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BLAP-Tags, TUBEs and DUB-Chips: Combined Novel Technologies will Advance Molecular Epithelial Physiology.

Hamilton KL - Front Physiol (2012)

Bottom Line: After the specific modulators have been identified, further experimentation is required to assess the downstream use as potential clinical targets for a particular disease.The first step is to identify the specific modulators.This perspective highlights a multi-prong technologies approach that uses three novel technologies (BLAP-tagged proteins, TUBES, and DUB-Chips) that can rapidly identify a number of potential candidates that modulate ubiquitylation and deubiquitylation of cellular proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Otago School of Medical Sciences, University of Otago Dunedin, New Zealand.

ABSTRACT
The field of ubiquitylation and deubiquitylation of proteins in molecular physiology is growing at a rapid rate. Our understanding of molecular physiology of these processes may become limited by the advancement of technologies that scientists can employ. Therefore, it is important to approach physiological questions of ubiquitylation and deubiquitylation of proteins from a multiple methodological direction. Indeed, the role of ubiquitylation and deubiquitylation of proteins in cellular function has been implicated in the pathophysiology of human diseases including cancer, viral diseases, and neurodegenerative disorders. There are many modulators (activators and inhibitors) of ubiquitylation and deubiquitylation. Therefore, the link is being able to rapidly assess potential modulators of ubiquitylation and deubiquitylation and determine which specific modulators play a role(s) within a particular physiological setting. After the specific modulators have been identified, further experimentation is required to assess the downstream use as potential clinical targets for a particular disease. The first step is to identify the specific modulators. This perspective highlights a multi-prong technologies approach that uses three novel technologies (BLAP-tagged proteins, TUBES, and DUB-Chips) that can rapidly identify a number of potential candidates that modulate ubiquitylation and deubiquitylation of cellular proteins.

No MeSH data available.


Related in: MedlinePlus

DUB-Chip as a tool for identifying DUBs interacting with KCa3.1. (A) Immunofluorescence images of BLAP-KCa3.1 labeled with streptavidin–Alexa488 and incubated for 0, 90 min, or 3 h at 37°C. (B) Cells were lysed in the presence of GST-TUBE2, then the lysates were pulled down on GST beads, eluted and hybridized on a DUB panel. Interactions between the fluorescently tagged KCa3.1 and specific DUBs were quantified by measuring fluorescence intensity. (C) Dub-Chip data, expressed as relative fluorescence units (RFU), indicated that there were interactions between ubiquitylated KCa3.1 and ubiquitin specific protease 2 (UPS2), UPS 8 and a weaker interaction with AMSH [associated molecule with the SH3 domain of STAM; highlighted in red in (C)]. As can be seen, the interactions between the ubiquitylated KCa3.1 and the DUBs were transient, as interactions were not identified at 180 min. (D) This panel lists the various classes of DUBs, DeSUMOylases, and deNEDDylases impregnated on the DUB-Chip. This figure was used with permission from FASEB J. The figure legend was slightly modified from the original Figure 4A and 4B from Balut et al. (2011).
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Figure 1: DUB-Chip as a tool for identifying DUBs interacting with KCa3.1. (A) Immunofluorescence images of BLAP-KCa3.1 labeled with streptavidin–Alexa488 and incubated for 0, 90 min, or 3 h at 37°C. (B) Cells were lysed in the presence of GST-TUBE2, then the lysates were pulled down on GST beads, eluted and hybridized on a DUB panel. Interactions between the fluorescently tagged KCa3.1 and specific DUBs were quantified by measuring fluorescence intensity. (C) Dub-Chip data, expressed as relative fluorescence units (RFU), indicated that there were interactions between ubiquitylated KCa3.1 and ubiquitin specific protease 2 (UPS2), UPS 8 and a weaker interaction with AMSH [associated molecule with the SH3 domain of STAM; highlighted in red in (C)]. As can be seen, the interactions between the ubiquitylated KCa3.1 and the DUBs were transient, as interactions were not identified at 180 min. (D) This panel lists the various classes of DUBs, DeSUMOylases, and deNEDDylases impregnated on the DUB-Chip. This figure was used with permission from FASEB J. The figure legend was slightly modified from the original Figure 4A and 4B from Balut et al. (2011).

Mentions: So, the third novel methodology used by Devor and co-workers (Balut et al., 2011) was the DUB-Chip protein microarray and this was used in combination with the BLAP-tagged channels labeled with streptavidin–Alexa488 and the TUBEs to address the question of the which DUB(s) was (were) involved in the deubiquitylation of KCa3.1 during trafficking to the lysosome? Briefly, KCa3.1-BLAP channels were transfected into HEK cells and the channels were enzymatically biotinylated and streptavidin labeled at the plasma membrane and cells were then immediately lysed in the presence of GST-TUBE2 (time = 0), or returned to 37°C for various periods of time (90 min or 3 h) to allow endocytosis and then lysed in the presence of TUBEs (Balut et al., 2011). The TUBEs with their ubiquitylated proteins were pulled down on glutathione agarose, eluted, and then directly hybridized to the DUB-Chip. Since the only fluorescent protein in the sample was KCa3.1, an association of the channel with a DUB on the protein microarray could be evaluated by scanning arrays with a Typhoon Imager (9410 Imager, GE Life Sciences, Piscataway, NJ, USA; Balut et al., 2011). Channel-DUB interactions were verified after 90 min as there were notable interactions of KCa3.1 with USP2 and USP8 and a weaker association with AMSH (associated molecule with the SH3 domain of STAM; Figure 1). However, it was noted that these associations were transient in nature, as samples collected at 3 hr did not exhibit any association of KCa3.1 with the DUBs (Balut et al., 2011). Therefore, KCa3.1 is the first protein to be used to demonstrate a direct interaction between any protein and a DUB on the DUB-Chip protein microarray.


BLAP-Tags, TUBEs and DUB-Chips: Combined Novel Technologies will Advance Molecular Epithelial Physiology.

Hamilton KL - Front Physiol (2012)

DUB-Chip as a tool for identifying DUBs interacting with KCa3.1. (A) Immunofluorescence images of BLAP-KCa3.1 labeled with streptavidin–Alexa488 and incubated for 0, 90 min, or 3 h at 37°C. (B) Cells were lysed in the presence of GST-TUBE2, then the lysates were pulled down on GST beads, eluted and hybridized on a DUB panel. Interactions between the fluorescently tagged KCa3.1 and specific DUBs were quantified by measuring fluorescence intensity. (C) Dub-Chip data, expressed as relative fluorescence units (RFU), indicated that there were interactions between ubiquitylated KCa3.1 and ubiquitin specific protease 2 (UPS2), UPS 8 and a weaker interaction with AMSH [associated molecule with the SH3 domain of STAM; highlighted in red in (C)]. As can be seen, the interactions between the ubiquitylated KCa3.1 and the DUBs were transient, as interactions were not identified at 180 min. (D) This panel lists the various classes of DUBs, DeSUMOylases, and deNEDDylases impregnated on the DUB-Chip. This figure was used with permission from FASEB J. The figure legend was slightly modified from the original Figure 4A and 4B from Balut et al. (2011).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351676&req=5

Figure 1: DUB-Chip as a tool for identifying DUBs interacting with KCa3.1. (A) Immunofluorescence images of BLAP-KCa3.1 labeled with streptavidin–Alexa488 and incubated for 0, 90 min, or 3 h at 37°C. (B) Cells were lysed in the presence of GST-TUBE2, then the lysates were pulled down on GST beads, eluted and hybridized on a DUB panel. Interactions between the fluorescently tagged KCa3.1 and specific DUBs were quantified by measuring fluorescence intensity. (C) Dub-Chip data, expressed as relative fluorescence units (RFU), indicated that there were interactions between ubiquitylated KCa3.1 and ubiquitin specific protease 2 (UPS2), UPS 8 and a weaker interaction with AMSH [associated molecule with the SH3 domain of STAM; highlighted in red in (C)]. As can be seen, the interactions between the ubiquitylated KCa3.1 and the DUBs were transient, as interactions were not identified at 180 min. (D) This panel lists the various classes of DUBs, DeSUMOylases, and deNEDDylases impregnated on the DUB-Chip. This figure was used with permission from FASEB J. The figure legend was slightly modified from the original Figure 4A and 4B from Balut et al. (2011).
Mentions: So, the third novel methodology used by Devor and co-workers (Balut et al., 2011) was the DUB-Chip protein microarray and this was used in combination with the BLAP-tagged channels labeled with streptavidin–Alexa488 and the TUBEs to address the question of the which DUB(s) was (were) involved in the deubiquitylation of KCa3.1 during trafficking to the lysosome? Briefly, KCa3.1-BLAP channels were transfected into HEK cells and the channels were enzymatically biotinylated and streptavidin labeled at the plasma membrane and cells were then immediately lysed in the presence of GST-TUBE2 (time = 0), or returned to 37°C for various periods of time (90 min or 3 h) to allow endocytosis and then lysed in the presence of TUBEs (Balut et al., 2011). The TUBEs with their ubiquitylated proteins were pulled down on glutathione agarose, eluted, and then directly hybridized to the DUB-Chip. Since the only fluorescent protein in the sample was KCa3.1, an association of the channel with a DUB on the protein microarray could be evaluated by scanning arrays with a Typhoon Imager (9410 Imager, GE Life Sciences, Piscataway, NJ, USA; Balut et al., 2011). Channel-DUB interactions were verified after 90 min as there were notable interactions of KCa3.1 with USP2 and USP8 and a weaker association with AMSH (associated molecule with the SH3 domain of STAM; Figure 1). However, it was noted that these associations were transient in nature, as samples collected at 3 hr did not exhibit any association of KCa3.1 with the DUBs (Balut et al., 2011). Therefore, KCa3.1 is the first protein to be used to demonstrate a direct interaction between any protein and a DUB on the DUB-Chip protein microarray.

Bottom Line: After the specific modulators have been identified, further experimentation is required to assess the downstream use as potential clinical targets for a particular disease.The first step is to identify the specific modulators.This perspective highlights a multi-prong technologies approach that uses three novel technologies (BLAP-tagged proteins, TUBES, and DUB-Chips) that can rapidly identify a number of potential candidates that modulate ubiquitylation and deubiquitylation of cellular proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Otago School of Medical Sciences, University of Otago Dunedin, New Zealand.

ABSTRACT
The field of ubiquitylation and deubiquitylation of proteins in molecular physiology is growing at a rapid rate. Our understanding of molecular physiology of these processes may become limited by the advancement of technologies that scientists can employ. Therefore, it is important to approach physiological questions of ubiquitylation and deubiquitylation of proteins from a multiple methodological direction. Indeed, the role of ubiquitylation and deubiquitylation of proteins in cellular function has been implicated in the pathophysiology of human diseases including cancer, viral diseases, and neurodegenerative disorders. There are many modulators (activators and inhibitors) of ubiquitylation and deubiquitylation. Therefore, the link is being able to rapidly assess potential modulators of ubiquitylation and deubiquitylation and determine which specific modulators play a role(s) within a particular physiological setting. After the specific modulators have been identified, further experimentation is required to assess the downstream use as potential clinical targets for a particular disease. The first step is to identify the specific modulators. This perspective highlights a multi-prong technologies approach that uses three novel technologies (BLAP-tagged proteins, TUBES, and DUB-Chips) that can rapidly identify a number of potential candidates that modulate ubiquitylation and deubiquitylation of cellular proteins.

No MeSH data available.


Related in: MedlinePlus