The Proteasome is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

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Title: The Proteasome is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus
Authors: Hirano, Kiyoko; Zuber, Christian; Roth, MD, PhD, Jurgen; Ziak, Martin
Publisher: American Journal of Pathology
Date Published: July 01, 2003
Reference Number: 614
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Mutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t(1/2) of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t(1/2) = 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t(1/2) = 1.8 hours) but in a proteasome- and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants.

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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

T126M AQP2 and E258K AQP2 are two mutations of aquaporin 2 (AQP2) that result in NDI. Hirano, et al., studied these two mutations together with normal AQP2 to determine how they are formed, taken apart and distributed in the cell. They performed their experiments using laboratory cell cultures.

The researchers found that both the mutant and the normal AQP2 were formed in a part of the cell called the endoplasmic reticulum (ER). Proteins are measured in units of mass known as kilodaltons (kd). When the AQP2s under study are formed in their nonglycosylated forms (i.e., the form that does not have a linkage with a glycosyl group), they are slightly smaller than when they are synthesized in their glycosylated forms.

The mutants were dismantled by the cell much sooner than the normal AQP2. The nonglycosylated form of T126M was broken down by the cell over twice as quickly as the glycosylated form of T126M. The E258K AQP2 also was broken down by the cell far more quickly than the normal AQP2. However, where T126M was broken down by the enzyme, proteasome, E258K was broken down by both proteasome and lysosome. The E258K was directed to the lysosomes which disassembled it via an intracellular structure known as the Golgi apparatus, where it previously had been retained. T126M is retained in the ER. Both the ER and the Golgi apparatus function as a type of quality control center within the cell, holding misshapen proteins and either dismantling them or sending them elsewhere to be dismantled.

Though there were differences in how fast and where the two mutants were degraded, both were degraded by the cell. These differences could bear relevance to NDI research. For example, if researchers want to try and rescue the T126M from the ER and bring it to the cell surface, they should target the glycosylated form because it lasts longer. Also, since only proteasome seems to be involved in T126M, proteasome inhibitors might stabilize it and inhibit its dismantling by the cell.