Yong Tae Kwon

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.

A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

ABSTRACT A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1−/− UBR2 −/− mice die as early embryos, the rescued UBR1 −/− UBR2 −/− fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a ∼70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1 −/− UBR2 −/− fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.

The N-End Rule Pathway

The N-end rule pathway is a proteolytic system in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as essential components of degrons, called N-degrons. Known N-recognins in eukaryotes mediate protein ubiquitylation and selective proteolysis by the 26S proteasome. Substrates of N-recognins can be generated when normally embedded destabilizing residues are exposed at the N terminus by proteolytic cleavage. N-degrons can also be generated through modifications of posttranslationally exposed pro-N-degrons of otherwise stable proteins; such modifications include oxidation, arginylation, leucylation, phenylalanylation, and acetylation. Although there are variations in components, degrons, and hierarchical structures, the proteolytic systems based on generation and recognition of N-degrons have been observed in all eukaryotes and prokaryotes examined thus far. The N-end rule pathway regulates homeostasis of various physiological processes, in part, through interaction with small molecules. Here, we review the biochemical mechanisms, structures, physiological functions, and small-molecule-mediated regulation of the N-end rule pathway.

Effects of low dose quercetin: Cancer cell-specific inhibition of cell cycle progression

Quercetin is a flavonoid present in many vegetables, fruits, and beverages. Due to its anti‐oxidant, anti‐tumor, and anti‐inflammatory activity, quercetin has been studied extensively as a chemoprevention agent in several cancer models. Since most of these studies used higher doses of quercetin than clinically achievable, we focused on the effectiveness of physiologically relevant doses of quercetin. A low dose of quercetin exerted cancer cell‐specific inhibition of proliferation and this inhibition resulted from cell cycle arrest at the G1 phase. Quercetin induced p21 CDK inhibitor with a concomitant decrease of phosphorylation of pRb, which inhibits the G1/S cell cycle progression by trapping E2F1. A low dose of quercetin induced mild DNA damage and Chk2 activation, which is the main regulator of p21 expression by quercetin. In addition, quercetin down‐regulated the cyclin B1 and CDK1, essential components of G2/M cell cycle progression. Inhibition of the recruitment of key transcription factor NF‐Y to cyclin B1 gene promoter by quercetin led to transcriptional inhibition. This study proved that the chemo‐preventive efficacy of a physiologically relevant dose of quercetin can be achievable through the inhibition of cell cycle progression. J. Cell. Biochem. 106: 73–82, 2009.

Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3α) of the N-End Rule Pathway

ABSTRACT The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3α). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Δ S. cerevisiae. We also constructedUBR1−/− mouse strains that lacked the UBR1 protein. UBR1 −/− mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1 −/− mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1 −/−mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein,UBR1 −/− fibroblasts contained the N-end rule pathway. Thus, UBR1 −/− mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3α). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.

Female Lethality and Apoptosis of Spermatocytes in Mice Lacking the UBR2 Ubiquitin Ligase of the N-End Rule Pathway

ABSTRACT Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1−/− mice, which lacked the pathways ubiquitin ligase E3α, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2 −/− mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2 −/− genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2 −/− females died as embryos, whereas UBR2 −/− males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2 −/− testes was normal and spermatogonia were intact as well, but UBR2 −/− spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2 −/− spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.

The N-end rule pathway: emerging functions and molecular principles of substrate recognition.

The N-end rule defines the protein-destabilizing activity of a given amino-terminal residue and its post-translational modification. Since its discovery 25 years ago, the pathway involved in the N-end rule has been thought to target only a limited set of specific substrates of the ubiquitin–proteasome system. Recent studies have provided insights into the components, substrates, functions and structural basis of substrate recognition. The N-end rule pathway is now emerging as a major cellular proteolytic system, in which the majority of proteins are born with or acquire specific N-terminal degradation determinants through protein-specific or global post-translational modifications.

Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells

Previous studies have demonstrated that a small subset of cancer cells is capable of tumor initiation. The existence of tumor initiating cancer stem cells (CSCs) has several implications in terms of future cancer treatment and therapies. However, recently, several researchers proposed that differentiated cancer cells (non-CSCs) can convert to stem-like cells to maintain equilibrium. These results imply that removing CSCs may prompt non-CSCs in the tumor to convert into stem cells to maintain the equilibrium. Interleukin-6 (IL-6) has been found to play an important role in the inducible formation of CSCs and their dynamic equilibrium with non-stem cells. In this study, we used CSC-like human breast cancer cells and their alternate subset non-CSCs to investigate how IL-6 regulates the conversion of non-CSCs to CSCs. MDA-MB-231 and MDA-MB-453 CSC-like cells formed mammospheres well, whereas most of non-stem cells died by anoikis and only part of the remaining non-stem cells produced viable mammospheres. Similar results were observed in xenograft tumor formation. Data from cytokine array assay show that IL-6 was secreted from non-CSCs when cells were cultured in ultra-low attachment plates. IL-6 regulates CSC-associated OCT-4 gene expression through the IL-6-JAK1-STAT3 signal transduction pathway in non-CSCs. Inhibiting this pathway by treatment with anti-IL-6 antibody (1 μg/ml) or niclosamide (0.5-2 μM)/LLL12 (5-10 μM) effectively prevented OCT-4 gene expression. These results suggest that the IL-6-JAK1-STAT3 signal transduction pathway plays an important role in the conversion of non-CSCs into CSCs through regulation of OCT-4 gene expression.

Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway.

ABSTRACT The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouseAte1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster)Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p–GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them inate1Δ Saccharomyces cerevisiae in the presence of test substrates that included Asp-βgal (β-galactosidase) and Cys-βgal. Both forms of the mouse R-transferase conferred instability on Asp-βgal (but not on Cys-βgal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is ∼0.1 in the skeletal muscle, ∼0.25 in the spleen, ∼3.3 in the liver and brain, and ∼10 in the testis, suggesting that the two R-transferases are functionally distinct.

Altered Activity, Social Behavior, and Spatial Memory in Mice Lacking the NTAN1p Amidase and the Asparagine Branch of the N-End Rule Pathway

ABSTRACT The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by theUbr1-encoded E3α, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (NtN-amidase), encoded by the Ntan1 gene. In wild-type embryos,Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1 −/− mouse strains lacked the NtN-amidase activity but retained glutamine-specific NtQ-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1 −/− fibroblasts. TheNtan1 −/− mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.