Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

Wiedner, Susan D.; Anderson, Lindsey; Sadler, Natalie; Chrisler, William B.; Kodali, Vamsi; Smith, Richard; Wright, Aaron

doi:10.1002/anie.201309135

Cited by 38 publications

(30 citation statements)

References 30 publications

(30 reference statements)

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“…[30] In the current work, the interior localization of an amino-functionalized molecule in lysosomes in live cells is demonstrated. The application of two-color SIM on live HeLa (S3) cells transfected with Fusion-Red-LAMP and co-stained with compound 4 confirms that FusionRed-LAMP is anchored on the membrane of lysosomes, whereas compound 4 is confined in the lumen of lysosomes.…”

Section: Resultsmentioning

confidence: 99%

“…[29] Wright and co-workers have recently shown that the weakly basic 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP)-derived protein activity probes are encapsulated in lysosomes in fixed cells using SIM. [30] In the current work, the interior localization of an amino-functionalized molecule in lysosomes in live cells is demonstrated. The application of two-color SIM on live HeLa (S3) cells transfected with Fusion-Red-LAMP and co-stained with compound 4 confirms that Fu-sionRed-LAMP is anchored on the membrane of lysosomes, whereas compound 4 is confined in the lumen of lysosomes.…”

Section: Structured Illumination Microscopy (Sim)mentioning

confidence: 87%

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A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)‐Enhanced Photostability and a Narrow Band of Emission

Sreenath

Zhao

Allen

et al. 2014

Chemistry A European J

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We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.

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Section: Resultsmentioning

confidence: 99%

Section: Structured Illumination Microscopy (Sim)mentioning

confidence: 87%

A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)‐Enhanced Photostability and a Narrow Band of Emission

Sreenath

Zhao

Allen

et al. 2014

Chemistry A European J

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“…[13][14][15][16][17] The peptidic part of such covalent modifiers accounts for sufficient affinity to be accommodated in the active site of the target protease due to specific non-covalent interactions mainly with the S1-S4 binding pockets. In particular, epoxide derivatives and acyloxymethyl ketones have been successfully devolved to activity-based probes for cysteine cathepsins.…”

Section: Introductionmentioning

confidence: 99%

Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors

et al. 2015

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Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 μM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe's concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 μM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes’ lipophilicity, and the obtained

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“…45,46 While relatively unexplored in an autophagy context compared with the methods outlined in previous sections, chemical proteomics techniques such as bio-orthogonal noncanonical amino acid tagging (BONCAT) and affinity-based protein profiling (ABPP) have already been successfully used for novel and pioneering studies in autophagy and will be further discussed in the corresponding section later in this review. [47][48][49][50]…”

Section: Chemical Proteomicsmentioning

confidence: 99%

Recent advances in quantitative and chemical proteomics for autophagy studies

et al. 2017

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Macroautophagy/autophagy is an evolutionarily well-conserved cellular degradative process with important biological functions that is closely implicated in health and disease. In recent years, quantitative mass spectrometry-based proteomics and chemical proteomics have emerged as important tools for the study of autophagy, through large-scale unbiased analysis of the proteome or through highly specific and accurate analysis of individual proteins of interest. At present, a variety of approaches have been successfully applied, including (i) expression and interaction proteomics for the study of protein post-translational modifications, (ii) investigating spatio-temporal dynamics of protein synthesis and degradation, and (iii) direct determination of protein activity and profiling molecular targets in the autophagic process. In this review, we attempted to provide an overview of principles and techniques relevant to the application of quantitative and chemical proteomics methods to autophagy, and outline the current landscape as well as future outlook of these methods in autophagy research.

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Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

Cited by 38 publications

References 30 publications

A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)‐Enhanced Photostability and a Narrow Band of Emission

A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)‐Enhanced Photostability and a Narrow Band of Emission

Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors

Recent advances in quantitative and chemical proteomics for autophagy studies

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