Desumoylation prevents the endoplasmic reticulum-associated degradation of DF508-CFTR
(2010) 24th annual North American Cystic Fibrosis Conference — Location: baltimore, MD, USA (21.October.2010)
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- Abstract
- Sumoylation is the covalent and reversible post-translational conjugation of the small ubiquitin-like modifier (SUMO) protein to lysine residues of protein. The enzymatic machinery mediating sumoylation is similar to that of the ubiquitin system, including an E1 activating enzyme, an E2 conjugating enzyme, E3 ligases and desumoylating enzymes (Sentrin/SUMOspecific proteases, or SENPs). Sumoylation has been shown to play a role in transcription, cell division, DNA repair, and, more recently, in protein quality control and ion channel function. We have reported earlier that over-expression of the SUMO-conjugating enzyme Ubc9 decreased the steady-state level of CFTR, whereas the overexpression of the desumoylating enzyme SENP1 increased it; therefore we have initiated a systematic analysis of the effect of sumoylation on CFTR biogenesis. Increasing the intracellular level of SUMO1, SUMO2, and SUMO3 accelerated the intracellular degradation of ΔF508 CFTR in HEK293 cells. Over-expression of SUMO1 or SUMO3 reduced the half-life of ΔF508 CFTR by half, and the over-expression of SUMO3 had a smaller effect. The general increase in sumoylation observed during hypoxia presumably has a similar effect, making sumoylation physiologically relevant in CF patients. Next, we performed RT-PCR reactions using total RNA isolated from human bronchial epithelial (HBE) cells, in order to ascertain which SENPs are expressed in a cell type physiologically relevant to CF. With the exception of SENP3, messenger RNA of each SENP could be detected in HBE cells from four different non-CF patients. We detected mRNA encoding each of the six SENPs in three frequently used cell lines, Calu-3 cells, HEK293 cells, and HeLa cells. Co-expression of each of the six SENPs with ΔF508 CFTR in HEK293 cells increased the level of ΔF508 CFTR to varying degrees. On the other hand, SENPs had varying effects on the steady-state level of WT CFTR: SENP1, SENP2, and SENP6 increased the level of WT CFTR, whereas the others had no effect. Comparing the effect of each SENP on ΔF508 CFTR with that on WT CFTR revealed that three of them increased the level of ΔF508 CFTR whereas they had no effect on WT CFTR, indicating that they might be able to distinguish between the two CFTR species. Most of the research on SENPs focuses on nuclear events and extranuclear localization has been recognized for only a few of them. In order to establish the possibility of a physical interaction between CFTR and the SENPs we over-expressed each SENP in HEK293 cells and performed a crude fractionation of the cells to nuclear, cytosolic, and membrane fractions. The bulk of each SENP was detected in the nuclear or the non-nuclear membrane fraction, and only in the case of SENP5 was some protein detected in the cytosolic fraction. This data clearly indicate that the SENPs can be found in (on) non-nuclear membranes; therefore they may interact with CFTR directly. In summary, these data point to desumoylating enzymes having therapeutic potential in CF and warrant further study of the interactions of SENPs with ΔF508 CFTR.
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Schmidt, B., Noël, S., Gong, X., Ahner, A., & Frizzell, R. (2010). Desumoylation prevents the endoplasmic reticulum-associated degradation of DF508-CFTR. Pediatric Pulmonology, 45(S33), 227. https://hdl.handle.net/2078.5/42543 (Original work published 2010)