Aaron Ciechanover

Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntingtons disease (HD), Alzheimers disease (AD), Parkinsons disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy

The conjugation of the 76 amino acid protein ubiquitin to other proteins can alter the metabolic stability or non-proteolytic functions of the substrate. Once attached to a substrate (monoubiquitination), ubiquitin can itself be ubiquitinated on any of its seven lysine (Lys) residues or its N-terminal methionine (Met1). A single ubiquitin polymer may contain mixed linkages and/or two or more branches. In addition, ubiquitin can be conjugated with ubiquitin-like modifiers such as SUMO or small molecules such as phosphate. The diverse ways to assemble ubiquitin chains provide countless means to modulate biological processes. We overview here the complexity of the ubiquitin code, with an emphasis on the emerging role of linkage-specific degradation signals (degrons) in the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (hereafter autophagy).

Protein Quality Control by Molecular Chaperones in Neurodegeneration

Protein homeostasis (proteostasis) requires the timely degradation of misfolded proteins and their aggregates by protein quality control (PQC), of which molecular chaperones are an essential component. Compared with other cell types, PQC in neurons is particularly challenging because they have a unique cellular structure with long extensions. Making it worse, neurons are postmitotic, i.e., cannot dilute toxic substances by division, and, thus, are highly sensitive to misfolded proteins, especially as they age. Failure in PQC is often associated with neurodegenerative diseases, such as Huntingtons disease (HD), Alzheimers disease (AD), Parkinsons disease (PD), and prion disease. In fact, many neurodegenerative diseases are considered to be protein misfolding disorders. To prevent the accumulation of disease-causing aggregates, neurons utilize a repertoire of chaperones that recognize misfolded proteins through exposed hydrophobic surfaces and assist their refolding. If such an effort fails, chaperones can facilitate the degradation of terminally misfolded proteins through either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). If soluble, the substrates associated with chaperones, such as Hsp70, are ubiquitinated by Ub ligases and degraded through the proteasome complex. Some misfolded proteins carrying the KFERQ motif are recognized by the chaperone Hsc70 and delivered to the lysosomal lumen through a process called, chaperone-mediated autophagy (CMA). Aggregation-prone misfolded proteins that remain unprocessed are directed to macroautophagy in which cargoes are collected by adaptors, such as p62/SQSTM-1/Sequestosome-1, and delivered to the autophagosome for lysosomal degradation. The aggregates that have survived all these refolding/degradative processes can still be directly dissolved, i.e., disaggregated by chaperones. Studies have shown that molecular chaperones alleviate the pathogenic symptoms by neurodegeneration-causing protein aggregates. Chaperone-inducing drugs and anti-aggregation drugs are actively exploited for beneficial effects on symptoms of disease. Here, we discuss how chaperones protect misfolded proteins from aggregation and mediate the degradation of terminally misfolded proteins in collaboration with cellular degradative machinery. The topics also include therapeutic approaches to improve the expression and turnover of molecular chaperones and to develop anti-aggregation drugs.

KPC1-Mediated Ubiquitination and Proteasomal Processing of NF-κB1 p105 to p50 Restricts Tumor Growth

NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.

p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis

Macroautophagy mediates the selective degradation of proteins and non-proteinaceous cellular constituents. Here, we show that the N-end rule pathway modulates macroautophagy. In this mechanism, the autophagic adapter p62/SQSTM1/Sequestosome-1 is an N-recognin that binds type-1 and type-2 N-terminal degrons (N-degrons), including arginine (Nt-Arg). Both types of N-degrons bind its ZZ domain. By employing three-dimensional modeling, we developed synthetic ligands to p62 ZZ domain. The binding of Nt-Arg and synthetic ligands to ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62 interaction with LC3, leading to the delivery of p62 and its cargoes to the autophagosome. Upon binding to its ligand, p62 acts as a modulator of macroautophagy, inducing autophagosome biogenesis. Through these dual functions, cells can activate p62 and induce selective autophagy upon the accumulation of autophagic cargoes. We also propose that p62 mediates the crosstalk between the ubiquitin-proteasome system and autophagy through its binding Nt-Arg and other N-degrons.Soluble misfolded proteins that fail to be degraded by the ubiquitin proteasome system (UPS) are redirected to autophagy via specific adaptors, such as p62. Here the authors show that p62 recognises N-degrons in these proteins, acting as a N-recognin from the proteolytic N-end rule pathway, and targets these cargos to autophagosomal degradation.

p62- and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome

Significance Whereas the role of the ubiquitin system in protein degradation is well established, little is known regarding the regulation of its own components, including its catalytic arm, the 26S proteasome. Here we show that in stressed mammalian cells, the proteasome is targeted by autophagy, which requires site-specific ubiquitination of its ubiquitin receptors. The process is mediated by the p62/SQSTM1 adapter and requires its ubiquitin-associated domain. Independently, p62 serves also as a shuttling protein for ubiquitinated substrates, using its PB1 domain. This places p62 in a pivotal position where under certain conditions it binds to the proteasome as a protease, whereas in other conditions it recognizes the proteasome as a prey. The regulation of this intricate “decision making” process remains elusive. The ubiquitin-proteasome system and autophagy are the two main proteolytic systems involved in, among other functions, the maintenance of cell integrity by eliminating misfolded and damaged proteins and organelles. Both systems remove their targets after their conjugation with ubiquitin. An interesting, yet incompletely understood problem relates to the fate of the components of the two systems. Here we provide evidence that amino acid starvation enhances polyubiquitination on specific sites of the proteasome, a modification essential for its targeting to the autophagic machinery. The uptake of the ubiquitinated proteasome is mediated by its interaction with the ubiquitin-associated domain of p62/SQSTM1, a process that also requires interaction with LC3. Importantly, deletion of the PB1 domain of p62, which is important for the targeting of ubiquitinated substrates to the proteasome, has no effect on stress-induced autophagy of this proteolytic machinery, suggesting that the domain of p62 that binds to the proteasome determines the function of p62 in either targeting substrates to the proteasome or targeting the proteasome to autophagy.

Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination

Significance A substrate-conjugated polyubiquitin chain is accepted as the “canonical” proteasomal degradation signal. Using a cellular (human and yeast) proteomic screen in the exclusive presence of nonpolymerizable ubiquitin, we show that a large group of proteins is degraded by the proteasome following monoubiquitination. The screen also unraveled polyubiquitin-dependent substrates, as they are stabilized in the presence of this ubiquitin mutant. Notably, monoubiquitination- and polyubiquitination-dependent substrates display distinct important characteristics. Monoubiquitinated proteins are of lower molecular mass and of lesser structural disorder. The two groups can be assigned to defined cellular pathways. Furthermore, some of the characteristics are confined to either human or yeast cells, suggesting that the mechanism of action/recognition of the ubiquitin system in the two organisms are different somehow. The “canonical” proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established—in both human and yeast cells—a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes.

The endoplasmic reticulum–residing chaperone BiP is short-lived and metabolized through N-terminal arginylation

N-terminal arginylation in response to various stresses causes the ER chaperone BiP to relocate to and be degraded in the cytosol. Instability of the ER-residing proteins Some ER proteins are subjected to a posttranslational modification known as N-terminal arginylation. Shim et al. found that the ER chaperone BiP was unexpectedly short-lived and that N-terminal arginylation promoted its relocalization to the cytosol, where it was degraded. ER stress, particularly when combined with proteasomal inhibition, increased the N-terminal arginylation of BiP. This pathway was inhibited by HERP, a component of the ER degradation pathway. These results suggest that ER proteins are more unstable than was previously appreciated, which may enable cells to quickly return the abundance of ER chaperones to basal amounts after ER stress has been resolved. BiP and other endoplasmic reticulum (ER)–resident proteins are thought to be metabolically stable and to function primarily in the ER lumen. We sought to assess how the abundance of these proteins dynamically fluctuates in response to various stresses and how their subpopulations are relocated to non-ER compartments such as the cytosol. We showed that the molecular chaperone BiP (also known as GRP78) was short-lived under basal conditions and ER stress. The turnover of BiP was in part driven by its amino-terminal arginylation (Nt-arginylation) by the arginyltransferase ATE1, which generated an autophagic N-degron of the N-end rule pathway. ER stress elicited the formation of R-BiP, an effect that was increased when the proteasome was also inhibited. Nt-arginylation correlated with the cytosolic relocalization of BiP under the types of stress tested. The cytosolic relocalization of BiP did not require the functionality of the unfolded protein response or the Sec61- or Derlin1-containing translocon. A key inhibitor of the turnover and Nt-arginylation of BiP was HERP (homocysteine-responsive ER protein), a 43-kDa ER membrane–integrated protein that is an essential component of ER-associated protein degradation. Pharmacological inhibition of the ER-Golgi secretory pathway also suppressed R-BiP formation. Finally, we showed that cytosolic R-BiP induced by ER stress and proteasomal inhibition was routed to autophagic vacuoles and possibly additional metabolic fates. These results suggest that Nt-arginylation is a posttranslational modification that modulates the function, localization, and metabolic fate of ER-resident proteins.

N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis

Significance Conjugation of the amino acid l-arginine (l-Arg) to the protein N termini is a universal posttranslational modification in eukaryotes, yet its functions remain poorly understood. Previous studies showed that the N-terminal Arg of arginylated substrates is bound by N-recognins to induce substrate ubiquitination and proteasomal degradation via the N-end rule pathway of the ubiquitin (Ub)-proteasome system (UPS). Here, we show that the same Nt-Arg residues of arginylated proteins modulate proteolytic flux via macroautophagy when misfolded proteins accumulate beyond the UPS’s capacity. Their Nt-Arg residues bind and allosterically activate the autophagic adaptor p62/STQSM/Sequestosome-1, facilitating cargo collection and lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux when the UPS is in trouble. The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D101-CDC6 and D1156-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62–cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.

Glioma‐derived cancer stem cells are hypersensitive to proteasomal inhibition

Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem cells derived from gliomas, GSCs, are up to 1,000‐fold more sensitive to PIs (IC50, 27–70 nM) compared with their differentiated controls (IC50, 47 to »100 μM). The stemness of GSCs correlates to increased ubiquitination, whose misregulation readily triggers apoptosis. PI‐induced apoptosis of GSCs is independent of NF‐κB but involves the phosphorylation of c‐Jun N‐terminal kinase as well as the transcriptional activation of endoplasmic reticulum (ER) stress‐associated proapoptotic mediators. In contrast to the general notion that ER stress‐associated apoptosis is signaled by prolonged unfolded protein response (UPR), GSC‐selective apoptosis is instead counteracted by the UPR. ATF3 is a key mediator in GSC‐selective apoptosis. Pharmaceutical uncoupling of the UPR from its downstream apoptosis sensitizes GSCs to PIs in vitro and during tumorigenesis in mice. Thus, a combinational treatment of a PI with an inhibitor of UPR‐coupled apoptosis may enhance targeting of stem cells in gliomas.