Cytomegalovirus promoter up-regulation is the major cause of increased protein levels of unstable reporter proteins after treatment of living cells with proteasome inhibitors.

Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E–associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

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TRIB1 downregulates hepatic lipogenesis and glycogenesis via multiple molecular interactions

Journal of Molecular Endocrinology ◽

10.1530/jme-13-0243 ◽

2014 ◽

Vol 52 (2) ◽

pp. 145-158 ◽

Cited By ~ 30

Author(s):

Yuumi Ishizuka ◽

Kazuhiro Nakayama ◽

Ayumi Ogawa ◽

Saho Makishima ◽

Supichaya Boonvisut ◽

...

Keyword(s):

Molecular Interactions ◽

Molecular Mechanisms ◽

Protein A ◽

Proteasome Inhibitors ◽

Adenovirus Vector ◽

Hepatic Lipogenesis ◽

Enhancer Activity ◽

Alcoholic Fatty Liver ◽

Hepatic Lipid Accumulation ◽

Protein Levels

Mammalian tribbles homolog 1 (TRIB1) regulates hepatic lipogenesis and is genetically associated with plasma triglyceride (TG) levels and cholesterol, but the molecular mechanisms remain obscure. We explored these mechanisms in mouse livers transfected with a TRIB1 overexpression, a shRNA template or a control (LacZ) adenovirus vector. The overexpression of TRIB1 reduced, whereas induction of the shRNA template increased, plasma glucose, TG, and cholesterol and simultaneously hepatic TG and glycogen levels. The involvement of TRIB1 in hepatic lipid accumulation was supported by the findings of a human SNP association study. A TRIB1 SNP, rs6982502, was identified in an enhancer sequence, modulated enhancer activity in reporter gene assays, and was significantly (P=9.39×10−7) associated with ultrasonographically diagnosed non-alcoholic fatty liver disease in a population of 5570 individuals. Transcriptome analyses of mouse livers revealed significant modulation of the gene sets involved in glycogenolysis and lipogenesis. Enforced TRIB1 expression abolished CCAAT/enhancer binding protein A (CEBPA), CEBPB, and MLXIPL proteins, whereas knockdown increased the protein level. Levels of TRIB1 expression simultaneously affected MKK4 (MAP2K4), MEK1 (MAP2K1), and ERK1/2 (MAPK1/3) protein levels and the phosphorylation of JNK, but not of ERK1/2. Pull-down and mammalian two-hybrid analyses revealed novel molecular interaction between TRIB1 and a hepatic lipogenic master regulator, MLXIPL. Co-expression of TRIB1 and CEBPA or MLXIPL reduced their protein levels and proteasome inhibitors attenuated the reduction. These data suggested that the modulation of TRIB1 expression affects hepatic lipogenesis and glycogenesis through multiple molecular interactions.

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Recent Advances in the Discovery of Novel Peptide Inhibitors Targeting 26S Proteasome

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180813120012 ◽

2019 ◽

Vol 18 (12) ◽

pp. 1656-1673

Author(s):

Xinjie Gu ◽

Shutao Ma

Keyword(s):

Cancer Therapy ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Peptide Inhibitors ◽

26S Proteasome ◽

Design Strategies ◽

Intracellular Protein ◽

Primary Tumors ◽

Protein Levels ◽

Ubiquitin Proteasome

Background: The 26S proteasome is a proteolytic complex of multimeric protease, which operates at the executive end of the Ubiquitin-Proteasome System (UPS) and degrades the polyubiquitylated proteins. Methods: After a brief introduction of 26S proteasome and Ubiquitin-Proteasome System (UPS), this review focuses on the structure and function of the 26S proteasome in intracellular protein level regulation. Then, physiological regulation mechanisms and processes are elaborated. In addition, the advantages and defects of approved 26S proteasome inhibitors were discussed. Finally, we summarized the novel peptide 26S proteasome inhibitors according to their structural classifications, highlighting their design strategies, inhibitory activity and Structure-Activity Relationships (SARs). Results: Cellular function maintenance relies on the proteasome metabolizing intracellular proteins to control intracellular protein levels, which is especially important for cancer cells to survive and proliferate. In primary tumors, proteasomes had a higher level and more potent activity. Currently, the approved small peptide inhibitors have proved their specific 26S proteasome inhibitory effects and considerable antitumor activities, but with obvious defects. Increasingly, novel peptide inhibitors are emerging and possess promising values in cancer therapy. Conclusion: Overall, the 26S proteasome is an efficient therapeutic target and novel 26S proteasome inhibitors hold potency for cancer therapy.

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Proteasome Inhibiters Up-Regulate TRAIL/Apo2L and Its Receptors Significantly Contributing to Proteasome Inhibitor-Induced Apoptosis in Primary Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽

10.1182/blood.v104.11.2810.2810 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2810-2810

Author(s):

Albert F. Kabore ◽

Kristin McCrea ◽

James B. Johnston ◽

Spencer B. Gibson

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Proteasome Inhibitor ◽

Substantial Reduction ◽

Proteasome Inhibitors ◽

Lymphocytic Leukemia ◽

Apoptotic Pathway ◽

Protein Levels ◽

Trail Receptors ◽

Induced Apoptosis ◽

Primary Chronic Lymphocytic Leukemia

Abstract The proteasome inhibitor, bortezomib has recently emerged as a new therapeutic treatment for refractory multiple myeloma and is presently being evaluated for other hematological malignancies either alone or in combination with other antitumor agents. Proteasome inhibitors cause the accumulation of many proteins but the precise mechanism responsible for their antitumor effect is unclear. In the present study, we have determined that cytotoxic effect the proteasome inhibitor MG-132 in primary chronic lymphocytic leukemia (CLL) cells is through the activation of the TRAIL (tumor necrosis factor-related apoptosis inducing ligand) apoptotic pathway. MG-132 induced apoptosis in approximately 70% of primary CLL cells as measured by annexin V staining. Addition of DR4:Fc that prevents TRAIL ligation with its receptors decreased the amount of MG-132 induced apoptosis by approximately 40% suggesting MG-132 caused activation of the TRAIL apoptotic pathway. MG-132 also up-regulated both the mRNA and protein levels of TRAIL and protein levels of TRAIL receptors DR4 and DR5. This upregulation correlated with activation of caspase 8 and cleavage of pro-apoptotic Bcl-2 family member Bid. Moreover, MG-132 treatment also induced a substantial reduction in the FLICE-like inhibitory protein (c-FLIP) protein levels. In contrast to CLL cells, proteasome inhibitors failed to activate the TRAIL apoptotic pathway in normal B-cells. This indicates that proteasome inhibitors are inducing apoptosis in primary CLL cells through activation of the TRAIL apoptotic signaling pathway through up-regulation of TRAIL and its cognate receptors and reduced FLIP expression. Thus, proteasome inhibitors may have a therapeutic role in CLL, either when used alone or in combination with TRAIL or antibodies against DR4/DR5.

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Gfi1 Protein Turnover Is Regulated by the Ubiquitin Ligase Triad1.

Blood ◽

10.1182/blood.v108.11.1173.1173 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1173-1173

Author(s):

Laurens T. van der Meer ◽

Jurgen A.F. Marteijn ◽

Theo M. de Witte ◽

Joop H. Jansen ◽

Bert A. van der Reijden

Keyword(s):

Ubiquitin Ligase ◽

Half Life ◽

Proteasome Inhibitors ◽

Ring Finger ◽

Ubiquitin Ligases ◽

Proteasomal Degradation ◽

Mrna Levels ◽

Yeast Two Hybrid ◽

Protein Levels ◽

Two Hybrid

Abstract The transcriptional repressor Growth factor independence-1 (Gfi1) plays an essential role during various stages of hematopoiesis. It is crucial for the self-renewal and long-term reconstituting potential of stem cells, essential for neutrophilic differentiation, and it plays an important role in T-cell and dendritic cell development. Gfi1 has also been implicated in malignant hematopoeisis because the Gfi1 gene is a common proviral integration site in murine leukemia models. We recently found that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Although Gfi1 mRNA levels are low in primary human monocytes, the protein levels are high due to low proteasomal degradation. Conversely, in mature granulocytes Gfi1 mRNA levels are high but protein levels are low due to strong proteasome-mediated turnover. Because Gfi1 plays an important role in normal and malignant hematopoiesis it will be of great interest to identify the ubiquitin ligases that regulate its turnover. Previously, we showed that the RING finger ubiquitin ligase Triad1 regulates myeloid cell proliferation. Using yeast-two-hybrid assays we found that Triad1 binds the zinc finger region of Gfi1. This interaction was confirmed in co-immunoprecipitation experiments. To study whether the turnover of Gfi1 is regulated by Triad1 we performed ubiquitination assays. To our suprise we found that instead of promoting ubiquitination, Triad1 inhibited Gfi1 protein ubiquitination, also in the presence of proteasome inhibitors. RNAi mediated down regulation of Triad1 protein levels stimulated Gfi1 ubiquitination. Importantly, expression of a Triad1 point mutant (H158A) that fails to bind the ubiquitin conjugating enzyme UbcH7 also inhibited Gfi1 ubiquitination. To study whether the observed diminished ubiquitination by Triad1 affected the turnover of Gfi1 we analyzed Gfi1 protein half-life using the protein synthesis inhibitor cycloheximide. This showed that Triad1 co-expression prolonged the half-life of Gfi1 significantly. We conclude that Triad1 inhibits Gfi1 ubiquitination, resulting in decreased turnover of the protein. As this inhibition also occurs in the presence of proteasome inhibitors and is independent of the ubiquitin ligase activity of Triad1, these data support a model in which Triad1 competes for Gfi1 binding with other ubiquitin ligases that do mark Gfi1 for proteasomal degradation. Currently, we are testing candidate ubiquitin ligases (RING finger and HECT proteins) that were found to associate with Gfi1 in yeast-two-hybrid assays to gain more insight in how the activity of this important transcription factor is regulated.

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WIP Is a Chaperone for Wiskott-Aldrich Syndrome Protein.

Blood ◽

10.1182/blood.v112.11.1471.1471 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1471-1471

Author(s):

Yoji Sasahara ◽

Miguel A de la Fuente ◽

Narayanaswamy Ramesh ◽

Shigeru Tsuchiya ◽

Raif S Geha

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Critical Role ◽

Proteasome Inhibitors ◽

Binding Domain ◽

Mrna Levels ◽

Missense Mutations ◽

Wiskott Aldrich Syndrome ◽

Protein Levels

Abstract WASP, the product of the gene mutated in Wiskott-Aldrich syndrome (WAS), plays a critical role in T cell activation and actin reorganization. WIP is a WASP-interacting protein that binds to Ena-VASP homology 1 (EVH1) domain of WASP. We previously reported functional roles of WIP in the recruitment and activation of WASP in T cells. To investigate a role of WIP in WASP degradation and understand the reason why most missense mutations in WAS patients are in EVH1 domain of WASP, we examined WASP levels in WIP knockout (WIP−/−) T cells and mechanisms of WASP degradation following T cell receptor (TCR) ligation. WASP protein levels, but not mRNA levels, were severely diminished in T cells from WIP−/− mice and were restored by introduction of full-length or WASP binding domain of WIP. A fraction of WASP was cleaved by Ca-dependent protease calpain, ubiquitinated and degraded by the proteasome following TCR ligation. Inducible expression of WASP binding domain of WIP increased WASP levels in vivo and recombinant WIP protein protected WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP protein levels in T cells from WIP−/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupted WIP binding. The calpain inhibitor calpeptin also increased WASP protein levels in activated T cells and B cells from the WAS patients. Despite its ability to increase WASP levels, proteasome inhibitors failed to correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient (published in PNAS, 104, p926–931, 2007). These results demonstrate that WIP stabilizes WASP in normal T cells and that WIP deficiency or impaired WASP-WIP interaction in WAS patients with missense mutation in WIP binding site results in WASP degradation.

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CDK9 Is Constitutively Expressed throughout the Cell Cycle, and Its Steady-State Expression Is Independent of SKP2

Molecular and Cellular Biology ◽

10.1128/mcb.23.15.5165-5173.2003 ◽

2003 ◽

Vol 23 (15) ◽

pp. 5165-5173 ◽

Cited By ~ 49

Author(s):

Judit Garriga ◽

Sabyasachi Bhattacharya ◽

Joaquim Calbó ◽

Renée M. Marshall ◽

May Truongcao ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Proteasome Inhibitors ◽

Elongation Factor ◽

Dependent Manner ◽

Protein Levels ◽

Ubiquitin Ligase Complex ◽

A Cell ◽

Cycle Dependent ◽

Interfering Rna

ABSTRACT CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCFSKP2 ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G0, despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t 1/2) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t 1/2 of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins, such as p27, but did not affect the expression of endogenous CDK9. Ectopic overexpression of SKP2 led to reduction of p27 protein levels but had no effect on the expression of endogenous CDK9. Finally, downregulation of endogenous SKP2 gene expression by interfering RNA had no effect on CDK9 protein levels, whereas p27 protein levels increased dramatically. Therefore, the SCFSKP2 ubiquitin ligase does not regulate CDK9 expression in a cell cycle-dependent manner.

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Myeloid cell factor–1 is a critical survival factor for multiple myeloma

Blood ◽

10.1182/blood.v99.6.1885 ◽

2002 ◽

Vol 99 (6) ◽

pp. 1885-1893 ◽

Cited By ~ 262

Author(s):

Bin Zhang ◽

Ivana Gojo ◽

Robert G. Fenton

Keyword(s):

Multiple Myeloma ◽

Cell Survival ◽

Actinomycin D ◽

Plasma Cells ◽

Proteasome Inhibitors ◽

Myeloid Cell ◽

Caspase Activation ◽

Protein Levels ◽

Survival Factor ◽

Induced Apoptosis

Abstract Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow caused primarily by failure of normal homeostatic mechanisms to prevent the expansion of postgerminal center plasma cells. We have examined the molecular mechanisms that promote the survival of MM cells and have identified a key role for myeloid cell factor–1 (Mcl-1), an antiapoptotic member of the Bcl-2 family. These experiments were initiated by the observation that MM cells were exquisitely sensitive to culture in the presence of actinomycin D: caspase activation occurred within 3 hours of treatment and cells were not protected by interleukin-6, the main MM cell growth and survival factor. Actinomycin D–induced apoptosis was blocked by proteasome inhibitors, suggesting that a labile protein was required for MM cell survival. Further analysis demonstrated that Mcl-1 was likely to be the labile factor governing MM cell survival. Mcl-1 protein levels decreased rapidly after culture in the presence of actinomycin D in concordance with effector caspase activation, but addition of proteasome inhibitors reversed the loss of Mcl-1 and maintained cell viability. The levels of other antiapoptotic proteins, including Bcl-2 and members of the inhibitors-of-apoptosis family, were unaffected by these interventions. Furthermore, Mcl-1 antisense oligonucleotides caused a rapid down-regulation of Mcl-1 protein levels and the coincident induction of apoptosis, whereas overexpression of Mcl-1 delayed actinomycin D–induced apoptosis with kinetics that correlated with expression levels of Mcl-1. These data indicate that Mcl-1 expression is required for the survival of MM cells and may represent an important target for future therapeutics.

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