A, Detection of PP2A-C subunit binding to TSPyV sT antigen with pull-down assay and Western blotting. Lane M shows Precision Plus Protein WesternC standards marker (Bio-Rad Laboratories Inc); lane 1, histidine-thioredoxin tag (HIS-TRX) bait plus HEK293 cell lysate as prey protein source; lane 2, HIS-TRX-TSPyV sT fusion bait protein plus HEK293 cell lysate as prey protein source; lane 3, HEK293 cell lysate. In lane 2, TSPyV sT protein (bait) and PP2A-C subunit protein interaction can be seen, and the 36-kDa–sized PP2A-C subunit protein (prey) was detected. In the protein extract of HEK293 cells (lane 3), the PP2A-C subunit protein was also detected. B, Detection of PP2A-A subunit binding to TSPyV sT antigen with pull-down experiment and Western blotting. Lane M shows Precision Plus Protein WesternC standards marker (Bio-Rad Laboratories Inc); lane 1, HIS-TRX only; lane 2, HIS-TRX-TSPyV sT fusion protein; lane 3, HEK293 cell protein. In lane 2, interaction between TSPyV sT protein and PP2A-A subunit protein can be visualized (65-kDa–sized PP2A-Aα subunit was detected). In the protein extract of HEK293 cells (lane 3), the PP2A-A subunit protein was also seen. Plus sign indicates detected, and minus sign, not detected.
Trichodysplasia spinulosa–associated polyomavirus sT protein expressed in HEK293 cells was incubated with mouse anti–PP2A-C monoclonal antibody (clone ID 6, EMD Millipore) in the immunoprecipitation (IP) step (lane 1). The coimmunoprecipitated proteins were separated on 12% sodium dodecyl sulfate–polyacrylamide gel. Western blot detection was performed with custom-made mouse monoclonal antibody against a TSPyV sT antigen epitope (clone 2E10B11). Coimmunoprecipitated TSPyV sT protein with a mass of approximately 23 kDa can be seen in lane 1. When control mouse IgG (Santa Cruz Biotechnology Inc) was used in the IP step (lane 2), no TSPyV sT protein can be seen. Lane 3 shows tetracycline-induced HEK293 cells expressing approximately 23-kDa–sized TSPyV sT protein, and lane M, Precision Plus Protein WesternC standards marker (Bio-Rad Laboratories Inc). When noninduced HEK293 cell lysate was used (lane 4), no TSPyV sT protein was detected. Asterisks denote heavy and light chains of antibody. Plus sign indicates detected, and minus sign, not detected.
Nguyen HP, Patel A, Simonette RA, Rady P, Tyring SK. Binding of the Trichodysplasia Spinulosa–Associated Polyomavirus Small T Antigen to Protein Phosphatase 2A: Elucidation of a Potential Pathogenic Mechanism in a Rare Skin Disease. JAMA Dermatol. 2014;150(11):1234-1236. doi:10.1001/jamadermatol.2014.1095
Trichodysplasia spinulosa is a disfiguring skin disease caused by the trichodysplasia spinulosa–associated polyomavirus (TSPyV).1 Like other polyomaviruses, TSPyV expresses the large T and small T (sT) antigens, but how these proteins regulate trichodysplasia spinulosa pathogenesis is unknown. In the closely related human pathogen Merkel cell polyomavirus, sT acts as a transforming oncoprotein in vitro because it alone is sufficient to transform rodent fibroblast cells.2 Interestingly, this mechanism does not seem to involve protein phosphatase 2A (PP2A), which is a characteristic target of other polyomaviruses’ sT antigens. Because PP2A regulates important cellular pathways, the inactivation is one of the critical steps in the polyomavirus pathomechanism. The primary purpose of this study was to determine whether TSPyV sT antigen was capable of binding PP2A; if positive, this finding would implicate specific pathways in trichodysplasia spinulosa pathogenesis.
Institutional review board requirements were waived for the present work.
The target TSPyV sT sequence was subcloned into a LentiVector containing a red fluorescent protein–blasticidin selection marker (GenTarget Inc), and the vector was then used to generate a tetracycline-inducible cell line expressing TSPyV sT antigen in HEK293 host cells. The expression of the TSPyV sT antigen was detected by Western blotting using a mouse monoclonal antibody (clone ID 2E10B11, GenScript Inc, 1:100). The antibody was developed against a selected TSPyV sT peptide (KEYFGKKKYDENVI).
Prokaryotic histidine-thioredoxin-sT fusion protein was produced, immobilized on HisPur cobalt resin (Thermo Fisher Scientific), and used as a bait protein. The source of the prey proteins was HEK293 cell protein extract. The pull-down assay was performed following the manufacturer’s protocol (Pierce Pull-Down PolyHis Protein:Protein Interaction Kit, Thermo Fisher Scientific). The eluted prey proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (4%-12% gel, Novex system, Invitrogen–Life Technologies) and Western blotting. The PP2A-A subunit was detected using a rat monoclonal antibody (clone 6G3, Santa Cruz Biotechnology Inc, 1:200). The PP2A-C subunit was detected with application of a mouse monoclonal antibody (clone 1D6, Millipore, 1:2000). Following application of appropriate horseradish peroxidase–labeled secondary antibodies (Santa Cruz Biotechnology Inc), chemiluminescent detection was performed using a SuperSignal West Femto kit (Thermo Fisher Scientific) and an Alpha Innotech image workstation 8900.
Trichodysplasia spinulosa–associated polyomavirus sT-expressing HEK293 cell protein extract was prepared as described. The protein extract was incubated with 2 μg anti–PP2A-C mouse monoclonal antibody (clone 1D6, 1:2000). Protein-antibody complexes were collected on protein G agarose beads (Roche Applied Science). After washing steps were completed, the protein complexes were dissociated by means of the addition of SDS loading buffer and heating. Proteins were separated on SDS-PAGE (12%, Novex) and transferred to polyvinylidene fluoride membrane (Invitrogen–Life Technologies). For the detection, anti-sT mouse monoclonal antibody (clone ID 2E10B11, 1:100) was used. Chemiluminescent detection was performed as described in the Pull-Down Assay subsection of the Methods section.
Physical interactions were observed between TSPyV sT and PP2A-C, as well as PP2A-A, subunits in pull-down assays (Figure 1). Protein-protein interaction was detected between TSPyV sT and PP2A-C in the coimmunoprecipitation experiment (Figure 2).
Many tumor viruses, including polyomaviruses, interact with PP2A in various ways in order to deregulate key cellular pathways.3 Simian virus 40 sT binds the A and C subunits of PP2A, and this interaction inhibits PP2A activity.4
It has been previously shown that the TSPyV sT antigen contains putative PP2A binding motifs.1 On the basis of this prediction, we used protein-protein interaction assays to demonstrate that TSPyV sT is indeed capable of binding PP2A in vitro (Figure 1 and Figure 2). This binding activity may inhibit PP2A-modulated protein kinase signaling pathways. This interaction also might lead to the hyperphosphorylation of the pocket proteins (pRb, p107, p130) and dysregulation of the cell cycle.5 These processes likely underlie the histological observations of excessive proliferative cells in enlarged hair follicles that are characteristically observed in trichodysplasia spinulosa.6
Accepted for Publication: April 28, 2014.
Corresponding Author: Stephen K. Tyring, MD, PhD, MBA, University of Texas Health Science Center, 6431 Fannin St, Houston, TX 77030 (firstname.lastname@example.org).
Published Online: August 27, 2014. doi:10.1001/jamadermatol.2014.1095.
Author Contributions: Drs Tyring and Rady had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Nguyen, Rady, Tyring.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Nguyen, Patel, Simonette, Rady.
Critical revision of the manuscript for important intellectual content: Nguyen, Rady, Tyring.
Obtained funding: Tyring.
Administrative, technical, or material support: All authors.
Study supervision: Nguyen, Tyring.
Conflict of Interest Disclosures: None reported.