Comprehensive Evaluation of Rapamycin’s Specificity as an mTOR Inhibitor

Artoni, Filippo; Grützmacher, Nina; Demetriades, Constantinos

doi:10.1101/2022.12.10.519872

Cited by 2 publications

(4 citation statements)

References 105 publications

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“…Rapamycin is a bacterial macrolide initially used as an antifungal agent. However, due to its inhibitory effect on mTOR signaling, it is widely used as an autophagy inducer agent [24]. Activation of autophagy was confirmed using western blotting for assessment of LC3II/LC3I and p62 levels (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Identification of Thioredoxin1 interacting proteins in neuronal cytoskeletal organization during autophagy

Islam,

Sultana,

Padmanabhan

et al. 2024

Preprint

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Thioredoxin1 (Trx1) is a major cytoplasmic thiol oxidoreductase protein involved in redox signaling. This function is rendered by a rapid electron transfer reaction during which Trx1 reduces its substrate and itself becomes oxidized. In this reaction, Trx1 forms a transient disulfide bond with the substrate which is unstable and therefore identification of Trx1 substrates is technically challenging. This process maintains the cellular proteins in a balanced redox state and ensures cellular homeostasis. Trx1 levels are reduced in some neurodegenerative diseases; therefore, understanding the interactions between Trx1 and its substrates in neurons could have significant therapeutic implications. We utilized a transgenic mouse model expressing a Flag-tagged mutant form of Trx1 that can form stable disulfide bonds with its substrates allowing identification of the Trx1 interacting proteins. The involvement of Trx1 has been suggested in autophagy, we aimed to investigate Trx1 substrate after pharmacologic induction of autophagy in primary hippocampal neurons. Treatment of primary neurons by rapamycin, a standard autophagy inducer, caused significant reduction of neurite outgrowth and alterations in the cytoskeleton. Through immunoprecipitation and mass spectrometry, we have identified 77 Trx1 interacting proteins which were associated with a wide range of cellular functions including a major impact on cytoskeletal organization. The results were confirmed in Trx1 knocked-down cells and in nucleofected primary neurons. Our study suggests a novel role for Trx1 in regulation of neuronal cytoskeleton organization, marking the first investigation of Trx1-interacting proteins in primary neurons and confirming the multifaceted role of Trx1 in physiological and pathological states.

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Section: Resultsmentioning

confidence: 99%

“…Rapamycin is a bacterial macrolide initially used as an antifungal agent. However, due to its inhibitory effect on mTOR signaling, it is widely used as an autophagy inducer agent [24].…”

Section: Rapamycin Induces Autophagy In Primary Cortical Neuronsmentioning

confidence: 99%

Identification of Thioredoxin1 interacting proteins in neuronal cytoskeletal organization during autophagy

Islam,

Sultana,

Padmanabhan

et al. 2024

Preprint

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“…To investigate whether this mTORC1-dependent gene expression program depends on the presence of the Rags, we performed RNA-seq experiments and tested the expression of selected target genes for each of these TFs, comparing WT and RagA/B KO HEK293FT cells. As a control, we also re-analysed our recently published RNA-seq dataset from Rapamycin-vs. DMSO-treated HEK293FT cells, in which mTORC1 is inhibited and S6K is dephosphorylated 59 . These analyses showed that selected SREBP, HIF1 and ATF4 target genes were consistently downregulated upon mTORC1 inhibition with rapamycin, confirming that the activity of these TFs is regulated downstream of mTORC1 (Ext.…”

Section: Lysosomal and Non-lysosomal Mtorc1 Complexes Regulate Differ...mentioning

confidence: 99%

“…To assess mRNA expression of selected SREBP, HIF1, ATF4, and TFEB target genes, we used two RNA-seq datasets: from rapamycin-or DMSO-treated HEK293FT cells (described in 59 ; NCBI Sequence Read Archive PRJNA872474), and from RagA/B KO versus control HEK293FT cells. Target genes of each transcription factor were retrieved using the Harmonizome 3.0 online platform (https://maayanlab.cloud/Harmonizome/) 105 .…”

Section: Gene Expression Analysismentioning

confidence: 99%

Spatially and Functionally Distinct mTORC1 Entities Orchestrate the Cellular Response to Amino Acid Availability

Fernandes,

Angelidaki,

Nüchel

et al. 2023

Preprint

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Amino acid (AA) availability is a robust determinant of cell growth, through controlling mTORC1 activity1. According to the predominant model in the field, AA sufficiency drives the recruitment and activation of mTORC1 on the lysosomal surface by the heterodimeric Rag GTPases, from where it coordinates the majority of cellular processes (reviewed in2,3). Importantly, however, 15 years after its initial discovery, the teleonomy of the proposed lysosomal regulation of mTORC1, and where mTORC1 acts on its effector proteins remain enigmatic4. Here, by using multiple pharmacological and genetic means to perturb the lysosomal AA sensing and protein recycling machineries, we describe the spatial separation of mTORC1 regulation and downstream functions in mammalian cells, with lysosomal and non-lysosomal mTORC1 phosphorylating distinct substrates in response to different AA sources. Moreover, we reveal that a fraction of mTOR localizes at lysosomes due to basal lysosomal proteolysis that locally supplies new AAs, even in cells grown in the presence of extracellular nutrients, whereas cytoplasmic mTORC1 is regulated by exogenous AAs. Overall, our study substantially expands our knowledge about the topology of mTORC1 regulation by AAs, and hints at the existence of distinct, Rag- and lysosome-independent mechanisms that control its activity at other subcellular locations. Given the importance of mTORC1 signalling and AA sensing for human ageing and disease2, our findings will likely open new directions toward the identification of function-specific mTORC1 regulators, and suggest new targets for drug discovery against conditions with dysregulated mTORC1 activity in the future.

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Comprehensive Evaluation of Rapamycin’s Specificity as an mTOR Inhibitor

Cited by 2 publications

References 105 publications

Identification of Thioredoxin1 interacting proteins in neuronal cytoskeletal organization during autophagy

Identification of Thioredoxin1 interacting proteins in neuronal cytoskeletal organization during autophagy

Spatially and Functionally Distinct mTORC1 Entities Orchestrate the Cellular Response to Amino Acid Availability

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