Rapid cell type-specific nascent proteome labeling in Drosophila
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Villalobos-Cantor et. al. describe a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. The method is potentially interesting but needs further characterization to be able to assess its use.
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Abstract
Controlled protein synthesis is required to regulate gene expression and is often carried out in a cell type-specific manner. Protein synthesis is commonly measured by labeling the nascent proteome with amino acid analogs or isotope-containing amino acids. These methods have been difficult to implement in vivo as they require lengthy amino acid replacement procedures. O-propargyl-puromycin (OPP) is a puromycin analog that incorporates into nascent polypeptide chains. Through its terminal alkyne, OPP can be conjugated to a fluorophore-azide for directly visualizing nascent protein synthesis, or to a biotin-azide for capture and identification of newly-synthesized proteins. To achieve cell type-specific OPP incorporation, we developed phenylacetyl-OPP (PhAc-OPP), a puromycin analog harboring an enzyme-labile blocking group that can be removed by penicillin G acylase (PGA). Here, we show that cell type-specific PGA expression in Drosophila can be used to achieve OPP labeling of newly-synthesized proteins in targeted cell populations within the brain. Following a brief 2 hr incubation of intact brains with PhAc-OPP, we observe robust imaging and affinity purification of OPP-labeled nascent proteins in PGA-targeted cell populations. We apply this method to show a pronounced age-related decline in neuronal protein synthesis in the fly brain, demonstrating the capability of PhAc-OPP to quantitatively capture in vivo protein synthesis states. This method, which we call POPPi ( P GA-dependent OPP i ncorporation), should be applicable for rapidly visualizing protein synthesis and identifying nascent proteins synthesized under diverse physiological and pathological conditions with cellular specificity in vivo.
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eLife assessment
Villalobos-Cantor et. al. describe a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. The method is potentially interesting but needs further characterization to be able to assess its use.
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Reviewer #1 (Public Review):
Villalobos-Cantor et al. describe a chemical/genetic strategy to enable cell-type-specific labeling of nascent proteins in living tissues (called POPPi). O-propargyl-puromycin (OPP) is a commonly used compound to label nascent proteins in cells and tissue, however, its application is limited in vivo because it can not be targeted to individual cell types, tissues, or organs. Using Drosophila as a genetically tractable in vivo model organism, Villalobos-Cantor et al. incubate live tissue with a puromycin analog called phenylacetyl-OPP (PhAc-OPP) in combination with cell-type expression of Penicillin G acylase (PGA), which converts PhAc-OPP to OPP. As PGA is under the control of the Gal4/UAS system, a vast library of tissue-specific Gal4 lines can in theory be used to conduct labeling experiments in vivo.
The …
Reviewer #1 (Public Review):
Villalobos-Cantor et al. describe a chemical/genetic strategy to enable cell-type-specific labeling of nascent proteins in living tissues (called POPPi). O-propargyl-puromycin (OPP) is a commonly used compound to label nascent proteins in cells and tissue, however, its application is limited in vivo because it can not be targeted to individual cell types, tissues, or organs. Using Drosophila as a genetically tractable in vivo model organism, Villalobos-Cantor et al. incubate live tissue with a puromycin analog called phenylacetyl-OPP (PhAc-OPP) in combination with cell-type expression of Penicillin G acylase (PGA), which converts PhAc-OPP to OPP. As PGA is under the control of the Gal4/UAS system, a vast library of tissue-specific Gal4 lines can in theory be used to conduct labeling experiments in vivo.
The major strength of the methods and results is the demonstration that labeling can occur in specific cell types of the dissected brain - neurons and glia. For example, protein synthesis in individual dopamine neurons in the brain can be visualized and distinguished from neighboring cells, a remarkable achievement and striking image. These results in dissected brains nicely demonstrate that PhAc-OPP can penetrate into brain tissue, diffuse to internal locations, pass through the cell membrane, and become converted to OPP and label nascent proteins. A major weakness of the methods and results is the lack of exploration of POPPi in tissues other than the brain, as well as in non-dissected living animals. For example, the authors do not test if PhAc-OPP delivery can occur by feeding animals, or if PhAc-OPP can penetrate into various dissected tissues. Results from these experiments would be of great importance to others interested in applying this technique in non-brain tissues, and would properly support the authors' claims in the title and abstract that this is a general method (not only for the brain).
Assuming that PhAc-OPP can penetrate various dissected tissues, this method would have a significant impact on tissue-specific measurements of protein synthesis and could be a valuable new molecular reporter for gene function analysis (e.g. tissue-specific gene knockout + POPPi). If PhAc-OPP could be ingested by flies, perfuse through the body, and label nascent proteins in a cell-type specific manner, then POPPi could be incredibly useful for tissue-specific proteome profiling (i.e. mass spectrometry) in an in vivo living animal (non-dissected), similar to the BioID system.
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Reviewer #2 (Public Review):
In this manuscript, Villalobos-Cantor et. al. described a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. PhAc-OPP contains a blocking group that prevents it from incorporating into the growing peptide, and the blocking group can be cleaved off by the enzyme PGA, which is expressed in the cell type of interest.
The authors validated POPPi in different cell types in the Drosophila brain and showed that this method could be used to image general translation or to biochemically enrich …
Reviewer #2 (Public Review):
In this manuscript, Villalobos-Cantor et. al. described a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. PhAc-OPP contains a blocking group that prevents it from incorporating into the growing peptide, and the blocking group can be cleaved off by the enzyme PGA, which is expressed in the cell type of interest.
The authors validated POPPi in different cell types in the Drosophila brain and showed that this method could be used to image general translation or to biochemically enrich nascent peptides in a cell-type-specific manner. They also showed that with an optimized labeling protocol, it is possible to achieve efficient labeling with minimum effect on animal viability and health. The authors further used POPPi to provide independent support for a previously known phenomenon: age-dependent decline in general translation in the neurons. The results of this work are solid, and the main conclusions are well supported by the data presented. The manuscript is very well written with a clear logic flow and is very easy to read.
What is less clear is how generally useful POPPi will be to the community. The authors pointed out two major cell-type specific applications of POPPi, 1) imaging general translation and 2) biochemically purifying nascent peptides. For application #1, although POPPi might be a more desirable method in some cases, a combination of non-cell-type specific labeling using OPP, and marking the cell type of interest by a fluorescent protein might be simpler. Because labeling with OPP eliminates the enzymatic step that converts non-reactive PhAc-OPP to reactive OPP, the labeling kinetics can be improved, and the toxicity associated with PGA expression can be avoided. For application #2, a currently widely used strategy for a similar purpose is various types of ribosome profiling techniques. Ribosome profiling may be easier to perform than POPPi, and because proteins cannot be amplified, a very large quantity of starting materials will be needed if one wants to use POPPi to characterize cell-type specific nascent proteome. In fact, in this manuscript, the authors used western blots to detect candidate proteins and did not use mass spec to characterize the nascent proteome.
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Reviewer #3 (Public Review):
In this manuscript, Villalobos-Cantor et al. have implemented the method for monitoring cellular proteome that their lab has established in cell culture models of Drosophila brains. The method uses a puromycin analog (O-propargyl-puromycin, OPP) that is locked by the addition of phenylacetyl group (PhAc-OPP) that can be unlocked by expression of Penicillin G acetylase (PGA) to tag the proteins translated in a specific cell type. When unlocked, OPP can get incorporated into the newly translating nascent peptide, and abort translation while allowing click chemistry addition of various tags, such as fluorophore-azide to visualize or biotin-azide to immunopurify polypeptides. The authors demonstrate the use of the method in adult drosophila brains expressing PGA in neurons or glia, showing that the addition of …
Reviewer #3 (Public Review):
In this manuscript, Villalobos-Cantor et al. have implemented the method for monitoring cellular proteome that their lab has established in cell culture models of Drosophila brains. The method uses a puromycin analog (O-propargyl-puromycin, OPP) that is locked by the addition of phenylacetyl group (PhAc-OPP) that can be unlocked by expression of Penicillin G acetylase (PGA) to tag the proteins translated in a specific cell type. When unlocked, OPP can get incorporated into the newly translating nascent peptide, and abort translation while allowing click chemistry addition of various tags, such as fluorophore-azide to visualize or biotin-azide to immunopurify polypeptides. The authors demonstrate the use of the method in adult drosophila brains expressing PGA in neurons or glia, showing that the addition of OPP is indeed PGA dependent and the proteins are only tagged in the cells that express PGA. The authors also show that when fluorophore azide is used to visualize the proteome and the samples are run on a gel, bands of various sizes can be observed to have incorporated OPP, arguing the method labels the proteome indiscriminately. The authors also optimized the protocol by titrating the amount of PhAc-OPP to use to minimize cellular stress. Also, they show that driving the expression of PGA with elav-Gal4 or repo-Gal4 is not toxic and does not cause phenotypes although Actin-Gal4 driven expression causes phenotypes. Finally the authors demonstrate the use of the technique to show that there is an age-induced decrease in total protein synthesis in the fly brain. This is a nice technique to implement in fly but the characterization of the technique is not complete in its current state. It is not clear what percentage of the nascent peptides are tagged, and whether the cells in the tissue are equally represented in the lysates for immunopurification.
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