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.

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.

Effects of the combination of a proteasome inhibitor (PSI) and an inhibitor of ubiquitin-ligases (Leu-Ala) on the ultrastructure of human leukemic U937 cells.

We have used the dipeptide Leu-Ala in an attempt to prevent the formation of ubiquitin-protein conjugates in U937 cells by inhibition of cellular E3 enzymes (ubiquitin ligases). Proteasome inhibitors induce the formation of perinuclear aggregates of ubiquitinated proteins and proteasomes (aggresomes) in the area of the proteolytic center of the cell. Leu-Ala did not prevent the forrmation of those aggregates under the action of PSI (peptidyl aldehyde, selective inhibitor of the chymotrypsin-like activity of the proteasome), however it induced an accumulation of lipid droplets in treated cells, suggesting a previously unknown involvement of Leu-Ala in lipid metabolism. We conclude, that either Leu-Ala is not able to completely inhibit the cellular E3 enzymes or some of those enzymes are insensitive to this dipeptide, allowing therefore the build-up of ubiquitin-conjugates in the proteolytic centre of the cell.

Proteasome localization and ultrastructure of spermatozoa from patients with varicocele--immunoelectron microscopic study.

Localization of proteasomes in spermatozoa from patients with varicocele-associated sterility was studied by means of immunolabeling with the MPC21 monoclonal antibody detecting the C3 subunit of the 20S proteasome. The reaction was visualized for electron microscopy using the secondary Nano-Gold-coupled antibody with Gold-Enhancement in pre-embedding technique. We found that semen samples from varicocele patients contained a large amount of abnormal spermatozoa characterized by the presence of dispersed chromatin and large residual bodies (cytoplasmic droplets) as well as spermatids at various stages of spermiogenesis. In normal spermatozoa, the immunolabeling was found in the acrosome, postacrosomal regions, nuclear vacuoles, in the neck and in the middle-piece as well as in the residual bodies, while chromatin remained unlabeled. In varicocele spermatozoa, the immunolabeling was also associated with chromatin and large residual bodies (cytoplasmic droplets). In contrast to normal, mature spermatozoa, the chromatin of the cells at earlier stages of spermiogenesis was strongly immunolabeled. The association of proteasomes with sperm chromatin and large residual bodies can be the sign of abnormality and disturbances in spermatogenesis associated with varicocele.

Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study.

The ultrastructural localization of a proteasomal antigen in human spermatozoa was studied by means of immunolabeling with the MPC21 monoclonal antibody and secondary gold labeled antibody with 1.4 nm gold particles in combination with silver enhancement reaction using pre-embedding technique. The labeling was found in the acrosomal and postacrosomal regions, in the connecting-piece (neck) and, in some cases, in the middle-piece and also in the residual bodies. There was no significant reaction in condensed chromatin. In some abnormal forms of spermatozoa, in which the chromatin was not well condensed, the labeling in nuclei was present. The nuclear vacuoles with looser chromatin were usually strongly labeled. The nuclei of cells representing different stages of spermatogenesis, that were present in semen samples, were also labeled.