Biochemical and Biophysical Research Communications
Prevention of aberrant protein aggregation by anchoring the molecular chaperone αB-crystallin to the endoplasmic reticulum
Introduction
The molecular chaperone αBC is a member of the small heat shock protein and operates in cellular response to various stresses such as heat shock, ischemia and oxidation [1]. It prevents protein aggregation by binding to improperly folded proteins. Several mutants of αBC have been reported [2], [3], [4], among which the R120G mutant is the first to be identified to cause α-crystallinopathy. The illness is an autosomal dominant muscular disease characterized by compromised functions of cardiac and skeletal muscles. Histologically, a typical feature of α-crystallinopathy is the accumulation of protein aggregates containing αBC and desmin, an intermediate filament protein. Transfection of the R120G mutant into non-muscular cell lines resulted in aggregate formation, as observed in the specimens from α-crystallinopathy patients [4], [5]. Although not yet clearly understood, defective chaperone activity of the R120G mutant is expected to trigger the accumulation of protein aggregates and underlie the development of α-crystallinopathy [6], [7]. α-Crystallinopathy thus reflects the failure of protein quality control and is classified as a protein deposition disease [8], [9], including Alzheimer’s disease and Parkinson’s disease.
The ER is intimately involved in protein quality control and has been recognized as a key regulator of stress responses [10]. It has been reported that misfolded proteins are sequestered to ER-associated subcellular compartments such as Q-body [11], JUNQ [12] and ERAC [13], further supporting the view that the ER deals with potentially harmful proteins. We recently have found that ultraviolet C-induced cell death is attenuated by tethering αBC to the ER, revealing a link between this organelle and the DNA damage response [14]. The finding led us to reason by analogy that other cellular responses could also be controlled by manipulating the ER using αBC as a biological tool.
Here we have demonstrated that ER-anchored αBC remarkably suppresses aggregate formation mediated by the disease-causing αBC mutant. Based on the results, we propose that modulation of the micromilieu surrounding the ER membrane is effective in preventing the accumulation of protein aggregates, a possible cause of the protein deposition disease.
Section snippets
Cell culture and transfection
HeLa cells were maintained in α-MEM (nakalai tesque) supplemented with 10% fetal bovine serum (FBS). The expression constructs and siRNA were transfected into the cells using polyethylenimine and HiPerfect transfection reagent (Qiagen), respectively.
Expression vectors and small interfering RNAs
The construction of the following expression vectors were described previously [14]: pcDNA4/myc-αBC (WTαBC), pcDNA4/myc-TMαBC (TMαBC), pcDNA4/myc-TMαBCn (TMαBCn). Knockdown experiments were performed with the siRNA against the ATG5 mRNA sequence
ER-anchored αBC represses aggregate formation mediated by the R120G mutant
Expression of the R120G mutant C-terminally tagged with GFP in HeLa cells, a human cervical cancer cell line, led to formation of protein aggregates (Fig. 1A, vector). We then determine whether tethering αBC to the ER affects the R120G mutant-mediated protein aggregation. Upon co-transfection with either an empty vector or the myc-tagged wild-type αBC construct (WTαBC [14]), aggregates were found in ∼30% of the GFP-positive cells (Fig. 1A and B, vector and WTαBC). Meanwhile, the rate dropped to
Discussion
We have revealed that aggregation of the disease-causing R120G mutant is remarkably suppressed by TMαBC. It was highly expected that the reduction of the abundance of the R120G mutant diminishes burden on the protein disposal system. Indeed, it has been known that aggregate formation is closely associated with disturbance of protein homeostasis [18]. We therefore postulated that TMαBC corrects the imbalance between the quantity of aberrant proteins and the capacity of protein disposal system,
Acknowledgments
This work was supported by JSPS KAKENHI Grant Numbers 24590081 and 26350502, and by A Strategic Project for Innovative Research “iPUT” from Tokushima University.
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