Changes in spatio-temporal localization of tripeptidyl peptidase II (TPPII) in murine colon adenocarcinoma cells during aggresome formation: a microscopy study based on a novel fluorescent proteasome inhibitor

L.P. Bialy¹, U. Kuckelkorn², P. Henklein², J. Fayet³, G.M. Wilczyński⁴, A. Kamiński⁵ and I. Mlynarczuk-Bialy<sup>1

1</sup>Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, Warszawa, Poland, ²Institut für Biochemie, Charité - Universitätsmedizin Berlin, Berlin, Germany, ³Department of Ophthalmology, First Medical Faculty, Medical University of Warsaw, ⁴Laboratory of Molecular and Systemic Neuromorphology, Department of Neurophysiology, Nencki Institute of Experimental Biology PAS Warsaw and ⁵Department of Transplantology and Central Tissue Bank, Center for Biostructure Research, Medical University of Warsaw, Warszawa, Poland

Offprint requests to: Izabela Mlynarczuk-Bialy MD, PhD, Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, Cha?ubi?skiego 5, 02-004 Warszawa, Poland. e-mal: izamlynar@esculap.pl or imlynarczuk@wum.edu.pl

Summary. Extralysosomal proteolysis is a multistep process involving the Ubiquitin- Proteasome System (UPS) and supplementary peptidases. Tripeptidyl peptidase II (TPPII) is the most extensively characterized enzyme, supplementing and sometimes substituting for proteasomal functions. In response to proteasome inhibition, polyubiquitinated proteins acting as proteasome substrates aggregate with proteasomes and form aggresomes. Several proteasome inhibitors are used as anti-cancer drugs. Thus, in our study, we used a novel fluorescent-tagged proteasome inhibitor BSc2118 to induce aggresome formation in C26 murine colon adenocarcinoma cells. It allowed us to obtain effective, inhibitor-based, proteasome staining in vivo. This method has been validated by standard post-fixed indirect immunostaining and also allowed co-immunodetection of TPPII and polyubiquitinated proteins under laser scanning confocal microscopy. We found that in the absence of the inhibitor, TPPII is diffusely dispersed within the cytoplasm of C26 cells. The proteasome and ubiquitin-rich perinuclear region failed to display enhanced TPPII staining. However, when proteasome function was impaired by the inhibitor, TPPII associated more closely with both the proteasome and polyubiquitinated proteins via TPPII recruitment to the perinuclear region and subsequently into emerging aggresomal structures. Furthermore, we have demonstrated the dynamic recruitment of TPPII into the developing aggresome: TPPII in the early aggresome was dispersed within the central part but subsequently aggregated on the surface of this structure. In the mature aggresome of C26 cells TPPII formed a spherical mantle, which surrounded the round core containing proteasomes and polyubiquitinated proteins. Our morphological data indicate that TPPII displays spatial localization with proteasomes especially upon proteasome inhibition in aggresomes of C26 cells. Histol Histopathol 34, 359-372 (2019)

Key words: TPPII, Proteasome, Ubiquitin, Aggresome, Proteasome inhibitor BSc2118

DOI: 10.14670/HH-18-042