Subsequent Proteasome-dependent Degradation Research Articles

NEMO Nuances NF-κB

See related article, pages 133–144 Stress-induced adverse remodeling of the myocardium is a major mechanism leading to heart failure, a leading and rapidly escalating source of morbidity and mortality worldwide.1,2 As a result, much work is underway to dissect molecular mechanisms governing cardiac remodeling in hopes of identifying novel therapeutic targets. In recent years, much of this work has focused on the hypertrophic growth response of the cardiac myocyte. Initially adaptive, cardiac hypertrophy compensates for declines in cardiac performance and increases in wall stress; sustained hypertrophy, however, is a major risk factor for emergence of systolic dysfunction and clinical heart failure.3 On the bright side, numerous preclinical studies have demonstrated that abrogation of the hypertrophic response is well tolerated, and even beneficial.4 One potential target of therapy in the pathologically remodeled, hypertrophied heart is the transcription factor nuclear factor (NF)-κB. First discovered more than 20 years ago, NF-κB has been linked to numerous neurohormonal, pathophysiological, and stress stimuli responses, and it has been characterized most extensively in the immune system. In the heart, activation of NF-κB-dependent transcription has been detected in numerous disease contexts, including hypertrophy, ischemia/reperfusion injury, myocardial infarction, allograft rejection, myocarditis, apoptosis, and more.5,6 Within coronary vessels, NF-κB has been implicated in atherosclerosis and restenosis.5,6 However, parsing the specific role(s) of NF-κB in these diverse disease processes has been hampered by the embryonic lethality of inactivation of several NF-κB components.7–9 In heart, the NF-κB family of transcription factors comprises 4 members: p50, p52, p65, and RelB. All are capable of multimerization, forming either homo- or heterodimers, but the ubiquitously expressed p50 and p65 (herein termed NF-κB) are responsible for the majority of NF-κB binding activity in the myocardium. Activation of cytoplasmic NF-κB requires phosphorylation and subsequent proteasome-dependent degradation of its repressor, …

Lysine 269 is essential for cyclin D1 ubiquitylation by the SCFFbx4/αB-crystallin ligase and subsequent proteasome-dependent degradation

Protein ubiquitylation is a complex enzymatic process that results in the covalent attachment of ubiquitin, via Gly-76 of ubiquitin, to an ε-NH2-group of an internal lysine residue in a given substrate. While E3 ligases frequently utilize lysines adjacent to the degron within the substrate, many substrates can be targeted to the proteasome via polyubiquitylation of any lysine. We have assessed the role of lysine residues proximal to the cyclin D1 phosphodegron for ubiquitylation by the SCFFbx4/αB-crystallin ubiquitin ligase and subsequent proteasome-dependent degradation of cyclin D1. The work described herein reveals a requisite role for Lys-269 (K269) for the rapid, poly-ubiquitin mediated degradation of cyclin D1. Mutation of lysine 269, which is proximal to the phosphodegron sequence surrounding Thr-286 in cyclin D1, not only stabilizes cyclin D1, but also triggers cyclin D1 accumulation within the nucleus thereby promoting cell transformation. In addition, D1-K269R is resistant to genotoxic stress induced degradation, similar to non-phosphorylatable D1-T286A, supporting the critical role for the post-translational regulation of cyclin D1 in the response to DNA damaging agents. Strikingly, while mutation of lysine 269 to arginine inhibits cyclin D1 degradation, it does not inhibit cyclin D1 ubiquitylation in vivo demonstrating that ubiquitylation of a specific lysine can influence substrate targeting to the 26S proteasome.

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.

Two different mechanisms control ethylene sensitivity in Arabidopsis via the regulation of EBF2 expression

EIN3 is a key transcription factor in the ethylene signaling pathway in Arabidopsis and its action is controlled by SCFEBF1/2 complex-mediated ubiquitination and subsequent 26S proteasome-dependent degradation. Our recent study revealed that ethylene signaling is regulated by a negative feedback system involving the direct activation of the EBF2 promoter by EIN3. This negative feedback loop likely modulates both the magnitude of the response to ethylene and the recovery after its withdrawal. The sequence downstream of the EBF2 stop codon was also found to play a role in determining the EBF2 expression levels independently of ethylene signaling. The reduction in EBF2 expression via the function of this sequence was suggested to be important for coupling the EBF2 expression levels to growth control. Two different mechanisms thus appear to be involved in determining ethylene sensitivity in Arabidopsis.Addendum to: Konishi M, Yanagisawa S. Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2 expression by EIN3. Plant J 2008; In press.

Ajuba negatively regulates the Wnt signaling pathway by promoting GSK-3β-mediated phosphorylation of β-catenin

The Wnt signaling pathway is essential for embryonic development and carcinogenesis. Upon Wnt stimulation, beta-catenin is stabilized and associates with T-cell factor or lymphoid enhancing factor, thereby activating transcription of target genes. In the absence of Wnt stimulation, the level of beta-catenin is reduced via glycogen synthase kinase (GSK)-3beta-mediated phosphorylation and subsequent proteasome-dependent degradation. Here, we report the identification of Ajuba as a negative regulator of the Wnt signaling pathway. Ajuba is a member of LIM domain-containing proteins that contribute to cell fate determination and regulate cell proliferation and differentiation. We found that enforced expression of Ajuba destabilized beta-catenin and suppressed target gene expression. Ajuba promoted GSK-3beta-mediated phosphorylation of beta-catenin by reinforcing the association between beta-catenin and GSK-3beta. Furthermore, Wnt stimulation induced both accumulation of beta-catenin and destabilization of Ajuba. Our findings suggest that Ajuba is important for regulation of the Wnt signaling pathway.

NUP98 Dysregulation in Myeloid Leukemogenesis

Nucleoporin 98 (NUP98) is a component of the nuclear pore complex that facilitates mRNA export from the nucleus. It is mapped to 11p15.5 and is fused to a number of distinct partners, including nine members of the homeobox family as a consequence of leukemia-associated chromosomal translocations. NUP98-HOXA9 is associated with the t(7;11)(p15;p15) translocation in acute myeloid leukemia (AML), myelodysplastic syndrome, and blastic crisis of chronic myeloid leukemia. Expression of NUP98-HOXA9 in murine bone marrow resulted in a myeloproliferative disease progressing to AML by 7-8 months. Transduction of NUP98 fusion genes into human CD34(+) cells confers a proliferative advantage in long-term cytokine-stimulated and stromal cocultures and in NOD-SCID engrafted mice, associated with a five- to eight-fold increase in hematopoietic stem cells. NUP98-HOXA9 expression inhibited erythroid and myeloid differentiation but enhanced serial progenitor replating. NUP98-HOXA9 upregulated a number of homeobox genes of the A and B cluster as well as MEIS1 and Pim-1, and downmodulated globin genes and C/EBPalpha. The HOXA9 component of the NUP98-HOXA9 fusion protein was protected from cullin-4A-mediated ubiquitination and subsequent proteasome-dependent degradation. In NUP98-HOX-transduced CD34(+) cells and cells from AML patients with t(7;11)(p15;p15) NUP98 was no longer associated with the nuclear pore complex but formed intranuclear aggregation bodies. Analysis of NUP98 allelic expression in AML and myelodysplastic syndrome showed loss of heterozygosity observed in 29% of the former and 8% of the latter. This was associated with poor prognosis.

Enforced Expression of NUP98-HOXA9 in Human CD34+ Cells Enhances Stem Cell Proliferation

The t(7;11)(p15;p15) translocation, observed in acute myelogenous leukemia and myelodysplastic syndrome, generates a chimeric gene where the 5' portion of the sequence encoding the human nucleoporin NUP98 protein is fused to the 3' region of HOXA9. Here, we show that retroviral-mediated enforced expression of the NUP98-HOXA9 fusion protein in cord blood-derived CD34(+) cells confers a proliferative advantage in both cytokine-stimulated suspension cultures and stromal coculture. This advantage is reflected in the selective expansion of hematopoietic stem cells as measured in vitro by cobblestone area-forming cell assays and in vivo by competitive repopulation of nonobese diabetic/severe combined immunodeficient mice. NUP98-HOXA9 expression inhibited erythroid progenitor differentiation and delayed neutrophil maturation in transduced progenitors but strongly enhanced their serial replating efficiency. Analysis of the transcriptosome of transduced cells revealed up-regulation of several homeobox genes of the A and B cluster as well as of Meis1 and Pim-1 and down-modulation of globin genes and of CAAT/enhancer binding protein alpha. The latter gene, when coexpressed with NUP98-HOXA9, reversed the enhanced proliferation of transduced CD34(+) cells. Unlike HOXA9, the NUP98-HOXA9 fusion was protected from ubiquitination mediated by Cullin-4A and subsequent proteasome-dependent degradation. The resulting protein stabilization may contribute to the leukemogenic activity of the fusion protein.

Retinoic acid attenuates promyelocytic leukemia protein-induced cell death in breast cancer cells by activation of the ubiquitin–proteasome pathway

All- trans-retinoic acid and the tumor suppressor promyelocytic leukemia protein (PML) are potent regulators of the growth of cancer cells. This study investigates the individual and combined effects of PML, when overexpressed by the recombinant PML adenovirus, and all- trans-retinoic acid on the proliferation of human estrogen-receptor negative SKBR-3 and estrogen-receptor positive MCF-7 breast cancer cell lines. All- trans-retinoic acid caused a significant degree of cell death in SKBR-3 cells and MCF-7 cells, and PML elicited a similar incidence of or slightly more cell death in MCF-7 cells. Dual-treated cells displayed significantly less cell death than did single-treated cells in the same cell line. We concluded that PML and all- trans-retinoic acid cause cell death by different pathways: PML activates ERK1/2, p38 MAPK, and p21; arrests the cell cycle; and later causes cell death; and all- trans-retinoic acid activates proteasome function, caspase cleavage, and apoptosis. The combined use of all- trans-retinoic acid and PML gene therapy may not be the best treatment for patients with cancer, because the ubiquitinylation of PML and its subsequent proteasome-dependent degradation by retinoic acids occur before overexpressed PML exhibits tumor-suppressive activity.

Modulation of Hsp90 function in neurodegenerative disorders: a molecular-targeted therapy against disease-causing protein

Abnormal accumulation of disease-causing protein is a commonly observed characteristic in chronic neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and polyglutamine (polyQ) diseases. A therapeutic approach that could selectively eliminate would be a promising remedy for neurodegenerative disorders. Spinal and bulbar muscular atrophy (SBMA), one of the polyQ diseases, is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. The pathogenic gene product is polyQ-expanded androgen receptor (AR), which belongs to the heat shock protein (Hsp) 90 client protein family. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a novel Hsp90 inhibitor, is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-AAG is now in phase II clinical trials as a potential anti-cancer agent because of its ability to selectively degrade several oncoproteins. We have recently demonstrated the efficacy and safety of 17-AAG in a mouse model of SBMA. The administration of 17-AAG significantly ameliorated polyQ-mediated motor neuron degeneration by reducing the total amount of mutant AR. 17-AAG accomplished the preferential reduction of mutant AR mainly through Hsp90 chaperone complex formation and subsequent proteasome-dependent degradation. 17-AAG induced Hsp70 and Hsp40 in vivo as previously reported; however, its ability to induce HSPs was limited, suggesting that the HSP induction might support the degradation of mutant protein. The ability of 17-AAG to preferentially degrade mutant protein would be directly applicable to SBMA and other neurodegenerative diseases in which the disease-causing proteins also belong to the Hsp90 client protein family. Our proposed therapeutic approach, modulation of Hsp90 function by 17-AAG treatment, has emerged as a candidate for molecular-targeted therapies for neurodegenerative diseases. This review will consider our research findings and discuss the possibility of a clinical application of 17-AAG to SBMA and other neurodegenerative diseases.

Activation of HIF-prolyl Hydroxylases by R59949, an Inhibitor of the Diacylglycerol Kinase

Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

Mechanisms of Reactive Oxygen Species–Dependent Downregulation of Insulin Receptor Substrate-1 by Angiotensin II

Angiotensin II has been implicated in the pathogenesis of the vascular complications of insulin resistance. Recently, serine phosphorylation and degradation of insulin receptor substrate-1 (IRS-1) were shown to inhibit Akt activation and reduce glucose uptake. Therefore, we examined the effects of chronic angiotensin II treatment on IRS-1 phosphorylation and protein expression in vascular smooth muscle cells (VSMCs). Using Western analysis, we found that angiotensin II (100 nmol/L; 18 hours) caused a 61+/-5% degradation of IRS-1 and abolished insulin-induced activation of Akt. Phosphorylation of IRS-1 on Ser307, which leads to subsequent IRS-1 degradation, was stimulated by angiotensin II. This phosphorylation was blocked by the Src inhibitor PP1 and by the antioxidants N-acetylcysteine and ebselen. Stable overexpression of catalase abrogated angiotensin II-induced IRS-1 phosphorylation and IRS-1 degradation. Similarly, a mutant phosphoinositide-dependent kinase-1 (PDK1) that cannot associate with Src abolished IRS-1 phosphorylation and degradation induced by angiotensin II. Proteasome inhibitors also prevented IRS-1 degradation. Thus, angiotensin II decreases IRS-1 protein levels in VSMCs via Src, PDK1, and reactive oxygen species-mediated phosphorylation of IRS-1 on Ser307 and subsequent proteasome-dependent degradation. These events impair insulin signaling and provide a molecular basis for understanding the clinical observation that angiotensin II type 1 receptor antagonists improve insulin resistance and its associated vasculopathies.

Induction of β-Transducin Repeat-containing Protein by JNK Signaling and Its Role in the Activation of NF-κB

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed.

Adaptation of the ubiquitin-proteasome proteolytic pathway in cancer cachexia.

The ubiquitin-proteasome proteolytic pathway is of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitinylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyze recent findings in the regulation of ubiquitinylation of protein substrates and of their subsequent proteasome-dependent degradation in animal models of cancer cachexia. In particular, we discuss the influence of various mediators (anorexia, hormones, prostaglandins, cytokines, and proteolysis-inducing factor) in signaling the activation of ubiquitin-proteasome proteolysis in skeletal muscle. These findings have lead to new concepts that are starting to be used for preventing cachexia in cancer and other wasting diseases.