Abstract
Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5.
Highlights
NatB, in protein clearance via mTOR-Akt signaling, it should be noted that Ard1, a catalytic subunit of the NatA complex, affects mTOR signaling by N-acetylation of TSC2 [57], which is a GAP (GTPase-activating protein) for Rheb (Ras homolog enriched in brain), a Ras family GTPase, and the active form of
We used polyQ aggregates as a model of neurodegenerative conditions to evaluate whether Mdm20 regulates protein clearance and/or aggregate formation in neurons as it does in budding yeast
We found that in neurons, Mdm20 promotes protein aggregate formation, and the loss of Mdm20 function leads to protein clearance by inducing autophagy through reducing the phosphorylation level of Akt-Ser473
Summary
Mdm20-deletion construct and the association with Nat in HEK293 cells. Forty-eight hours post-transfection, the cells were immunoprecipitated (IP) with an anti-Flag antibody and immunoblotted with anti-Flag and anti-Nat antibodies. In Mdm or Nat5-OE cells, the amount of LC3II was not affected by these chemical treatments (data not shown) These results suggest that loss of Mdm induces and up-regulates the autophagy pathway, possibly leading to the stimulation of protein aggregate clearance. The overexpression of Akt-WT and AktS473D increased polyQ aggregate formation in both control and Mdm20-KD cells (Figure 8A) These data indicate that regulation of the phosphorylation level of Ser473 by Mdm affects the progression of polyQ aggregation possibly through inhibiting autophagy induction. Rapamycin and Akt-inhibitor-VIII induce autophagy (Figures 6D and 7C)), they reduced the phosphorylation level of Akt (Figure 8B) These chemical treatments suppressed the polyQ aggregate formation in mock or control cells (Figure 8B). NatB, in protein clearance via mTOR-Akt signaling, it should be noted that Ard, a catalytic subunit of the NatA complex, affects mTOR signaling by N-acetylation of TSC2 [57], which is a GAP (GTPase-activating protein) for Rheb (Ras homolog enriched in brain), a Ras family GTPase, and the active form of
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