scholarly journals SPM1 Stabilizes Subpellicular Microtubules in Toxoplasma gondii

2011 ◽  
Vol 11 (2) ◽  
pp. 206-216 ◽  
Author(s):  
Johnson Q. Tran ◽  
Catherine Li ◽  
Alice Chyan ◽  
Lawton Chung ◽  
Naomi S. Morrissette
Keyword(s):  
Amino Acid ◽  
Toxoplasma Gondii ◽  
Protozoan Parasite ◽  
Content Type ◽  
Amino Terminal ◽  
Novel Proteins ◽  
Amino Acid Repeats ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

ABSTRACTWe have identified two novel proteins that colocalize with thesubpellicularmicrotubules in the protozoan parasiteToxoplasma gondiiand named these proteins SPM1 and SPM2. These proteins have basic isoelectric points and both have homologs in other apicomplexan parasites. SPM1 contains six tandem copies of a 32-amino-acid repeat, whereas SPM2 lacks defined protein signatures. Alignment ofToxoplasmaSPM2 with apparentPlasmodiumSPM2 homologs indicates that the greatest degree of conservation lies in the carboxy-terminal half of the protein. Analysis ofPlasmodiumhomologs of SPM1 indicates that while the central 32-amino-acid repeats have expanded to different degrees (7, 8, 9, 12, or 13 repeats), the amino- and carboxy-terminal regions remain conserved. In contrast, although theCryptosporidiumSPM1 homolog has a conserved carboxy tail, the five repeats are considerably diverged, and it has a smaller amino-terminal domain. SPM1 is localized along the full length of the subpellicular microtubules but does not associate with the conoid or spindle microtubules. SPM2 has a restricted localization along the middle region of the subpellicular microtubules. Domain deletion analysis indicates that four or more copies of the SPM1 repeat are required for localization to microtubules, and the amino-terminal 63 residues of SPM2 are required for localization to the subpellicular microtubules. Gene deletion studies indicate that neither SPM1 nor SPM2 is essential for tachyzoite viability. However, loss of SPM1 decreases overall parasite fitness and eliminates the stability of subpellicular microtubules to detergent extraction.

Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

scholarly journals Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains.

1989 ◽  
Vol 9 (8) ◽  
pp. 3418-3428 ◽  
Author(s):  
W Gu ◽  
N J Cowan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tubulin Isotype ◽  
The Stability ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo.

scholarly journals Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210.

1993 ◽  
Vol 178 (6) ◽  
pp. 2237-2242 ◽  
Author(s):  
R E Nickowitz ◽  
H J Worman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Primary Biliary Cirrhosis ◽  
Antigenic Determinants ◽  
Nuclear Pore ◽  
Biliary Cirrhosis ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Patients with primary biliary cirrhosis (PBC) frequently have autoantibodies against a 210-kD integral glycoprotein of the nuclear envelope pore membrane. This protein, termed gp210, has a 1,783-amino acid amino-terminal domain located in the perinuclear space, a 20-amino acid transmembrane segment, and a 58-amino acid cytoplasmic carboxy-terminal tail. We now demonstrate that autoantibodies from 25 patients with PBC that recognize gp210 react with the cytoplasmic carboxy-terminal tail while none react with unmodified linear epitopes in the amino-terminal domain. The epitope(s) recognized by autoantibodies from all 25 patients is contained within a stretch of 15 amino acids. The recognized amino acid sequence is homologous to the protein products of the Escherichia coli mutY gene and Salmonella typhimurium mutB gene with an exact identity of six consecutive amino acids, suggesting that anti-gp210 antibodies may arise by molecular mimicry of bacterial antigenic determinants.

scholarly journals Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.

1991 ◽  
Vol 11 (1) ◽  
pp. 63-74 ◽  
Author(s):  
P M Lieberman ◽  
M C Schmidt ◽  
C C Kao ◽  
A J Berk
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Conformational Change ◽  
Tata Box ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.

Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change

1991 ◽  
Vol 11 (1) ◽  
pp. 63-74
Author(s):  
P M Lieberman ◽  
M C Schmidt ◽  
C C Kao ◽  
A J Berk
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Conformational Change ◽  
Tata Box ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.

scholarly journals Hsp60 Is Targeted to a Cryptic Mitochondrion-Derived Organelle (“Crypton”) in the Microaerophilic Protozoan Parasite Entamoeba histolytica

1999 ◽  
Vol 19 (3) ◽  
pp. 2198-2205 ◽  
Author(s):  
Zhiming Mai ◽  
Sudip Ghosh ◽  
Marta Frisardi ◽  
Ben Rosenthal ◽  
Rick Rogers ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Entamoeba Histolytica ◽  
Protozoan Parasite ◽  
Coding Region ◽  
Amino Terminal ◽  
Chaperonin 60 ◽  
Carboxy Terminal ◽  
Alcohol Dehydrogenase 1

ABSTRACT Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of anE. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42°C. 5′ and 3′ rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shockEscherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolyticaHsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named “crypton,” as its structure was previously hidden and its function is still cryptic.

scholarly journals Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Xiaoyu Hu ◽  
William J. O’Shaughnessy ◽  
Tsebaot G. Beraki ◽  
Michael L. Reese
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Kinases ◽  
Daughter Cell ◽  
Protozoan Parasite ◽  
Productive Infection ◽  
Centrosome Duplication ◽  
Loss Of Function ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Parasite Replication

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

Distinct domains of a nucleolar protein mediate protein kinase binding, interaction with nucleic acids and nucleolar localization

1998 ◽  
Vol 111 (17) ◽  
pp. 2615-2623 ◽  
Author(s):  
A. Das ◽  
J.H. Park ◽  
C.B. Hagen ◽  
M. Parsons
Keyword(s):  
Protein Kinase ◽  
Nucleic Acids ◽  
Protozoan Parasite ◽  
In Vitro Transcription ◽  
Nucleic Acid Binding ◽  
Binding Interaction ◽  
Amino Terminal ◽  
Amino Acid Region ◽  
Carboxy Terminal

Nopp44/46 is a phosphoprotein of the protozoan parasite Trypanosoma brucei that is localized to the nucleolus. Based on the primary sequence, Nopp44/46 appears to be a protein composed of distinct domains. This communication describes the relationship of these domains to the known functional interactions of the molecule and suggests that the amino-terminal region defines a novel homology region that functions in nucleolar targeting. We have previously shown that Nopp44/46 is capable of interacting with nucleic acids and associating with a protein kinase. Using in vitro transcription and translation, we now demonstrate that the nucleic acid binding function maps to the carboxy-terminal domain of the molecule, a region rich in arginine-glycine-glycine motifs. Our experiments reveal that a central region containing a high proportion of acidic residues is required for association with the protein kinase. Analysis of transfectants expressing epitope-tagged Nopp44/46 deletion constructs showed that the amino-terminal 96 amino acids allowed nuclear and nucleolar accumulation of the protein. This region of the molecule shows homology to several recently described nucleolar proteins. Deletion of a 27-amino-acid region within this domain abrogated nucleolar, but not nuclear, localization. These studies show that Nopp44/46 is composed of distinct modules, each of which plays a different role in molecular interactions. We suggest that this protein could facilitate interactions between sets of nucleolar molecules.

scholarly journals The Protozoan Parasite Toxoplasma gondii Selectively Reprograms the Host Cell Translatome

2018 ◽  
Vol 86 (9) ◽  
Author(s):  
Louis-Philippe Leroux ◽  
Julie Lorent ◽  
Tyson E. Graber ◽  
Visnu Chaparro ◽  
Laia Masvidal ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Activation ◽  
Immune Cell ◽  
Mrna Translation ◽  
Protozoan Parasite ◽  
Translational Efficiency ◽  
Type I ◽  
Content Type ◽  
Infection Inhibition

ABSTRACT The intracellular parasite Toxoplasma gondii promotes infection by targeting multiple host cell processes; however, whether it modulates mRNA translation is currently unknown. Here, we show that infection of primary murine macrophages with type I or II T. gondii strains causes a profound perturbation of the host cell translatome. Notably, translation of transcripts encoding proteins involved in metabolic activity and components of the translation machinery was activated upon infection. In contrast, the translational efficiency of mRNAs related to immune cell activation and cytoskeleton/cytoplasm organization was largely suppressed. Mechanistically, T. gondii bolstered mechanistic target of rapamycin (mTOR) signaling to selectively activate the translation of mTOR-sensitive mRNAs, including those with a 5′-terminal oligopyrimidine (5′ TOP) motif and those encoding mitochondrion-related proteins. Consistent with parasite modulation of host mTOR-sensitive translation to promote infection, inhibition of mTOR activity suppressed T. gondii replication. Thus, selective reprogramming of host mRNA translation represents an important subversion strategy during T. gondii infection.

scholarly journals Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Tomokazu Tamura ◽  
Takasuke Fukuhara ◽  
Takuro Uchida ◽  
Chikako Ono ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Viral Life Cycle ◽  
Luciferase Gene ◽  
Content Type ◽  
Recombinant Viruses ◽  
Nanoluc Luciferase ◽  
The Stability ◽  
Split Luciferase

ABSTRACTThe familyFlaviviridaeconsists of four genera,Flavivirus,Pestivirus,Pegivirus, andHepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of theFlaviviridaeviruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamicsin vivo. Taken together, our findings indicate that the recombinantFlaviviridaeviruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals againstFlaviviridaeviruses.IMPORTANCEThe construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinantFlaviviridaeviruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable toin vitroandin vivoexperiments, suggesting that these recombinantFlaviviridaeviruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals againstFlaviviridaeviruses.

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