Rapid assay to detect possible natural substrates of proteases in living cells

Boonacker, Emil; Elferink, Sjoerd; Bardai, Abdennasser; Wormmeester, J.; Noorden, C. J. F. Van

doi:10.2144/03354st07

“…We first measured if HERPUD1 stability affects the range of lysosomal pH, using the pH-sensitive lysosomal dye LysoTracker Red, which accumulates and emits red fluorescence in acidic compartments with pH < 6.5 ( De Duve and Christian, 1974 ; Chou et al, 2001 ). Then, we measured CATHEPSIN-B activity using the Magic Red assay ( Boonacker et al, 2003 ). As shown in Figure 6C , expression of HERPUD1-ΔUBL causes a significant increase in the number of LysoTracker positive structures (211.95 ± 78.90), compared to control cells (117.85 ± 48.97) ( Figures 6C,D ).…”

Section: Resultsmentioning

confidence: 99%

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival

Vargas

¹

,

Peña

²

,

Arias‐Muñoz

³

et al. 2022

Front. Cell Dev. Biol.

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Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

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“…We first measured if HERPUD1 stability affects the range of lysosomal pH, using the pH-sensitive lysosomal dye LysoTracker Red, which accumulates and emits red fluorescence in acidic compartments with pH <6.5 (De Duve et al 1974; Chou, Paul Krapcho, and Hacker 2001). Then, we measured CATHEPSIN-B activity using the Magic Red assay (Boonacker et al 2003). As shown in Figure 6C, expression of HERPUD1-ΔUBL causes a significant increase in the number of LysoTracker positive structures (211.95 ± 78.90), compared to control cells (117.85 ± 48.97) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Negative modulation of macroautophagy by HERPUD1 is counteracted by an increased ER-lysosomal network with impact in drug-induced stress cell survival

Vargas

¹

,

Oh

²

,

En

³

et al. 2021

Preprint

0

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Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation but independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER protein of short-half life and a well-known substrate of the proteasome. We found that increased HERPUD1 stability by deletion of its ubiquitin-like domain (UBL) plays a negative role on basal and induced macroautophagy. Moreover, we found it triggers ER expansion by reordering the ER in crystalloid structures, but in the absence of unfolded protein response activation. Surprisingly, we found ER expansion led to an increase in the number and function of lysosomes establishing a tight network with the presence of membrane-contact sites. Importantly, a phosphomimetic S59D mutation within the UBL mimics UBL deletion on its stability and the ER-lysosomal network expansion revealing an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in cell stress survival.

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“…Proteolytic activity appeared to be regulated posttranslationally. It was also applied to detect natural substrates of dipeptidyl peptidase IV by determining fluorescence (cresyl violet) production in the presence or absence of candidate substrates such as b-casomorphin (Boonacker et al 2003b). The caspase-3 substrate allows the detection of apoptotic cells (Lee et al 2003).…”

Section: Magic Red Substrates the Cresyl Violet-based Magicmentioning

confidence: 99%

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

Noorden

¹

2010

J Histochem Cytochem.

Self Cite

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S U M M A R Y For the understanding of functions of proteins in biological and pathological processes, reporter molecules such as fluorescent proteins have become indispensable tools for visualizing the location of these proteins in intact animals, tissues, and cells. For enzymes, imaging their activity also provides information on their function or functions, which does not necessarily correlate with their location. Metabolic mapping enables imaging of activity of enzymes. The enzyme under study forms a reaction product that is fluorescent or colored by conversion of either a fluorogenic or chromogenic substrate or a fluorescent substrate with different spectral characteristics. Most chromogenic staining methods were developed in the latter half of the twentieth century but still find new applications in modern cell biology and pathology. Fluorescence methods have rapidly evolved during the last decade. This review critically evaluates the methods that are available at present for metabolic mapping in living animals, unfixed cryostat sections of tissues, and living cells, and refers to protocols of the methods of choice. (J Histochem Cytochem 58:481-497, 2010)

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Rapid assay to detect possible natural substrates of proteases in living cells

Cited by 6 publications

References 33 publications

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival

Negative modulation of macroautophagy by HERPUD1 is counteracted by an increased ER-lysosomal network with impact in drug-induced stress cell survival

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

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