Overexpressed TTC3 Protein Tends to be Cleaved into Fragments and Form Aggregates in the Nucleus
Human tetratricopeptide repeat domain 3 (TTC3) is a gene on 21q22.2 within the Down syndrome critical region (DSCR). Earlier studies suggest that TTC3 may be an important regulator in individual development, especially in neural development. As an E3 ligase, TTC3 binds to phosphorylated Akt and silence its activity via proteasomal cascade. Several groups also reported the involvement of TTC3 in familial Alzheimer’s disease recently. In addition, our previous work shows that TTC3 also regulates the degradation of DNA polymerase gamma and over-expressed TTC3 protein tends to form insoluble aggregates in cells. In this study, we focus on the solubility and intracellular localization of TTC3 protein. Over-expressed TTC3 tends to form insoluble aggregates over time. The proteasome inhibitor MG132 treatment resulted in more TTC3 aggregates in a short period of time. We fused the fluorescent protein to either terminus of the TTC3 protein and found that the intracellular localization of fluorescent signals are different between the N-terminal tagged and C-terminal tagged proteins. Western blotting revealed that the TTC3 protein is cleaved into fragments of different sizes at multiple sites. The N-terminal sub-fragments of TTC3 are prone to from nuclear aggregates and the TTC3 nuclear import is mediated by signals within the N-terminal 1 to 650 residues. Moreover, over-expressed TTC3 induced a considerable degree of cytotoxicity, and its N-terminal sub-fragments are more potent inhibitors of cell proliferation than full-length protein. Considering the prevalent proteostasis dysregulation in neurodegenerative diseases, these findings may relate to the pathology of such diseases.
KeywordsTTC3 Aggregation Proteotoxicity Nuclear localization signal Alzheimer’s disease Down syndrome
The authors will appreciate the support from Experimental Technology Center for Life Sciences, College of Life Sciences, BNU.
FD and YG conceived and designed the study. YG, KW, S-PX and PM performed the experiments. FD and YG wrote the paper. FD, WL and YS reviewed and edited the manuscript. All authors read and approved the manuscript. YG and KW contributed equally to this work.
This work was supported by grants from the National Natural Science Foundation of China (NSFC) (31370768, 30970610) and the Open Research Fund of the State Key Laboratory of Cognitive Neuroscience and Learning.
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Conflict of interest
The authors declare that they have no competing interests.
Glossary and Abbreviation
The artificially designated fragment which covers a certain region of TTC3 protein and is constructed into expression vectors.
The proteolytic product of full-length TTC3 or its sub-fragments. Full-length TTC3 and the sub-fragments are cleaved by the intracellular proteases.
The fragment which primarily covers the region near the N-terminus of full-length TTC3 protein.
The fragment which primarily covers the region near the C-terminus of full-length TTC3 protein.
The tag peptide which is fused to the N-terminus of full-length TTC3 or its sub fragments.
The tag peptide which is fused to the C-terminus of full-length TTC3 or its sub fragments.
The fluorescent signal which is derived from the N-terminal tag or fluorescent protein fused to the N-terminus of full-length TTC3 or its sub-fragments.
The fluorescent signal which is derived from the C-terminal tag or fluorescent protein fused to the C-terminus of full-length TTC3 or its sub-fragments.
The RIPA-soluble components of the cells. The cells are lysed with RIPA buffer, and the supernatant is obtained after centrifugation.
The pellets. After centrifugation, the RIPA-insoluble components were made into pellets.
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