Sequestering sequestosome 1 via S-acylation in autophagy, Huntington disease, and beyond

ABSTRACT

Protein mislocalization and aggregation are hallmark features in neurodegeneration. As proteins mislocalize, proteostasis deficiency and protein aggregation typically follow. Autophagy is a crucial pathway for the removal of protein aggregates to maintain neuronal health, but is impaired in various neurodegenerative diseases, including Huntington disease (HD). We identified S-acylation, a reversible lipid modification of proteins, as an important regulator in protein trafficking and autophagy. SQSTM1 (sequestosome 1/p62) is an essential selective autophagy receptor for the sequestration of ubiquitinated cargoes within autophagosomes and subsequent delivery into lysosomes for degradation. Recently, we reported that S-acylation of SQSTM1 at the di-cysteine motif C289,290 directs SQSTM1 to lysosomes. We further showed that SQSTM1 S-acylation is significantly reduced in brains from both HD patients and mouse HD model, which may result in the cargo sequestration defect within autophagosomes in HD. Treatment with palmostatin B, a deacylation inhibitor, significantly increases SQSTM1 localization to lysosomes. Our work highlights SQSTM1 S-acylation as a novel potential therapeutic strategy in HD. As a crucial autophagy component, our work suggests S-acylation of SQSTM1 may have a broader role in neurodegeneration.

Regardless of the etiology, whether genetic, environmental or of unknown origin, protein mislocalization is often the first step in neurodegeneration. For various reasons, mislocalization is typically followed by protein aggregation. Although the toxicity of protein aggregates in various diseases has been a topic of debate, aggregation is an indicator of proteostasis deficiency and often demarks an impairment in protein turnover. While the proteasome is essential for removing misfolded proteins, in diseases characterized by large aggregates the proteasome becomes overwhelmed. Autophagy becomes the bulk pathway for the removal of misfolded and aggregating proteins. Thus, defining the mechanisms regulating protein localization is critical to understand neuronal cell biology and disease pathogenesis to develop effective therapeutics targeted to the earliest stages of disease progression. We recently showed that protein mislocalization, proteostasis deficiency, and autophagy are linked by S-acylation^[1].

S-acylation is a vital post-translational modification characterized by the reversible attachment of a fatty acid, most commonly palmitate, to cysteine residues of the target proteins via a labile thioester bond. Akin to phosphorylation, S-acylation is mediated by enzymes that add (writers) or remove (erasers) the fatty acid (Figure 1). Addition of the lipid moiety is mediated by ZDHHC enzymes, named after their conserved DHHC active site and zinc finger domain. Deacylation is facilitated by a growing group of serine hydrolases and thioesterases known as α/β-hydrolase domain (ABHD) and acyl protein thioesterases (APT), respectively. The hydrophobic fatty acid enhances membrane association and, due to its reversibility, dynamically regulates the trafficking of cytosolic proteins to and from membranes. Disruption of S-acylation can lead to protein mislocalization, ultimately impairing essential cellular processes, especially those that are highly membrane-associated such as autophagy.

Figure 1.

SQSTM1 S-acylation is a target in HD. S-acylation involves the transfer of fatty acids from Coenzyme A (CoA) to free cysteine residues on proteins, a post-translational modification mediated by ZDHHC enzymes or removed by APT/ABHD enzymes. A) reduced S-acylation of proteins involved in cargo-loading, including SQSTM1 and mHTT, in autophagy may contribute to the cargo loading failure in HD. The addition of specific deacylation inhibitors may be an effective therapeutic in increasing the S-acylation of autophagy proteins by decreasing their deacylation. B) restoring S-acylation of SQSTM1 supports healthy cargo-loading in autophagy in wild-type cells. Figure created in Biorender.

Autophagy is the major intracellular clearance mechanism responsible for degrading protein aggregates and damaged organelles. In Huntington disease (HD), a neurodegenerative disorder caused by a CAG expansion that encodes a polyglutamine (PolyQ) extension at the N-terminus of huntingtin (HTT) protein, autophagy is impaired at several steps. In particular, there is a cargo-loading defect in HD that leads to empty autophagosomes and decreased turnover of autophagic cargo that builds up in the cytoplasm. Sequestration of cellular waste, particularly protein aggregates, into autophagosomes for degradation in autolysosomes is facilitated by selective autophagy receptors such as sequestosome 1 (SQSTM1, also known as p62). Selective receptors such as SQSTM1 bind to cargo and recruit the autophagy machinery, leading to the in situ formation of a phagophore that expands around the cargo, which is also facilitated by the SQSTM1-LC3-II interaction. Ultimately, SQSTM1 and its cargo, like mutant HTT, are degraded in the lysosome. HTT is an S-acylated protein. Previously, we showed that mutant HTT is less S-acylated compared to wild-type HTT (Figure 1B) and that increasing palmitoylation with palmostatin B, a broad-spectrum deacylation inhibitor, was protective in HD.

In the present study, we used an unbiased proteomics approach to assess the effect of palmostatin B on autophagy^[1]. The levels of multiple autophagy regulators, including LC3B and SQSTM1, significantly increased in the presence of palmostatin B. LC3B does not contain any cysteines. Therefore, LC3 levels are more likely related to the effect of palmostatin B on autophagy overall. Given its shared roles in autophagy and HD, we predicted that SQSTM1 S-acylation was crucial for its function and that it may be disrupted in HD. We confirmed that SQSTM1 is S-acylated at the C289,290 di-cysteine motif, and we showed that SQSTM1 S-acylation directs SQSTM1 to the lysosome where it is degraded. S-acylation of SQSTM1 significantly increased in the presence of bafilomycin A1, which blocks degradation in lysosomes. Mutation of the Cysteines 289 and 290 to Serine (C289,290S) significantly reduced S-acylation, indicating that S-acylated SQSTM1 is the active form delivered to lysosomes. However, S-acylation was not completely blocked in C289,290S, suggesting additional sites of S-acylation in SQSTM1. Of particular importance, we found that SQSTM1 S-acylation is significantly reduced in the brains of HD patients. This effect was recapitulated in the YAC128 HD mouse model. Fasting-induced autophagy rescued SQSTM1 S-acylation in YAC128 mice brains. This suggests that S-acylating enzymes may be activated during fasting-induced autophagy. However, because fasting is impractical for HD patients, we next investigated pharmacological approaches which could rescue SQSTM1 S-acylation, thereby autophagy.

In addition, we showed that palmostatin B significantly increases SQSTM1 delivery to lysosomes in HeLa cells. Treatment with palmostatin B also increased LC3-II levels, indicating increased autophagic flux. However, SQSTM1 levels were also increased, which is inconsistent with increased autophagic flux, since SQSTM1 is degraded in autolysosomes, and should decrease in its levels. This suggests that while palmostatin B may stimulate autophagy, it may also induce a partial block in the pathway. Given its broad activity, palmostatin B likely affects the S-acylation of multiple autophagy- and lysosome-related proteins, contributing to these mixed effects. Therefore, the significance of our findings lies not in endorsing palmostatin B as a therapeutic agent, but in highlighting SQSTM1 S-acylation as a novel and promising therapeutic target in HD. To advance this strategy, more selective deacylation inhibitors will be needed to rescue autophagy without the unintended effects. Ultimately, our work suggests that selectively increasing S-acylation in HD is beneficial. Finally, because SQSTM1 is essential for removing autophagic cargo, including physiological protein aggregates, through nonselective and selective (i.e. aggrephagy) autophagy, our work suggests that S-acylation of SQSTM1 may play a broader and important role in neurodegeneration. Indeed, many mutations in SQSTM1 are linked with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Consequently, it will be important to assess the role of SQSTM1 S-acylation in neurodegeneration overall.

Funding Statement

D.D.O.M. and F.A. were supported by a Natural Sciences and Engineering Research Council (NSERC) Discovery Grant [RGPIN-2019–04617]. Y.A. is supported by a Canada Graduate Scholarship-Master’s (CGS-M) from the Canadian Institutes of Health Research (CIHR).

Disclosure statement

The authors declare that they have no conflicts of interest.

Data availability statement

Data sharing is not applicable to this article as no data were created or analyzed in this study.

List of Abbreviations

APT

Acyl protein thioesterase

ABHD

α/β-hydrolase domain

C

Cysteine

CAG

Cytosine Adenine Guanine

HD

Huntington disease (HD),

HTT

huntingtin

PolyQ

polyglutamine

S

Serine

SQSTM1

sequestosome 1

ZDHHC

zinc-finger and aspartate – histidine – histidine – cysteine (DHHC) motif-containing enzyme