Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue
<i>Trypanosoma brucei</i> (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimu...
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MDPI AG
2021-06-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/13/6776 |
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| author | Adélle Burger Paula Macucule-Tinga Stephen John Bentley Michael Hans Ludewig Ndumiso Nhlakanipho Mhlongo Addmore Shonhai Aileen Boshoff |
| author_facet | Adélle Burger Paula Macucule-Tinga Stephen John Bentley Michael Hans Ludewig Ndumiso Nhlakanipho Mhlongo Addmore Shonhai Aileen Boshoff |
| author_sort | Adélle Burger |
| collection | DOAJ |
| description | <i>Trypanosoma brucei</i> (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimulates the ATPase activity of TbHsp70.c. In the current study, immunofluorescence confirmed that TbHsp70.c is both a cytosolic and a nuclear protein. Furthermore, in silico analysis was used to elucidate an atypical linker and hydrophobic pocket. Tellingly, TbHsp70.c lacks the EEVD and GGMP motifs, both of which are implicated in substrate selectivity and co-chaperone binding in canonical Hsp70s. Far western analysis revealed that TbSTi1 interacts directly with TbHsp70 and TbHsp70.4, but does not bind TbHsp70.c. We further investigated the effect of quercetin and methylene blue on the Tbj2-driven ATPase activity of TbHsp70.c. We established that quercetin inhibited, whilst methylene blue enhanced, the Tbj2-stimulated ATPase activity of TbHsp70.c. Furthermore, these inhibitors were lethal to parasites. Lastly, we used molecular docking to show that quercetin and methylene blue may bind the nucleotide binding pocket of TbHsp70.c. Our findings suggest that small molecule inhibitors that target TbHsp70.c could be developed to serve as possible drug candidates against <i>T. brucei</i>. |
| first_indexed | 2024-03-10T10:06:58Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T10:06:58Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-d64f492e02874da7ad9423a156209d642023-11-22T01:30:57ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-06-012213677610.3390/ijms22136776Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene BlueAdélle Burger0Paula Macucule-Tinga1Stephen John Bentley2Michael Hans Ludewig3Ndumiso Nhlakanipho Mhlongo4Addmore Shonhai5Aileen Boshoff6Department of Biochemistry, University of Venda, Private Bag X5050, Thohoyandou 0950, South AfricaBiotechnology Innovation Centre, Faculty of Science, Rhodes University, PO Box 94, Makhanda/Grahamstown 6140, South AfricaBiotechnology Innovation Centre, Faculty of Science, Rhodes University, PO Box 94, Makhanda/Grahamstown 6140, South AfricaBiotechnology Innovation Centre, Faculty of Science, Rhodes University, PO Box 94, Makhanda/Grahamstown 6140, South AfricaDepartment of Medical Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South AfricaDepartment of Biochemistry, University of Venda, Private Bag X5050, Thohoyandou 0950, South AfricaBiotechnology Innovation Centre, Faculty of Science, Rhodes University, PO Box 94, Makhanda/Grahamstown 6140, South Africa<i>Trypanosoma brucei</i> (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimulates the ATPase activity of TbHsp70.c. In the current study, immunofluorescence confirmed that TbHsp70.c is both a cytosolic and a nuclear protein. Furthermore, in silico analysis was used to elucidate an atypical linker and hydrophobic pocket. Tellingly, TbHsp70.c lacks the EEVD and GGMP motifs, both of which are implicated in substrate selectivity and co-chaperone binding in canonical Hsp70s. Far western analysis revealed that TbSTi1 interacts directly with TbHsp70 and TbHsp70.4, but does not bind TbHsp70.c. We further investigated the effect of quercetin and methylene blue on the Tbj2-driven ATPase activity of TbHsp70.c. We established that quercetin inhibited, whilst methylene blue enhanced, the Tbj2-stimulated ATPase activity of TbHsp70.c. Furthermore, these inhibitors were lethal to parasites. Lastly, we used molecular docking to show that quercetin and methylene blue may bind the nucleotide binding pocket of TbHsp70.c. Our findings suggest that small molecule inhibitors that target TbHsp70.c could be developed to serve as possible drug candidates against <i>T. brucei</i>.https://www.mdpi.com/1422-0067/22/13/6776<i>Trypanosoma brucei</i>chaperoneTbHsp70.cTbHsp70TbSTi1Tbj2 |
| spellingShingle | Adélle Burger Paula Macucule-Tinga Stephen John Bentley Michael Hans Ludewig Ndumiso Nhlakanipho Mhlongo Addmore Shonhai Aileen Boshoff Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue International Journal of Molecular Sciences <i>Trypanosoma brucei</i> chaperone TbHsp70.c TbHsp70 TbSTi1 Tbj2 |
| title | Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue |
| title_full | Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue |
| title_fullStr | Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue |
| title_full_unstemmed | Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue |
| title_short | Characterization of an Atypical <i>Trypanosoma brucei</i> Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue |
| title_sort | characterization of an atypical i trypanosoma brucei i hsp70 demonstrates its cytosolic nuclear localization and modulation by quercetin and methylene blue |
| topic | <i>Trypanosoma brucei</i> chaperone TbHsp70.c TbHsp70 TbSTi1 Tbj2 |
| url | https://www.mdpi.com/1422-0067/22/13/6776 |
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