Jong Bok Yoon

Regional and temporal specialization in the nucleus: a transcriptionally‐active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle

PTF (PSE‐binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter ∼1.3 μm) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription ‘factories’ where Br‐UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only ∼30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes.

Expression of Heat Shock Protein 27 in Human Atherosclerotic Plaques and Increased Plasma Level of Heat Shock Protein 27 in Patients With Acute Coronary Syndrome

Background— We intended to identify proteins that are differentially expressed in human atherosclerotic plaques. Methods and Results— Comparative 2-dimensional electrophoretic analysis on carotid atherosclerotic endarterectomy specimens (n=10) revealed that heat shock protein 27 (Hsp27) expression was significantly increased in the nearby normal-appearing area compared with the plaque core area from the same vessel specimen, which was further confirmed by Western blot analysis. The Hsp27 expression in the adjacent normal-appearing vessel areas was much higher than that in nonatherosclerotic reference arteries. The phosphorylation of Hsp27 showed a gradation in the degree of phosphorylation: greatest in the reference arteries, intermediate in the adjacent normal-appearing area, and lowest in plaque core area. Immunohistochemical analysis showed that the phosphorylation of Hsp27 of smooth muscle cells in the carotid endarterectomy specimens was decreased compared with that in the reference artery specimen. The mean plasma level of Hsp27 was significantly higher in patients with acute coronary syndrome (ACS) (n=27; 106.1±74.1 ng/mL) than in the normal reference subjects (n=29; 45.8±29.5 ng/mL; P<0.005). The plasma levels of Hsp27 were significantly correlated with those of heat shock protein 70 (Hsp70) (r=0.422, P<0.0005), with adjustment for ACS/reference status. Conclusions— In the atherosclerotic lesion, Hsp27 expression is increased in the normal-appearing vessel adjacent to atherosclerotic plaque, whereas levels in the plaque itself are significantly decreased. Both plaque and adjacent artery show decreased Hsp27 phosphorylation compared with reference vessel. In ACS, plasma Hsp27 and Hsp70 are increased, and levels of Hsp27 correlate with Hsp70, C-reactive protein, and CD40L levels.

Receptor interacting protein is ubiquitinated by cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) in vitro

Receptor interacting protein (RIP) is recruited to tumor necrosis factor‐α receptor 1 (TNFR1) complex upon stimulation and plays a crucial role in the receptor‐mediated NF‐κB activation. Among the components of the TNFR1 complex are proteins that possess ubiquitin‐protein isopeptide ligase (E3) activities, such as TNFR1‐associated factor 2 (TRAF2), cellular inhibitor of apoptosis proteins (c‐IAPs) namely, c‐IAP1 and c‐IAP2. Here, we showed that ectopically expressed RIP is ubiquitinated, and either the intermediate or death domain of RIP is required for this modification. Expression of c‐IAP1 and c‐IAP2 decreased the steady‐state level of RIP, which was blocked by inhibition of the 26S proteasome. RIP degradation requires intact c‐IAP2 containing the RING domain. Our in vitro ubiquitination assay revealed that while TRAF2 had no effect, both c‐IAP1 and c‐IAP2‐mediated RIP ubiquitination with similar efficiency, indicating that c‐IAPs can function as E3 toward RIP.

Ras Stabilization Through Aberrant Activation of Wnt/β-Catenin Signaling Promotes Intestinal Tumorigenesis

Crosstalk between the Wnt/β-catenin and the MAPK pathways contributes to colon cancer. Blocking Ras Degradation in Colon Cancer Both the Wnt/β-catenin pathway and the Ras-activated mitogen-activated protein kinase (MAPK) pathway can contribute to cancer. Jeong et al. report an interaction between these two pathways through the β-catenin destruction complex and the E3 ubiquitin ligase adaptor β-TrCP. Ras was phosphorylated by glycogen synthase kinase 3β, a component of the β-catenin destruction complex, and this enabled the phosphorylation-dependent recruitment of β-TrCP, which increased ubiquitin- and proteasome-mediated degradation of Ras, thereby providing a brake on Ras-mediated activation of the MAPK pathway. Conditions that compromised the function of the destruction complex, such as the presence of Wnt or genetic mutations or deficiency in components of the destruction complex, stabilized Ras and increased MAPK pathway activity. The importance of this regulatory crosstalk was verified in colon cancer samples from patients and mouse models of colon cancer, suggesting that targeting both the hyperactive Wnt pathway and the Ras pathway may be an effective combination therapy. Although the guanosine triphosphate/guanosine diphosphate loading switch is a major regulatory mechanism that controls the activity of the guanosine triphosphatase Ras, we report a distinct mechanism for regulating Ras activity through phosphorylation-mediated degradation and describe the role of this second regulatory mechanism in the suppression of cellular transformation and tumors induced by Ras mutations. We found that negative regulators of Wnt/β-catenin signaling contributed to the polyubiquitin-dependent degradation of Ras after its phosphorylation by glycogen synthase kinase 3β (GSK3β) and the subsequent recruitment of β-TrCP–E3 ligase. We found a positive association between tumorigenesis and Ras stabilization resulting from the aberrant activation of Wnt/β-catenin signaling in adenomas from two mouse models of colon cancer, human colonic tumors from various stages, and colon polyps of patients with familial adenomatous polyposis. Our results indicated that GSK3β plays an essential role in Ras degradation and that inhibition of this degradation pathway by aberrant Wnt/β-catenin signaling may contribute to Ras-induced transformation in colorectal tumorigenesis.

Herp enhances ER-associated protein degradation by recruiting ubiquilins

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3delta, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3delta was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.

Parkin Directly Modulates 26S Proteasome Activity

Parkinsons disease (PD) is a common neurodegenerative disease that involves the deterioration of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD remains poorly understood, recent genetic, postmortem, and experimental evidence shows that abnormal protein accumulation and subsequent aggregate formation are prominent features of both sporadic and familial PD. While proteasome dysfunction is observed in PD, diverse mutations in the parkin gene are linked to early-onset autosomal-recessive forms of familial PD. We demonstrate that parkin, an E3 ubiquitin ligase, activates the 26S proteasome in an E3 ligase activity-independent manner. Furthermore, an N-terminal ubiquitin-like domain within parkin is critical for the activation of the 26S proteasome through enhancing the interaction between 19S proteasomal subunits, whereas the PD-linked R42P mutant abolishes this action. The current findings point to a novel role for parkin for 26S proteasome assembly and suggest that parkin mutations contribute to the proteasomal dysfunction in PD.

Hypoxia-induced methylation of a pontin chromatin remodeling factor

Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Although Pontin is frequently overexpressed in human cancers of various types and implicated in oncogenic functions, the upstream signaling network leading to the regulation of Pontin that in turn affects transcription of downstream target genes has not been extensively studied. Here, we identify Pontin is methylated by G9a/GLP methyltransferases in hypoxic condition and potentiates HIF-1α-mediated activation by increasing the recruitment of p300 coactivator to a subset of HIF-1α target promoters. Intriguingly, Pontin methylation results in the increased invasive and migratory properties by activating downstream target gene, Ets1. In contrast, inhibition of Pontin methylation results in the suppression of tumorigenic and metastatic properties. Together, our data provide new approaches by targeting Pontin methylation and its downstream targets for the development of therapeutic agents for human cancers.

Proteasomal ATPase-Associated Factor 1 Negatively Regulates Proteasome Activity by Interacting with Proteasomal ATPases

ABSTRACT The 26S proteasome, composed of the 20S core and the 19S regulatory complex, plays a central role in ubiquitin-dependent proteolysis by catalyzing degradation of polyubiquitinated proteins. In a search for proteins involved in regulation of the proteasome, we affinity purified the 19S regulatory complex from HeLa cells and identified a novel protein of 43 kDa in size as an associated protein. Immunoprecipitation analyses suggested that this protein specifically interacted with the proteasomal ATPases. Hence the protein was named proteasomal ATPase-associated factor 1 (PAAF1). Immunoaffinity purification of PAAF1 confirmed its interaction with the 19S regulatory complex and further showed that the 19S regulatory complex bound with PAAF1 was not stably associated with the 20S core. Overexpression of PAAF1 in HeLa cells decreased the level of the 20S core associated with the 19S complex in a dose-dependent fashion, suggesting that PAAF1 binding to proteasomal ATPases inhibited the assembly of the 26S proteasome. Proteasomal degradation assays using reporters based on green fluorescent protein revealed that overexpression of PAAF1 inhibited the proteasome activity in vivo. Furthermore, the suppression of PAAF1 expression that is mediated by small inhibitory RNA enhanced the proteasome activity. These results suggest that PAAF1 functions as a negative regulator of the proteasome by controlling the assembly/disassembly of the proteasome.

The HECT Domain of TRIP12 Ubiquitinates Substrates of the Ubiquitin Fusion Degradation Pathway

The ubiquitin fusion degradation (UFD) pathway is a proteolytic system conserved in yeast and mammals in which an uncleavable ubiquitin moiety linked to the N terminus of a protein functions as a degradation signal of the fusion protein. Although key components of the UFD pathway in yeast have been identified, the E3 enzyme of the human UFD pathway has not been studied. In this work, we show that TRIP12 is the E3 enzyme of the human UFD pathway. Thus, TRIP12 catalyzes in vitro ubiquitination of UFD substrates in conjunction with E1, E2, and E4 enzymes. Knockdown of TRIP12 stabilizes not only artificial UFD substrates but a physiological substrate UBB+1. Moreover, TRIP12 knockdown reduces UBB+1-induced cell death in human neuroblastoma cells. Surprisingly, complementation of TRIP12 knockdown cells with the TRIP12 HECT domain mostly restores efficient degradation of UFD substrates, indicating that the TRIP12 HECT domain can act as the E3 enzyme for the UFD pathway in human cells. The TRIP12 HECT domain directs ubiquitination of UFD substrates in vitro and can be specifically cross-linked to the ubiquitin moiety of the substrates in vivo, suggesting that the TRIP12 HECT domain possesses a noncovalent ubiquitin-binding site. In addition, we demonstrate that UbΔGG, a mutant ubiquitin that cannot be conjugated to other proteins, is a substrate of the TRIP12 HECT domain both in vivo and in vitro, indicating that the C-terminal extension fused to the uncleavable ubiquitin is not required for substrate recognition in the UFD pathway. These results provide new insights into the mechanism of the mammalian UFD pathway and the functional nonequivalence of different HECT domains.

Osmotic stress inhibits proteasome by p38 MAPK-dependent phosphorylation.

Osmotic stress causes profound perturbations of cell functions. Although the adaptive responses required for cell survival upon osmotic stress are being unraveled, little is known about the effects of osmotic stress on ubiquitin-dependent proteolysis. We now report that hyperosmotic stress inhibits proteasome activity by activating p38 MAPK. Osmotic stress increased the level of polyubiquitinated proteins in the cell. The selective p38 inhibitor SB202190 decreased osmotic stress-associated accumulation of polyubiquitinated proteins, indicating that p38 MAPK plays an inhibitory role in the ubiquitin proteasome system. Activated p38 MAPK stabilized various substrates of the proteasome and increased polyubiquitinated proteins. Proteasome preparations purified from cells expressing activated p38 MAPK had substantially lower peptidase activities than control proteasome samples. Proteasome phosphorylation sites dependent on p38 were identified by measuring changes in the extent of proteasome phosphorylation in response to p38 MAPK activation. The residue Thr-273 of Rpn2 is the major phosphorylation site affected by p38 MAPK. The mutation T273A in Rpn2 blocked the proteasome inhibition that is mediated by p38 MAPK. These results suggest that p38 MAPK negatively regulates the proteasome activity by phosphorylating Thr-273 of Rpn2.