregulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites.

scholarly journals Molecular Tuning of an EF-Hand-like Calcium Binding Loop

1997 ◽  
Vol 110 (2) ◽  
pp. 173-184 ◽  
Author(s):  
Steven K. Drake ◽  
Michael A. Zimmer ◽  
Craig Kundrot ◽  
Joseph J. Falke
Keyword(s):  
Calcium Binding ◽  
Ion Selectivity ◽  
Side Chain ◽  
Binding Motif ◽  
Side Chains ◽  
Binding Parameters ◽  
Size Selectivity ◽  
The Third ◽  
Ef Hand ◽  
Binding Loop

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.

scholarly journals Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions

2014 ◽  
Vol 13 (9) ◽  
pp. 1232-1240 ◽  
Author(s):  
Zhenqiu Huang ◽  
Sabine Kaltenbrunner ◽  
Eva Šimková ◽  
David Stanĕk ◽  
Julius Lukeš ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Yellow Fluorescent Protein ◽  
Rna Binding Protein ◽  
Mitochondrial Rna ◽  
Content Type ◽  
Petite Mutant

ABSTRACT There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei . Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo . In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi . The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

scholarly journals The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

2010 ◽  
Vol 30 (11) ◽  
pp. 2724-2736 ◽  
Author(s):  
Benoît Gilquin ◽  
Brian R. Cannon ◽  
Arnaud Hubstenberger ◽  
Boualem Moulouel ◽  
Elin Falk ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Nuclear Translocation ◽  
P53 Protein ◽  
Binding Domain ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Aaa Atpase ◽  
Calcium Dependent ◽  
Ef Hand ◽  
Cell Growth And Differentiation

ABSTRACT S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.

scholarly journals Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei

2015 ◽  
Vol 35 (23) ◽  
pp. 3945-3961 ◽  
Author(s):  
Suzanne M. McDermott ◽  
Jason Carnes ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Bloodstream Form ◽  
Single Amino Acid ◽  
Wild Type ◽  
Rnase Iii ◽  
Content Type ◽  
Life Cycle Stages

KREPB5 is an essential component of ∼20S editosomes inTrypanosoma bruceiwhich contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional nullT. bruceibloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ∼20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages ofT. bruceithat differentially edit mRNAs.

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