Characterization of two T. gondii CK1 isoforms
| Autor: | Laurent Meijer, Tanya Zhong, Robert G. K. Donald, Paul A. Liberator |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Gene isoform
Cytoplasm Blotting Western Molecular Sequence Data Antiprotozoal Agents Biology Leishmania mexicana law.invention 03 medical and health sciences 0302 clinical medicine Affinity chromatography law Cyclin-dependent kinase Casein Kinase I parasitic diseases Animals Immunoprecipitation Amino Acid Sequence Cloning Molecular Enzyme Inhibitors Molecular Biology 030304 developmental biology 0303 health sciences Adenine Cell Membrane Toxoplasma gondii Casein Kinase Ialpha biology.organism_classification Molecular biology 3. Good health Isoenzymes Biochemistry 030220 oncology & carcinogenesis Recombinant DNA biology.protein Parasitology Casein kinase 1 Sequence Alignment Toxoplasma |
| Zdroj: | Molecular and biochemical parasitology. 141(1) |
| ISSN: | 0166-6851 |
| Popis: | Previous affinity chromatography experiments have described the unexpected binding of an isoform of casein kinase I (CK1) from Leishmania mexicana, Trypanosoma cruzi, Plasmodium falciparum and Toxoplasma gondii to an immobilized cyclin-dependent kinase (CDK) inhibitor (purvalanol B). In order to further evaluate CK1 as a potential anti-parasitic target, two T. gondii CK1 genes were cloned by PCR using primers derived from a putative CK1 gene fragment identified from a T. gondii EST database. The genes are predicted to encode a smaller polypeptide of 38 kDa (TgCK1alpha) and larger 49 kDa isoform bearing a C-terminal extension (TgCK1beta). Enzymatically active recombinant FLAG-epitope tagged TgCK1alpha and TgCK1beta enzymes were immuno-precipitated from transiently transfected T. gondii parasites. While TgCK1alpha expression was found to be cytosolic, TgCK1beta was expressed predominantly at the plasma membrane. Deletion mapping showed that the C-terminal domain of TgCK1beta confers this membrane-association. Recombinant TgCK1alpha and TgCK1beta isoforms were also expressed in E. coli and biochemically characterized. A 38kDa native CK1 activity was partially purified from T. gondii tachyzoites by ion-exchange and hydrophobic interaction chromatography with biochemical and serological properties closely resembling those of recombinant TgCK1alpha. In contrast, we were not able to identify a native CK1 activity corresponding to the larger TgCK1beta 49 kDa isoform in tachyzoite lysates. Purvalanol B and the related compound aminopurvalanol A selectively inhibit TgCK1alpha, confirming the existence of potentially exploitable structural differences between host and parasite CK1 enzymes. Since the more cell-permeable aminopurvalanol also inhibits parasite growth, these results provide further impetus to investigate inhibitors of CK1 as anti-parasitic agents. |
| Databáze: | OpenAIRE |
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