Fluorescent probes for selective protein labeling in lysosomes: a case of α‐galactosidase A

Abstract

Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC-tag) detection. We tested the efficiency of halogen-substituted biarsenical probes to interact with C-terminally TC-tagged GALA peptide at pH 4.5 and identified F2FlAsH-EDT as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS-7 cells transiently expressing TC-tagged GALA. The investigated fluorescence-based LCI technology of TC-tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.-Bohl, C., Pomorski, A., Seemann, S., Knospe, A.-M., Zheng, C., Krężel, A., Rolfs, A., Lukas, J. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

Optimized allosteric inhibition of engineered protein tyrosine phosphatases with an expanded palette of biarsenical small molecules

“…Specifically, we used as HePTP, under conditions in which FlAsH gives only partial inhibition, to test a panel of previously reported FlAsH derivatives that contain halogen, alkyl-group, or carboxyl-group modifications appended to FlAsH’s fluorescein scaffold (see Supplementary Figure 4 for structures). 15,28 We found, not surprisingly, that all of the FlAsH derivatives are as HePTP inhibitors (Figure 7). Although F4FLAsH, 5-CrAsH, and 5-BuCrAsH were no more effective (or only marginally more effective) than the parent compound FlAsH, we identified the 2′,7′-derivatives, Cl2FlAsH, F2FlAsH and Et2FlAsH, as as HePTP inhibitors with increased potency.…”

Strategies for organelle targeting of fluorescent probes

“…The most common lysosomal markers include those in the lysosomal associated membrane protein LAMP class, especially LAMP1 and LAMP2, which account for 50% of all lysosomal membrane proteins (Table S5). 73 It is also possible to fuse FPs with lysosomal hydrolases, 74 but these may not be distributed evenly amongst all lysosomes due to varied nutrient availability. The main issue with using protein biomarkers for the lysosome is that other digestive vesicles may also be labelled as the proteins are transported through the fusion events leading to lysosome formation and maturation.…”

Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy

“…The three potential LIR peptides, LIR1, LIR2, LIR3 and the corresponding variants were prepared by Solid-phase synthesis using the Fmoc strategy on TentaGel RAM resin essentially as described before [26]. Peptides were synthesized using a fully automated Liberty 1 microwave-assisted synthesizer (CEM).…”