Abstract
We examined the feedback between the major protein degradation pathway, the ubiquitin-proteasome system (UPS), and protein synthesis in rat and mouse neurons. When protein degradation was inhibited, we observed a coordinate dramatic reduction in nascent protein synthesis in neuronal cell bodies and dendrites. The mechanism for translation inhibition involved the phosphorylation of eIF2α, surprisingly mediated by eIF2α kinase 1, or heme-regulated kinase inhibitor (HRI). Under basal conditions, neuronal expression of HRI is barely detectable. Following proteasome inhibition, HRI protein levels increase owing to stabilization of HRI and enhanced translation, likely via the increased availability of tRNAs for its rare codons. Once expressed, HRI is constitutively active in neurons because endogenous heme levels are so low; HRI activity results in eIF2α phosphorylation and the resulting inhibition of translation. These data demonstrate a novel role for neuronal HRI that senses and responds to compromised function of the proteasome to restore proteostasis.
Highlights
The concept of an optimal protein concentration and its associated regulatory mechanisms is known as ‘proteostasis’
To determine whether the processes of neuronal protein synthesis and degradation are coupled, we examined the effect of blocking proteasomal degradation on nascent protein synthesis in cultured hippocampal neurons (Figure 1A)
Blocking proteasome activity with either of two chemically distinct inhibitors (MG-132, 10 mM; Lactacystin, 10 mM; Figure 1A–E and Figure 1—figure supplement 1A–F) for 2 hr led to a dramatic decrease in protein synthesis, measured with two different labeling methods: the non-canonical amino acid azidohomoalanine (AHA) and BONCAT (Dieterich et al, 2006; Figure 1B–C) or metabolic labeling with puromycin (Figure 1D,E and Figure 1—figure supplement 1E)
Summary
The concept of an optimal protein concentration and its associated regulatory mechanisms is known as ‘proteostasis’. Protein degradation, mediated by the ubiquitin proteasome system (UPS), plays an important role in regulating synaptic function (Bingol and Schuman, 2006; Tai and Schuman, 2008; Ramachandran and Margolis, 2017). While it is clear that changes in synaptic transmission involve extensive regulation of the synaptic proteome via the regulated synthesis and degradation of proteins, it is not well understood how these two processes are coordinately regulated to achieve the desired level of individual proteins at synapses. To address this question, we studied the impact of proteasome inhibition on protein synthesis in mature neurons. The activity and expression of neuronal HRI is regulated in a biologically clever manner, expression is kept low by a short- half-life and a translational control mechanism that is relieved by proteasome inhibition- augmenting HRI expression and leading to the coordinate regulation of protein synthesis
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