Protein Quality Control by Molecular Chaperones in Neurodegeneration

Ciechanover, Aaron,Kwon, Yong Tae Frontiers Media S.A. 2017 Frontiers in neuroscience Vol.11 No.-

<P>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 Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's 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.</P>

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

Aaron Ciechanover,권용태 생화학분자생물학회 2015 Experimental and molecular medicine Vol.47 No.-

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 lysosomalhydrolases. 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 Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s 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

Kwon, Yong Tae,Ciechanover, Aaron Elsevier 2017 Trends in biochemical sciences Vol.42 No.11

<P>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).</P> <P><B>Trends</B></P> <P>Monoubiquitination has been thought to be non-proteolytic and regulate the interactions and activities of substrates. Emerging evidence shows that monoubiquitin(s) of substrates functions as the degron in the UPS and autophagy.</P> <P>Atypical linkages such as Lys11 and Lys29 linkages serve as proteasomal degrons, alone or in combination with other linkages such as Lys48 and/or Lys63.</P> <P>Ubiquitin chains such as Lys63 linkages mediate autophagic protein quality control through the recognition by autophagic adaptors such as p62.</P> <P>Ubiquitin chains, mainly Lys63 linkages, function as a <I>trans</I>-degron for autophagic removal of cellular organelles and structures in mitophagy, pexophagy, ER-phagy, ribophagy, and lipophagy.</P> <P>Ubiquitin is the substrate of small-molecule post-translational modifications such as phosphorylation and acetylation.</P>

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

Cohen-Kaplan, Victoria,Livneh, Ido,Avni, Noa,Fabre, Bertrand,Ziv, Tamar,Kwon, Yong Tae,Ciechanover, Aaron National Academy of Sciences 2016 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.113 No.47

<P>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.</P>

Diverse fate of ubiquitin chain moieties: The proximal is degraded with the target, and the distal protects the proximal from removal and recycles

Sun, Hao,Mali, Sachitanand M.,Singh, Sumeet K.,Meledin, Roman,Brik, Ashraf,Kwon, Yong Tae,Kravtsova-Ivantsiv, Yelena,Bercovich, Beatrice,Ciechanover, Aaron National Academy of Sciences 2019 Proceedings of the National Academy of Sciences Vol.116 No.16

<P><B>Significance</B></P><P>The canonical targeting signal for degrading proteins by the ubiquitin (Ub) system—a chain composed of multiple Ub moieties—has remained a mystery. The structure of the proteasome, the enzyme that recognizes the signal and degrades the target substrate cannot explain why such a long chain is needed. To better understand this problem, we synthesized α-globin to which chains with different number of Ubs were attached. In long adducts, the proximal Ub remains on the substrate, likely securing its attachment to the proteasome, and is degraded with it. The distal Ub protects the proximal from removal by deubiquitinating enzymes and is then removed and recycled. In short adducts, the Ub moieties are rapidly removed, and the substrate remains stable.</P><P>One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K<SUP>48</SUP>-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.</P>

Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/GRP78/BiP

Cha-Molstad, Hyunjoo,Yu, Ji Eun,Lee, Su Hyun,Kim, Jung Gi,Sung, Ki Sa,Hwang, Joonsung,Yoo, Young Dong,Lee, Yoon Jee,Kim, Sung Tae,Lee, Dae Hee,Ciechanover, Aaron,Kim, Bo Yeon,Kwon, Yong Tae Informa UK (TaylorFrancis) 2016 AUTOPHAGY Vol.12 No.2

<P>The N-end rule pathway is a proteolytic system, in which single N-terminal residues act as a determinant of a class of degrons, called N-degrons. In the ubiquitin (Ub)-proteasome system, specific recognition components, called N-recognins, recognize N-degrons and accelerate polyubiquitination and proteasomal degradation of the substrates. In this study, we show that the pathway regulates the activity of the macroautophagic receptor SQSTM1/p62 (sequestosome 1) through N-terminal arginylation (Nt-arginylation) of endoplasmic reticulum (ER)-residing molecular chaperones, including HSPA5/GRP78/BiP, CALR (calreticulin), and PDI (protein disulfide isomerase). The arginylation is co-induced with macroautophagy (hereafter autophagy) as part of innate immunity to cytosolic DNA and when misfolded proteins accumulate under proteasomal inhibition. Following cytosolic relocalization and arginylation, Nt-arginylated HSPA5 (R-HSPA5) is targeted to autophagosomes and degraded by lysosomal hydrolases through the interaction of its N-terminal Arg (Nt-Arg) with ZZ domain of SQSTM1. Upon binding to Nt-Arg, SQSTM1 undergoes a conformational change, which promotes SQSTM1 self-polymerization and interaction with LC3, leading to SQSTM1 targeting to autophagosomes. Cargoes of R-HSPA5 include cytosolic misfolded proteins destined to be degraded through autophagy. Here, we discuss the mechanisms by which the N-end rule pathway regulates SQSTM1-dependent selective autophagy.</P>

The N-Degron Pathway Mediates ER-phagy

Ji, Chang Hoon,Kim, Hee Yeon,Heo, Ah Jung,Lee, Su Hyun,Lee, Min Ju,Kim, Su Bin,Srinivasrao, Ganipisetti,Mun, Su Ran,Cha-Molstad, Hyunjoo,Ciechanover, Aaron,Choi, Cheol Yong,Lee, Hee Gu,Kim, Bo Yeon,Kw Elsevier 2019 Molecular cell Vol.75 No.5

<P><B>Summary</B></P> <P>The endoplasmic reticulum (ER) is susceptible to wear-and-tear and proteotoxic stress, necessitating its turnover. Here, we show that the N-degron pathway mediates ER-phagy. This autophagic degradation initiates when the transmembrane E3 ligase TRIM13 (also known as RFP2) is ubiquitinated via the lysine 63 (K63) linkage. K63-ubiquitinated TRIM13 recruits p62 (also known as sequestosome-1), whose complex undergoes oligomerization. The oligomerization is induced when the ZZ domain of p62 is bound by the N-terminal arginine (Nt-Arg) of arginylated substrates. Upon activation by the Nt-Arg, oligomerized TRIM13-p62 complexes are separated along with the ER compartments and targeted to autophagosomes, leading to lysosomal degradation. When protein aggregates accumulate within the ER lumen, degradation-resistant autophagic cargoes are co-segregated by ER membranes for lysosomal degradation. We developed synthetic ligands to the p62 ZZ domain that enhance ER-phagy for ER protein quality control and alleviate ER stresses. Our results elucidate the biochemical mechanisms and pharmaceutical means that regulate ER homeostasis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The autophagic adaptor p62 mediates autophagic degradation of the ER (ER-phagy) </LI> <LI> The ER membrane E3 ligase TRIM13 is a ubiquitin-dependent ER-phagy receptor to p62 </LI> <LI> N-terminal arginylation is an ER-phagy degron via binding to the ZZ domain of p62 </LI> <LI> p62-TRIM13-Nt-Arg circuit mediates ER protein quality control and homeostasis </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Regulation of autophagic proteolysis by the N-recognin SQSTM1/p62 of the N-end rule pathway

Cha-Molstad, Hyunjoo,Lee, Su Hyun,Kim, Jung Gi,Sung, Ki Woon,Hwang, Joonsung,Shim, Sang Mi,Ganipisetti, Srinivasrao,McGuire, Terry,Mook-Jung, Inhee,Ciechanover, Aaron,Xie, Xiang-Qun,Kim, Bo Yeon,Kwon, Informa UK (TaylorFrancis) 2018 AUTOPHAGY Vol.14 No.2

<P>In macroautophagy/autophagy, cargoes are collected by specific receptors, such as SQSTM1/p62 (sequestosome 1), and delivered to phagophores for lysosomal degradation. To date, little is known about how cells modulate SQSTM1 activity and autophagosome biogenesis in response to accumulating cargoes. In this study, we show that SQSTM1 is an N-recognin whose ZZ domain binds N-terminal arginine (Nt-Arg) and other N-degrons (Nt-Lys, Nt-His, Nt-Trp, Nt-Phe, and Nt-Tyr) of the N-end rule pathway. The substrates of SQSTM1 include the endoplasmic reticulum (ER)-residing chaperone HSPA5/GRP78/BiP. Upon N-end rule interaction with the Nt-Arg of arginylated HSPA5 (R-HSPA5), SQSTM1 undergoes self-polymerization via disulfide bonds of Cys residues including Cys113, facilitating cargo collection. In parallel, Nt-Arg-bound SQSTM1 acts as an inducer of autophagosome biogenesis and autophagic flux. Through this dual regulatory mechanism, SQSTM1 plays a key role in the crosstalk between the ubiquitin (Ub)-proteasome system (UPS) and autophagy. Based on these results, we employed 3D-modeling of SQSTM1 and a virtual chemical library to develop small molecule ligands to the ZZ domain of SQSTM1. These autophagy inducers accelerated the autophagic removal of mutant HTT (huntingtin) aggregates. We suggest that SQSTM1 can be exploited as a novel drug target to modulate autophagic processes in pathophysiological conditions.</P>

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

Yoo, Young Dong,Mun, Su Ran,Ji, Chang Hoon,Sung, Ki Woon,Kang, Keum Young,Heo, Ah Jung,Lee, Su Hyun,An, Jee Young,Hwang, Joonsung,Xie, Xiang-Qun,Ciechanover, Aaron,Kim, Bo Yeon,Kwon, Yong Tae National Academy of Sciences 2018 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.115 No.12

<P>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 Ntarginylation, 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 cellularwastes tomacroautophagy 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.</P>

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

Kravtsova-Ivantsiv, Y.,Shomer, I.,Cohen-Kaplan, V.,Snijder, B.,Superti-Furga, G.,Gonen, H.,Sommer, T.,Ziv, T.,Admon, A.,Naroditsky, I.,Jbara, M.,Brik, A.,Pikarsky, E.,Kwon, Y.,Doweck, I.,Ciechanover, Cell Press ; MIT Press 2015 Cell Vol.161 No.2

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.