| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- 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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| 2. |
- Hwang, Hui Yun, et al.
(författare)
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Profiling the Protein Targets of Unmodified Bio-Active Molecules with Drug Affinity Responsive Target Stability and Liquid Chromatography/Tandem Mass Spectrometry
- 2020
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 20:9
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Forskningsöversikt (refereegranskat)abstract
- Identifying the target proteins of bioactive small molecules is a key step in understanding mode-of-action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand-modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity-based protein profiling. Target proteins are then enriched by performing a pull-down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read-out). This review highlights recent proteomic approaches utilizing data-dependent analysis and data-independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio-active molecules can be further validated.
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| 3. |
- Kim, Dongyoung, et al.
(författare)
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FK506, an Immunosuppressive Drug, Induces Autophagy by Binding to the V-ATPase Catalytic Subunit A in Neuronal Cells
- 2017
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 16:1, s. 55-64
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Tidskriftsartikel (refereegranskat)abstract
- The drug FK506 (tacrolimus, fujimycin) exerts its immunosuppressive effects by regulating the nuclear factor of the activated T-cell (NFAT) family of transcription factors. However, FK506 also exhibits neuroprotective effects, but its direct target proteins that mediate these effects have not been determined. To identify the target proteins responsible for FK506's neuroprotective effects, the drug affinity responsive target stability (DARTS) method was performed using label-free FK506, and LC-MS/MS analysis of the FK506-treated proteome was also performed. Using DARTS and LC-MS/MS analyses in combination with reference studies, V-ATPase catalytic subunit A (ATP6V1A) was identified as a new target protein of FK506. The biological relevance of ATP6V1A in mediating the neuroprotective effects of FK506 was validated by analyzing FK506 activity with respect to autophagy via acridine orange staining and transcription factor EB (TFEB) translocation assay. These analyses demonstrated that the binding of FK506 with ATP6V1A induces autophagy by activating the translocation of TFEB from the cytosol into the nucleus. Because autophagy has been identified as a mechanism for treating neurodegenerative diseases and because we have demonstrated that FK506 induces autophagy, this study demonstrates that FK506 is a possible new therapy for treating neurodegenerative diseases.
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