Biomedical subjects

Klaus Fortschegger

Publications and source records attributed to Klaus Fortschegger.

3 recordsLinked to original sources

PubMed2005

SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly.

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human-yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein-protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56-74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.

Amino Acid Sequence↗

PubMed2005

Comparison of early passage, senescent and hTERT immortalized endothelial cells.

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.

Cell Differentiation↗

PubMed2005

Interaction of U-box E3 ligase SNEV with PSMB4, the beta7 subunit of the 20 S proteasome.

Recognition of specific substrates for degradation by the ubiquitin-proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the beta7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast beta7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin-proteasome system and the spliceosome.

Amino Acid Sequence↗