Young Dong Yoo

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.

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.

Characterization of Arginylation Branch of N-end Rule Pathway in G-protein-mediated Proliferation and Signaling of Cardiomyocytes

Background: ATE1 transfers Arg to protein N termini, generating the degron for the N-end rule pathway. Results: ATE1-deficient cardiomyocytes are impaired in the PLC/PKC-MEK1-ERK axis of Gαq-mediated cardiac signaling. Conclusion: The arginine branch of the N-end rule pathway controls G-protein signaling in cardiomyocytes in part through hypoxia-sensitive degradation of GAP proteins. Significance: This study provides a cellular mechanism underlying cardiovascular defects observed in ATE1-deficient mice. The N-end rule pathway is a proteolytic system in which destabilizing N-terminal amino acids of short lived proteins are recognized by recognition components (N-recognins) as an essential element of degrons, called N-degrons. In eukaryotes, the major way to generate N-degrons is through arginylation by ATE1 arginyl-tRNA-protein transferases, which transfer Arg from aminoacyl-tRNA to N-terminal Asp and Glu (and Cys as well in mammals). We have shown previously that ATE1-deficient mice die during embryogenesis with defects in cardiac and vascular development. Here, we characterized the arginylation-dependent N-end rule pathway in cardiomyocytes. Our results suggest that the cardiac and vascular defects in ATE1-deficient embryos are independent from each other and cell-autonomous. ATE1-deficient myocardium and cardiomyocytes therein, but not non-cardiomyocytes, showed reduced DNA synthesis and mitotic activity ∼24 h before the onset of cardiac and vascular defects at embryonic day 12.5 associated with the impairment in the phospholipase C/PKC-MEK1-ERK axis of Gαq-mediated cardiac signaling pathways. Cardiac overexpression of Gαq rescued ATE1-deficient embryos from thin myocardium and ventricular septal defect but not from vascular defects, genetically dissecting vascular defects from cardiac defects. The misregulation in cardiovascular signaling can be attributed in part to the failure in hypoxia-sensitive degradation of RGS4, a GTPase-activating protein for Gαq. This study is the first to characterize the N-end rule pathway in cardiomyocytes and reveals the role of its arginylation branch in Gαq-mediated signaling of cardiomyocytes in part through N-degron-based, oxygen-sensitive proteolysis of G-protein regulators.

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.

Effects of Different Sensitization Events on HLA Alloimmunization in Solid Organ Transplantation Patients

OBJECTIVE HLA alloimmunization is caused by various sensitization events, such as transfusion, pregnancy, or organ transplantation. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using an identical test method. We evaluated how different sensitization events affect the panel-reactive antibody (PRA) status in solid organ transplantation candidates. METHODS PRA identification tests were performed on 674 patients (354 males and 320 females) using Luminex assay kits (LIFECODES, Gen-Probe, Stamford, CT, United States). PRA-positive rates (HLA-A, B, or DR antibodies of median fluorescence intensity [MFI] values of ≥1000) and antibody strengths in PRA-positive cases were analyzed according to the different sensitization events and gender. RESULTS PRA (class I and/or II)-positive rates were significantly higher in patients with transfusion (33.0%; P = .001), pregnancy (71.4%; P < .001), or transplantation events (76.9%; P < .001) than in controls without any identifiable sensitization events (5.6%). Transplantation had the strongest immunization effect, especially for class II HLA antigens. Female compared with male patients (60.3% vs 34.2%; P < .001) and retransplantation compared with first transplantation candidates of kidney transplantation (80.2% vs 41.1%; P < .001) showed a significantly higher PRA-positive rate. Retransplantation candidates (MFI 14,164) showed significantly stronger antibody strength than first transplantation candidates (MFI 5456) and those with single sensitization events of transfusion (MFI 4185) or pregnancy (MFI 5548; P < .001 for each). CONCLUSION Solid organ transplantation appears to have the strongest HLA alloimmunization effect followed by pregnancy and transfusion, especially for class II HLA antigens.

UBR2 of the N-end rule pathway is required for chromosome stability via histone ubiquitylation in spermatocytes and somatic cells.

The N-end rule pathway is a proteolytic system in which its recognition components (N-recognins) recognize destabilizing N-terminal residues of short-lived proteins as an essential element of specific degrons, called N-degrons. The RING E3 ligases UBR2 and UBR1 are major N-recognins that share size (200 kDa), conserved domains and substrate specificities to N-degrons. Despite the known function of the N-end rule pathway in degradation of cytosolic proteins, the major phenotype of UBR2-deficient male mice is infertility caused by arrest of spermatocytes at meiotic prophase I. UBR2-deficient spermatocytes are impaired in transcriptional silencing of sex chromosome-linked genes and ubiquitylation of histone H2A. In this study we show that the recruitment of UBR2 to meiotic chromosomes spatiotemporally correlates to the induction of chromatin-associated ubiquitylation, which is significantly impaired in UBR2-deficient spermatocytes. UBR2 functions as a scaffold E3 that promotes HR6B/UbcH2-dependent ubiquitylation of H2A and H2B but not H3 and H4, through a mechanism distinct from typical polyubiquitylation. The E3 activity of UBR2 in histone ubiquitylation is allosterically activated by dipeptides bearing destabilizing N-terminal residues. Insufficient monoubiquitylation and polyubiquitylation on UBR2-deficient meiotic chromosomes correlate to defects in double strand break (DSB) repair and other meiotic processes, resulting in pachytene arrest at stage IV and apoptosis. Some of these functions of UBR2 are observed in somatic cells, in which UBR2 is a chromatin-binding protein involved in chromatin-associated ubiquitylation upon DNA damage. UBR2-deficient somatic cells show an array of chromosomal abnormalities, including hyperproliferation, chromosome instability, and hypersensitivity to DNA damage-inducing reagents. UBR2-deficient mice enriched in C57 background die upon birth with defects in lung expansion and neural development. Thus, UBR2, known as the recognition component of a major cellular proteolytic system, is associated with chromatin and controls chromatin dynamics and gene expression in both germ cells and somatic cells.

Cloning and characterization of the gene encoding an extracellular alkaline serine protease from Vibrio metschnikovii strain RH530.

The gene vapF, encoding VapT, one of the extracellular sodium dodecyl sulfate (SDS)-resistant alkaline serine proteases (Serp) from the Gram- Vibrio metschnikovii strain RH530 has been cloned in Escherichia coli. The recombinant E. coli produced a protease which co-migrated with VapT on gelatin polyacrylamide gels. The nucleotide (nt) sequence of the cloned vapT revealed a single open reading frame of 1641 bp encoding 547 amino acids (aa) (58,961 Da). Upon analysis of the N-terminal aa sequence, VapT was shown to be processed properly in recombinant E. coli and to consist of 428 aa (45,626 Da). The deduced aa sequence of VapT showed significant sequence homology to subtilisin Carlsberg from Bacillus licheniformis, particularly in the regions containing active site residues and calcium-binding sites. VapT had an intervening region of approx. 149 aa between the His and Ser residues of the active site, as compared with other Serp.

The N-end rule proteolytic system in autophagy

The N-end rule pathway is a cellular proteolytic system that utilizes specific N-terminal residues as degradation determinants, called N-degrons. N-degrons are recognized and bound by specific recognition components (N-recognins) that mediate polyubiquitination of low-abundance regulators and selective proteolysis through the proteasome. Our earlier work identified UBR4/p600 as one of the N-recognins that promotes N-degron-dependent proteasomal degradation. In this study, we show that UBR4 is associated with cellular cargoes destined to autophagic vacuoles and is degraded by the lysosome. UBR4 loss causes multiple misregulations in autophagic pathways, including an increased formation of LC3 puncta. UBR4-deficient mice die during embryogenesis primarily due to defective vascular development in the yolk sac (YS), wherein UBR4 is associated with a bulk lysosomal degradation system that absorbs maternal proteins from the YS cavity and digests them into amino acids. Our results suggest that UBR4 plays a role not only in selective proteolysis of short-lived regulators through the proteasome, but also bulk degradation through the lysosome. Here, we discuss a possible mechanism of UBR4 as a regulatory component in the delivery of cargoes destined to interact with the autophagic core machinery.

Development and Characterization of Monomeric N‑End Rule Inhibitors through In Vitro Model Substrates

In the N-end rule pathway, a set of N-terminal amino acids, called N-degrons, are recognized and ubiquitinated by the UBR proteins. Here we examined various N-end rule inhibitors to identify essential structural components of the system. Our study using in vitro biochemical assay indicated that the l-conformation and protonated α-amino group of the first residue were critical for N-degrons to properly interact with the UBR proteins. The monomeric molecules with minimum interacting motifs showed endopeptidase resistance and better inhibitory activities than traditional dipeptide inhibitors. Collectively, our study identifies a pharmacophore of N-end rule inhibitors, which provides a structural platform to improve the efficiency and druggable properties of inhibitors. Considering that the N-end rule has been implicated in many pathophysiological processes in cells, inhibitors of this pathway, such as p-chloroamphetamine, are potentially of clinical interest in a novel aspect of action mechanisms.

Molecular mechanisms controlling asymmetric and symmetric self-renewal of cancer stem cells

Cancer stem cells (CSCs), or alternatively called tumor initiating cells (TICs), are a subpopulation of tumor cells, which possesses the ability to self-renew and differentiate into bulk tumor mass. An accumulating body of evidence suggests that CSCs contribute to the growth and recurrence of tumors and the resistance to chemo- and radiotherapy. CSCs achieve self-renewal through asymmetric division, in which one daughter cell retains the self-renewal ability, and the other is destined to differentiation. Recent studies revealed the mechanisms of asymmetric division in normal stem cells (NSCs) and, to a limited degree, CSCs as well. Asymmetric division initiates when a set of polarity-determining proteins mark the apical side of mother stem cells, which arranges the unequal alignment of mitotic spindle and centrosomes along the apical-basal polarity axis. This subsequently guides the recruitment of fate-determining proteins to the basal side of mother cells. Following cytokinesis, two daughter cells unequally inherit centrosomes, differentiation-promoting fate determinants, and other proteins involved in the maintenance of stemness. Modulation of asymmetric and symmetric division of CSCs may provide new strategies for dual targeting of CSCs and the bulk tumor mass. In this review, we discuss the current understanding of the mechanisms by which NSCs and CSCs achieve asymmetric division, including the functions of polarity- and fate-determining factors.