Rapamycin inhibits trypanosome cell growth by preventing TOR complex 2 formation

Barquilla, Antonio; Crespo, José L.; Navarro, Miguel

doi:10.1073/pnas.0802668105

Cited by 121 publications

(163 citation statements)

References 30 publications

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“…851 Thus, in each case care needs to be applied and the use of the protein to monitor autophagy validated. In addition, due to possible divergence in the upstream signaling kinases, classical inhibitors such as 3-MA, or inducers like rapamycin, which are not as potent for trypanosomes 852 or apicomplexan parasites as in mammalian cells or yeast, must be used with caution (Coppens I, personal communication); 201 however, RNAi knockdown of TORC1 (e.g., TOR1 or RPTOR) is effective in inducing autophagy.…”

Section: Do Not Distributementioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Section: Do Not Distributementioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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“…For example, T. brucei, by this methodological definition, contains one predicted orthologue and three paralogues of S. cerevisiae Tor1/Tor2. Nevertheless, experimental data show that two T. brucei proteins have TOR activity 116 and therefore, by some definitions based on functional analysis (see text), may both be considered as orthologues. Asterisks indicate the presence of homologous sequences that could not be reliably predicted as paralogous or not.…”

Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 99%

“…Like some other unicellular eukaryotes, trypanosomes contain two TOR kinases, TOR1 and TOR2; however, two additional TOR-related proteins (TOR-like 1 and TOR-like 2) have also been described. 116 TOR1 and TOR2 participate in the complexes TORC1 and TORC2, respectively, which differ in function, subcellular localization and rapamycin sensitivity in trypanosomes. 116 As in yeast, TORC1 and TORC2 are associated with the orthologues KOG1/raptor and AVO3/rictor.…”

Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 99%

“…116 TOR1 and TOR2 participate in the complexes TORC1 and TORC2, respectively, which differ in function, subcellular localization and rapamycin sensitivity in trypanosomes. 116 As in yeast, TORC1 and TORC2 are associated with the orthologues KOG1/raptor and AVO3/rictor. TOR1 is predominantly nuclear, whereas TOR2 is excluded from the nucleus and is associated with the ER and the mitochondrion.…”

Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 99%

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Autophagy in protists

et al. 2011

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Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles, and defense against parasitic invaders. During the last 10-20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target

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“…(Protein synthesis rate was linear for the duration of the assay and 35 Sincorporation was similar at two different cell densities.) Although reduced protein synthesis may reflect the slowgrowth rate of RBP42-depleted cells, it may be a direct effect of RBP42 on mRNA translation (Barquilla et al 2008;Freire et al 2011). …”

Section: Rbp42 Is a Cytoplasmic Protein And Associates With Polysomesmentioning

confidence: 99%

The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism

Das

Morales

Banday

et al. 2012

RNA

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RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.

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Rapamycin inhibits trypanosome cell growth by preventing TOR complex 2 formation

Cited by 121 publications

References 30 publications

Guidelines for the use and interpretation of assays for monitoring autophagy

Guidelines for the use and interpretation of assays for monitoring autophagy

Autophagy in protists

The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism

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