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Sample records for promoting proteasomal degradation

  1. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    PubMed

    Comyn, Sophie A; Young, Barry P; Loewen, Christopher J; Mayor, Thibault

    2016-07-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  2. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

    PubMed Central

    Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

    2016-01-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  3. Skp2 inhibits osteogenesis by promoting ubiquitin-proteasome degradation of Runx2.

    PubMed

    Thacker, Gatha; Kumar, Yogesh; Khan, Mohd Parvez; Shukla, Nidhi; Kapoor, Isha; Kanaujiya, Jitendra Kumar; Lochab, Savita; Ahmed, Shakil; Sanyal, Sabyasachi; Chattopadhyay, Naibedya; Trivedi, Arun Kumar

    2016-04-01

    Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene. PMID:26778333

  4. Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana.

    PubMed

    Ding, Yezhang; Dommel, Matthew; Mou, Zhonglin

    2016-04-01

    Proteasome-mediated turnover of the transcription coactivator NPR1 is pivotal for efficient activation of the broad-spectrum plant immune responses known as localized acquired resistance (LAR) and systemic acquired resistance (SAR) in adjacent and systemic tissues, respectively, and requires the CUL3-based E3 ligase and its adaptor proteins, NPR3 and NPR4, which are receptors for the signaling molecule salicylic acid (SA). It has been shown that SA prevents NPR1 turnover under non-inducing and LAR/SAR-inducing conditions, but how cellular NPR1 homeostasis is maintained remains unclear. Here, we show that the phytohormone abscisic acid (ABA) and SA antagonistically influence cellular NPR1 protein levels. ABA promotes NPR1 degradation via the CUL3(NPR) (3/) (NPR) (4) complex-mediated proteasome pathway, whereas SA may protect NPR1 from ABA-promoted degradation through phosphorylation. Furthermore, we demonstrate that the timing and strength of SA and ABA signaling are critical in modulating NPR1 accumulation and target gene expression. Perturbing ABA or SA signaling in adjacent tissues alters the temporal dynamic pattern of NPR1 accumulation and target gene transcription. Finally, we show that sequential SA and ABA treatment leads to dynamic changes in NPR1 protein levels and target gene expression. Our results revealed a tight correlation between sequential SA and ABA signaling and dynamic changes in NPR1 protein levels and NPR1-dependent transcription in plant immune responses. PMID:26865090

  5. RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect

    PubMed Central

    Zhang, Chen-Song; Liu, Qi; Li, Mengqi; Lin, Shu-Yong; Peng, Yongying; Wu, Di; Li, Terytty Yang; Fu, Qiang; Jia, Weiping; Wang, Xinjun; Ma, Teng; Zong, Yue; Cui, Jiwen; Pu, Chengfei; Lian, Guili; Guo, Huiling; Ye, Zhiyun; Lin, Sheng-Cai

    2015-01-01

    Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα. PMID:26215701

  6. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    PubMed

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms.

  7. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    SciTech Connect

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing; Gu, Jianxin

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  8. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444, isse 4)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-21

    Highlights: • The 2000–2634aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  9. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444 issue 3)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-14

    Highlights: • The 2000–2634 aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  10. Pupylation-dependent and -independent proteasomal degradation in mycobacteria.

    PubMed

    Imkamp, Frank; Ziemski, Michal; Weber-Ban, Eilika

    2015-08-01

    Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway. PMID:26352358

  11. Cysteine string protein promotes proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) by increasing its interaction with the C terminus of Hsp70-interacting protein and promoting CFTR ubiquitylation.

    PubMed

    Schmidt, Béla Z; Watts, Rebecca J; Aridor, Meir; Frizzell, Raymond A

    2009-02-13

    Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/Hsp70 and CHIP-mediated CFTR degradation.

  12. Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

    PubMed Central

    Haller, Martina; Hock, Andreas K; Giampazolias, Evangelos; Oberst, Andrew; Green, Douglas R; Debnath, Jayanta; Ryan, Kevin M; Vousden, Karen H; Tait, Stephen W G

    2015-01-01

    During macroautophagy, conjugation of ATG12 to ATG5 is essential for LC3 lipidation and autophagosome formation. Additionally, ATG12 has ATG5-independent functions in diverse processes including mitochondrial fusion and mitochondrial-dependent apoptosis. In this study, we investigated the regulation of free ATG12. In stark contrast to the stable ATG12–ATG5 conjugate, we find that free ATG12 is highly unstable and rapidly degraded in a proteasome-dependent manner. Surprisingly, ATG12, itself a ubiquitin-like protein, is directly ubiquitinated and this promotes its proteasomal degradation. As a functional consequence of its turnover, accumulation of free ATG12 contributes to proteasome inhibitor-mediated apoptosis, a finding that may be clinically important given the use of proteasome inhibitors as anticancer agents. Collectively, our results reveal a novel interconnection between autophagy, proteasome activity, and cell death mediated by the ubiquitin-like properties of ATG12. PMID:25629932

  13. Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation*

    PubMed Central

    Goitea, Victor E.; Hallak, Marta E.

    2015-01-01

    Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm. PMID:25969538

  14. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    PubMed Central

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  15. N-terminal residues regulate proteasomal degradation of AANAT.

    PubMed

    Huang, Zheping; Liu, Tiecheng; Borjigin, Jimo

    2010-04-01

    Serotonin N-acetyltransferase (AANAT) catalyzes the conversion of serotonin to N-acetylserotonin, which is the immediate precursor for formation of melatonin. Although it is known that AANAT is degraded via the proteasomal proteolysis, detailed mechanisms are not defined. In this paper, we tested the in vivo role of proteasome inhibition on AANAT activity and melatonin release and examined the amino acid residues in AANAT that contribute to the proteasome degradation. We have shown that inhibition of proteasome activities in vivo in the intact pineal gland fails to prevent the light-induced suppression of melatonin secretion. Furthermore, in cell lines stably expressing AANAT, inhibition of proteasomal proteolysis, which resulted in a large accumulation of AANAT protein, similarly failed to increase AANAT enzyme activity proportional to the amount of proteins accumulated. Site-directed mutagenesis analysis of AANAT revealed that the AANAT degradation is independent of lysine and the two surface cysteine residues. Deletion analysis of N-terminus identified the second amino acid leucine (L2) as the key residue that contributes to the proteasomal proteolysis of AANAT protein. These results suggest that rat AANAT protein is degraded via the N-end rule pathway of proteasomal proteolysis and the leucine at the N-terminus appears to be the key residue recognized by N-end rule pathway.

  16. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    SciTech Connect

    Cho, Eun-Ah; Juhnn, Yong-Sung

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  17. E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

    PubMed

    Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

    2016-10-01

    C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response.

  18. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

    The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5Δ as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5Δ mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome. PMID:20008553

  19. Aberrant Splicing Promotes Proteasomal Degradation of L-type CaV1.2 Calcium Channels by Competitive Binding for CaVβ Subunits in Cardiac Hypertrophy

    PubMed Central

    Hu, Zhenyu; Wang, Jiong-Wei; Yu, Dejie; Soon, Jia Lin; de Kleijn, Dominique P. V.; Foo, Roger; Liao, Ping; Colecraft, Henry M.; Soong, Tuck Wah

    2016-01-01

    Decreased expression and activity of CaV1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of CaV1.2 channel, named CaV1.2e21+22, that contained the pair of mutually exclusive exons 21 and 22. This variant was highly expressed in neonatal hearts. The abundance of this variant was gradually increased by 12.5-folds within 14 days of transverse aortic banding that induced cardiac hypertrophy in adult mouse hearts and was also elevated in left ventricles from patients with dilated cardiomyopathy. Although this variant did not conduct Ca2+ ions, it reduced the cell-surface expression of wild-type CaV1.2 channels and consequently decreased the whole-cell Ca2+ influx via the CaV1.2 channels. In addition, the CaV1.2e21+22 variant interacted with CaVβ subunits significantly more than wild-type CaV1.2 channels, and competition of CaVβ subunits by CaV1.2e21+22 consequently enhanced ubiquitination and subsequent proteasomal degradation of the wild-type CaV1.2 channels. Our findings show that the resurgence of a specific neonatal splice variant of CaV1.2 channels in adult heart under stress may contribute to heart failure. PMID:27731386

  20. Proteasome Inhibition Promotes Parkin-Ubc13 Interaction and Lysine 63-Linked Ubiquitination

    PubMed Central

    Ng, Xiao-Hui; Henry-Basil, Adeline; Sim, Roy W. X.; Tan, Jeanne M. M.; Chai, Chou; Lim, Kah-Leong

    2013-01-01

    Disruption of the ubiquitin-proteasome system, which normally identifies and degrades unwanted intracellular proteins, is thought to underlie neurodegeneration. Supporting this, mutations of Parkin, a ubiquitin ligase, are associated with autosomal recessive parkinsonism. Remarkably, Parkin can protect neurons against a wide spectrum of stress, including those that promote proteasome dysfunction. Although the mechanism underlying the preservation of proteasome function by Parkin is hitherto unclear, we have previously proposed that Parkin-mediated K63-linked ubiquitination (which is usually uncoupled from the proteasome) may serve to mitigate proteasomal stress by diverting the substrate load away from the machinery. By means of linkage-specific antibodies, we demonstrated here that proteasome inhibition indeed promotes K63-linked ubiquitination of proteins especially in Parkin-expressing cells. Importantly, we further demonstrated that the recruitment of Ubc13 (an E2 that mediates K63-linked polyubiquitin chain formation exclusively) by Parkin is selectively enhanced under conditions of proteasomal stress, thus identifying a mechanism by which Parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress. This mode of ubiquitination appears to facilitate the subsequent clearance of Parkin substrates via autophagy. Consistent with the proposed protective role of K63-linked ubiquitination in times of proteolytic stress, we found that Ubc13-deficient cells are significantly more susceptible to cell death induced by proteasome inhibitors compared to their wild type counterparts. Taken together, our study suggests a role for Parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is burdened or impaired. PMID:24023840

  1. Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

    ERIC Educational Resources Information Center

    Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

    2006-01-01

    This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

  2. Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.

    PubMed

    Lee, Jin-Gu; Takahama, Shokichi; Zhang, Guofeng; Tomarev, Stanislav I; Ye, Yihong

    2016-07-01

    To safeguard proteomic integrity, cells rely on the proteasome to degrade aberrant polypeptides, but it is unclear how cells remove defective proteins that have escaped degradation owing to proteasome insufficiency or dysfunction. Here we report a pathway termed misfolding-associated protein secretion, which uses the endoplasmic reticulum (ER)-associated deubiquitylase USP19 to preferentially export aberrant cytosolic proteins. Intriguingly, the catalytic domain of USP19 possesses an unprecedented chaperone activity, allowing recruitment of misfolded proteins to the ER surface for deubiquitylation. Deubiquitylated cargos are encapsulated into ER-associated late endosomes and secreted to the cell exterior. USP19-deficient cells cannot efficiently secrete unwanted proteins, and grow more slowly than wild-type cells following exposure to a proteasome inhibitor. Together, our findings delineate a protein quality control (PQC) pathway that, unlike degradation-based PQC mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process. PMID:27295555

  3. Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

    PubMed Central

    Fang, Nancy N.; Zhu, Mang; Rose, Amalia; Wu, Kuen-Phon; Mayor, Thibault

    2016-01-01

    Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions. PMID:27698423

  4. The 19S proteasome activator promotes human cytomegalovirus immediate early gene expression through proteolytic and nonproteolytic mechanisms.

    PubMed

    Winkler, Laura L; Kalejta, Robert F

    2014-10-01

    Proteasomes are large, multisubunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed that this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation but also plays a 20S CP-independent role promoting transcription. Here, we present a nonproteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the major immediate early promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and nonproteolytic roles to promote HCMV lytic infection. Importance: Proteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function toward therapeutic effect is an incomplete capitalization on the potential of this approach. Here, we provide an additional example of nonproteolytic 19S RP function in promoting HCMV transcription. These data provide a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during

  5. DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation.

    PubMed

    Yi, Juan; Lu, Guang; Li, Li; Wang, Xiaozhen; Cao, Li; Lin, Ming; Zhang, Sha; Shao, Genze

    2015-05-19

    The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.

  6. JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis

    PubMed Central

    Deng, Shulin; Jang, In-Cheol; Su, Linlin; Xu, Jun; Chua, Nam-Hai

    2016-01-01

    H3K9 methylation is usually associated with DNA methylation, and together they symbolize transcriptionally silenced heterochromatin. A number of proteins involved in epigenetic processes have been characterized. However, how the stability of these proteins is regulated at the post-translational level is largely unknown. Here, we show that an Arabidopsis JmjC domain protein, JMJ24, possesses ubiquitin E3 ligase activity. JMJ24 directly targets a DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), for proteasomal degradation to initiate destabilization of the heterochromatic state of endogenous silenced loci. Our results uncover an additional connection between two conserved epigenetic modifications: histone modification and DNA methylation. PMID:26798133

  7. Truncated ERG Oncoproteins from TMPRSS2-ERG Fusions Are Resistant to SPOP-Mediated Proteasome Degradation.

    PubMed

    An, Jian; Ren, Shancheng; Murphy, Stephen J; Dalangood, Sumiya; Chang, Cunjie; Pang, Xiaodong; Cui, Yangyan; Wang, Liguo; Pan, Yunqian; Zhang, Xiaowei; Zhu, Yasheng; Wang, Chenji; Halling, Geoffrey C; Cheng, Liang; Sukov, William R; Karnes, R Jeffrey; Vasmatzis, George; Zhang, Qing; Zhang, Jun; Cheville, John C; Yan, Jun; Sun, Yinghao; Huang, Haojie

    2015-09-17

    SPOP mutations and TMPRSS2-ERG rearrangements occur collectively in up to 65% of human prostate cancers. Although the two events are mutually exclusive, it is unclear whether they are functionally interrelated. Here, we demonstrate that SPOP, functioning as an E3 ubiquitin ligase substrate-binding protein, promotes ubiquitination and proteasome degradation of wild-type ERG by recognizing a degron motif at the N terminus of ERG. Prostate cancer-associated SPOP mutations abrogate the SPOP-mediated degradation function on the ERG oncoprotein. Conversely, the majority of TMPRSS2-ERG fusions encode N-terminal-truncated ERG proteins that are resistant to the SPOP-mediated degradation because of degron impairment. Our findings reveal degradation resistance as a previously uncharacterized mechanism that contributes to elevation of truncated ERG proteins in prostate cancer. They also suggest that overcoming ERG resistance to SPOP-mediated degradation represents a viable strategy for treatment of prostate cancers expressing either mutated SPOP or truncated ERG.

  8. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation.

    PubMed

    Choi, Won Hoon; de Poot, Stefanie A H; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min Jae

    2016-01-01

    When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼ 200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells. PMID:26957043

  9. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation

    PubMed Central

    Choi, Won Hoon; de Poot, Stefanie A. H.; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min Jae

    2016-01-01

    When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells. PMID:26957043

  10. cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins.

    PubMed

    Lokireddy, Sudarsanareddy; Kukushkin, Nikolay Vadimovich; Goldberg, Alfred Lewis

    2015-12-29

    Although rates of protein degradation by the ubiquitin-proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the proteasome's capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of proteasomes by this mechanism may be useful in treating proteotoxic diseases.

  11. Calcium-dependent proteasome activation is required for axonal neurofilament degradation.

    PubMed

    Park, Joo Youn; Jang, So Young; Shin, Yoon Kyung; Suh, Duk Joon; Park, Hwan Tae

    2013-12-25

    Even though many studies have identified roles of proteasomes in axonal degeneration, the molecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were significantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration.

  12. Disease-proportional proteasomal degradation of missense dystrophins.

    PubMed

    Talsness, Dana M; Belanto, Joseph J; Ervasti, James M

    2015-10-01

    The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin. PMID:26392559

  13. Disease-proportional proteasomal degradation of missense dystrophins

    PubMed Central

    Talsness, Dana M.; Belanto, Joseph J.; Ervasti, James M.

    2015-01-01

    The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin. PMID:26392559

  14. Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

    PubMed

    Belogurov, Alexey; Kudriaeva, Anna; Kuzina, Ekaterina; Smirnov, Ivan; Bobik, Tatyana; Ponomarenko, Natalia; Kravtsova-Ivantsiv, Yelena; Ciechanover, Aaron; Gabibov, Alexander

    2014-06-20

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

  15. Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

    PubMed Central

    Singh Gautam, Amit Kumar; Balakrishnan, Satish; Venkatraman, Prasanna

    2012-01-01

    Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases

  16. ORF2 protein of porcine circovirus type 2 promotes phagocytic activity of porcine macrophages by inhibiting proteasomal degradation of complement component 1, q subcomponent binding protein (C1QBP) through physical interaction.

    PubMed

    Choi, Chang-Yong; Oh, Hae-Na; Lee, Suk Jun; Chun, Taehoon

    2015-11-01

    Defining how each ORF of porcine circovirus type 2 (PCV2) manipulates the host immune system may be helpful to understand the disease progression of post-weaning multisystemic wasting syndrome. In this study, we demonstrated a direct interaction between the PCV2 ORF2 and complement component 1, q subcomponent binding protein (C1QBP) within the cytoplasm of host macrophages. The physical interaction between PCV2 ORF2 and C1QBP inhibited ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of C1QBP by the interaction with PCV2 ORF2 further enhanced the phagocytic activity of porcine macrophages through the phosphoinositol 3-kinase signalling pathway. This may explain the molecular basis of how PCV2 ORF2 enhances the phagocytic activity of host macrophages. PMID:26361775

  17. ORF2 protein of porcine circovirus type 2 promotes phagocytic activity of porcine macrophages by inhibiting proteasomal degradation of complement component 1, q subcomponent binding protein (C1QBP) through physical interaction.

    PubMed

    Choi, Chang-Yong; Oh, Hae-Na; Lee, Suk Jun; Chun, Taehoon

    2015-11-01

    Defining how each ORF of porcine circovirus type 2 (PCV2) manipulates the host immune system may be helpful to understand the disease progression of post-weaning multisystemic wasting syndrome. In this study, we demonstrated a direct interaction between the PCV2 ORF2 and complement component 1, q subcomponent binding protein (C1QBP) within the cytoplasm of host macrophages. The physical interaction between PCV2 ORF2 and C1QBP inhibited ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of C1QBP by the interaction with PCV2 ORF2 further enhanced the phagocytic activity of porcine macrophages through the phosphoinositol 3-kinase signalling pathway. This may explain the molecular basis of how PCV2 ORF2 enhances the phagocytic activity of host macrophages.

  18. Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

    PubMed

    Raynes, Rachel; Pomatto, Laura C D; Davies, Kelvin J A

    2016-08-01

    The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators.

  19. Curcumin inhibits HIV-1 by promoting Tat protein degradation

    PubMed Central

    Ali, Amjad; Banerjea, Akhil C.

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  20. Curcumin inhibits HIV-1 by promoting Tat protein degradation.

    PubMed

    Ali, Amjad; Banerjea, Akhil C

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  1. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

    PubMed

    Sbardella, Diego; Tundo, Grazia Raffaella; Sciandra, Francesca; Bozzi, Manuela; Gioia, Magda; Ciaccio, Chiara; Tarantino, Umberto; Brancaccio, Andrea; Coletta, Massimo; Marini, Stefano

    2015-01-01

    Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

  2. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution

    PubMed Central

    Sbardella, Diego; Tundo, Grazia Raffaella; Sciandra, Francesca; Bozzi, Manuela; Gioia, Magda; Ciaccio, Chiara; Tarantino, Umberto; Brancaccio, Andrea; Coletta, Massimo; Marini, Stefano

    2015-01-01

    Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism. PMID:26186340

  3. SOCS3 Drives Proteasomal Degradation of TBK1 and Negatively Regulates Antiviral Innate Immunity

    PubMed Central

    Liu, Dong; Sheng, Chunjie; Gao, Shijuan; Yao, Chen; Li, Jiandong; Jiang, Wei; Chen, Huiming; Wu, Jiaoxiang; Pan, Changchuan

    2015-01-01

    TANK-binding kinase 1 (TBK1)-mediated induction of type I interferon (IFN) plays a critical role in host antiviral responses and immune homeostasis. The negative regulation of TBK1 activity is largely unknown. We report that suppressor of cytokine signaling 3 (SOCS3) inhibits the IFN-β signaling pathway by promoting proteasomal degradation of TBK1. Overexpression and knockdown experiments indicated that SOCS3 is a negative regulator of IFN regulatory factor 3 (IRF3) phosphorylation and IFN-β transcription. Moreover, SOCS3 directly associates with TBK1, and they colocalize in the cytoplasm. SOCS3 catalyzes K48-linked polyubiquitination of TBK1 at Lys341 and Lys344 and promotes subsequent TBK1 degradation. On the contrary, SOCS3 knockdown markedly increases the abundance of TBK1. Interestingly, both the BOX domain of SOCS3 and Ser172 phosphorylation of TBK1 are indispensable for the processes of ubiquitination and degradation. Ectopic expression of SOCS3 significantly inhibits vesicular stomatitis virus (VSV) and influenza A virus strain A/WSN/33 (WSN)-induced IRF3 phosphorylation and facilitates the replication of WSN virus by detecting the transcription of its viral RNA (vRNA). Knockdown of SOCS3 represses WSN replication. Collectively, these results demonstrate that SOCS3 acts as a negative regulator of IFN-β signal by ubiquitinating and degrading TBK1, shed light on the understanding of antiviral innate immunity, and provide a potential target for developing antiviral agents. PMID:25939384

  4. Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

    PubMed Central

    Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung

    2009-01-01

    Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic α- and β-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy in coping with crisis of misfolded proteins. PMID:19682429

  5. Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

    SciTech Connect

    Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung

    2009-10-16

    Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic {alpha}- and {beta}-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.

  6. Fludarabine Downregulates Indoleamine 2,3-Dioxygenase in Tumors via a Proteasome-Mediated Degradation Mechanism

    PubMed Central

    Hanafi, Laïla-Aïcha; Gauchat, Dominique; Godin-Ethier, Jessica; Possamaï, David; Duvignaud, Jean-Baptiste; Leclerc, Denis; Grandvaux, Nathalie; Lapointe, Réjean

    2014-01-01

    Indoleamine 2,3-dioxygenase (IDO) is found in multiple malignancies and exerts immunosuppressive effects that are central in protecting tumors from host T lymphocyte rejection. IDO is an enzyme involved in the catabolism of tryptophan resulting in inhibition of T lymphocyte function. While inhibition of IDO enzymatic activity results in tumor rejection, it is still unknown how we can directly target IDO expression within tumors using drugs. We have chosen to interfere with IDO expression by targeting the key-signaling event signal transducer and activator of transcription 1 (STAT1). We evaluated the efficacy of fludarabine, previously described to inhibit STAT1 phosphorylation. Interestingly, fludarabine was efficient in suppressing protein expression and consequently IDO activity in two different cell lines derived from breast cancer and melanoma when IDO was activated with interferon-gamma (IFN-γ) or supernatants prepared from activated T lymphocytes. However, fludarabine had no inhibitory effect on STAT1 phosphorylation. Other IFN-γ-responsive genes were only marginally inhibited by fludarabine. The level of IDO transcript was unaffected by this inhibitor, suggesting the involvement of post-transcriptional control. Strikingly, we have found that the inhibition of proteasome partially protected IDO from fludarabine-induced degradation, indicating that fludarabine induces IDO degradation through a proteasome-dependent pathway. Currently used in the clinic to treat some malignancies, fludarabine has the potential for use in the treatment of human tumors through induction of IDO degradation and consequently, for the promotion of T cell-mediated anti-tumor response. PMID:24911872

  7. Is insulin signaling molecules misguided in diabetes for ubiquitin-proteasome mediated degradation?

    PubMed

    Balasubramanyam, Muthuswamy; Sampathkumar, Rangasamy; Mohan, Viswanathan

    2005-07-01

    Recent mining of the human and mouse genomes, use of yeast genetics, and detailed analyses of several biochemical pathways, have resulted in the identification of many new roles for ubiquitin-proteasome mediated degradation of proteins. In the context of last year's award of Noble Prize (Chemistry) work, the ubiquitin and ubiquitin-like modifications are increasingly recognized as key regulatory events in health and disease. Although the ATP-dependent ubiquitin-proteasome system has evolved as premier cellular proteolytic machinery, dysregulation of this system by several different mechanisms leads to inappropriate degradation of specific proteins and pathological consequences. While aberrations in the ubiquitin-proteasome pathway have been implicated in certain malignancies and neurodegenerative disorders, recent studies indicate a role for this system in the pathogenesis of diabetes and its complications. Inappropriate degradation of insulin signaling molecules such as insulin receptor substrates (IRS-1 and IRS-2) has been demonstrated in experimental diabetes, mediated in part through the up-regulation of suppressors of cytokine signaling (SOCS). It appears that altered ubiquitin-proteasome system might be one of the molecular mechanisms of insulin resistance in many pathological situations. Drugs that modulate the SOCS action and/or proteasomal degradation of proteins could become novel agents for the treatment of insulin resistance and Type 2 diabetes.

  8. Bacterial proteasome and PafA, the pup ligase, interact to form a modular protein tagging and degradation machine.

    PubMed

    Forer, Nadav; Korman, Maayan; Elharar, Yifat; Vishkautzan, Marina; Gur, Eyal

    2013-12-17

    Proteasome-containing bacteria possess a tagging system that directs proteins to proteasomal degradation by conjugating them to a prokaryotic ubiquitin-like protein (Pup). A single ligating enzyme, PafA, is responsible for Pup conjugation to lysine side chains of protein substrates. As Pup is recognized by the regulatory subunit of the proteasome, Pup functions as a degradation tag. Pup presents overlapping regions for binding of the proteasome and PafA. It was, therefore, unclear whether Pup binding by the proteasome regulatory subunit, Mpa, and by PafA are mutually exclusive events. The work presented here provides evidence for the simultaneous interaction of Pup with both Mpa and PafA. Surprisingly, we found that PafA and Mpa can form a complex both in vitro and in vivo. Our results thus suggest that PafA and the proteasome can function as a modular machine for the tagging and degradation of cytoplasmic proteins. PMID:24228735

  9. Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.

    PubMed

    Khedgikar, V; Kushwaha, P; Gautam, J; Verma, A; Changkija, B; Kumar, A; Sharma, S; Nagar, G K; Singh, D; Trivedi, P K; Sangwan, N S; Mishra, P R; Trivedi, R

    2013-01-01

    Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA

  10. Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.

    PubMed

    Khedgikar, V; Kushwaha, P; Gautam, J; Verma, A; Changkija, B; Kumar, A; Sharma, S; Nagar, G K; Singh, D; Trivedi, P K; Sangwan, N S; Mishra, P R; Trivedi, R

    2013-01-01

    Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA

  11. TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation

    PubMed Central

    Weng, Pei-Lun; Yeh, Te-Huei

    2016-01-01

    Tumor susceptibility gene 101 (TSG101) elicits an array of cellular functions, including promoting cytokinesis, cell cycle progression and proliferation, as well as facilitating endosomal trafficking and viral budding. TSG101 protein is highly and aberrantly expressed in various human cancers. Specifically, a TSG101 splicing variant missing nucleotides 154 to 1054 (TSGΔ154-1054), which is linked to progressive tumor-stage and metastasis, has puzzled investigators for more than a decade. TSG101-associated E3 ligase (Tal)- and MDM2-mediated proteasomal degradation are the two major routes for posttranslational regulation of the total amount of TSG101. We reveal that overabundance of TSG101 results from TSGΔ154-1054 stabilizing the TSG101 protein by competitively binding to Tal, but not MDM2, thereby perturbing the Tal interaction with TSG101 and impeding subsequent polyubiquitination and proteasomal degradation of TSG101. TSGΔ154-1054 therefore specifically enhances TSG101-stimulated cell proliferation, clonogenicity, and tumor growth in nude mice. This finding shows the functional significance of TSGΔ154-1054 in preventing the ubiquitin-proteasome proteolysis of TSG101, which increases tumor malignancy and hints at its potential as a therapeutic target in cancer treatment. PMID:26811492

  12. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  13. Protein Degradation by Ubiquitin-Proteasome System in Formation and Labilization of Contextual Conditioning Memory

    ERIC Educational Resources Information Center

    Fustiñana, María Sol; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-01-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this…

  14. Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways

    PubMed Central

    Delgado, M E; Dyck, L; Laussmann, M A; Rehm, M

    2014-01-01

    Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death. PMID:24457955

  15. Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase: Signal transduction and synaptic transmission.

    PubMed

    Rezvani, Khosrow; Baalman, Kelli; Teng, Yanfen; Mee, Maureen P; Dawson, Simon P; Wang, Hongmin; De Biasi, Mariella; Mayer, R John

    2012-07-01

    The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.

  16. The Hypoxia-controlled FBXL14 Ubiquitin Ligase Targets SNAIL1 for Proteasome Degradation*

    PubMed Central

    Viñas-Castells, Rosa; Beltran, Manuel; Valls, Gabriela; Gómez, Irene; García, José Miguel; Montserrat-Sentís, Bàrbara; Baulida, Josep; Bonilla, Félix; de Herreros, Antonio García; Díaz, Víctor M.

    2010-01-01

    The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase β-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3β. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3β, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3β. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia. PMID:19955572

  17. Proteasome-mediated degradation antagonizes critical levels of the apoptosis-inducing C1D protein

    PubMed Central

    Rothbarth, Karsten; Stammer, Hermann; Werner, Dieter

    2002-01-01

    The C1D gene is expressed in a broad spectrum of mammalian cells and tissues but its product induces apoptotic cell death when exceeding a critical level. Critical levels are achieved in a fraction of cells by transient transfection with EGFP-tagged C1D expression constructs. However, transfected cells expressing sub-critical levels of C1D(EGFP) escape apoptotic cell death by activation of a proteasome-mediated rescue mechanism. Inhibition of the proteasome-dependent degradation of the C1D(EGFP) protein results in a parallel increase of the intracellular C1D level and in the fraction of apoptotic cells. PMID:12379155

  18. HUWE1 ubiquitinates MyoD and targets it for proteasomal degradation

    SciTech Connect

    Noy, Tahel; Suad, Oded; Taglicht, Daniel; Ciechanover, Aaron

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer HUWE1 ubiquitinates MyoD in vitro and in cells. Black-Right-Pointing-Pointer The ubiquitination by HUWE1 targets MyoD for proteasomal degradation. Black-Right-Pointing-Pointer HUWE1 can modify MyoD on its N-terminal residue. -- Abstract: MyoD is a tissue-specific transcriptional activator that acts as a master switch for muscle development. It activates a broad array of muscle-specific genes, which leads to conversion of proliferating myoblasts into mature myotubes. The ubiquitin proteasome system (UPS) plays an important role in controlling MyoD. Both its N-terminal residue and internal lysines can be targeted by ubiquitin, and both modifications appear to direct it for proteasomal degradation. The protein is short-lived and has a half-life of {approx}45 min in different cells. It was reported that MyoD can be ubiquitinated by MAFbx/AT-1, but accumulating lines of experimental evidence showed that other ligase(s) may also participate in its targeting. Here we describe the involvement of HUWE1 in the ubiquitination and proteasomal degradation of MyoD. Furthermore, we show that the ligase can ubiquitinate the protein in its N-terminal residue.

  19. Controlled access of p53 to the nucleus regulates its proteasomal degradation by MDM2.

    PubMed

    Davis, James R; Mossalam, Mohanad; Lim, Carol S

    2013-04-01

    The tumor suppressor p53 can be sent to the proteasome for degradation by placing its nucleo-cytoplasmic shuttling under ligand control. Endogenous p53 is ubiquitinated by MDM2 in the nucleus, and controlling the access of p53 to the nuclear compartment regulates its ubiquitination and proteasomal degradation. This was accomplished by the use of a protein switch that places nuclear translocation under the control of externally applied dexamethasone. Fluorescence microscopy revealed that sending protein switch p53 (PS-p53) to the nucleus produces a distinct punctate distribution in both the cytoplasm and nucleus. The nuclear role in accessing the proteasome was investigated by inhibiting classical nuclear export with leptomycin B. Trapping PS-p53 in the nucleus only allows this punctate staining in that compartment, suggesting that PS-p53 must translocate first to the nuclear compartment for cytoplasmic punctate staining to occur. The role of MDM2 binding was explored by inhibiting MDM2/p53 binding with nutlin-3. Inhibition of this interaction blocked both nuclear export and cytoplasmic and nuclear punctate staining, providing evidence that any change in localization after nuclear translocation is due to MDM2 binding. Further, blocking the proteolytic activity of the proteasome maintained the nuclear localization of the construct. Truncations of p53 were made to determine smaller constructs still capable of interacting with MDM2, and their subcellular localization and degradation potential was observed. PS-p53 and a smaller construct containing the two MDM2 binding regions of p53 (Box I + V) were indeed degraded by the proteasome as measured by loss of enhanced green fluorescent protein that was also fused to the construct. The influence of these constructs on p53 gene transactivation function was assessed and revealed that PS-p53 decreased gene transactivation, while PS-p53(Box I + V) did not significantly change baseline gene transactivation.

  20. 20S proteasome activation promotes life span extension and resistance to proteotoxicity in Caenorhabditis elegans.

    PubMed

    Chondrogianni, Niki; Georgila, Konstantina; Kourtis, Nikos; Tavernarakis, Nektarios; Gonos, Efstathios S

    2015-02-01

    Protein homeostasis (proteostasis) is one of the nodal points that need to be preserved to retain physiologic cellular/organismal balance. The ubiquitin-proteasome system (UPS) is responsible for the removal of both normal and damaged proteins, with the proteasome being the downstream effector. The proteasome is the major cellular protease with progressive impairment of function during aging and senescence. Despite the documented age-retarding properties of proteasome activation in various cellular models, simultaneous enhancement of the 20S core proteasome content, assembly, and function have never been reported in any multicellular organism. Consequently, the possible effects of the core proteasome modulation on organismal life span are elusive. In this study, we have achieved activation of the 20S proteasome at organismal level. We demonstrate enhancement of proteasome levels, assembly, and activity in the nematode Caenorhabditis elegans, resulting in life span extension and increased resistance to stress. We also provide evidence that the observed life span extension is dependent on the transcriptional activity of Dauer formation abnormal/Forkhead box class O (DAF-16/FOXO), skinhead-1 (SKN-1), and heat shock factor-1 (HSF-1) factors through regulation of downstream longevity genes. We further show that the reported beneficial effects are not ubiquitous but they are dependent on the genetic context. Finally, we provide evidence that proteasome core activation might be a potential strategy to minimize protein homeostasis deficiencies underlying aggregation-related diseases, such as Alzheimer's disease (AD) or Huntington's disease (HD). In summary, this is the first report demonstrating that 20S core proteasome up-regulation in terms of both content and activity is feasible in a multicellular eukaryotic organism and that in turn this modulation promotes extension of organismal health span and life span. PMID:25395451

  1. The proteasome.

    PubMed

    Dalton, William S

    2004-12-01

    The proteasome is an abundant multicatalytic enzyme complex present in the cytoplasm and nucleus of all eukaryotic cells. The primary function of the proteasome is to degrade proteins. While it was once thought to act primarily as a cellular "garbage disposal" that removed damaged or misfolded proteins from cells, the proteasome is now known to also remove various short-lived proteins that regulate the cell cycle, cell growth, and differentiation. By regulating the turnover of these proteins via timely degradation and recycling, the proteasome plays a critical role in the maintenance of cellular homeostasis. Substrates of the proteasome include cell-cycle regulators, signaling molecules, tumor suppressors, transcription factors, and antiapoptotic proteins; over 80% of all cellular proteins are recycled through the proteasome. This article discusses the structure and function of the proteasome, and its role in malignant cells and as a therapeutic target.

  2. Ubiquitination and proteasome degradation of the E6 proteins of human papillomavirus types 11 and 18.

    PubMed

    Stewart, Deborah; Kazemi, Shirin; Li, Suiyang; Massimi, Paola; Banks, Lawrence; Koromilas, Antonis E; Matlashewski, Greg

    2004-06-01

    Human papillomaviruses (HPVs) are aetiological agents for genital warts and cervical cancer, the different pathologies of which are dependent on the type of HPV infection. Oncogenic HPV types associated with cancer are carcinogens by virtue of their oncogene products, which target key regulators of cell proliferation and apoptosis. The viral E6 protein from oncogenic HPV types plays a central role in carcinogenesis by exploiting the cellular proteasome degradation pathway in order to mediate the degradation of cellular proteins, most notably the prototype tumour suppressor protein p53. Much less is known about the cellular targets of E6 from the non-oncogenic HPV types associated with genital warts. It is also unclear what factors influence the level and stability of the viral E6 proteins in cells. This report demonstrates that both oncogenic and non-oncogenic HPV E6 proteins (from types 18 and 11, respectively) are ubiquitinated and targeted for degradation by the 26S proteasome. E6 domains required for the induction of p53 or DLG degradation, or E6AP binding, are not involved in proteasome-mediated degradation of HPV-18 E6. These results provide insight into the cellular modulation of E6 protein levels from both high-risk and low-risk HPV types. PMID:15166424

  3. The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT61[OPEN

    PubMed Central

    2015-01-01

    Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. PMID:25739698

  4. The ubiquitin+proteasome protein degradation pathway as a therapeutic strategy in the treatment of solid tumor malignancies.

    PubMed

    Driscoll, James J; Minter, Alex; Driscoll, Daniel A; Burris, Jason K

    2011-02-01

    A concept that currently steers the development of cancer therapies has been that agents directed against specific proteins that facilitate tumorigenesis or maintain a malignant phenotype will have greater efficacy, less toxicity and a more sustained response relative to traditional cytotoxic chemotherapeutic agents. The clinical success of the targeted agent Imatinib mesylate as an inhibitor of the tyrosine kinase associated with the breakpoint cluster region-Abelson oncogene locus (BCR-ABL) in the treatment of Philadelphia-positive chronic myelogenous leukemia (CML) has served as a paradigm. While intellectually gratifying, the selective targeting of a single driver event by a small molecule, e.g., kinase inhibitor, to dampen a tumor-promoting pathway in the treatment of solid tumors is limited by many factors. Focus can alternatively be placed on targeting fundamental cellular processes that regulate multiple events, e.g., protein degradation, through the Ubiquitin (Ub)+Proteasome System (UPS). The UPS plays a critical role in modulating numerous cellular proteins to regulate cellular processes such as signal transduction, growth, proliferation, differentiation and apoptosis. Clinical success with the proteasome inhibitor bortezomib revolutionized treatment of B-cell lineage malignancies such as Multiple Myeloma (MM). However, many patients harbor primary resistance and do not respond to bortezomib and those that do respond inevitably develop resistance (secondary resistance). The lack of clinical efficacy of proteasome inhibitors in the treatment of solid tumors may be linked mechanistically to the resistance detected during treatment of hematologic malignancies. Potential mechanisms of resistance and means to improve the response to proteasome inhibitors in solid tumors are discussed.

  5. Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

    PubMed Central

    Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

    2010-01-01

    Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation. PMID:20478262

  6. Molecular Design, Synthesis, and Evaluation of SNIPER(ER) That Induces Proteasomal Degradation of ERα.

    PubMed

    Okuhira, Keiichiro; Demizu, Yosuke; Hattori, Takayuki; Ohoka, Nobumichi; Shibata, Norihito; Kurihara, Masaaki; Naito, Mikihiko

    2016-01-01

    Manipulation of protein stability using small molecules has a great potential for both basic research and clinical therapy. Based on our protein knockdown technology, we recently developed a novel small molecule SNIPER(ER) that targets the estrogen receptor alpha (ERα) for degradation via the ubiquitin-proteasome system. This chapter describes the design and synthesis of SNIPER(ER) compounds, and methods for the evaluation of their activity in cellular system.

  7. Yeast Pah1p phosphatidate phosphatase is regulated by proteasome-mediated degradation.

    PubMed

    Pascual, Florencia; Hsieh, Lu-Sheng; Soto-Cardalda, Aníbal; Carman, George M

    2014-04-01

    Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.

  8. Direct cellular delivery of human proteasomes to delay tau aggregation.

    PubMed

    Han, Dong Hoon; Na, Hee-Kyung; Choi, Won Hoon; Lee, Jung Hoon; Kim, Yun Kyung; Won, Cheolhee; Lee, Seung-Han; Kim, Kwang Pyo; Kuret, Jeff; Min, Dal-Hee; Lee, Min Jae

    2014-01-01

    The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins. PMID:25476420

  9. VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle

    PubMed Central

    Cantarero, Lara; Sanz-García, Marta; Vinograd-Byk, Hadar; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A.

    2015-01-01

    Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics. PMID:26068304

  10. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

    PubMed Central

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K.; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-01

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. PMID:26609072

  11. Assaying Proteasomal Degradation in a Cell-free System in Plants

    PubMed Central

    García-Cano, Elena; Zaltsman, Adi; Citovsky, Vitaly

    2014-01-01

    The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions. The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions. PMID:24747194

  12. Regulation of Ubiquitin-Proteasome System–mediated Degradation by Cytosolic Stress

    PubMed Central

    Kelly, Sean M.; VanSlyke, Judy K.

    2007-01-01

    ER-associated, ubiquitin-proteasome system (UPS)-mediated degradation of the wild-type (WT) gap junction protein connexin32 (Cx32) is inhibited by mild forms of cytosolic stress at a step before its dislocation into the cytosol. We show that the same conditions (a 30-min, 42°C heat shock or oxidative stress induced by arsenite) also reduce the endoplasmic reticulum (ER)-associated turnover of disease-causing mutants of Cx32 and the cystic fibrosis transmembrane conductance regulator (CFTR), as well as that of WT CFTR and unassembled Ig light chain. Stress-stabilized WT Cx32 and CFTR, but not the mutant/unassembled proteins examined, could traverse the secretory pathway. Heat shock also slowed the otherwise rapid UPS-mediated turnover of the cytosolic proteins myoD and GFPu, but not the degradation of an ubiquitination-independent construct (GFP-ODC) closely related to the latter. Analysis of mutant Cx32 from cells exposed to proteasome inhibitors and/or cytosolic stress indicated that stress reduces degradation at the level of substrate polyubiquitination. These findings reveal a new link between the cytosolic stress-induced heat shock response, ER-associated degradation, and polyubiquitination. Stress-denatured proteins may titer a limiting component of the ubiquitination machinery away from pre-existing UPS substrates, thereby sparing the latter from degradation. PMID:17699585

  13. Cyclic AMP signaling reduces sirtuin 6 expression in non-small cell lung cancer cells by promoting ubiquitin-proteasomal degradation via inhibition of the Raf-MEK-ERK (Raf/mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase) pathway.

    PubMed

    Kim, Eui-Jun; Juhnn, Yong-Sung

    2015-04-10

    The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gαs plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.

  14. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    SciTech Connect

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo

    2014-08-08

    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

  15. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  16. Ribosomal proteins produced in excess are degraded by the ubiquitin-proteasome system.

    PubMed

    Sung, Min-Kyung; Reitsma, Justin M; Sweredoski, Michael J; Hess, Sonja; Deshaies, Raymond J

    2016-09-01

    Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin-proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment. PMID:27385339

  17. UV Irradiation Triggers Cylindromatosis Translocation, Modification, and Degradation in a Proteasome-Independent Manner.

    PubMed

    Zhou, Ping; Hao, Ziwei; Wang, Xincheng; Gao, Jinmin; Li, Dengwen; Xie, Songbo; Zhang, Tong-Cun

    2016-03-01

    The tumor suppressor, cylindromatosis (CYLD), is a negative regulator of NF-κB signaling by removing lysine 63-linked ubiquitin chains from multiple NF-κB signaling components, including TRAF2, TRAF6, and NEMO. How CYLD itself is regulated, however, remains yet to be characterized. In this study, we present the first evidence that UV irradiation is able to induce CYLD translocation from the cytoplasm to microtubules and that the cytoskeleton-associated CYLD is subject to posttranslational modification and degradation in a proteasome-independent manner. By immunostaining, we found that CYLD displayed microtubule-like filament localization under ultraviolet (UV) irradiation. Further studies revealed that the cytoskeleton-associated CYLD underwent posttranslational modification, which in turn contributed to CYLD degradation in an unknown manner, distinct from proteasome-mediated degradation under normal conditions. Collectively, our data suggest that UV-induced CYLD degradation might serve as an underlying mechanism for UV-induced NF-κB pathway activation. PMID:26717101

  18. UV Irradiation Triggers Cylindromatosis Translocation, Modification, and Degradation in a Proteasome-Independent Manner.

    PubMed

    Zhou, Ping; Hao, Ziwei; Wang, Xincheng; Gao, Jinmin; Li, Dengwen; Xie, Songbo; Zhang, Tong-Cun

    2016-03-01

    The tumor suppressor, cylindromatosis (CYLD), is a negative regulator of NF-κB signaling by removing lysine 63-linked ubiquitin chains from multiple NF-κB signaling components, including TRAF2, TRAF6, and NEMO. How CYLD itself is regulated, however, remains yet to be characterized. In this study, we present the first evidence that UV irradiation is able to induce CYLD translocation from the cytoplasm to microtubules and that the cytoskeleton-associated CYLD is subject to posttranslational modification and degradation in a proteasome-independent manner. By immunostaining, we found that CYLD displayed microtubule-like filament localization under ultraviolet (UV) irradiation. Further studies revealed that the cytoskeleton-associated CYLD underwent posttranslational modification, which in turn contributed to CYLD degradation in an unknown manner, distinct from proteasome-mediated degradation under normal conditions. Collectively, our data suggest that UV-induced CYLD degradation might serve as an underlying mechanism for UV-induced NF-κB pathway activation.

  19. The proteasomal de-ubiquitinating enzyme POH1 promotes the double-strand DNA break response

    PubMed Central

    Butler, Laura R; Densham, Ruth M; Jia, Junying; Garvin, Alexander J; Stone, Helen R; Shah, Vandna; Weekes, Daniel; Festy, Frederic; Beesley, James; Morris, Joanna R

    2012-01-01

    The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome. PMID:22909820

  20. Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

    PubMed Central

    Liao, Chih-Kai; Jeng, Chung-Jiuan; Wang, Hwai-Shi; Wang, Shu-Huei; Wu, Jiahn-Chun

    2013-01-01

    The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. PMID:24236122

  1. Paeoniflorin inhibits human glioma cells via STAT3 degradation by the ubiquitin–proteasome pathway

    PubMed Central

    Nie, Xiao-hu; Ou-yang, Jia; Xing, Ying; Li, Dan-yan; Dong, Xing-yu; Liu, Ru-en; Xu, Ru-xiang

    2015-01-01

    We investigated the underlying mechanism for the potent proapoptotic effect of paeoniflorin (PF) on human glioma cells in vitro, focusing on signal transducer and activator of transcription 3 (STAT3) signaling. Significant time- and dose-dependent apoptosis and inhibition of proliferation were observed in PF-treated U87 and U251 glioma cells. Expression of STAT3, its active form phosphorylated STAT3 (p-STAT3), and several downstream molecules, including HIAP, Bcl-2, cyclin D1, and Survivin, were significantly downregulated upon PF treatment. Overexpression of STAT3 induced resistance to PF, suggesting that STAT3 was a critical target of PF. Interestingly, rapid downregulation of STAT3 was consistent with its accelerated degradation, but not with its dephosphorylation or transcriptional modulation. Using specific inhibitors, we demonstrated that the prodegradation effect of PF on STAT3 was mainly through the ubiquitin–proteasome pathway rather than via lysosomal degradation. These findings indicated that PF-induced growth suppression and apoptosis in human glioma cells through the proteasome-dependent degradation of STAT3. PMID:26508835

  2. Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation

    PubMed Central

    Li, Sheng; Yang, Li-juan; Wang, Ping; He, Yu-jiao; Huang, Jun-mei; Liu, Han-wei; Shen, Xiao-fei; Wang, Fei

    2016-01-01

    Background Type I interferons (IFN-α/β) have broad and potent immunoregulatory and antiproliferative activities. However, it is still known whether the dietary flavonoids exhibit their antiviral and anticancer properties by modulating the function of type I IFNs. Objective This study aimed at determining the role of apigenin, a dietary plant flavonoid abundant in common fruits and vegetables, on the type I IFN-mediated inhibition of cancer cell viability. Design Inhibitory effect of apigenin on human 26S proteasome, a known negative regulator of type I IFN signaling, was evaluated in vitro. Molecular docking was conducted to know the interaction between apigenin and subunits of 26S proteasome. Effects of apigenin on JAK/STAT pathway, 26S proteasome-mediated interferon receptor stability, and cancer cells viability were also investigated. Results Apigenin was identified to be a potent inhibitor of human 26S proteasome in a cell-based assay. Apigenin inhibited the chymotrypsin-like, caspase-like, and trypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Results from computational modeling of the potential interactions of apigenin with the chymotrypsin site (β5 subunit), caspase site (β1 subunit), and trypsin site (β2 subunit) of the proteasome were consistent with the observed proteasome inhibitory activity. Apigenin enhanced the phosphorylation of signal transducer and activator of transcription proteins (STAT1 and STAT2) and promoted the endogenous IFN-α-regulated gene expression. Apigenin inhibited the IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Apigenin also sensitized the inhibitory effect of IFN-α on viability of cervical carcinoma HeLa cells. Conclusion These results suggest that apigenin potentiates the inhibitory effect of IFN-α on cancer cell viability by activating JAK/STAT signaling pathway through inhibition of 26S proteasome

  3. Hepatitis C virus core protein inhibits E6AP expression via DNA methylation to escape from ubiquitin-dependent proteasomal degradation.

    PubMed

    Kwak, Juri; Shim, Joo Hee; Tiwari, Indira; Jang, Kyung Lib

    2016-09-28

    The E6-associated protein (E6AP) is a ubiquitin ligase that mediates ubiquitination and proteasomal degradation of hepatitis C virus (HCV) core protein. Given the role of HCV core protein as a major component of the viral nucleocapsid, as well as a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis, HCV has likely evolved a strategy to counteract the host anti-viral defense mechanism of E6AP and maximize its potential to produce infectious virus particles. In the present study, we found that HCV core protein derived from either ectopic expression or HCV infection inhibits E6AP expression via promoter hypermethylation in human hepatocytes. As a result, the potential of E6AP to ubiquitinate and degrade HCV core protein through the ubiquitin-proteasome system was severely impaired, which in turn led to stimulation of virus propagation. The effects of HCV core protein were almost completely abolished when the E6AP level was restored by ectopic expression of E6AP, treatment with a universal DNA methyltransferase (DNMT) inhibitor, 5-Aza-2'dC, or knock-down of DNMT1. In conclusion, HCV core protein inhibits E6AP expression via DNA methylation to protect itself from ubiquitin-dependent proteasomal degradation and stimulate virus propagation, providing a potential target for the development of anti-viral drugs against HCV.

  4. Ikaros is degraded by proteasome-dependent mechanism in the early phase of apoptosis induction

    SciTech Connect

    He, Li-Cai; Xu, Han-Zhang; Gu, Zhi-Min; Liu, Chuan-Xu; Chen, Guo-Qiang; Wang, Yue-Fei; Wen, Dong-Hua; Wu, Ying-Li

    2011-03-18

    Research highlights: {yields} Chemotherapeutic drugs or UV treatment reduces Ikaros prior to caspase-3 activation. {yields} Etoposide treatment does not alter the mRNA but shortens the half-life of Ikaros. {yields} MG132 or epoxomicin but not calpeptin inhibits etoposide-induced Ikaros degradation. {yields} Overexpression of Ikaros accelerates etoposide-induced apoptosis in NB4 cells. -- Abstract: Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. In this work, we found that chemotherapeutic drugs or ultraviolet radiation (UV) treatment could reduce the expression of full-length Ikaros (IK1) protein in less than 3 h in leukemic NB4, Kasumi-1 and Jurkat cells, prior to the activation of caspase-3. Etoposide treatment could not alter the mRNA level of IK1 but it could shorten the half-life of IK1. Co-treatment with the proteasome inhibitor MG132 or epoxomicin but not calpain inhibitor calpeptin inhibited etoposide-induced Ikaros downregulation. Overexpression of IK1 could accelerate etoposide-induced apoptosis in NB4 cells, as evidenced by the increase of Annexin V positive cells and the more early activation of caspase 3. To our knowledge, this is the first report to show that upon chemotherapy drugs or UV treatment, IK1 could be degraded via the proteasome system in the early phase of apoptosis induction. These data might shed new insight on the role of IK1 in apoptosis and the post-translational regulation of IK1.

  5. Geldanamycin-induced degradation of Chk1 is mediated by proteasome

    SciTech Connect

    Nomura, M.; E-mail: nomura413jp@yahoo.co.jp; Nomura, N.; Yamashita, J.

    2005-09-30

    Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3 h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24 h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells.

  6. Control of Death-associated Protein Kinase (DAPK) Activity by Phosphorylation and Proteasomal Degradation*

    PubMed Central

    Jin, Yijun; Blue, Emily K.; Gallagher, Patricia J.

    2010-01-01

    Activation of death-associated protein kinase (DAPK) occurs via dephosphorylation of Ser-308 and subsequent association of calcium/calmodulin. In this study, we confirmed the existence of the alternatively spliced human DAPK-β, and we examined the levels of DAPK autophosphorylation and DAPK catalytic activity in response to tumor necrosis factor or ceramide. It was found that DAPK is rapidly dephosphorylated in response to tumor necrosis factor or ceramide and then subsequently degraded via proteasome activity. Dephosphorylation and activation of DAPK are shown to temporally precede its subsequent degradation. This results in an initial increase in kinase activity followed by a decrease in DAPK expression and activity. The decline in DAPK expression is paralleled with increased caspase activity and cell apoptosis. These results suggest that the apoptosis regulatory activities mediated by DAPK are controlled both by phosphorylation status and protein stability. PMID:17056602

  7. Inhibition of Stat5a/b enhances proteasomal degradation of androgen receptor liganded by antiandrogens in prostate cancer

    PubMed Central

    Hoang, David T.; Gu, Lei; Liao, Zhiyong; Talati, Pooja G.; Shen, Feng; Koptyra, Mateusz; Tan, Shyh-Han; Ellsworth, Elyse; Gupta, Shilpa; Montie, Heather; Dagvadorj, Ayush; Savolainen, Saija; Leiby, Benjamin; Mirtti, Tuomas; Merry, Diane E.; Nevalainen, Marja T.

    2015-01-01

    Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, Bicalutamide, Flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the Prostate Specific Antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b. PMID:25552366

  8. Histone deacetylase 4 promotes ubiquitin-dependent proteasomal degradation of Sp3 in SH-SY5Y cells treated with di(2-ethylhexyl)phthalate (DEHP), determining neuronal death

    SciTech Connect

    Guida, Natascia; Laudati, Giusy; Galgani, Mario; Santopaolo, Marianna; Montuori, Paolo; Triassi, Maria; Di Renzo, Gianfranco; Canzoniero, Lorella M.T.; Formisano, Luigi

    2014-10-01

    Phthalates, phthalic acid esters, are widely used as plasticizers to produce polymeric materials in industrial production of plastics and daily consumable products. Animal studies have shown that di(2-ethylhexyl)phthalate (DEHP) may cause toxic effects in the rat brain. In the present study, chronic exposure to DEHP (0.1–100 μM) caused dose-dependent cell death via the activation of caspase-3 in neuroblastoma cells. Intriguingly, this harmful effect was prevented by the pan-histone deacetylase (HDAC) inhibitor trichostatin A, by the class II HDAC inhibitor MC-1568, but not by the class I HDAC inhibitor MS-275. Furthermore, DEHP reduced specificity protein 3 (Sp3) gene expression, but not Sp3 mRNA, after 24 and 48 h exposures. However, Sp3 protein reduction was prevented by pre-treatment with MC-1568, suggesting the involvement of class II HDACs in causing this effect. Then, we investigated the possible relationship between DEHP-induced neuronal death and the post-translational mechanisms responsible for the down-regulation of Sp3. Interestingly, DEHP-induced Sp3 reduction was associated to its deacetylation and polyubiquitination. Co-immunoprecipitation studies showed that Sp3 physically interacted with HDAC4 after DEHP exposure, while HDAC4 inhibition by antisense oligodeoxynucleotide reverted the DEHP-induced degradation of Sp3. Notably, Sp3 overexpression was able to counteract the detrimental effect induced by DEHP. Taken together, these results suggest that DEHP exerts its toxic effect by inducing deacetylation of Sp3 via HDAC4, and afterwards, Sp3-polyubiquitination. - Highlights: • Di(2-ethylhexyl)phthalate (DEHP) is cytotoxic to SH-SY5Y cells and cortical neurons. • DEHP-induced cytotoxicity is mediated by apoptosis. • DEHP-induced apoptotic cell death is inhibited by class II HDAC MC-1568. • DEHP neurotoxicity is caused by HDAC4-mediated Sp3 degradation by ubiquitin.

  9. Glycogen synthase kinase 3beta phosphorylates p21WAF1/CIP1 for proteasomal degradation after UV irradiation.

    PubMed

    Lee, Ji Young; Yu, Su Jin; Park, Yun Gyu; Kim, Joon; Sohn, Jeongwon

    2007-04-01

    UV irradiation has been reported to induce p21(WAF1/CIP1) protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3beta (GSK-3beta) is required for its degradation in response to UV irradiation and that GSK-3beta activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3beta activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3beta. ser-114, located within the putative GSK-3beta target sequence, was phosphorylated by GSK-3beta upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3beta is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3beta as the missing link between UV-induced ATR activation and p21 degradation.

  10. Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

    PubMed Central

    Metz, Philippe; Chiramel, Abhilash; Chatel-Chaix, Laurent; Alvisi, Gualtiero; Bankhead, Peter; Mora-Rodríguez, Rodrigo; Long, Gang; Hamacher-Brady, Anne

    2015-01-01

    ABSTRACT Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. IMPORTANCE Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the

  11. Histone deacetylase 4 promotes ubiquitin-dependent proteasomal degradation of Sp3 in SH-SY5Y cells treated with di(2-ethylhexyl)phthalate (DEHP), determining neuronal death.

    PubMed

    Guida, Natascia; Laudati, Giusy; Galgani, Mario; Santopaolo, Marianna; Montuori, Paolo; Triassi, Maria; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2014-10-01

    Phthalates, phthalic acid esters, are widely used as plasticizers to produce polymeric materials in industrial production of plastics and daily consumable products. Animal studies have shown that di(2-ethylhexyl)phthalate (DEHP) may cause toxic effects in the rat brain. In the present study, chronic exposure to DEHP (0.1-100μM) caused dose-dependent cell death via the activation of caspase-3 in neuroblastoma cells. Intriguingly, this harmful effect was prevented by the pan-histone deacetylase (HDAC) inhibitor trichostatin A, by the class II HDAC inhibitor MC-1568, but not by the class I HDAC inhibitor MS-275. Furthermore, DEHP reduced specificity protein 3 (Sp3) gene expression, but not Sp3 mRNA, after 24 and 48h exposures. However, Sp3 protein reduction was prevented by pre-treatment with MC-1568, suggesting the involvement of class II HDACs in causing this effect. Then, we investigated the possible relationship between DEHP-induced neuronal death and the post-translational mechanisms responsible for the down-regulation of Sp3. Interestingly, DEHP-induced Sp3 reduction was associated to its deacetylation and polyubiquitination. Co-immunoprecipitation studies showed that Sp3 physically interacted with HDAC4 after DEHP exposure, while HDAC4 inhibition by antisense oligodeoxynucleotide reverted the DEHP-induced degradation of Sp3. Notably, Sp3 overexpression was able to counteract the detrimental effect induced by DEHP. Taken together, these results suggest that DEHP exerts its toxic effect by inducing deacetylation of Sp3 via HDAC4, and afterwards, Sp3-polyubiquitination.

  12. DBC2 resistance is achieved by enhancing 26S proteasome-mediated protein degradation.

    PubMed

    Collado, Denise; Yoshihara, Takashi; Hamaguchi, Masaaki

    2007-08-31

    Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.

  13. REGγ regulates ERα degradation via ubiquitin–proteasome pathway in breast cancer

    SciTech Connect

    Chai, Fan; Liang, Yan; Bi, Jiong; Chen, Li; Zhang, Fan; Cui, Youhong; Jiang, Jun

    2015-01-02

    Highlights: • High expression of REGγ is correlated with ERα status and poor clinical features. • Cell growth, mobility and invasion are significantly impaired by REGγ knockdown. • REGγ indirectly regulates ERα protein expression. - Abstract: REGγ is a proteasome coactivator which regulates proteolytic activity in eukaryotic cells. Abundant lines of evidence have showed that REGγ is over expressed in a number of human carcinomas. However, its precise role in the pathogenesis of cancer is still unclear. In this study, by examining 200 human breast cancer specimens, we demonstrated that REGγ was highly expressed in breast cancers, and the expression of REGγ was positively correlated with breast cancer patient estrogen receptor alpha (ERα) status. Moreover, the expression of REGγ was found positively associated with poor clinical features and low survival rates in ERα positive breast cancer patients. Further cell culture studies using MCF7 and BT474 breast cancer cell lines showed that cell proliferation, motility, and invasion capacities were decreased significantly by REGγ knockdown. Lastly, we demonstrated that REGγ indirectly regulates the degradation of ERα protein via ubiquitin–proteasome pathway. In conclusion, our findings provide the evidence that REGγ expression was positively correlated with ERα status and poor clinical prognosis in ERα positive breast cancer patients. As well, we disclose a new connection between the two molecules that are both highly expressed in most breast cancer cases.

  14. Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components*

    PubMed Central

    Lee, Kyoung-Hee; Biswas, Amlan; Liu, Yuen-Joyce; Kobayashi, Koichi S.

    2012-01-01

    The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock. PMID:23019338

  15. Involvement of protein degradation by the ubiquitin proteasome system in opiate addictive behaviors.

    PubMed

    Massaly, Nicolas; Dahan, Lionel; Baudonnat, Mathieu; Hovnanian, Caroline; Rekik, Khaoula; Solinas, Marcello; David, Vincent; Pech, Stéphane; Zajac, Jean-Marie; Roullet, Pascal; Mouledous, Lionel; Frances, Bernard

    2013-03-01

    Plastic changes in the nucleus accumbens (NAcc), a structure occupying a key position in the neural circuitry related to motivation, are among the critical cellular processes responsible for drug addiction. During the last decade, it has been shown that memory formation and related neuronal plasticity may rely not only on protein synthesis but also on protein degradation by the ubiquitin proteasome system (UPS). In this study, we assess the role of protein degradation in the NAcc in opiate-related behaviors. For this purpose, we coupled behavioral experiments to intra-accumbens injections of lactacystin, an inhibitor of the UPS. We show that protein degradation in the NAcc is mandatory for a full range of animal models of opiate addiction including morphine locomotor sensitization, morphine conditioned place preference, intra-ventral tegmental area morphine self-administration and intra-venous heroin self-administration but not for discrimination learning rewarded by highly palatable food. This study provides the first evidence of a specific role of protein degradation by the UPS in addiction.

  16. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

    PubMed Central

    Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  17. E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models

    PubMed Central

    Prideaux, Matt; Allen, Steve; Buttle, David J.; Pitsillides, Andrew A.; Farquharson, Colin

    2015-01-01

    The transmembrane glycoprotein E11 is considered critical in early osteoblast–osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO‐A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post‐translational degradation. We found that exogenous treatment of MLO‐A5 and osteocytic IDG‐SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome‐selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin‐A) produced similar dose‐dependent increases in E11 protein levels in MLO‐A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO‐A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome‐mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. J. Cell

  18. mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy.

    PubMed

    Zhao, Jinghui; Zhai, Bo; Gygi, Steven P; Goldberg, Alfred Lewis

    2015-12-29

    Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440-443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth. PMID:26669439

  19. Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

    SciTech Connect

    Strom, Bjorn O.; Paulsen, Ragnhild E.

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer NGFI-B is a molecular target for some anti-cancer drugs. Black-Right-Pointing-Pointer NGFI-B turnover may be important for their anti-cancer action. Black-Right-Pointing-Pointer NGFI-B is degraded by the proteasome. Black-Right-Pointing-Pointer NGFI-B is stabilized by treatment with EGF. Black-Right-Pointing-Pointer Mimicking the EGF-induced phosphorylation also stabilizes the protein. -- Abstract: NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

  20. Proteasome Activators

    PubMed Central

    Stadtmueller, Beth M.; Hill, Christopher P.

    2011-01-01

    Summary Proteasomes degrade a multitude of protein substrates in the cytosol and nucleus, and thereby are essential for many aspects of cellular function. Because the proteolytic sites are sequestered in a closed barrel-shaped structure, activators are required to facilitate substrate access. Structural and biochemical studies of two activator families, 11S and Blm10, have provided insights to proteasome activation mechanisms, although the biological functions of these factors remain obscure. Recent advances have improved our understanding of the third activator family, including the 19S activator, which targets polyubiquitylated proteins for degradation. PMID:21211719

  1. Iron-dependent degradation of apo-IRP1 by the ubiquitin-proteasome pathway.

    PubMed

    Wang, Jian; Fillebeen, Carine; Chen, Guohua; Biederbick, Annette; Lill, Roland; Pantopoulos, Kostas

    2007-04-01

    Iron regulatory protein 1 (IRP1) controls the translation or stability of several mRNAs by binding to "iron-responsive elements" within their untranslated regions. In iron-replete cells, IRP1 assembles a cubane iron-sulfur cluster (ISC) that inhibits RNA-binding activity and converts the protein to cytosolic aconitase. We show that the constitutive IRP1(C437S) mutant, which fails to form an ISC, is destabilized by iron. Thus, exposure of H1299 cells to ferric ammonium citrate reduced the half-life of transfected IRP1(C437S) from approximately 24 h to approximately 10 h. The iron-dependent degradation of IRP1(C437S) involved ubiquitination, required ongoing transcription and translation, and could be efficiently blocked by the proteasomal inhibitors MG132 and lactacystin. Similar results were obtained with overexpressed wild-type IRP1, which predominated in the apo-form even in iron-loaded H1299 cells, possibly due to saturation of the ISC assembly machinery. Importantly, inhibition of ISC biogenesis in HeLa cells by small interfering RNA knockdown of the cysteine desulfurase Nfs1 sensitized endogenous IRP1 for iron-dependent degradation. Collectively, these data uncover a mechanism for the regulation of IRP1 abundance as a means to control its RNA-binding activity, when the ISC assembly pathway is impaired.

  2. Nitric oxide-independent vasodilator rescues heme-oxidized soluble guanylate cyclase from proteasomal degradation.

    PubMed

    Meurer, Sabine; Pioch, Sylke; Pabst, Tatjana; Opitz, Nils; Schmidt, Peter M; Beckhaus, Tobias; Wagner, Kristina; Matt, Simone; Gegenbauer, Kristina; Geschka, Sandra; Karas, Michael; Stasch, Johannes-Peter; Schmidt, Harald H H W; Müller-Esterl, Werner

    2009-07-01

    Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the alphabeta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both alpha and beta subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC. PMID:19478201

  3. Celastrol induces proteasomal degradation of FANCD2 to sensitize lung cancer cells to DNA crosslinking agents.

    PubMed

    Wang, Gui-Zhen; Liu, Yong-Qiang; Cheng, Xin; Zhou, Guang-Biao

    2015-07-01

    The Fanconi anemia (FA) pathway plays a key role in interstrand crosslink (ICL) repair and maintenance of the genomic stability, while inhibition of this pathway may sensitize cancer cells to DNA ICL agents and ionizing radiation (IR). The active FA core complex acts as an E3 ligase to monoubiquitinate FANCD2, which is a functional readout of an activated FA pathway. In the present study, we aimed to identify FANCD2-targeting agents, and found that the natural compound celastrol induced degradation of FANCD2 through the ubiquitin-proteasome pathway. We demonstrated that celastrol downregulated the basal and DNA damaging agent-induced monoubiquitination of FANCD2, followed by proteolytic degradation of the substrate. Furthermore, celastrol treatment abrogated the G2 checkpoint induced by IR, and enhanced the ICL agent-induced DNA damage and inhibitory effects on lung cancer cells through depletion of FANCD2. These results indicate that celastrol is a FANCD2 inhibitor that could interfere with the monoubiquitination and protein stability of FANCD2, providing a novel opportunity to develop FA pathway inhibitor and combinational therapy for malignant neoplasms.

  4. The HIV-1 Protein Vpr Targets the Endoribonuclease Dicer for Proteasomal Degradation to Boost Macrophage Infection

    PubMed Central

    Klockow, Laurieann Casey; Sharifi, Hamayun J.; Wen, Xiaoyun; Flagg, Meg; Furuya, Andrea K. M.; Nekorchuk, Michael; de Noronha, Carlos M.

    2013-01-01

    The HIV-1 protein Vpr enhances macrophage infection, triggers G2 cell cycle arrest, and targets cells for NK-cell killing. Vpr acts through the CRL4DCAF1 ubiquitin ligase complex to cause G2 arrest and trigger expression of NK ligands. Corresponding ubiquitination targets have not been identified. UNG2 and SMUG1 are the only known substrates for Vpr-directed depletion through CRL4DCAF1. Here we identify the endoribonuclease Dicer as a target of HIV-1 Vpr-directed proteasomal degradation through CRL4DCAF1. We show that HIV-1 Vpr inhibits short hairpin RNA function as expected upon reduction of Dicer levels. Dicer inhibits HIV-1 replication in T cells. We demonstrate that Dicer also restricts HIV-1 replication in human monocyte-derived macrophages (MDM) and that reducing Dicer expression in MDMs enhances HIV-1 infection in a Vpr-dependent manner. Our results support a model in which Vpr complexes with human Dicer to boost its interaction with the CRL4DCAF1 ubiquitin ligase complex and its subsequent degradation. PMID:23849790

  5. Evasion from proteasomal degradation by mutated Fos proteins expressed from FBJ-MSV and FBR-MSV osteosarcomatogenic retroviruses.

    PubMed

    Acquaviva, Claire; Bossis, Guillaume; Ferrara, Patrizia; Brockly, Frédérique; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2002-09-01

    c-Fos proto-oncoprotein is highly unstable, which is crucial for rapid gene expression shut-off and control of its intrinsic oncogenic potential. It is massively degraded by the proteasome in vivo in various situations. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for recognition and subsequent hydrolysis by the proteasome in vivo is still lacking. Moreover, genetic analysis have also indicated that c-Fos can be addressed to the proteasome via different mechanisms depending on the conditions studied. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of a C-terminal c-Fos destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to (an) unidentified proteolytic system(s). This observation is consistent with the idea that Fos-expressing viruses have evolved gene expression controls that avoid high protein accumulation-linked apoptosis.

  6. Proteasome-mediated degradation of IκBα and processing of p105 in Crohn disease and ulcerative colitis

    PubMed Central

    Visekruna, Alexander; Joeris, Thorsten; Seidel, Daniel; Kroesen, Anjo; Loddenkemper, Christoph; Zeitz, Martin; Kaufmann, Stefan H.E.; Schmidt-Ullrich, Ruth; Steinhoff, Ulrich

    2006-01-01

    Enhanced NF-κB activity is involved in the pathology of both forms of inflammatory bowel disease (IBD), Crohn disease (CD) and ulcerative colitis (UC). Here we analyzed the mechanism of proteasome-mediated NF-κB activation in CD and UC. Our studies demonstrate that the subunit composition and the proteolytic function of proteasomes differ between UC and CD. High expression of the immunoproteasome subunits β1i and β2i is characteristic of the inflamed mucosa of CD. In line with this, we found enhanced processing of NF-κB precursor p105 and degradation of inhibitor of NF-κB, IκBα, by immunoproteasomes isolated from the mucosa of CD patients. In comparison with healthy controls and CD patients, UC patients exhibited an intermediate phenotype regarding the proteasome-mediated processing/degradation of NF-κB components. Finally, increased expression of the NF-κB family member c-Rel in the inflamed mucosa of CD patients suggests that p50/c-Rel is important for IFN-γ–mediated induction of immunoproteasomes via IL-12–driven Th1 responses. These findings suggest that distinct proteasome subunits influence the intensity of NF-κB–mediated inflammation in IBD patients. PMID:17124531

  7. Could inhibition of the proteasome cause mad cow disease?

    PubMed

    Hooper, Nigel M

    2003-04-01

    The proteasome is the cellular machinery responsible for the degradation of normal and misfolded proteins. Inhibitors of the proteasome are being evaluated as therapeutic agents and recent work suggests that such inhibition might promote the neurotoxic properties of the prion protein (the causative agent of mad cow disease) and its conformational conversion to the infectious form, thus raising the question as to whether proteasome inhibitors might facilitate the development of prion diseases. PMID:12679058

  8. 26S Proteasome: Hunter and Prey in Auxin Signaling.

    PubMed

    Kong, Xiangpei; Zhang, Liangran; Ding, Zhaojun

    2016-07-01

    Auxin binds to TRANSPORT INHIBITOR RESPONSE 1 and AUXIN SIGNALLING F-BOX proteins (TIR1/AFBs) and promotes the degradation of Aux/IAA transcriptional repressors. The proteasome regulator PROTEASOME REGULATOR1 (PTRE1) has now been shown to be required for auxin-mediated repression of 26S proteasome activity, thus providing new insights into the fine-tuning of the homoeostasis of Aux/IAA proteins and auxin signaling. PMID:27246455

  9. Could inhibition of the proteasome cause mad cow disease?

    PubMed

    Hooper, Nigel M

    2003-04-01

    The proteasome is the cellular machinery responsible for the degradation of normal and misfolded proteins. Inhibitors of the proteasome are being evaluated as therapeutic agents and recent work suggests that such inhibition might promote the neurotoxic properties of the prion protein (the causative agent of mad cow disease) and its conformational conversion to the infectious form, thus raising the question as to whether proteasome inhibitors might facilitate the development of prion diseases.

  10. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

    SciTech Connect

    Wu, Di Wu, Shian

    2013-04-19

    Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  11. Proteasomal Degradation of γ-Aminobutyric AcidB Receptors Is Mediated by the Interaction of the GABAB2 C Terminus with the Proteasomal ATPase Rtp6 and Regulated by Neuronal Activity*

    PubMed Central

    Zemoura, Khaled; Benke, Dietmar

    2014-01-01

    Regulation of cell surface expression of neurotransmitter receptors is crucial for determining synaptic strength and plasticity, but the underlying mechanisms are not well understood. We previously showed that proteasomal degradation of GABAB receptors via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery determines the number of cell surface GABAB receptors and thereby GABAB receptor-mediated neuronal inhibition. Here, we show that proteasomal degradation of GABAB receptors requires the interaction of the GABAB2 C terminus with the proteasomal AAA-ATPase Rpt6. A mutant of Rpt6 lacking ATPase activity prevented degradation of GABAB receptors but not the removal of Lys48-linked ubiquitin from GABAB2. Blocking ERAD activity diminished the interaction of Rtp6 with GABAB receptors resulting in increased total as well as cell surface expression of GABAB receptors. Modulating neuronal activity affected proteasomal activity and correspondingly the interaction level of Rpt6 with GABAB2. This resulted in altered cell surface expression of the receptors. Thus, neuronal activity-dependent proteasomal degradation of GABAB receptors by the ERAD machinery is a potent mechanism regulating the number of GABAB receptors available for signaling and is expected to contribute to homeostatic neuronal plasticity. PMID:24482233

  12. The Kaposi's sarcoma-associated herpesvirus ORF34 protein binds to HIF-1α and causes its degradation via the proteasome pathway.

    PubMed

    Haque, Muzammel; Kousoulas, Konstantin G

    2013-02-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761-6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway. PMID:23221556

  13. The tumor suppressor ING3 is degraded by SCF(Skp2)-mediated ubiquitin-proteasome system.

    PubMed

    Chen, G; Wang, Y; Garate, M; Zhou, J; Li, G

    2010-03-11

    The inhibitor of growth family member 3 (ING3) has been shown to modulate transcription, cell cycle control and apoptosis. We previously reported that nuclear ING3 expression was remarkably reduced in melanomas, which correlated with a poorer patient survival, suggesting that decreased ING3 expression may be associated with melanoma progression. However, the mechanism of diminished ING3 expression in melanoma is not clear. Here we show that ING3 level was decreased in metastatic melanoma cells because of a rapid degradation. Furthermore, we showed that ING3 undergoes degradation through the ubiquitin-proteasome pathway. ING3 physically interacts with subunits of E3 ligase Skp1-Cullin-F-box protein complex (SCF complex). Knockdown of F-box protein S-phase kinase-associated protein 2 (Skp2) reduces the ubiquitination of ING3 and significantly stabilizes ING3 in melanoma cells. In addition, lysine 96 residue is essential for ING3 ubiquitination as its mutation to arginine dramatically abrogated ING3 degradation. Disruption of ING3 degradation stimulated ING3-induced G1 cell-cycle arrest and enhanced ultraviolet-induced apoptosis. Taken together, our data show that ING3 is degraded by the ubiquitin-proteasome pathway through the SCF(Skp2) complex and interruption of ING3 degradation enhances the tumor-suppressive function of ING3, which provides a potential cancer therapeutic approach by interfering ING3 degradation. PMID:19935701

  14. Complement modulates the function of the ubiquitin-proteasome system and endoplasmic reticulum-associated degradation in glomerular epithelial cells.

    PubMed

    Kitzler, Thomas M; Papillon, Joan; Guillemette, Julie; Wing, Simon S; Cybulsky, Andrey V

    2012-05-01

    In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin-proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFP(u) (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFP(u) levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFP(u) was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFP(u) degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.

  15. Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation

    PubMed Central

    Lobbestael, E.; Civiero, L.; De Wit, T.; Taymans, J.-M.; Greggio, E.; Baekelandt, V.

    2016-01-01

    Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson’s disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies. PMID:27658356

  16. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals.

    PubMed

    Karabinova, Pavla; Kubelka, Michal; Susor, Andrej

    2011-10-01

    Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.

  17. Protein degradation by ubiquitin–proteasome system in formation and labilization of contextual conditioning memory

    PubMed Central

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro

    2014-01-01

    The ubiquitin–proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  18. Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

    PubMed Central

    Schwartzkopff, Benjamin; Platta, Harald W.; Hasan, Sohel; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-01-01

    Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p. PMID:26182377

  19. Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory.

    PubMed

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-09-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates.

  20. Ubiquitin proteasome-dependent degradation of the transcriptional coactivator PGC-1{alpha} via the N-terminal pathway.

    PubMed

    Trausch-Azar, Julie; Leone, Teresa C; Kelly, Daniel P; Schwartz, Alan L

    2010-12-17

    PGC-1α is a potent, inducible transcriptional coactivator that exerts control on mitochondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1α levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Here, we demonstrate that PGC-1α is rapidly degraded in the nucleus (t(½ 0.3 h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, are nuclear and rapidly degraded (t(½) 0.5 h), consistent with degradation via the N terminus-dependent ubiquitin subpathway. Both PGC-1α and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally occurring N-terminal splice variant of 270 residues, NT-PGC-1α is cytoplasmic and stable (t(½>7 h), providing additional evidence that PGC-1α is degraded in the nucleus. These results strongly suggest that the nuclear N terminus-dependent ubiquitin proteasome pathway governs PGC-1α cellular degradation. In contrast, the cellular localization of NT-PCG-1α results in a longer-half-life and possible distinct temporal and potentially biological actions.

  1. Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms

    PubMed Central

    Locatelli, Denise; Terao, Mineko; Kurosaki, Mami; Zanellati, Maria Clara; Pletto, Daniela Rita; Finardi, Adele; Colciaghi, Francesca; Garattini, Enrico; Battaglia, Giorgio Stefano

    2015-01-01

    The key pathogenic steps leading to spinal muscular atrophy (SMA), a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN), the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN. PMID:26214005

  2. Design Principles Involving Protein Disorder Facilitate Specific Substrate Selection and Degradation by the Ubiquitin-Proteasome System.

    PubMed

    Guharoy, Mainak; Bhowmick, Pallab; Tompa, Peter

    2016-03-25

    The ubiquitin-proteasome system (UPS) regulates diverse cellular pathways by the timely removal (or processing) of proteins. Here we review the role of structural disorder and conformational flexibility in the different aspects of degradation. First, we discuss post-translational modifications within disordered regions that regulate E3 ligase localization, conformation, and enzymatic activity, and also the role of flexible linkers in mediating ubiquitin transfer and reaction processivity. Next we review well studied substrates and discuss that substrate elements (degrons) recognized by E3 ligases are highly disordered: short linear motifs recognized by many E3s constitute an important class of degrons, and these are almost always present in disordered regions. Substrate lysines targeted for ubiquitination are also often located in neighboring regions of the E3 docking motifs and are therefore part of the disordered segment. Finally, biochemical experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially unfolded region to facilitate substrate entry into the proteasomal core.

  3. A Novel Role for ATM in Regulating Proteasome-Mediated Protein Degradation through Suppression of the ISG15 Conjugation Pathway

    PubMed Central

    Wood, Laurence M.; Sankar, Surendran; Reed, Ryan E.; Haas, Arthur L.; Liu, Leroy F.; McKinnon, Peter; Desai, Shyamal D.

    2011-01-01

    Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients. PMID:21298066

  4. Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function.

    PubMed

    Otero, Maria G; Alloatti, Matías; Cromberg, Lucas E; Almenar-Queralt, Angels; Encalada, Sandra E; Pozo Devoto, Victorio M; Bruno, Luciana; Goldstein, Lawrence S B; Falzone, Tomás L

    2014-04-01

    Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

  5. Cystic Fibrosis Transmembrane Conductance Regulator Controls Lung Proteasomal Degradation and Nuclear Factor-κB Activity in Conditions of Oxidative Stress

    PubMed Central

    Boncoeur, Emilie; Roque, Telma; Bonvin, Elise; Saint-Criq, Vinciane; Bonora, Monique; Clement, Annick; Tabary, Olivier; Henrion-Caude, Alexandra; Jacquot, Jacky

    2008-01-01

    Cystic fibrosis is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, resulting in progressive oxidative lung damage. In this study, we evaluated the role of CFTR in the control of ubiquitin-proteasome activity and nuclear factor (NF)-κB/IκB-α signaling after lung oxidative stress. After a 64-hour exposure to hyperoxia-mediated oxidative stress, CFTR-deficient (cftr−/−) mice exhibited significantly elevated lung proteasomal activity compared with wild-type (cftr+/+) animals. This was accompanied by reduced lung caspase-3 activity and defective degradation of NF-κB inhibitor IκB-α. In vitro, human CFTR-deficient lung cells exposed to oxidative stress exhibited increased proteasomal activity and decreased NF-κB-dependent transcriptional activity compared with CFTR-sufficient lung cells. Inhibition of the CFTR Cl− channel by CFTRinh-172 in the normal bronchial immortalized cell line 16HBE14o− increased proteasomal degradation after exposure to oxidative stress. Caspase-3 inhibition by Z-DQMD in CFTR-sufficient lung cells mimicked the response profile of increased proteasomal degradation and reduced NF-κB activity observed in CFTR-deficient lung cells exposed to oxidative stress. Taken together, these results suggest that functional CFTR Cl− channel activity is crucial for regulation of lung proteasomal degradation and NF-κB activity in conditions of oxidative stress. PMID:18372427

  6. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-11-06

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

  7. ADD66, a Gene Involved in the Endoplasmic Reticulum-associated Degradation of α-1-Antitrypsin-Z in Yeast, Facilitates Proteasome Activity and Assembly

    PubMed Central

    Scott, Craig M.; Kruse, Kristina B.; Schmidt, Béla Z.; Perlmutter, David H.; McCracken, Ardythe A.

    2007-01-01

    Antitrypsin deficiency is a primary cause of juvenile liver disease, and it arises from expression of the “Z” variant of the α-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retrotranslocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed previously, and a gene, ADD66, was identified that facilitates A1PiZ turnover. We report here that ADD66 encodes an ∼30-kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Δ mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also seems to be a proteasome substrate. Consistent with its role in ER-associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC), and they provide the first link between PAC activity and ERAD. PMID:17634286

  8. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    SciTech Connect

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J. . E-mail: hjw14@columbia.edu

    2006-12-29

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1{alpha} associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome.

  9. Proteasomal degradation of retinoid X receptor α reprograms transcriptional activity of PPARγ in obese mice and humans

    PubMed Central

    Lefebvre, Bruno; Benomar, Yacir; Guédin, Aurore; Langlois, Audrey; Hennuyer, Nathalie; Dumont, Julie; Bouchaert, Emmanuel; Dacquet, Catherine; Pénicaud, Luc; Casteilla, Louis; Pattou, Francois; Ktorza, Alain; Staels, Bart; Lefebvre, Philippe

    2010-01-01

    Obese patients have chronic, low-grade inflammation that predisposes to type 2 diabetes and results, in part, from dysregulated visceral white adipose tissue (WAT) functions. The specific signaling pathways underlying WAT dysregulation, however, remain unclear. Here we report that the PPARγ signaling pathway operates differently in the visceral WAT of lean and obese mice. PPARγ in visceral, but not subcutaneous, WAT from obese mice displayed increased sensitivity to activation by its agonist rosiglitazone. This increased sensitivity correlated with increased expression of the gene encoding the ubiquitin hydrolase/ligase ubiquitin carboxyterminal esterase L1 (UCH-L1) and with increased degradation of the PPARγ heterodimerization partner retinoid X receptor α (RXRα), but not RXRβ, in visceral WAT from obese humans and mice. Interestingly, increased UCH-L1 expression and RXRα proteasomal degradation was induced in vitro by conditions mimicking hypoxia, a condition that occurs in obese visceral WAT. Finally, PPARγ-RXRβ heterodimers, but not PPARγ-RXRα complexes, were able to efficiently dismiss the transcriptional corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) upon agonist binding. Increasing the RXRα/RXRβ ratio resulted in increased PPARγ responsiveness following agonist stimulation. Thus, the selective proteasomal degradation of RXRα initiated by UCH-L1 upregulation modulates the relative affinity of PPARγ heterodimers for SMRT and their responsiveness to PPARγ agonists, ultimately activating the PPARγ-controlled gene network in visceral WAT of obese animals and humans. PMID:20364085

  10. Disturbance of proteasomal and autophagic protein degradation pathways by amyotrophic lateral sclerosis-linked mutations in ubiquilin 2.

    PubMed

    Osaka, Mayuko; Ito, Daisuke; Suzuki, Norihiro

    2016-04-01

    Ubiquilin (UBQLN), a member of the ubiquitin-like (UBL)-ubiquitin-associated (UBA) family, is a dual regulator of both the proteasomal and autophagic branches of the cellular protein degradation system. Mutations in the UBQLN2 gene encoding ubiquilin 2 cause X-linked amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), and UBQLN2-positive inclusions have been identified in ALS patients with UBQLN2 mutations as well as in cases of both familial and sporadic ALS without UBQLN2 mutations. Compelling evidence links UBQLN2 to disturbance of the protein quality control network in neurons, but the pathomechanisms remain obscure. This study aimed to clarify how ALS-linked mutations in UBQLN2 affect the protein degradation system. Overexpression of a UBQLN2 with ALS-associated mutations resulted in the accumulation of polyubiquitinated proteins in neuronal cells, including the ALS-associated protein TDP-43. This effect was dependent on the UBA domain but not on inclusion formation. Immunocytochemistry and protein fractionation analysis of IVm-UBQLN2 cellular distribution indicated that it sequesters ubiquitinated substrates from both the proteasomal and autophagic branches of the protein degradation system, resulting in accumulation of polyubiquitinated substrates. These findings provide a molecular basis for the development of ALS/FTD-associated proteinopathy and establish novel therapeutic targets for ALS.

  11. Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer.

    PubMed

    Patrick, B A; Jaiswal, A K

    2012-10-01

    Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in the stabilization of p63 against 20S proteasomal degradation resulting in thinning of the epithelium and chemical-induced skin cancer (Oncogene (2011) 30, 1098-1107). Current studies have demonstrated that NQO1 control of CCAAT-enhancer binding protein (C/EBPα) against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of the NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and chromatin immunoprecipitation assays identified a C/EBPα-binding site between nucleotide position -185 and -174 that bound to C/EBPα and upregulated p63 gene expression. Co-immunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to the chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein, resulting in an increase in p63 RNA and protein in wild-type but not in NQO1-/- mice. Collectively, the current data combined with previous data suggest that stress induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation, leading to protection against chemical-induced skin carcinogenesis. The studies are significant as 2-4% human individuals are homozygous and 23% are heterozygous for the NQO1P187S mutation and might be susceptible to stress-induced skin diseases.

  12. Ubiquitin proteasome pathway-mediated degradation of proteins: effects due to site-specific substrate deamidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The accumulation, aggregation, and precipitation of proteins are etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome p...

  13. Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis.

    PubMed

    Deruyffelaere, Carine; Bouchez, Isabelle; Morin, Halima; Guillot, Alain; Miquel, Martine; Froissard, Marine; Chardot, Thierry; D'Andrea, Sabine

    2015-07-01

    In oleaginous seeds, lipids--stored in organelles called oil bodies (OBs)--are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1-OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1-OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub(2)) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub(2)-oleosin aggregates. These results indicate that K48Ub(2)-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization.

  14. Silymarin induces cyclin D1 proteasomal degradation via its phosphorylation of threonine-286 in human colorectal cancer cells.

    PubMed

    Eo, Hyun Ji; Park, Gwang Hun; Song, Hun Min; Lee, Jin Wook; Kim, Mi Kyoung; Lee, Man Hyo; Lee, Jeong Rak; Koo, Jin Suk; Jeong, Jin Boo

    2015-01-01

    Silymarin from milk thistle (Silybum marianum) plant has been reported to show anti-cancer, anti-inflammatory, antioxidant and hepatoprotective effects. For anti-cancer activity, silymarin is known to regulate cell cycle progression through cyclin D1 downregulation. However, the mechanism of silymarin-mediated cyclin D1 downregulation still remains unanswered. The current study was performed to elucidate the molecular mechanism of cyclin D1 downregulation by silymarin in human colorectal cancer cells. The treatment of silymarin suppressed the cell proliferation in HCT116 and SW480 cells and decreased cellular accumulation of exogenously-induced cyclin D1 protein. However, silymarin did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated silymarin-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with silymarin. In addition, silymarin increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated silymarin-mediated cyclin D1 downregulation. Inhibition of NF-κB by a selective inhibitor, BAY 11-7082 suppressed cyclin D1 phosphorylation and downregulation by silymarin. From these results, we suggest that silymarin-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via NF-κB activation. The current study provides new mechanistic link between silymarin, cyclin D1 downregulation and cell growth in human colorectal cancer cells. PMID:25479723

  15. Silymarin induces cyclin D1 proteasomal degradation via its phosphorylation of threonine-286 in human colorectal cancer cells.

    PubMed

    Eo, Hyun Ji; Park, Gwang Hun; Song, Hun Min; Lee, Jin Wook; Kim, Mi Kyoung; Lee, Man Hyo; Lee, Jeong Rak; Koo, Jin Suk; Jeong, Jin Boo

    2015-01-01

    Silymarin from milk thistle (Silybum marianum) plant has been reported to show anti-cancer, anti-inflammatory, antioxidant and hepatoprotective effects. For anti-cancer activity, silymarin is known to regulate cell cycle progression through cyclin D1 downregulation. However, the mechanism of silymarin-mediated cyclin D1 downregulation still remains unanswered. The current study was performed to elucidate the molecular mechanism of cyclin D1 downregulation by silymarin in human colorectal cancer cells. The treatment of silymarin suppressed the cell proliferation in HCT116 and SW480 cells and decreased cellular accumulation of exogenously-induced cyclin D1 protein. However, silymarin did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated silymarin-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with silymarin. In addition, silymarin increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated silymarin-mediated cyclin D1 downregulation. Inhibition of NF-κB by a selective inhibitor, BAY 11-7082 suppressed cyclin D1 phosphorylation and downregulation by silymarin. From these results, we suggest that silymarin-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via NF-κB activation. The current study provides new mechanistic link between silymarin, cyclin D1 downregulation and cell growth in human colorectal cancer cells.

  16. The Adenovirus E4-ORF3 Protein Stimulates SUMOylation of General Transcription Factor TFII-I to Direct Proteasomal Degradation

    PubMed Central

    Bridges, Rebecca G.; Sohn, Sook-Young; Wright, Jordan

    2016-01-01

    ABSTRACT Modulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad) early region 4 open reading frame 3 (E4-ORF3) protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I). Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection. PMID:26814176

  17. Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition

    PubMed Central

    van Baarsel, Eric D.; Metz, Patrick J.; Fisch, Kathleen; Widjaja, Christella E.; Kim, Stephanie H.; Lopez, Justine; Chang, Aaron N.; Geurink, Paul P.; Florea, Bogdan I.; Overkleeft, Hermen S.; Ovaa, Huib; Bui, Jack D.; Yang, Jing; Chang, John T.

    2016-01-01

    The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy. PMID:26930717

  18. NRAGE promotes cell proliferation by stabilizing PCNA in a ubiquitin-proteasome pathway in esophageal carcinomas.

    PubMed

    Yang, Qingyuan; Ou, Chao; Liu, Mei; Xiao, Weifan; Wen, Chuanjun; Sun, Fenyong

    2014-07-01

    Neurotrophin receptor-interacting melanoma antigen-encoding gene homolog (NRAGE) is generally recognized as a tumor suppressor as it induces cell apoptosis and suppresses cell metastasis. However, it has recently been reported that NRAGE is overexpressed in lung cancer, melanoma and colon cancer, implicating a complicated role of NRAGE as we have expected. In the study, we aim to elucidate the functional roles and molecular mechanisms of NRAGE in esophageal carcinoma. We found that both NRAGE mRNA and protein were significantly overexpressed in esophageal tumor tissues. Consistently, both in vivo and in vitro analyses demonstrated that knockdown of NRAGE apparently inhibited cell growth, and cell cycle analysis further demonstrated that NRAGE knockdown cells were mainly arrested in G2M cell phase, accompanied with an apparent reduction of S phase. In the process of exploring molecular mechanisms, we found that either knockdown in vitro or knockout in vivo of NRAGE reduced proliferating cell nuclear antigen (PCNA) protein, expression of which could completely rescue the inhibited proliferation in NRAGE defective cells. Furthermore, NRAGE physically interacted with PCNA in esophageal cancer cells through DNA polymerase III subunit, and knockdown of NRAGE facilitated PCNA K48-linked polyubiquitination, leading PCNA to the proteasome-dependent degradation and a ubiquitin-specific protease USP10 was identified to be a key regulator in the process of K48 polyubiquitination in NRAGE-deleted cells. In conclusion, our study highlights a unique role of NRAGE and implies that NRAGE is likely to be an attractive oncotarget in developing novel genetic anticancer therapeutic strategies for esophageal squamous cell carcinomas.

  19. Induced degradation of Tat by nucleocapsid (NC) via the proteasome pathway and its effect on HIV transcription.

    PubMed

    Hong, Hye-Won; Lee, Seong-Wook; Myung, Heejoon

    2013-04-01

    Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 Tat protein upregulates transcriptional transactivation. The nucleocapsid protein NC of HIV-1 is a component of virion and plays a key role in genome packaging. Herein, we have demonstrated the interaction between NC and Tat by means of a yeast two-hybrid assay, GST pull-down analysis, co-immunoprecipitation and subcellular colocalization analysis. We observed that the level of Tat was significantly reduced in the presence of NC. But NC did not affect mRNA expression level of Tat. The level of Tat in the presence of NC was increased by treating cells with a proteasome inhibitor, MG132. The ubiquitination state of Tat was not seen to increase in the presence of NC, suggesting the proteasomal degradation was independent of ubiquitination. Lowered level of Tat in the presence of NC led to a decrease in Tat-mediated transcriptional transactivation. PMID:23611845

  20. Induced Degradation of Tat by Nucleocapsid (NC) via the Proteasome Pathway and Its Effect on HIV Transcription

    PubMed Central

    Hong, Hye-Won; Lee, Seong-Wook; Myung, Heejoon

    2013-01-01

    Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 Tat protein upregulates transcriptional transactivation. The nucleocapsid protein NC of HIV-1 is a component of virion and plays a key role in genome packaging. Herein, we have demonstrated the interaction between NC and Tat by means of a yeast two-hybrid assay, GST pull-down analysis, co-immunoprecipitation and subcellular colocalization analysis. We observed that the level of Tat was significantly reduced in the presence of NC. But NC did not affect mRNA expression level of Tat. The level of Tat in the presence of NC was increased by treating cells with a proteasome inhibitor, MG132. The ubiquitination state of Tat was not seen to increase in the presence of NC, suggesting the proteasomal degradation was independent of ubiquitination. Lowered level of Tat in the presence of NC led to a decrease in Tat-mediated transcriptional transactivation. PMID:23611845

  1. Rapid Proteasomal Degradation of Mutant Proteins Is the Primary Mechanism Leading to Tumorigenesis in Patients With Missense AIP Mutations

    PubMed Central

    Hernández-Ramírez, Laura C.; Martucci, Federico; Morgan, Rhodri M. L.; Trivellin, Giampaolo; Tilley, Daniel; Ramos-Guajardo, Nancy; Iacovazzo, Donato; D'Acquisto, Fulvio; Prodromou, Chrisostomos

    2016-01-01

    Context: The pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. Objective: This study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters. Design and Setting: Half-life and protein–protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution. Patients: Data were obtained from our cohort of pituitary adenoma patients and literature-reported cases. Interventions: Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected. Main Outcome Measures: Half-life of wild-type and mutant AIP proteins and its correlation with clinical parameters. Results: Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7–92.9 h), and those with short (median, 27 h; IQR, 21.6–28.7 h) or very short (median, 7.7 h; IQR, 5.6–10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1–CUL1–F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP. Conclusions: AIP is a stable protein, driven to ubiquitination by the SKP1–CUL1–F-box protein complex

  2. Fucoidan inhibition of lung cancer in vivo and in vitro : role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation.

    PubMed

    Hsu, Hsien-Yeh; Lin, Tung-Yi; Wu, Yu-Chung; Tsao, Shu-Ming; Hwang, Pai-An; Shih, Yu-Wei; Hsu, Jason

    2014-09-15

    Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor β (TGFβ) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFβ stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFβ receptors.

  3. Strabismus Promotes Recruitment and Degradation of Farnesylated Prickle in Drosophila melanogaster Planar Polarity Specification

    PubMed Central

    Strutt, Helen; Thomas-MacArthur, Vickie; Strutt, David

    2013-01-01

    The core planar polarity proteins are required to specify the orientation of structures that are polarised in the plane of the epithelium. In the Drosophila melanogaster wing, the core proteins localise asymmetrically at either proximal or distal cell edges. Asymmetric localisation is thought to be biased by long-range cues, causing asymmetric complexes to become aligned with the tissue axes. Core proteins are then thought to participate in feedback interactions that are necessary to amplify asymmetry, and in order for such feedback interactions to operate correctly, the levels of the core proteins at junctions must be tightly regulated. We have investigated regulation of the core protein Prickle (Pk) in the pupal wing. The core protein Strabismus (Stbm) is required to recruit Pk into asymmetric complexes at proximal cell ends, and we report here that it also promotes proteasomal degradation of excess Pk, probably via a Cullin-1 dependent process. We also show for the first time that Pk is farnesylated in vivo, and this is essential for Pk function in the wing. Notably, farnesylation of Pk is necessary for it to be recruited into asymmetric complexes and function in feedback amplification, probably by reinforcing weak direct interactions between Stbm and Pk. Furthermore, farnesylation is also required for Stbm to promote proteasomal degradation of Pk. We propose that Stbm recruits farnesylated Pk into asymmetric complexes, but also promotes degradation of excess Pk that would otherwise perturb feedback amplification. PMID:23874239

  4. Propofol Prevents Hippocampal Neuronal Loss and Memory Impairment in Cerebral Ischemia Injury Through Promoting PTEN Degradation.

    PubMed

    Chen, Xin; Du, Ye-Mu; Xu, Feng; Liu, Dai; Wang, Yuan-Lin

    2016-09-01

    Neuroprotective effect of propofol against cerebral ischemia injury was widely investigated. However, its mechanisms remain unclear. Phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is supposed as a cell survival pathway, and phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a negative regulator of AKT phosphorylation. Whether PTEN was involved in the protective effect of propofol against cerebral ischemia injury was not elucidated. In this study, the function of PTEN in the acute phase of cerebral ischemia injury was investigated. Our data showed that propofol promoted the PTEN degradation in the acute phase of cerebral ischemia injury and concurrently activated AKT phosphorylation. The increase of ubiquitinated PTEN caused by cerebral ischemia injury were degraded in propofol-pretreated rats. Moreover, we evidenced that proteasome activity was stimulated in propofol-treated rats. These data pointed that PTEN degradation was facilitated in the acute phase after propofol treatment possibly through activating ubiquitin-proteasome system. Therefore, we applied PTEN inhibitor-bpV before cerebral ischemia injury. Like propofol, bpV pretreatment also mitigated cerebral ischemia injury-induced cell loss in CA1 region and memory impairment. Taken together, our data suggest that PTEN degradation is neuroprotective against cerebral ischemia injury and propofol facilitates PTEN degradation to prevent hippocampal neuronal loss and memory deficit in cerebral ischemia injury.

  5. Propofol Prevents Hippocampal Neuronal Loss and Memory Impairment in Cerebral Ischemia Injury Through Promoting PTEN Degradation.

    PubMed

    Chen, Xin; Du, Ye-Mu; Xu, Feng; Liu, Dai; Wang, Yuan-Lin

    2016-09-01

    Neuroprotective effect of propofol against cerebral ischemia injury was widely investigated. However, its mechanisms remain unclear. Phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is supposed as a cell survival pathway, and phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a negative regulator of AKT phosphorylation. Whether PTEN was involved in the protective effect of propofol against cerebral ischemia injury was not elucidated. In this study, the function of PTEN in the acute phase of cerebral ischemia injury was investigated. Our data showed that propofol promoted the PTEN degradation in the acute phase of cerebral ischemia injury and concurrently activated AKT phosphorylation. The increase of ubiquitinated PTEN caused by cerebral ischemia injury were degraded in propofol-pretreated rats. Moreover, we evidenced that proteasome activity was stimulated in propofol-treated rats. These data pointed that PTEN degradation was facilitated in the acute phase after propofol treatment possibly through activating ubiquitin-proteasome system. Therefore, we applied PTEN inhibitor-bpV before cerebral ischemia injury. Like propofol, bpV pretreatment also mitigated cerebral ischemia injury-induced cell loss in CA1 region and memory impairment. Taken together, our data suggest that PTEN degradation is neuroprotective against cerebral ischemia injury and propofol facilitates PTEN degradation to prevent hippocampal neuronal loss and memory deficit in cerebral ischemia injury. PMID:27480093

  6. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    DOE PAGESBeta

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; et al

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and proteinmore » degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.« less

  7. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    SciTech Connect

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.

  8. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    PubMed Central

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-01-01

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens. PMID:25831519

  9. Genetics of proteasome diseases.

    PubMed

    Gomes, Aldrin V

    2013-01-01

    The proteasome is a large, multiple subunit complex that is capable of degrading most intracellular proteins. Polymorphisms in proteasome subunits are associated with cardiovascular diseases, diabetes, neurological diseases, and cancer. One polymorphism in the proteasome gene PSMA6 (-8C/G) is associated with three different diseases: type 2 diabetes, myocardial infarction, and coronary artery disease. One type of proteasome, the immunoproteasome, which contains inducible catalytic subunits, is adapted to generate peptides for antigen presentation. It has recently been shown that mutations and polymorphisms in the immunoproteasome catalytic subunit PSMB8 are associated with several inflammatory and autoinflammatory diseases including Nakajo-Nishimura syndrome, CANDLE syndrome, and intestinal M. tuberculosis infection. This comprehensive review describes the disease-related polymorphisms in proteasome genes associated with human diseases and the physiological modulation of proteasome function by these polymorphisms. Given the large number of subunits and the central importance of the proteasome in human physiology as well as the fast pace of detection of proteasome polymorphisms associated with human diseases, it is likely that other polymorphisms in proteasome genes associated with diseases will be detected in the near future. While disease-associated polymorphisms are now readily discovered, the challenge will be to use this genetic information for clinical benefit. PMID:24490108

  10. Genetics of Proteasome Diseases

    PubMed Central

    Gomes, Aldrin V.

    2013-01-01

    The proteasome is a large, multiple subunit complex that is capable of degrading most intracellular proteins. Polymorphisms in proteasome subunits are associated with cardiovascular diseases, diabetes, neurological diseases, and cancer. One polymorphism in the proteasome gene PSMA6 (−8C/G) is associated with three different diseases: type 2 diabetes, myocardial infarction, and coronary artery disease. One type of proteasome, the immunoproteasome, which contains inducible catalytic subunits, is adapted to generate peptides for antigen presentation. It has recently been shown that mutations and polymorphisms in the immunoproteasome catalytic subunit PSMB8 are associated with several inflammatory and autoinflammatory diseases including Nakajo-Nishimura syndrome, CANDLE syndrome, and intestinal M. tuberculosis infection. This comprehensive review describes the disease-related polymorphisms in proteasome genes associated with human diseases and the physiological modulation of proteasome function by these polymorphisms. Given the large number of subunits and the central importance of the proteasome in human physiology as well as the fast pace of detection of proteasome polymorphisms associated with human diseases, it is likely that other polymorphisms in proteasome genes associated with diseases will be detected in the near future. While disease-associated polymorphisms are now readily discovered, the challenge will be to use this genetic information for clinical benefit. PMID:24490108

  11. Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.

    PubMed

    Shibata, Norihito; Ohoka, Nobumichi; Sugaki, Yusuke; Onodera, Chiaki; Inoue, Mizuho; Sakuraba, Yoshiyuki; Takakura, Daisuke; Hashii, Noritaka; Kawasaki, Nana; Gondo, Yoichi; Naito, Mikihiko

    2015-11-20

    During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders. PMID:26442586

  12. Homeodomain-interacting protein kinase 2 (HIPK2) targets {beta}-catenin for phosphorylation and proteasomal degradation

    SciTech Connect

    Kim, Eun-A; Kim, Ji Eon; Sung, Ki Sa; Choi, Dong Wook; Lee, Byeong Jae; Choi, Cheol Yong

    2010-04-16

    The regulation of intracellular {beta}-catenin levels is central in the Wnt/{beta}-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/{beta}-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of {beta}-catenin and results in the accumulation of {beta}-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of {beta}-catenin. HIPK2 phosphorylates {beta}-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of {beta}-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of {beta}-catenin, thereby potentiated {beta}-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of {beta}-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of {beta}-catenin through its phosphorylation and proteasomal degradation.

  13. Salt stress-induced disassembly of Arabidopsis cortical microtubule arrays involves 26S proteasome-dependent degradation of SPIRAL1.

    PubMed

    Wang, Songhu; Kurepa, Jasmina; Hashimoto, Takashi; Smalle, Jan A

    2011-09-01

    The dynamic instability of cortical microtubules (MTs) (i.e., their ability to rapidly alternate between phases of growth and shrinkage) plays an essential role in plant growth and development. In addition, recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions. The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress, the underlying molecular mechanism has remained largely unknown. Here, we show that the MT-associated protein SPIRAL1 (SPR1) plays a key role in salt stress-induced MT disassembly. SPR1, a microtubule stabilizing protein, is degraded by the 26S proteasome, and its degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance.

  14. Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

    PubMed Central

    Hampton, R Y; Gardner, R G; Rine, J

    1996-01-01

    3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the

  15. Bortezomib Amplifies Effect on Intracellular Proteasomes by Changing Proteasome Structure.

    PubMed

    Pitcher, David S; de Mattos-Shipley, Kate; Tzortzis, Konstantinos; Auner, Holger W; Karadimitris, Anastasios; Kleijnen, Maurits F

    2015-07-01

    The proteasome inhibitor Bortezomib is used to treat multiple myeloma (MM). Bortezomib inhibits protein degradation by inactivating proteasomes' active-sites. MM cells are exquisitely sensitive to Bortezomib - exhibiting a low-nanomolar IC(50) - suggesting that minimal inhibition of degradation suffices to kill MM cells. Instead, we report, a low Bortezomib concentration, contrary to expectation, achieves severe inhibition of proteasome activity in MM cells: the degree of inhibition exceeds what one would expect from the small proportion of active-sites that Bortezomib inhibits. Our data indicate that Bortezomib achieves this severe inhibition by triggering secondary changes in proteasome structure that further inhibit proteasome activity. Comparing MM cells to other, Bortezomib-resistant, cancer cells shows that the degree of proteasome inhibition is the greatest in MM cells and only there leads to proteasome stress, providing an explanation for why Bortezomib is effective against MM but not other cancers.

  16. Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription?

    PubMed

    Nawaz, Zafar; O'Malley, Bert W

    2004-03-01

    The importance of the ubiquitin proteasome pathway in higher eukaryotes has been well established in cell cycle regulation, signal transduction, and cell differentiation, but has only recently been linked to nuclear hormone receptor-regulated gene transcription. Characterization of a number of ubiquitin proteasome pathway enzymes as coactivators and observations that several nuclear receptors are ubiquitinated and degraded in the course of their nuclear activities provide evidence that ubiquitin proteasome-mediated protein degradation plays an integral role in eukaryotic transcription. In addition to receptors, studies have revealed that coactivators are ubiquitinated and degraded via the proteasome. The notion that the ubiquitin proteasome pathway is involved in gene transcription is further strengthened by the fact that ubiquitin proteasome pathway enzymes are recruited to the promoters of target genes and that proteasome-dependent degradation of nuclear receptors is required for efficient transcriptional activity. These findings suggest that protein degradation is coupled with nuclear receptor coactivation activity. It is possible that the ubiquitin proteasome pathway modulates transcription by promoting remodeling and turnover of the nuclear receptor-transcription complex. In this review, we discus the possible role of the ubiquitin proteasome pathway in nuclear hormone receptor-regulated gene transcription.

  17. A Novel Retinoblastoma Protein (RB) E3 Ubiquitin Ligase (NRBE3) Promotes RB Degradation and Is Transcriptionally Regulated by E2F1 Transcription Factor.

    PubMed

    Wang, Yingshuang; Zheng, Zongfang; Zhang, Jingyi; Wang, You; Kong, Ruirui; Liu, Jiangying; Zhang, Ying; Deng, Hongkui; Du, Xiaojuan; Ke, Yang

    2015-11-20

    Retinoblastoma protein (RB) plays critical roles in tumor suppression and is degraded through the proteasomal pathway. However, E3 ubiquitin ligases responsible for proteasome-mediated degradation of RB are largely unknown. Here we characterize a novel RB E3 ubiquitin ligase (NRBE3) that binds RB and promotes RB degradation. NRBE3 contains an LXCXE motif and bound RB in vitro. NRBE3 interacted with RB in cells when proteasome activity was inhibited. NRBE3 promoted RB ubiquitination and degradation via the ubiquitin-proteasome pathway. Importantly, purified NRBE3 ubiquitinated recombinant RB in vitro, and a U-box was identified as essential for its E3 activity. Surprisingly, NRBE3 was transcriptionally activated by E2F1/DP1. Consequently, NRBE3 affected the cell cycle by promoting G1/S transition. Moreover, NRBE3 was up-regulated in breast cancer tissues. Taken together, we identified NRBE3 as a novel ubiquitin E3 ligase for RB that might play a role as a potential oncoprotein in human cancers.

  18. ATP-DEPENDENT STEPS IN THE BINDING OF UBIQUITIN CONJUGATES TO THE 26S PROTEASOME THAT COMMIT TO DEGRADATION

    PubMed Central

    Peth, Andreas; Uchiki, Tomoaki; Goldberg, Alfred L.

    2010-01-01

    Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high affinity binding of ubiquitin chains, but in their absence ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2-4 fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP) to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded. PMID:21095592

  19. Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL–p97 complex

    PubMed Central

    Braunstein, Ilana; Zach, Lolita; Allan, Susanne; Kalies, Kai-Uwe; Stanhill, Ariel

    2015-01-01

    The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97–AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97’s role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin. PMID:26337389

  20. Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation

    PubMed Central

    Wang, Tao; Darwin, K. Heran; Li, Huilin

    2010-01-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analogue of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein bearing little sequence or structural resemblance to the highly structured ubiquitin. Thus it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled-coils that recognize Pup. Mpa binds unstructured Pup via hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an α-helix in Pup. Our work revealed a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This critical difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment of tuberculosis. PMID:20953180

  1. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    SciTech Connect

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  2. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    SciTech Connect

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  3. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation.

    PubMed

    Xu, Dongqing; Jiang, Yan; Li, Jigang; Lin, Fang; Holm, Magnus; Deng, Xing Wang

    2016-07-01

    BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX)-containing protein, has been previously identified as a positive regulator of light signaling; however, the precise role of BBX21 in regulating seedling photomorphogenesis remains largely unclear. In this study, we report that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) interacts with BBX21 in vivo and is able to ubiquitinate BBX21 in vitro. Thus, BBX21 is targeted for 26S proteasome-mediated degradation in dark-grown Arabidopsis seedlings in a COP1-dependent manner. Moreover, we show that BBX21 binds to the T/G-box in the ELONGATED HYPOCOTYL 5 (HY5) promoter and directly activates HY5 expression in the light. Transgenic seedlings overexpressing BBX21 exhibit dramatically shortened hypocotyls in the light, and this phenotype is dependent on a functional HY5. Taken together, our data suggest a molecular base underlying BBX21-mediated seedling photomorphogenesis, indicating that BBX21 is a pivotal component involved in the COP1-HY5 regulatory hub. PMID:27325768

  4. Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis[C][W][OA

    PubMed Central

    Liu, Xiaomin; Qin, Tao; Ma, Qianqian; Sun, Jingbo; Liu, Ziqiang; Yuan, Ming; Mao, Tonglin

    2013-01-01

    Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation. PMID:23653471

  5. Methyllysine reader plant homeodomain (PHD) finger protein 20-like 1 (PHF20L1) antagonizes DNA (cytosine-5) methyltransferase 1 (DNMT1) proteasomal degradation.

    PubMed

    Estève, Pierre-Olivier; Terragni, Jolyon; Deepti, Kanneganti; Chin, Hang Gyeong; Dai, Nan; Espejo, Alexsandra; Corrêa, Ivan R; Bedford, Mark T; Pradhan, Sriharsa

    2014-03-21

    Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

  6. Human cytomegalovirus inhibits apoptosis by proteasome-mediated degradation of Bax at endoplasmic reticulum-mitochondrion contacts.

    PubMed

    Zhang, Aiping; Hildreth, Richard L; Colberg-Poley, Anamaris M

    2013-05-01

    Human cytomegalovirus (HCMV) encodes the UL37 exon 1 protein (pUL37x1), which is the potent viral mitochondrion-localized inhibitor of apoptosis (vMIA), to increase survival of infected cells. HCMV vMIA traffics from the endoplasmic reticulum (ER) to ER subdomains, which are physically linked to mitochondria known as mitochondrion-associated membranes (MAM), and to mitochondria. The antiapoptotic function of vMIA is thought to primarily result from its ability to inhibit Bax-mediated permeabilization of the outer mitochondrial membrane (OMM). Here, we establish that vMIA retargets Bax to the MAM as well as to the OMM from immediate early through late times of infection. However, MAM localization of Bax results in its increased ubiquitination and proteasome-mediated degradation. Surprisingly, HCMV infection does not increase OMM-associated degradation (OMMAD) of Bax, even though the ER and mitochondria are physically connected at the MAM. It was recently found that lipid rafts at the plasma membrane can connect extrinsic and intrinsic apoptotic pathways and can serve as sites of apoptosome assembly. In transfected permissive human fibroblasts, vMIA mediates, through its cholesterol affinity, association of Bax and apoptosome components with MAM lipid rafts. While Bax association with MAM lipid rafts was detected in HCMV-infected cells, association of apoptosome components was not. These results establish that Bax recruitment to the MAM and its MAM-associated degradation (MAMAD) are a newly described antiapoptotic mechanism used by HCMV infection to increase cell survival for its growth.

  7. Regulatory function of the P295-T311 motif of the estrogen receptor α - does proteasomal degradation of the receptor induce emergence of peptides implicated in estrogenic responses?

    PubMed Central

    Gallo, Dominique; Haddad, Iman; Laurent, Guy; Vinh, Joëlle; Jacquemotte, Françoise; Jacquot, Yves; Leclercq, Guy

    2008-01-01

    The way in which estrogen receptor α (ERα) mediates gene transcription and hormone-dependent cancer cell proliferation is now being largely reconsidered in view of several recent discoveries. ERα-mediated transcription appears to be a cyclic and transient process where the proteasome - and thus receptor degradation - plays a pivotal role. In view of our recent investigations, which demonstrate the estrogenic activity of a synthetic peptide corresponding to a regulatory motif of the receptor (ERα17p), we propose that ERα proteasomal degradation could induce the emergence of regulatory peptide(s). The latter would function as a signal and contribute to the ERα activation process, amplifying the initial hormonal stimulation and giving rise to sustained estrogenic response. PMID:18432312

  8. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    PubMed Central

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J.; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M.; Bunting, Samuel F.; Inuzuka, Hiroyuki; Zheng, X. F. Steven; Shen, Zhiyuan; Sabaawy, Hatem E.; Liu, LianXin; Pine, Sharon R.

    2016-01-01

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance. PMID:27566146

  9. The SPRY domain–containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation

    PubMed Central

    Kuang, Zhihe; Lewis, Rowena S.; Curtis, Joan M.; Zhan, Yifan; Saunders, Bernadette M.; Babon, Jeffrey J.; Kolesnik, Tatiana B.; Low, Andrew; Masters, Seth L.; Willson, Tracy A.; Kedzierski, Lukasz; Yao, Shenggen; Handman, Emanuela

    2010-01-01

    Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain–containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB–iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections. PMID:20603330

  10. RSK1 protects P-glycoprotein/ABCB1 against ubiquitin–proteasomal degradation by downregulating the ubiquitin-conjugating enzyme E2 R1

    PubMed Central

    Katayama, Kazuhiro; Fujiwara, Chiaki; Noguchi, Kohji; Sugimoto, Yoshikazu

    2016-01-01

    P-glycoprotein (P-gp) is a critical determinant of multidrug resistance in cancer. We previously reported that MAPK inhibition downregulates P-gp expression and that P-gp undergoes ubiquitin–proteasomal degradation regulated by UBE2R1 and SCFFbx15. Here, we investigated the crosstalk between MAPK inhibition and the ubiquitin–proteasomal degradation of P-gp. Proteasome inhibitors or knockdown of FBXO15 and/or UBE2R1 cancelled MEK inhibitor-induced P-gp downregulation. RSK1 phosphorylated Thr162 on UBE2R1 but did not phosphorylate FBXO15. MEK and RSK inhibitors increased UBE2R1-WT but not UBE2R1-T162D and -T162A expression. UBE2R1-T162D showed higher self-ubiquitination and destabilisation than UBE2R1-WT and -T162A. Unlike UBE2R1-WT and -T162A, UBE2R1-T162D did not induce P-gp ubiquitination. UBE2R1-WT or -T162A downregulated P-gp expression and upregulated rhodamine 123 level and sensitivity to vincristine and doxorubicin. However, UBE2R1-T162D did not confer any change in P-gp expression, rhodamine 123 accumulation and sensitivity to the drugs. These results suggest that RSK1 protects P-gp against ubiquitination by reducing UBE2R1 stability. PMID:27786305

  11. Withaferin a induces proteasome-dependent degradation of breast cancer susceptibility gene 1 and heat shock factor 1 proteins in breast cancer cells.

    PubMed

    Zhang, Xuan; Timmermann, Barbara; Samadi, Abbas K; Cohen, Mark S

    2012-01-01

    The purpose of this study was to examine the regulation of prosurvival factors heat shock factor 1 (HSF1) and breast cancer susceptibility gene 1 (BRCA1) by a natural withanolide withaferin A (WA) in triple negative breast cancer cell lines MDA-MB-231 and BT20. Western analysis was used to examine alternations in HSF1 and BRCA1 protein levels following WA treatment. A protein synthesis inhibitor cycloheximide and a proteasome inhibitor MG132 were used to investigate the mechanisms of HSF1 and BRCA1 regulation by WA. It was found that WA induced a dose-dependent decrease in HSF1 and BRCA1 protein levels. Further analysis showed that levels of HSF1 and BRCA1 proteins decreased rapidly after WA treatment, and this was attributed to WA-induced denaturation of HSF1 and BRCA1 proteins and subsequent degradation via proteasome-dependent, and protein-synthesis dependent mechanism. In summary, WA induces denaturation and proteasomal degradation of HSF1 and BRCA1 proteins. Further studies are warranted to examine the contribution of HSF1 and BRCA1 depletion to the anticancer effects of WA in breast cancer.

  12. Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production.

    PubMed

    Brehm, Anja; Liu, Yin; Sheikh, Afzal; Marrero, Bernadette; Omoyinmi, Ebun; Zhou, Qing; Montealegre, Gina; Biancotto, Angelique; Reinhardt, Adam; Almeida de Jesus, Adriana; Pelletier, Martin; Tsai, Wanxia L; Remmers, Elaine F; Kardava, Lela; Hill, Suvimol; Kim, Hanna; Lachmann, Helen J; Megarbane, Andre; Chae, Jae Jin; Brady, Jilian; Castillo, Rhina D; Brown, Diane; Casano, Angel Vera; Gao, Ling; Chapelle, Dawn; Huang, Yan; Stone, Deborah; Chen, Yongqing; Sotzny, Franziska; Lee, Chyi-Chia Richard; Kastner, Daniel L; Torrelo, Antonio; Zlotogorski, Abraham; Moir, Susan; Gadina, Massimo; McCoy, Phil; Wesley, Robert; Rother, Kristina I; Rother, Kristina; Hildebrand, Peter W; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

    2015-11-01

    Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production. PMID:26524591

  13. Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

    PubMed Central

    Brehm, Anja; Liu, Yin; Sheikh, Afzal; Marrero, Bernadette; Omoyinmi, Ebun; Zhou, Qing; Montealegre, Gina; Biancotto, Angelique; Reinhardt, Adam; Almeida de Jesus, Adriana; Pelletier, Martin; Tsai, Wanxia L.; Remmers, Elaine F.; Kardava, Lela; Hill, Suvimol; Kim, Hanna; Lachmann, Helen J.; Megarbane, Andre; Chae, Jae Jin; Brady, Jilian; Castillo, Rhina D.; Brown, Diane; Casano, Angel Vera; Gao, Ling; Chapelle, Dawn; Huang, Yan; Stone, Deborah; Chen, Yongqing; Sotzny, Franziska; Lee, Chyi-Chia Richard; Kastner, Daniel L.; Torrelo, Antonio; Zlotogorski, Abraham; Moir, Susan; Gadina, Massimo; McCoy, Phil; Wesley, Robert; Rother, Kristina; Hildebrand, Peter W.; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

    2015-01-01

    Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production. PMID:26524591

  14. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  15. S-nitrosylation-dependent proteasomal degradation restrains Cdk5 activity to regulate hippocampal synaptic strength

    PubMed Central

    Zhang, Peng; Fu, Wing-Yu; Fu, Amy K. Y.; Ip, Nancy Y.

    2015-01-01

    Precise regulation of synaptic strength requires coordinated activity and functions of synaptic proteins, which is controlled by a variety of post-translational modification. Here we report that S-nitrosylation of p35, the activator of cyclin-dependent kinase 5 (Cdk5), by nitric oxide (NO) is important for the regulation of excitatory synaptic strength. While blockade of NO signalling results in structural and functional synaptic deficits as indicated by reduced mature dendritic spine density and surface expression of glutamate receptor subunits, phosphorylation of numerous synaptic substrates of Cdk5 and its activity are aberrantly upregulated following reduced NO production. The results show that the NO-induced reduction in Cdk5 activity is mediated by S-nitrosylation of p35, resulting in its ubiquitination and degradation by the E3 ligase PJA2. Silencing p35 protein in hippocampal neurons partially rescues the NO blockade-induced synaptic deficits. These findings collectively demonstrate that p35 S-nitrosylation by NO signalling is critical for regulating hippocampal synaptic strength. PMID:26503494

  16. Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

    PubMed Central

    Doi, Kenichiro; Sharma, Arun K.; Wang, Hong-Gang; Amin, Shantu

    2013-01-01

    Background and purpose Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells. Experimental approach For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis. Key results The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC50 of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids. Conclusions and implications Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors. PMID:24223823

  17. Chromium-Insulin Reduces Insulin Clearance and Enhances Insulin Signaling by Suppressing Hepatic Insulin-Degrading Enzyme and Proteasome Protein Expression in KKAy Mice.

    PubMed

    Wang, Zhong Q; Yu, Yongmei; Zhang, Xian H; Komorowski, James

    2014-01-01

    JDS-chromium-insulin (CRI)-003 is a novel form of insulin that has been directly conjugated with chromium (Cr) instead of zinc. Our hypothesis was that CRI enhances insulin's effects by altering insulin-degrading enzyme (IDE) and proteasome enzymes. To test this hypothesis, we measured hepatic IDE content and proteasome parameters in a diabetic animal model. Male KKAy mice were randomly divided into three groups (n = 8/group); Sham (saline), human regular insulin (Reg-In), and chromium conjugated human insulin (CRI), respectively. Interventions were initiated at doses of 2 U insulin/kg body weight daily for 8-weeks. Plasma glucose and insulin were measured. Hepatic IDE, proteasome, and insulin signaling proteins were determined by western blotting. Insulin tolerance tests at week 7 showed that both insulin treatments significantly reduced glucose concentrations and increased insulin levels compared with the Sham group, CRI significantly reduced glucose at 4 and 6 h relative to Reg-In (P < 0.05), suggesting the effects of CRI on reducing glucose last longer than Reg-In. CRI treatment significantly increased hepatic IRS-1 and Akt1 and reduced IDE, 20S as well as 19S protein abundance (P < 0.01, P < 0.05, and P < 0.001, respectively), but Reg-In only significantly increased Akt1 (P < 0.05). Similar results were also observed in Reg-In- and CRI-treated HepG2 cells. This study, for the first time, demonstrates that CRI reduces plasma insulin clearance by inhibition of hepatic IDE protein expression and enhances insulin signaling as well as prevents degradation of IRS-1 and IRS-2 by suppressing ubiquitin-proteasome pathway in diabetic mice.

  18. PLK1 and HOTAIR accelerate proteasomal degradation of SUZ12 and ZNF198 during hepatitis B virus-induced liver carcinogenesis

    PubMed Central

    Zhang, Hao; Diab, Ahmed; Fan, Huitao; Mani, Saravana Kumar Kailasam; Hullinger, Ronald; Merle, Philippe; Andrisani, Ourania

    2015-01-01

    Elucidating mechanisms of hepatitis B virus (HBV)-mediated hepatocarcinogenesis is needed to gain insights into the etiology and treatment of liver cancer. Cells where HBV is replicating exhibit increased expression of Plk1 kinase and reduced levels of two transcription repression factors, SUZ12 and ZNF198. SUZ12 is an essential subunit of the transcription repressive complex PRC2. ZNF198 stabilizes the transcription repressive complex composed of LSD1, Co-REST and HDAC1. These two transcription repressive complexes are held together by binding the long noncoding RNA HOTAIR. In this study we linked these regulatory events mechanistically, by showing that Plk1 induces proteasomal degradation of SUZ12 and ZNF198 by site-specific phosphorylation. Plk1-dependent ubiquitination of SUZ12 and ZNF198 was enhanced by expression of HOTAIR, significantly reducing SUZ12 and ZNF198 stability. In cells expressing the HBV X protein (HBx) downregulation of SUZ12 and ZNF198 mediated global changes in histone modifications. In turn, HBx-expressing cells propagated an altered chromatin landscape after cell division, as exemplified by changes in histone modifications of the EpCAM promoter, a target of PRC2 and LSD1/Co-REST/HDAC1 complexes. Notably, liver tumors from X/cmyc bitransgenic mice exhibited downregulation of SUZ12 and ZNF198 along with elevated expression of Plk1, HOTAIR, and EpCAM. Clinically, similar effects were documented in a set of HBV-related liver tumors consistent with the likelihood that downregulation of SUZ12 and ZNF198 leads to epigenetic reprogramming of infected hepatocytes. Since both Plk1 and HOTAIR are elevated in many human cancers, we propose that their combined effects are involved in epigenetic reprogramming associated broadly with oncogenic transformation. PMID:25855382

  19. The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

    PubMed Central

    Zilfou, Jack T.; Hoffman, William H.; Sank, Michael; George, Donna L.; Murphy, Maureen

    2001-01-01

    While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14ARF, Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage. PMID:11359905

  20. Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

    PubMed

    Bomberger, Jennifer M; Ely, Kenneth H; Bangia, Naveen; Ye, Siying; Green, Kathy A; Green, William R; Enelow, Richard I; Stanton, Bruce A

    2014-01-01

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

  1. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

    PubMed

    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-01

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  2. APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint*

    PubMed Central

    Kikuchi, Ryo; Ohata, Hirokazu; Ohoka, Nobumichi; Kawabata, Atsushi; Naito, Mikihiko

    2014-01-01

    In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC. PMID:24302728

  3. Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells.

    PubMed

    Deroo, Bonnie J; Archer, Trevor K

    2002-06-01

    Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.

  4. Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting

    PubMed Central

    Ciechanover, Aaron

    2012-01-01

    Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code was transcribed to RNA and translated to proteins, but how proteins were degraded had remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis was largely non-lysosomal, but the mechanisms involved have remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs. PMID:23908826

  5. Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

    SciTech Connect

    Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.; Luo, Kunxin

    2001-09-11

    Smad proteins mediate transforming growth factor-b signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGFb signaling that functions to maintain the repressed state of TGFb target genes in the absence of ligand. Upon TGFb stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGFb target genes. Here we show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase promoting complex (APC) and the UbcH5 family of ubiquitin conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box-dependent manner. In addition to the destruction box, efficient degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGFb signaling. Our studies elucidate an important pathway for the degradation of SnoN and reveal a novel role of the APC in regulation of TGFb signaling.

  6. Nuclear Import of Yeast Proteasomes

    PubMed Central

    Burcoglu, Julianne; Zhao, Liang; Enenkel, Cordula

    2015-01-01

    Proteasomes are highly conserved protease complexes responsible for the degradation of aberrant and short-lived proteins. In highly proliferating yeast and mammalian cells, proteasomes are predominantly nuclear. During quiescence and cell cycle arrest, proteasomes accumulate in granules in close proximity to the nuclear envelope/ER. With prolonged quiescence in yeast, these proteasome granules pinch off as membraneless organelles, and migrate as stable entities through the cytoplasm. Upon exit from quiescence, the proteasome granules clear and the proteasomes are rapidly transported into the nucleus, a process reflecting the dynamic nature of these multisubunit complexes. Due to the scarcity of studies on the nuclear transport of mammalian proteasomes, we summarised the current knowledge on the nuclear import of yeast proteasomes. This pathway uses canonical nuclear localisation signals within proteasomal subunits and Srp1/Kap95, and the canonical import receptor, named importin/karyopherin αβ. Blm10, a conserved 240 kDa protein, which is structurally related to Kap95, provides an alternative import pathway. Two models exist upon which either inactive precursor complexes or active holo-enzymes serve as the import cargo. Here, we reconcile both models and suggest that the import of inactive precursor complexes predominates in dividing cells, while the import of mature enzymes mainly occurs upon exit from quiescence. PMID:26262643

  7. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    SciTech Connect

    Li Lihua; Yang Huanjie; Chen Di; Cui, Cindy; Ping Dou, Q.

    2008-06-01

    The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF-Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC{sub 50} value of 32 {mu}mol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF-Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF-Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF-Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.

  8. Proteasome inhibitors prevent the degradation of familial Alzheimer's disease-linked presenilin 1 and potentiate A beta 42 recovery from human cells.

    PubMed Central

    Marambaud, P.; Ancolio, K.; Lopez-Perez, E.; Checler, F.

    1998-01-01

    BACKGROUND: Several lines of evidence suggest that most of the early-onset forms of familial Alzheimer's disease (FAD) are due to inherited mutations borne by a chromosome 14-encoded protein, presenilin 1 (PS1). This is likely related to an increased production of amyloid beta-peptide (A beta) 42, one of the main components of the extracellular deposits called senile plaques that invade human cortical areas during the disease. MATERIALS AND METHODS: We set up stably transfected HEK293 cells overexpressing wild-type (wt) and various FAD-linked mutated PS1. By Western blot analysis, we examined the influence of specific proteasome inhibitors on PS1-like immunoreactivities. Furthermore, by means of metabolic labeling and immunoprecipitation with A beta 40 and A beta 42-directed specific antibodies, we assessed the effect of the inhibitors on the production of A beta s by wt and mutated PS1-expressing cells transiently transfected with beta APP751. RESULTS: We show that two distinct proteasome inhibitors, Z-IE (Ot-Bu)A-Leucinal and lactacystin, increase in a time- and dose-dependent manner the immunoreactivities of both wt and mutated PS1. Furthermore, we demonstrate that PS1 is polyubiquitinated in these cells. Other inhibitors, ineffective on the proteasome, fail to protect wt and mutated PS1-like immunoreactivities. We also establish that the FAD-linked mutations of PS1 trigger a selective increased formation of A beta 42 as reflected by higher A beta 42 over total A beta ratios when compared with wtPS1-expressing cells. Interestingly, this augmentation was further amplified by proteasome inhibitors in cells expressing mutated but not wtPS1. CONCLUSION: Altogether, our data indicate that PS1 undergoes polyubiquitination in HEK293 cells and that the proteasome contributes to the degradation of wt and FAD-linked PS1, thereby directly influencing the A beta production in human cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:9562973

  9. Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl CpG-/CpG domain-binding proteins in embryonic orofacial cells.

    PubMed

    Mukhopadhyay, Partha; Greene, Robert M; Pisano, M Michele

    2015-12-01

    Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80μg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting.

  10. HRD1 suppresses the growth and metastasis of breast cancer cells by promoting IGF-1R degradation.

    PubMed

    Xu, Yue-Mei; Wang, Hong-Jiang; Chen, Fang; Guo, Wan-Hua; Wang, Yan-Yang; Li, Hang-Yu; Tang, Jin-Hai; Ding, Ying; Shen, Ya-Chen; Li, Min; Xuan, Wen-Ying; Liu, Lin-Hui; Wang, Jia; Wang, Xue-Rong; Gao, Ze-Jun; Liang, Xiu-Bin; Su, Dong-Ming

    2015-12-15

    HRD1 (3-hydroxy-3-methylglutaryl reductase degradation) is an E3 ubiquitin ligase. We found that HRD1 was significantly downregulated in 170 breast cancer tissues. Low tumoral HRD1 expression was correlated with clinicopathological characteristics and a shorter survival in breast cancer patients. P65 specifically bound to the HRD1 promoter and inhibited HRD1 expression. Suppression of NF-κB activity reversed IL-6-induced downregulation of HRD1 expression. HRD1 interacted with IGF-1R and promoted its ubiquitination and degradation by the proteasome. Overexpression of HRD1 resulted in the inhibition of growth, migration and invasion of breast cancer cells in vitro and in vivo. Furthermore, HRD1 attenuated IL-6-induced epithelial-mesenchymal transition in MCF10A cells. These findings uncover a novel role for HRD1 in breast cancer.

  11. HRD1 suppresses the growth and metastasis of breast cancer cells by promoting IGF-1R degradation

    PubMed Central

    Ding, Ying; Shen, Ya-Chen; Li, Min; Xuan, Wen-Ying; Liu, Lin-Hui; Wang, Jia; Wang, Xue-Rong; Gao, Ze-Jun; Liang, Xiu-Bin; Su, Dong-Ming

    2015-01-01

    HRD1 (3-hydroxy-3-methylglutaryl reductase degradation) is an E3 ubiquitin ligase. We found that HRD1 was significantly downregulated in 170 breast cancer tissues. Low tumoral HRD1 expression was correlated with clinicopathological characteristics and a shorter survival in breast cancer patients. P65 specifically bound to the HRD1 promoter and inhibited HRD1 expression. Suppression of NF-κB activity reversed IL-6-induced downregulation of HRD1 expression. HRD1 interacted with IGF-1R and promoted its ubiquitination and degradation by the proteasome. Overexpression of HRD1 resulted in the inhibition of growth, migration and invasion of breast cancer cells in vitro and in vivo. Furthermore, HRD1 attenuated IL-6-induced epithelial-mesenchymal transition in MCF10A cells. These findings uncover a novel role for HRD1 in breast cancer. PMID:26536657

  12. HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans

    PubMed Central

    Kinet, Maxime J; Malin, Jennifer A; Abraham, Mary C; Blum, Elyse S; Silverman, Melanie R; Lu, Yun; Shaham, Shai

    2016-01-01

    Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates. DOI: http://dx.doi.org/10.7554/eLife.12821.001 PMID:26952214

  13. The Ubiquitin-Proteasome Pathway and Proteasome Inhibitors

    PubMed Central

    Myung, Jayhyuk; Kim, Kyung Bo

    2008-01-01

    The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases. PMID:11410931

  14. Deltex1 promotes protein kinase Cθ degradation and sustains Casitas B-lineage lymphoma expression.

    PubMed

    Hsu, Tzu-Sheng; Hsiao, Huey-Wen; Wu, Pei-Jung; Liu, Wen-Hsien; Lai, Ming-Zong

    2014-08-15

    The generation of T cell anergy is associated with upregulation of ubiquitin E3 ligases including Casitas B-lineage lymphoma (Cbl-b), Itch, gene related to anergy in lymphocyte, and deltex1 (DTX1). These E3 ligases attenuate T cell activation by targeting to signaling molecules. For example, Cbl-b and Itch promote the degradation of protein kinase Cθ (PKCθ) and phospholipase C-γ1 (PLC-γ1) in anergic Th1 cells. How these anergy-associated E3 ligases coordinate during T cell anergy remains largely unknown. In the current study, we found that PKCθ and PLC-γ1 are also downregulated by DTX1. DTX1 interacted with PKCθ and PLC-γ1 and stimulated the degradation of PKCθ and PLC-γ1. T cell anergy-induced proteolysis of PKCθ was prevented in Dtx1(-/-) T cells, supporting the essential role of DTX1 in PKCθ downregulation. Similar to Cbl-b and Itch, DTX1 promoted monoubiquitination of PKCθ. Proteasome inhibitor did not inhibit DTX1-directed PKCθ degradation, but instead DTX1 directed the relocalization of PKCθ into the lysosomal pathway. In addition, DTX1 interacted with Cbl-b and increased the protein levels of Cbl-b. We further demonstrated the possibility that, through the downregulation of PKCθ, DTX1 prevented PKCθ-induced Cbl-b degradation and increased Cbl-b protein stability. Our results suggest the coordination between E3 ligases during T cell anergy; DTX1 acts with Cbl-b to assure a more extensive silencing of PKCθ, whereas DTX1-mediated PKCθ degradation further stabilizes Cbl-b.

  15. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    PubMed Central

    Li, Lihua; Yang, Huanjie; Chen, Di; Cui, Cindy; Dou, Q. Ping

    2013-01-01

    The ubiquitinproteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF–Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value of 32 μmol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF–Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF–Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF–Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd. PMID:18304598

  16. The Pallbearer E3 Ligase Promotes Actin Remodeling via RAC in Efferocytosis by Degrading the Ribosomal Protein S6

    PubMed Central

    Xiao, Hui; Wang, Hui; Silva, Elizabeth; Thompson, James; Guillou, Aurélien; Yates, John R.; Buchon, Nicolas; Franc, Nathalie C.

    2014-01-01

    Clearance of apoptotic cells (efferocytosis) is achieved through phagocytosis by professional or amateur phagocytes. It is critical for tissue homeostasis and remodeling in all animals. Failure in this process can contribute to the development of inflammatory autoimmune or neurodegenerative diseases. We previously found that the PALL-SCF E3-Ubiquitin ligase complex promotes apoptotic cell clearance, yet it remained unclear as to how it did so. Here, we show that the F-Box protein PALL interacts with phosphorylated Ribosomal protein S6 (RpS6) to promote its ubiquitylation and proteasomal degradation. This leads to RAC2 GTPase up-regulation and activation and F-actin remodeling that promotes efferocytosis. We further show that the specific role of PALL in efferocytosis is driven by its apoptotic cell-induced nuclear export. Finding a role for RpS6 in negatively regulating efferocytosis provides the opportunity to develop new strategies to regulate this process. PMID:25533207

  17. Carnosic acid promotes degradation of the androgen receptor and is regulated by the unfolded protein response pathway in vitro and in vivo.

    PubMed

    Petiwala, Sakina M; Li, Gongbo; Bosland, Maarten C; Lantvit, Daniel D; Petukhov, Pavel A; Johnson, Jeremy J

    2016-08-01

    Androgen deprivation therapy in prostate cancer is extremely effective; however, due to the continuous expression and/or mutagenesis of androgen receptor (AR), the resistance to antihormonal therapy is a natural progression. Consequently, targeting the AR for degradation offers an alternate approach to overcome this resistance in prostate cancer. In this study, we demonstrate that carnosic acid, a benzenediol diterpene, binds the ligand-binding domain of the AR and degrades the AR via endoplasmic reticulum (ER) stress-mediated proteasomal degradative pathway. In vitro, carnosic acid treatment induced degradation of AR and decreased expression of prostate-specific antigen in human prostate cancer cell lines LNCaP and 22Rv1. Carnosic acid also promoted the expression of ER proteins including BiP and CHOP in a dose-dependent manner. Downregulation of CHOP by small interfering RNA somewhat restored expression of AR suggesting that AR degradation is dependent on ER stress pathway. Future studies will need to evaluate other aspects of the unfolded protein response pathway to characterize the regulation of AR degradation. Furthermore, cotreating cells individually with carnosic acid and proteasome inhibitor (MG-132) and carnosic acid and an ER stress modulator (salubrinal) restored protein levels of AR, suggesting that AR degradation is mediated by ER stress-dependent proteasomal degradation pathway. Degradation of AR and induction of CHOP protein were also evident in vivo along with a 53% reduction in growth of xenograft prostate cancer tumors. In addition, carnosic acid-induced ER stress in prostate cancer cells but not in normal prostate epithelial cells procured from patient biopsies. In conclusion, these data suggest that molecules such as carnosic acid could be further evaluated and optimized as a potential therapeutic alternative to target AR in prostate cancer. PMID:27267997

  18. Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

    PubMed Central

    Yeh, Wei-Lan; Shioda, Keiko; Coser, Kathryn R.; Rivizzigno, Danielle; McSweeney, Kristen R.; Shioda, Toshi

    2013-01-01

    Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant. PMID:23593342

  19. Hinokitiol inhibits vasculogenic mimicry activity of breast cancer stem/progenitor cells through proteasome-mediated degradation of epidermal growth factor receptor

    PubMed Central

    TU, DOM-GENE; YU, YUN; LEE, CHE-HSIN; KUO, YU-LIANG; LU, YIN-CHE; TU, CHI-WEN; CHANG, WEN-WEI

    2016-01-01

    Hinokitiol, alternatively known as β-thujaplicin, is a tropolone-associated natural compound with antimicrobial, anti-inflammatory and antitumor activity. Breast cancer stem/progenitor cells (BCSCs) are a subpopulation of breast cancer cells associated with tumor initiation, chemoresistance and metastatic behavior, and may be enriched by mammosphere cultivation. Previous studies have demonstrated that BCSCs exhibit vasculogenic mimicry (VM) activity via the epidermal growth factor receptor (EGFR) signaling pathway. The present study investigated the anti-VM activity of hinokitiol in BCSCs. At a concentration below the half maximal inhibitory concentration, hinokitiol inhibited VM formation of mammosphere cells derived from two human breast cancer cell lines. Hinokitiol was additionally indicated to downregulate EGFR protein expression in mammosphere-forming BCSCs without affecting the expression of messenger RNA. The protein stability of EGFR in BCSCs was also decreased by hinokitiol. The EGFR protein expression and VM formation capability of hinokitiol-treated BCSCs were restored by co-treatment with MG132, a proteasome inhibitor. In conclusion, the present study indicated that hinokitiol may inhibit the VM activity of BCSCs through stimulating proteasome-mediated EGFR degradation. Hinokitiol may act as an anti-VM agent, and may be useful for the development of novel breast cancer therapeutic agents. PMID:27073579

  20. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

    SciTech Connect

    Schlax, Peter E.; Zhang Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T. Glen . E-mail: tlawson@bates.edu

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

  1. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo.

    PubMed

    Schlax, Peter E; Zhang, Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T Glen

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination. PMID:17150238

  2. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs.

  3. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs. PMID:23053530

  4. Myeloid Translocation Gene-16 Co-Repressor Promotes Degradation of Hypoxia-Inducible Factor 1

    PubMed Central

    Kumar, Parveen; Gullberg, Urban; Olsson, Inge; Ajore, Ram

    2015-01-01

    The myeloid translocation gene 16 (MTG16) co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1) heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1). Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs) in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α. PMID:25974097

  5. Transcriptional directionality of the human insulin-degrading enzyme promoter.

    PubMed

    Zhang, Lang; Wang, Pan; Ding, Qingyang; Wang, Zhao

    2013-10-01

    Unidirectional promoters dominate among mammalian genomes. However, the mechanism through which the transcriptional directionality of promoters is accomplished remains to be clarified. Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc metalloprotease, whose promoter contains a CpG island. We previously showed that the basal promoter region of mouse IDE has bidirectional transcriptional activity, but an upstream promoter element blocks its antisense transcription. Therefore, we wonder whether the human IDE promoter contains an analogous element. Similarly, the basal promoter region of human IDE (-102 ~ +173 and -196 ~ +173 relative to the transcription start site) showed bidirectional transcriptional activity. However, the region from -348 to +173 could only be transcribed from the normal orientation, implying that an upstream promoter element between -348 and -196 blocks the antisense transcription of the human IDE promoter. Through promoter deletion and mutagenesis analysis, we mapped this element precisely and found that the upstream promoter element locates between -318 and -304. Furthermore, the transcription-blocking elements in the mouse and human IDE promoters inhibited the transcription of the SV40 promoter when put downstream of it. In conclusion, we identify an upstream promoter element which blocks the antisense transcription of the human IDE promoter. Our studies are helpful to clarify the transcriptional directionality of promoters.

  6. Murine Cytomegalovirus US22 Protein pM140 Protects Its Binding Partner, pM141, from Proteasome-Dependent but Ubiquitin-Independent Degradation

    PubMed Central

    Bolin, Lisa L.; Hanson, Laura K.; Slater, Jacquelyn S.; Kerry, Julie A.; Campbell, Ann E.

    2010-01-01

    Stable assembly of murine cytomegalovirus (MCMV) virions in differentiated macrophages is dependent upon the expression of US22 family gene M140. The M140 protein (pM140) exists in complex with products of neighboring US22 genes. Here we report that pM140 protects its binding partner, pM141, from ubiquitin-independent proteasomal degradation. Protection is conferred by a stabilization domain mapping to amino acids 306 to 380 within pM140, and this domain is functionally independent from the region that confers binding of pM140 to pM141. The M140 protein thus contains multiple domains that collectively confer a structure necessary to function in virion assembly in macrophages. PMID:19955315

  7. Novel sphingosine-containing analogues selectively inhibit sphingosine kinase (SK) isozymes, induce SK1 proteasomal degradation and reduce DNA synthesis in human pulmonary arterial smooth muscle cells

    PubMed Central

    Byun, Hoe-Sup; Pyne, Susan; MacRitchie, Neil; Pyne, Nigel J.

    2013-01-01

    Sphingosine 1-phosphate (S1P) is involved in hyper-proliferative diseases such as cancer and pulmonary arterial hypertension. We have synthesized inhibitors that are selective for the two isoforms of sphingosine kinase (SK1 and SK2) that catalyze the synthesis of S1P. A thiourea adduct of sphinganine (F02) is selective for SK2 whereas the 1-deoxysphinganines 55-21 and 77-7 are selective for SK1. (2S,3R)-1-Deoxysphinganine (55-21) induced the proteasomal degradation of SK1 in human pulmonary arterial smooth muscle cells and inhibited DNA synthesis, while the more potent SK1 inhibitors PF-543 and VPC96091 failed to inhibit DNA synthesis. These findings indicate that moderate potency inhibitors such as 55-21 are likely to have utility in unraveling the functions of SK1 in inflammatory and hyperproliferative disorders. PMID:24396570

  8. Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation.

    PubMed

    Dominguez-Brauer, Carmen; Hao, Zhenyue; Elia, Andrew J; Fortin, Jérôme M; Nechanitzky, Robert; Brauer, Patrick M; Sheng, Yi; Mana, Miyeko D; Chio, Iok In Christine; Haight, Jillian; Pollett, Aaron; Cairns, Robert; Tworzyanski, Leanne; Inoue, Satoshi; Reardon, Colin; Marques, Ana; Silvester, Jennifer; Cox, Maureen A; Wakeham, Andrew; Yilmaz, Omer H; Sabatini, David M; van Es, Johan H; Clevers, Hans; Sato, Toshiro; Mak, Tak W

    2016-08-01

    The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

  9. Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization.

    PubMed

    Moulédous, Lionel; Neasta, Jérémie; Uttenweiler-Joseph, Sandrine; Stella, Alexandre; Matondo, Mariette; Corbani, Maïthé; Monsarrat, Bernard; Meunier, Jean-Claude

    2005-08-01

    The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role. PMID:15901846

  10. Proteasome function shapes innate and adaptive immune responses.

    PubMed

    Kammerl, Ilona E; Meiners, Silke

    2016-08-01

    The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively. PMID:27343191

  11. Proteasome function shapes innate and adaptive immune responses.

    PubMed

    Kammerl, Ilona E; Meiners, Silke

    2016-08-01

    The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively.

  12. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    SciTech Connect

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  13. Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

    PubMed Central

    Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.; Luo, Kunxin

    2001-01-01

    Smad proteins mediate transforming growth factor-β (TGF-β) signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGF-β signaling that functions to maintain the repressed state of TGF-β target genes in the absence of ligand. On TGF-β stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGF-β target genes. We show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase-promoting complex (APC) and the UbcH5 family of ubiquitin-conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box (D box)-dependent manner. In addition to the D box, efficient ubiquitination and degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGF-β signaling. Our studies elucidate an important mechanism and pathway for the degradation of SnoN and more importantly, reveal a novel role of the APC in the regulation of TGF-β signaling. PMID:11691834

  14. The RNA-binding protein Musashi-1 regulates proteasome subunit expression in breast cancer- and glioma-initiating cells

    PubMed Central

    Lagadec, Chann; Vlashi, Erina; Frohnen, Patricia; Alhiyari, Yazeed; Chan, Mabel; Pajonk, Frank

    2014-01-01

    Cancer stem cells (CSCs) or tumor-initiating cells, similar to normal tissue stem cells, rely on developmental pathways, such as the Notch pathway, to maintain their stem cell state. One of the regulators of the Notch pathway is Musashi-1, a mRNA-binding protein. Musashi-1 promotes Notch signaling by binding to the mRNA of Numb, the negative regulator of Notch signaling, thus preventing its translation. Cancer stem cells have also been shown to down-regulate their 26S proteasome activity in several types of solid tumors, thus making them resistant to proteasome-inhibitors used as anti-cancer agents in the clinic. Interestingly, the Notch pathway can be inhibited by proteasomal degradation of the Notch intracellular domain (Notch-ICD), therefore down-regulation of the 26S proteasome activity can lead to stabilization of Notch-ICD. Here we present evidence that the down-regulation of the 26S proteasome in CSCs constitutes another level of control by which Musashi-1 promotes signaling through the Notch pathway and maintenance of the stem cell phenotype of this subpopulation of cancer cells. We demonstrate that Musashi-1 mediates the down-regulation of the 26S proteasome by binding to the mRNA of NF-YA, the transcriptional factor regulating 26S proteasome subunit expression, thus providing an additional route by which the degradation of Notch-ICD is prevented, and Notch signaling is sustained. PMID:24022895

  15. WSB1 promotes tumor metastasis by inducing pVHL degradation

    PubMed Central

    Kim, Jung Jin; Lee, Seung Baek; Jang, Jinsung; Yi, Sang-Yeop; Kim, Sun-Hyun; Han, Sang-Ah; Lee, Jong-Min; Tong, Seo-Yun; Vincelette, Nicole D.; Gao, Bowen; Yin, Ping; Evans, Debra; Choi, Dong Wook; Qin, Bo; Liu, Tongzheng; Zhang, Haoxing; Deng, Min; Jen, Jin; Zhang, Jun; Wang, Liewei; Lou, Zhenkun

    2015-01-01

    The von Hippel-Lindau tumor suppressor pVHL is an E3 ligase that targets hypoxia-inducible factors (HIFs). Mutation of VHL results in HIF up-regulation and contributes to processes related to tumor progression such as invasion, metastasis, and angiogenesis. However, very little is known with regard to post-transcriptional regulation of pVHL. Here we show that WD repeat and SOCS box-containing protein 1 (WSB1) is a negative regulator of pVHL through WSB1's E3 ligase activity. Mechanistically, WSB1 promotes pVHL ubiquitination and proteasomal degradation, thereby stabilizing HIF under both normoxic and hypoxic conditions. As a consequence, WSB1 up-regulates the expression of HIF-1α’s target genes and promotes cancer invasion and metastasis through its effect on pVHL. Consistent with this, WSB1 protein level negatively correlates with pVHL level and metastasis-free survival in clinical samples. This work reveals a new mechanism of pVHL's regulation by which cancer acquires invasiveness and metastatic tendency. PMID:26545811

  16. A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells.

    PubMed Central

    Shumway, Stuart D; Miyamoto, Shigeki

    2004-01-01

    Inducible activation of the transcription factor NF-kappaB (nuclear factor kappaB) is classically mediated by proteasomal degradation of its associated inhibitors, IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. However, certain B-lymphocytes maintain constitutively nuclear NF-kappaB activity (a p50-c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IkappaBalpha, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IkappaBalpha degradation and constitutive p50-c-Rel activity. Domain analysis of IkappaBalpha by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IkappaBalpha represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IkappaBbeta, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IkappaBbeta to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IkappaBalpha that was required for complete targeting. Nevertheless, the NF-kappaB pool associated with IkappaBbeta now became constitutively active by virtue of IkappaBbeta instability in these cells. These findings further support the notion that IkappaB instability governs the maintenance of constitutive p50-c-Rel activity in certain B-cells via a unique degradation pathway. PMID:14763901

  17. An evolutionarily conserved pathway controls proteasome homeostasis.

    PubMed

    Rousseau, Adrien; Bertolotti, Anne

    2016-08-11

    The proteasome is essential for the selective degradation of most cellular proteins, but how cells maintain adequate amounts of proteasome is unclear. Here we show that there is an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1, the inhibition of which induced all known yeast 19S regulatory particle assembly-chaperones (RACs), as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, acts to increase the supply of RACs and proteasome subunits under challenging conditions in order to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and ERK5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/ERK5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance in response to the rising needs of cells. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation. PMID:27462806

  18. [Proteasome inhibitors in cancer therapy].

    PubMed

    Romaniuk, Wioletta; Ołdziej, Agnieszka Ewa; Zińczuk, Justyna; Kłoczko, Janusz

    2015-01-01

    Proteasomes are multisubunit enzyme complexes. They contain three enzymatic active sites which are termed chymotrypsin-like, trypsin-like, and caspase-like. The elementary function of the proteasomes is degradation of damaged proteins. Proteasome inhibition leads to accumulation of damaged protein, which leads to caspase activation and cell death. This relationship is used in cancer therapy. Bortezomib is the first proteasome inhibitor approved by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma. Carfilzomib belongs to the second generation of drugs, which was approved by the US FDA in 2012. Currently in the study phase there are four new inhibitors: ixazomib (MLN9780/MLN2238), delanzomib (CEP-18770), oprozomib (ONX0912/PR-047) and marizomib (NPI-0052). PMID:27259216

  19. Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome.

    PubMed

    Reichard, Eden L; Chirico, Giavanna G; Dewey, William J; Nassif, Nicholas D; Bard, Katelyn E; Millas, Nickolas E; Kraut, Daniel A

    2016-08-26

    In eukaryotic cells, proteins are targeted to the proteasome for degradation by polyubiquitination. These proteins bind to ubiquitin receptors, are engaged and unfolded by proteasomal ATPases, and are processively degraded. The factors determining to what extent the proteasome can successfully unfold and degrade a substrate are still poorly understood. We find that the architecture of polyubiquitin chains attached to a substrate affects the ability of the proteasome to unfold and degrade the substrate, with K48- or mixed-linkage chains leading to greater processivity than K63-linked chains. Ubiquitin-independent targeting of substrates to the proteasome gave substantially lower processivity of degradation than ubiquitin-dependent targeting. Thus, even though ubiquitin chains are removed early in degradation, during substrate engagement, remarkably they dramatically affect the later unfolding of a protein domain. Our work supports a model in which a polyubiquitin chain associated with a substrate switches the proteasome into an activated state that persists throughout the degradation process. PMID:27405762

  20. Dual attenuation of proteasomal and autophagic BMAL1 degradation in ClockΔ19/+ mice contributes to improved glucose homeostasis

    PubMed Central

    Jeong, Kwon; He, Baokun; Nohara, Kazunari; Park, Noheon; Shin, Youngmin; Kim, Seonghwa; Shimomura, Kazuhiro; Koike, Nobuya; Yoo, Seung-Hee; Chen, Zheng

    2015-01-01

    Circadian clocks orchestrate essential physiology in response to various cues, yet their mechanistic and functional plasticity remains unclear. Here, we investigated ClockΔ19/+ heterozygous (Clk/+) mice, known to display lengthened periodicity and dampened amplitude, as a model of partially perturbed clocks. Interestingly, Clk/+ mice exhibited improved glycemic control and resistance to circadian period lengthening under high-fat diet (HFD). Furthermore, BMAL1 protein levels in Clk/+ mouse liver were upregulated compared with wild-type (WT) mice under HFD. Pharmacological and molecular studies showed that BMAL1 turnover entailed proteasomal and autophagic activities, and CLOCKΔ19 attenuated both processes. Consistent with an important role of BMAL1 in glycemic control, enhanced activation of insulin signaling was observed in Clk/+ mice relative to WT in HFD. Finally, transcriptome analysis revealed reprogramming of clock-controlled metabolic genes in Clk/+ mice. Our results demonstrate a novel role of autophagy in circadian regulation and reveal an unforeseen plasticity of circadian and metabolic networks. PMID:26228022

  1. Increased proteasome-dependent degradation of estrogen receptor-alpha by TGF-beta1 in breast cancer cell lines.

    PubMed

    Petrel, Trevor A; Brueggemeier, Robert W

    2003-01-01

    Normal mammary epithelial cells are rapidly induced to G(1) arrest by the widely expressed cytokine, transforming growth factor beta (TGF-beta1). Studies in established breast cancer cell lines that express the estrogen receptor alpha (ERalpha) have demonstrated loss of this responsiveness. This inverse correlation suggests interpathway signaling important to cell growth and regulation. The adenocarcinoma breast cell line BT474, which was not growth arrested by TGF-beta1, was used as a model of estrogen-inducible growth to explore interpathway crosstalk. Although BT474 cells were not growth-arrested by TGF-beta1 as determined by flow cytometry analysis and 5'-bromo-3'-deoxyuridine incorporation into DNA, estrogen receptor protein levels were attenuated by 100 pM TGF-beta1 after 6 h. This decrease in ERalpha reached 50% of untreated control levels by 24 h of treatment and was further supported by a 50% decrease in estrogen-inducible DNA synthesis. Inspection of ERalpha transcripts suggested that this decrease was primarily the result of altered ERalpha protein stability or availability. Use of the proteasome inhibitor, MG132, abolished all effects on ERalpha by TGF-beta1. Collectively, this data supports a role for TGF-beta1 in regulating the growth of otherwise insensitive breast cancer cells through modulation of ERalpha stability. PMID:12461787

  2. VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage.

    PubMed

    Monsalve, Diana M; Campillo-Marcos, Ignacio; Salzano, Marcella; Sanz-García, Marta; Cantarero, Lara; Lazo, Pedro A

    2016-04-01

    NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.

  3. Structure characterization of the 26S proteasome

    PubMed Central

    Kim, Ho Min; Yu, Yadong; Cheng, Yifan

    2010-01-01

    In all eukaryotic cells, 26S proteasome plays an essential role in the process of ATP-dependent protein degradation. In this review, we focus on structure characterization of the 26S proteasome. Although the progress towards a high-resolution structure of the 26S proteasome has been slow, the recently solved structures of various proteasomal subcomplexes have greatly enhanced our understanding of this large machinery. In addition to having an ATP-dependent proteolytic function, the 26S proteasome is also involved in many non-proteolytic cellular activities, which are often mediated by subunits in its 19S regulatory complex. Thus, we include a detailed discussion of the structures of 19S subunits, including proteasomal ATPases, ubiquitin receptors, deubiquitinating enzymes and subunits that contain PCI domain. PMID:20800708

  4. Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression.

    PubMed

    Bandiera, Silvio; Weidlich, Simone; Harth, Volker; Broede, Peter; Ko, Yun; Friedberg, Thomas

    2005-02-01

    Allelic variations in CYP1B1 are reported to modulate the incidence of several types of cancer. To provide a mechanistic basis for this association, we investigated the impact of nonsilent allelic changes on the intracellular levels and post-translational regulation of CYP1B1 protein. When transiently expressed in COS-1 cells, either in the presence or absence of recombinant cytochrome P450 reductase, the cellular level of the CYP1B1.4 allelic variant (containing a Ser at the amino acid position 453; Ser453) was 2-fold lower compared with the other four allelic CYP1B1 proteins (containing Asn453), as analyzed by both immunoblotting and ethoxyresorufin O-deethylase activity. This difference was caused by post-translational regulation; as in the presence of cycloheximide, the rate of degradation of immunodetectable and enzymatically active CYP1B1.4 was distinctly faster than that of CYP1B1.1. Pulse-chase analysis revealed that the half-life of CYP1B1.4 was a mere 1.6 h compared with 4.8 h for CYP1B1.1. The presence of the proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal] increased the stability not only of immunodetectable CYP1B1, but also--unexpectedly given the size of the proteasome access channel--increased the stability of enzymatically active CYP1B1. The data presented herein also demonstrate that CYP1B1 is targeted for its polymorphism-dependent degradation by polyubiquitination but not phosphorylation. Our results importantly provide a mechanism to explain the recently reported lower incidence of endometrial cancer in individuals carrying the CYP1B1*4 compared with the CYP1B1*1 haplo-type. In addition, the mechanistic paradigms revealed herein may explain the strong overexpression of CYP1B1 in tumors compared with nondiseased tissues. PMID:15486049

  5. Ubiquitin-independent- versus ubiquitin-dependent proteasomal degradation of the c-Fos and Fra-1 transcription factors: is there a unique answer?

    PubMed

    Basbous, Jihane; Jariel-Encontre, Isabelle; Gomard, Tiphanie; Bossis, Guillaume; Piechaczyk, Marc

    2008-02-01

    The Fos family of transcription factors comprises c-Fos, Fra-1, Fra-2 and FosB, which are all intrinsically unstable proteins. Fos proteins heterodimerize with a variety of other transcription factors to control genes encoding key cell regulators. Their best known partners are the Jun family proteins (c-Jun, JunB, and JunD). At the cellular level, Fos-involving dimers control proliferation, differentiation, apoptosis and responses to environmental cues. At the organism level, they play paramount parts in organogenesis, immune responses and cognitive functions, among others. fos family genes are subjected to exquisite, complex and intermingled transcriptional and post-transcriptional regulations, which are necessary to avoid pathological effects. In particular, the Fos proteins undergo to numerous post-translational modifications, such as phosphorylations and sumoylation, regulating their transcriptional activity, their subcellular localization and their turnover. The mechanisms whereby c-Fos and Fra-1 are degraded have been studied in detail. Contrasting with the classical scenario, according to which most unstable key cell regulators are hydrolyzed by the proteasome after conjugation of polyubiquitin chains, the bulk of c-Fos and Fra-1 can be hydrolyzed independently of any prior ubiquitylation in different situations. c-Fos and Fra-1 share a common destabilizing domain whose primary sequence is conserved in Fra-2 and FosB, suggesting that similar breakdown mechanisms might be at play in the latter two proteins. However, a database search indicates that this domain is not found in any other protein, suggesting that the mechanisms underlying Fos protein destruction may be specific to this family. Interestingly, under particular conditions, a fraction of cytoplasmic c-Fos is ubiquitylated, leading to faster turnover. This poses the question of the multiplicity of degradation pathways that can target the same substrate depending on its activation state, its protein

  6. Nitric Oxide and Interleukin-1β Stimulate the Proteasome-Independent Degradation of the Retinoic Acid Hydroxylase CYP2C22 in Primary Rat Hepatocytes

    PubMed Central

    Lee, Choon-myung; Lee, Bang-sub; Arnold, Samuel L.; Isoherranen, Nina

    2014-01-01

    CYP2C22 was recently described as a retinoic acid–metabolizing cytochrome P450 enzyme whose transcription is induced by all-trans-retinoic acid (atRA) in hepatoma cells (Qian L, Zolfaghari R, and Ross AC (2010) J Lipid Res 51:1781–1792). We identified CYP2C22 as a putative nitric oxide (NO)–regulated protein in a proteomic screen and raised specific polyclonal antibodies to CYP2C22 to study its protein expression. We found that CYP2C22 is a liver-specific protein that was not significantly induced by activators of the pregnane X receptor, constitutive androstane receptor, or peroxisome proliferator-activated receptor-α, but was downregulated to <25% of control by the aryl hydrocarbon receptor agonist β-naphthoflavone in cultured rat hepatocytes. CYP2C22 protein and its mRNA both were induced by atRA in hepatocytes, with EC50 of 100–300 nM, whereas the maximal extent of mRNA induction was twice that of the protein. CYP2C22 protein, but not its mRNA, was rapidly downregulated in hepatocytes by interleukin-1 (IL-1) or NO-donating compounds, and the downregulation by IL-1 was blocked by inhibition of NO synthases. The NO donor (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate reduced the half-life of CYP2C22 from 8.7 to 3.4 hours in the presence of cycloheximide, demonstrating that NO-dependent downregulation is due to stimulated proteolysis. No intermediate degradation products were detected. However, this degradation was insensitive to inhibitors of calpains or the canonical proteasomal or lysosomal pathways, indicating that NO-dependent degradation of CYP2C22 proceeds via a novel pathway. PMID:24144795

  7. Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation.

    PubMed

    Tang, Jiayu; Hu, Zhiping; Tan, Jieqiong; Yang, Sonlin; Zeng, Liuwang

    2016-01-01

    Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders. PMID:27597885

  8. Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation

    PubMed Central

    Tang, Jiayu; Hu, Zhiping; Tan, Jieqiong; Yang, Sonlin

    2016-01-01

    Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders. PMID:27597885

  9. Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation

    PubMed Central

    Tang, Jiayu; Hu, Zhiping; Tan, Jieqiong; Yang, Sonlin

    2016-01-01

    Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders.

  10. SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals.

    PubMed

    Xie, Qi; Guo, Hui-Shan; Dallman, Geza; Fang, Shengyun; Weissman, Allan M; Chua, Nam-Hai

    2002-09-12

    The plant hormone indole-3 acetic acid (IAA or auxin) controls many aspects of plant development, including the production of lateral roots. Ubiquitin-mediated proteolysis has a central role in this process. The genes AXR1 and TIR1 aid the assembly of an active SCF (Skp1/Cullin/F-box) complex that probably promotes degradation of the AUX/IAA transcriptional repressors in response to auxin. The transcription activator NAC1, a member of the NAM/CUC family of transcription factors, functions downstream of TIR1 to transduce the auxin signal for lateral root development. Here we show that SINAT5, an Arabidopsis homologue of the RING-finger Drosophila protein SINA, has ubiquitin protein ligase activity and can ubiquitinate NAC1. This activity is abolished by mutations in the RING motif of SINAT5. Overexpressing SINAT5 produces fewer lateral roots, whereas overexpression of a dominant-negative Cys49 --> Ser mutant of SINAT5 develops more lateral roots. These lateral root phenotypes correlate with the expression of NAC1 observed in vivo. Low expression of NAC1 in roots can be increased by treatment with a proteasome inhibitor, which indicates that SINAT5 targets NAC1 for ubiquitin-mediated proteolysis to downregulate auxin signals in plant cells.

  11. Extracellular Matrix Degradation and Tissue Remodeling in Periprosthetic Loosening and Osteolysis: Focus on Matrix Metalloproteinases, Their Endogenous Tissue Inhibitors, and the Proteasome

    PubMed Central

    Syggelos, Spyros A.; Aletras, Alexios J.; Smirlaki, Ioanna; Skandalis, Spyros S.

    2013-01-01

    The leading complication of total joint replacement is periprosthetic osteolysis, which often results in aseptic loosening of the implant, leading to revision surgery. Extracellular matrix degradation and connective tissue remodeling around implants have been considered as major biological events in the periprosthetic loosening. Critical mediators of wear particle-induced inflammatory osteolysis released by periprosthetic synovial cells (mainly macrophages) are inflammatory cytokines, chemokines, and proteolytic enzymes, mainly matrix metalloproteinases (MMPs). Numerous studies reveal a strong interdependence of MMP expression and activity with the molecular mechanisms that control the composition and turnover of periprosthetic matrices. MMPs can either actively modulate or be modulated by the molecular mechanisms that determine the debris-induced remodeling of the periprosthetic microenvironment. In the present study, the molecular mechanisms that control the composition, turnover, and activity of matrix macromolecules within the periprosthetic microenvironment exposed to wear debris are summarized and presented. Special emphasis is given to MMPs and their endogenous tissue inhibitors (TIMPs), as well as to the proteasome pathway, which appears to be an elegant molecular regulator of specific matrix macromolecules (including specific MMPs and TIMPs). Furthermore, strong rationale for potential clinical applications of the described molecular mechanisms to the treatment of periprosthetic loosening and osteolysis is provided. PMID:23862137

  12. Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

    PubMed Central

    Gu, Yuan; Körbel, Christina; Scheuer, Claudia; Nenicu, Anca; Menger, Michael D.; Laschke, Matthias W.

    2016-01-01

    Tubeimoside-1 (TBMS1) is a potent anti-tumor phytochemical. Its functional and molecular mode of action, however, remains elusive so far. Since angiogenesis is essential for tumor progression and metastasis, we herein investigated the anti-angiogenic effects of the compound. In a non-small cell lung cancer (NSCLC) xenograft model we found that treatment of CD1 nu/nu mice with TBMS1 (5mg/kg) significantly suppressed the growth and vascularization of NCI-H460 flank tumors. Moreover, TBMS1 dose-dependently reduced vascular sprouting in a rat aortic ring assay. In vitro, TBMS1 induced endothelial cell apoptosis without decreasing the viability of NSCLC tumor cells and inhibited the migration of endothelial cells by disturbing their actin filament organization. TBMS1 further stimulated the proteasomal degradation of vascular endothelial growth factor receptor-2 (VEGFR2) and Tie2 in endothelial cells, which down-regulated AKT/mTOR signaling. These findings indicate that TBMS1 represents a novel phytochemical for anti-angiogenic treatment of cancer and other angiogenesis-related diseases. PMID:26701724

  13. Hedgehog-dependent E3-ligase Midline1 regulates ubiquitin-mediated proteasomal degradation of Pax6 during visual system development.

    PubMed

    Pfirrmann, Thorsten; Jandt, Enrico; Ranft, Swantje; Lokapally, Ashwin; Neuhaus, Herbert; Perron, Muriel; Hollemann, Thomas

    2016-09-01

    Pax6 is a key transcription factor involved in eye, brain, and pancreas development. Although pax6 is expressed in the whole prospective retinal field, subsequently its expression becomes restricted to the optic cup by reciprocal transcriptional repression of pax6 and pax2 However, it remains unclear how Pax6 protein is removed from the eyestalk territory on time. Here, we report that Mid1, a member of the RBCC/TRIM E3 ligase family, which was first identified in patients with the X-chromosome-linked Opitz BBB/G (OS) syndrome, interacts with Pax6. We found that the forming eyestalk is a major domain of mid1 expression, controlled by the morphogen Sonic hedgehog (Shh). Here, Mid1 regulates the ubiquitination and proteasomal degradation of Pax6 protein. Accordantly, when Mid1 levels are knocked down, Pax6 expression is expanded and eyes are enlarged. Our findings indicate that remaining or misaddressed Pax6 protein is cleared from the eyestalk region to properly set the border between the eyestalk territory and the retina via Mid1. Thus, we identified a posttranslational mechanism, regulated by Sonic hedgehog, which is important to suppress Pax6 activity and thus breaks pax6 autoregulation at defined steps during the formation of the visual system. PMID:27555585

  14. Pri sORF peptides induce selective proteasome-mediated protein processing.

    PubMed

    Zanet, J; Benrabah, E; Li, T; Pélissier-Monier, A; Chanut-Delalande, H; Ronsin, B; Bellen, H J; Payre, F; Plaza, S

    2015-09-18

    A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

  15. Proteasome inhibition improves the muscle of laminin α2 chain-deficient mice.

    PubMed

    Carmignac, Virginie; Quéré, Ronan; Durbeej, Madeleine

    2011-02-01

    Muscle atrophy, a significant characteristic of congenital muscular dystrophy with laminin α2 chain deficiency (also known as MDC1A), occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) plays a key role in protein degradation in skeletal muscle cells. In order to identify new targets for drug therapy against MDC1A, we have investigated whether increased proteasomal degradation is a feature of MDC1A. Using the generated dy(3K)/dy(3K) mutant mouse model of MDC1A, we studied the expression of members of the ubiquitin-proteasome pathway in laminin α2 chain-deficient muscle, and we treated dy(3K)/dy(3K) mice with the proteasome inhibitor MG-132. We show that members of the UPS are upregulated and that the global ubiquitination of proteins is raised in dystrophic limb muscles. Also, phosphorylation of Akt is diminished in diseased muscles. Importantly, proteasome inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. Specifically, treatment with MG-132 increases lifespan, enhances locomotive activity, enlarges muscle fiber diameter, reduces fibrosis, restores Akt phosphorylation and decreases apoptosis. These studies promote better understanding of the disease process in mice and could lead to a drug therapy for MDC1A patients.

  16. Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation*

    PubMed Central

    Doi, Kenichiro; Li, Rongshi; Sung, Shen-Shu; Wu, Hongwei; Liu, Yan; Manieri, Wanda; Krishnegowda, Gowdahalli; Awwad, Andy; Dewey, Alden; Liu, Xin; Amin, Shantu; Cheng, Chunwei; Qin, Yong; Schonbrunn, Ernst; Daughdrill, Gary; Loughran, Thomas P.; Sebti, Said; Wang, Hong-Gang

    2012-01-01

    The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-XL, and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-XL-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1–2 μm. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation. PMID:22311987

  17. Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

    SciTech Connect

    Dul, J. L.; Davis, D. P.; Williamson, E. K.; Stevens, F. J.; Argon, Y.; Univ. of Chicago

    2001-02-19

    In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its {beta} sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

  18. Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

    PubMed Central

    Malo, Mackenzie E.; Postnikoff, Spike D.L.; Arnason, Terra G.; Harkness, Troy A.A.

    2016-01-01

    The Saccharomyces cerevisiae Forkhead Box (Fox) orthologs, Forkheads (Fkh) 1 and 2, are conserved transcription factors required for stress response, cell cycle progression and longevity. These yeast proteins play a key role in mitotic progression through activation of the ubiquitin E3 ligase Anaphase Promoting Complex (APC) via transcriptional control. Here, we used genetic and molecular analyses to demonstrate that the APC E3 activity is necessary for mitotic Fkh1 protein degradation and subsequent cell cycle progression. We report that Fkh1 protein degradation occurs specifically during mitosis, requires APCCdc20 and proteasome activity, and that a stable Fkh1 mutant reduces normal chronological lifespan, increases genomic instability, and increases sensitivity to stress. Our data supports a model whereby cell cycle progression through mitosis and G1 requires the targeted degradation of Fkh1 by the APC. This is significant to many fields as these results impact our understanding of the mechanisms underpinning the control of aging and cancer. PMID:27099939

  19. Intracellular Dynamics of the Ubiquitin-Proteasome-System.

    PubMed

    Chowdhury, Maisha; Enenkel, Cordula

    2015-01-01

    The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus. Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm. Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body's cells. PMID:26339477

  20. Differential regulation of the REGγ–proteasome pathway by p53/TGF-β signalling and mutant p53 in cancer cells

    PubMed Central

    Ali, Amjad; Wang, Zhuo; Fu, Junjiang; Ji, Lei; Liu, Jiang; Li, Lei; Wang, Hui; Chen, Jiwu; Caulin, Carlos; Myers, Jeffrey N.; Zhang, Pei; Xiao, Jianru; Zhang, Bianhong; Li, Xiaotao

    2013-01-01

    Proteasome activity is frequently enhanced in cancer to accelerate metastasis and tumorigenesis. REGγ, a proteasome activator known to promote p53/p21/p16 degradation, is often overexpressed in cancer cells. Here we show that p53/TGF-β signalling inhibits the REGγ–20S proteasome pathway by repressing REGγ expression. Smad3 and p53 interact on the REGγ promoter via the p53RE/SBE region. Conversely, mutant p53 binds to the REGγ promoter and recruits p300. Importantly, mutant p53 prevents Smad3/N-CoR complex formation on the REGγ promoter, which enhances the activity of the REGγ–20S proteasome pathway and contributes to mutant p53 gain of function. Depletion of REGγ alters the cellular response to p53/TGF-β signalling in drug resistance, proliferation, cell cycle progression and proteasome activity. Moreover, p53 mutations show a positive correlation with REGγ expression in cancer samples. These findings suggest that targeting REGγ–20S proteasome for cancer therapy may be applicable to human tumours with abnormal p53/Smad protein status. Furthermore, this study demonstrates a link between p53/TGF-β signalling and the REGγ–20S proteasome pathway, and provides insight into the REGγ/p53 feedback loop. PMID:24157709

  1. Role of Ubiquitin-Mediated Degradation System in Plant Biology

    PubMed Central

    Sharma, Bhaskar; Joshi, Deepti; Yadav, Pawan K.; Gupta, Aditya K.; Bhatt, Tarun K.

    2016-01-01

    Ubiquitin-mediated proteasomal degradation is an important mechanism to control protein load in the cells. Ubiquitin binds to a protein on lysine residue and usually promotes its degradation through 26S proteasome system. Abnormal proteins and regulators of many processes, are targeted for degradation by the ubiquitin-proteasome system. It allows cells to maintain the response to cellular level signals and altered environmental conditions. The ubiquitin-mediated proteasomal degradation system plays a key role in the plant biology, including abiotic stress, immunity, and hormonal signaling by interfering with key components of these pathways. The involvement of the ubiquitin system in many vital processes led scientists to explore more about the ubiquitin machinery and most importantly its targets. In this review, we have summarized recent discoveries of the plant ubiquitin system and its involvement in critical processes of plant biology. PMID:27375660

  2. Role of Ubiquitin-Mediated Degradation System in Plant Biology.

    PubMed

    Sharma, Bhaskar; Joshi, Deepti; Yadav, Pawan K; Gupta, Aditya K; Bhatt, Tarun K

    2016-01-01

    Ubiquitin-mediated proteasomal degradation is an important mechanism to control protein load in the cells. Ubiquitin binds to a protein on lysine residue and usually promotes its degradation through 26S proteasome system. Abnormal proteins and regulators of many processes, are targeted for degradation by the ubiquitin-proteasome system. It allows cells to maintain the response to cellular level signals and altered environmental conditions. The ubiquitin-mediated proteasomal degradation system plays a key role in the plant biology, including abiotic stress, immunity, and hormonal signaling by interfering with key components of these pathways. The involvement of the ubiquitin system in many vital processes led scientists to explore more about the ubiquitin machinery and most importantly its targets. In this review, we have summarized recent discoveries of the plant ubiquitin system and its involvement in critical processes of plant biology. PMID:27375660

  3. Mitotic phosphorylation of Bloom helicase at Thr182 is required for its proteasomal degradation and maintenance of chromosomal stability.

    PubMed

    Kharat, S S; Tripathi, V; Damodaran, A P; Priyadarshini, R; Chandra, S; Tikoo, S; Nandhakumar, R; Srivastava, V; Priya, S; Hussain, M; Kaur, S; Fishman, J B; Sengupta, S

    2016-02-25

    Mutations in Bloom helicase (BLM) lead to Bloom Syndrome (BS). BS is characterized by multiple clinical manifestations including predisposition to a wide spectrum of cancers. Studies have revealed the mechanism of BLM recruitment after stalled replication and its role during the repair of DNA damage. We now provide evidence that BLM undergoes K48-linked ubiquitylation and subsequent degradation during mitosis due to the E3 ligase, Fbw7α. Fbw7α carries out its function after GSK3β- and CDK2/cyclin A2-dependent phosphorylation events on Thr171 and Ser175 of BLM which lies within a well-defined phosphodegron, a sequence which is conserved in all primates. Phosphorylation on BLM Thr171 and Ser175 depends on prior phosphorylation at Thr182 by Chk1/Chk2. Thr182 phosphorylation not only controls BLM ubiquitylation and degradation during mitosis but is also a determinant for its localization on the ultrafine bridges. Consequently lack of Thr182 phosphorylation leads to multiple manifestations of chromosomal instability including increased levels of DNA damage, lagging chromatin, micronuclei formation, breaks and quadriradials. Hence Thr182 phosphorylation on BLM has two functions-it regulates BLM turnover during mitosis and also helps to maintain the chromosomal stability. PMID:26028025

  4. The recognition of ubiquitinated proteins by the proteasome.

    PubMed

    Grice, Guinevere L; Nathan, James A

    2016-09-01

    The ability of ubiquitin to form up to eight different polyubiquitin chain linkages generates complexity within the ubiquitin proteasome system, and accounts for the diverse roles of ubiquitination within the cell. Understanding how each type of ubiquitin linkage is correctly interpreted by ubiquitin binding proteins provides important insights into the link between chain recognition and cellular fate. A major function of ubiquitination is to signal degradation of intracellular proteins by the 26S proteasome. Lysine-48 (K48) linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation, but recent studies show a role for other ubiquitin linked chains in facilitating degradation by the 26S proteasome. Here, we review how different types of polyubiquitin linkage bind to ubiquitin receptors on the 26S proteasome, how they signal degradation and discuss the implications of ubiquitin chain linkage in regulating protein breakdown by the proteasome. PMID:27137187

  5. Blm10 facilitates nuclear import of proteasome core particles

    PubMed Central

    Weberruss, Marion H; Savulescu, Anca F; Jando, Julia; Bissinger, Thomas; Harel, Amnon; Glickman, Michael H; Enenkel, Cordula

    2013-01-01

    Short-lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT-like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP-CP assemblies are primarily nuclear and promote cell division. During quiescence, RP-CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10-bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG-rich nucleoporins and is dissociated from the CP by Ran-GTP. Thus, Blm10 represents the first CP-dedicated nuclear import receptor in yeast. PMID:23982732

  6. Proteasome as a Molecular Target of Microcystin-LR

    PubMed Central

    Zhu, Zhu; Zhang, Li; Shi, Guoqing

    2015-01-01

    Proteasome degrades proteins in eukaryotic cells. As such, the proteasome is crucial in cell cycle and function. This study proved that microcystin-LR (MC-LR), which is a toxic by-product of algal bloom, can target cellular proteasome and selectively inhibit proteasome trypsin-like (TL) activity. MC-LR at 1 nM can inhibit up to 54% of the purified 20S proteasome TL activity and 43% of the proteasome TL activity in the liver of the cyprinid rare minnow (Gobiocypris rarus). Protein degradation was retarded in GFP-CL1-transfected PC-3 cells because MC-LR inhibited the proteasome TL activity. Docking studies indicated that MC-LR blocked the active site of the proteasome β2 subunit; thus, the proteasome TL activity was inhibited. In conclusion, MC-LR can target proteasome, selectively inhibit proteasome TL activity, and retard protein degradation. This study may be used as a reference of future research on the toxic mechanism of MC-LR. PMID:26090622

  7. Anaphase promoting complex–dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae

    PubMed Central

    Ostapenko, Denis; Solomon, Mark J.

    2011-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is an essential ubiquitin ligase that targets cell cycle proteins for proteasome-mediated degradation in mitosis and G1. The APC regulates a number of cell cycle processes, including spindle assembly, mitotic exit, and cytokinesis, but the full range of its functions is still unknown. To better understand cellular pathways controlled by the APC, we performed a proteomic screen to identify additional APC substrates. We analyzed cell cycle–regulated proteins whose expression peaked during the period when other APC substrates were expressed. Subsequent analysis identified several proteins, including the transcriptional repressors Nrm1 and Yhp1, as authentic APC substrates. We found that APCCdh1 targeted Nrm1 and Yhp1 for degradation in early G1 through Destruction-box motifs and that the degradation of these repressors coincided with transcriptional activation of MBF and Mcm1 target genes, respectively. In addition, Nrm1 was stabilized by phosphorylation, most likely by the budding yeast cyclin–dependent protein kinase, Cdc28. We found that expression of stabilized forms of Nrm1 and Yhp1 resulted in reduced cell fitness, due at least in part to incomplete activation of G1-specific genes. Therefore, in addition to its known functions, APC-mediated targeting of Nrm1 and Yhp1 coordinates transcription of multiple genes in G1 with other cell cycle events. PMID:21562221

  8. Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation

    PubMed Central

    Zakaria, Siti Mariam; Frings, Oliver; Fahlén, Sara; Nilsson, Helén; Goodwin, Jacob; von der Lehr, Natalie; Su, Yingtao; Lüscher, Bernhard; Castell, Alina; Larsson, Lars-Gunnar

    2016-01-01

    The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27Kip1 (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157 - a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27KIP1 potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc. PMID:26701207

  9. Bacterial Proteasome Activator Bpa (Rv3780) Is a Novel Ring-Shaped Interactor of the Mycobacterial Proteasome

    PubMed Central

    Delley, Cyrille L.; Laederach, Juerg; Ziemski, Michal; Bolten, Marcel; Boehringer, Daniel; Weber-Ban, Eilika

    2014-01-01

    The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins. PMID:25469515

  10. Sodium nitroprusside promotes IRP2 degradation via an increase in intracellular iron and in the absence of S nitrosylation at C178.

    PubMed

    Wang, Jian; Fillebeen, Carine; Chen, Guohua; Andriopoulos, Bill; Pantopoulos, Kostas

    2006-03-01

    In iron-replete cells the posttranscriptional regulator IRP2 undergoes ubiquitination and proteasomal degradation. A similar response occurs in cells exposed to sodium nitroprusside (SNP), an NO-releasing drug. It has been proposed that nitroprusside ([Fe(CN)5NO]2-) fails to donate iron into cells and that it promotes IRP2 degradation via S nitrosylation at C178. This residue is located within a stretch of 73 amino acids, earlier proposed to define an iron-dependent degradation domain. Surprisingly, we show that IRP2 bearing a C178S mutation or a Delta73 deletion is sensitive to degradation not only by ferric ammonium citrate (FAC) but also by SNP. Moreover, FAC and SNP attenuate the RNA-binding activities of IRP2 and its homologue IRP1 with similar kinetics. Actinomycin D, cycloheximide, succinylacetone, and dimethyl-oxalylglycine antagonize IRP2 degradation in response to both FAC and SNP, suggesting a common mechanistic basis. IRP2 is not only sensitive to fresh, but also to photodegraded SNP and remains unaffected by S-nitrosoglutathione (GSNO), an established nitrosation agent. Importantly, both fresh and photodegraded SNP, but not GSNO, promote a >4-fold increase in the calcein-accessible labile iron pool. Collectively, these results suggest that IRP2 degradation by SNP does not require S nitrosylation but rather represents a response to iron loading.

  11. How the ubiquitin proteasome system regulates the regulators of transcription.

    PubMed

    Ee, Gary; Lehming, Norbert

    2012-01-01

    The ubiquitin proteasome system plays an important role in transcription. Monoubiquitination of activators is believed to aid their function, while the 26S proteasomal degradation of repressors is believed to restrict their function. What remains controversial is the question of whether the degradation of activators aids or restricts their function.

  12. HSP90 protects the human T-cell leukemia virus type 1 (HTLV-1) tax oncoprotein from proteasomal degradation to support NF-κB activation and HTLV-1 replication.

    PubMed

    Gao, Linlin; Harhaj, Edward William

    2013-12-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients.

  13. Inhibition of all-trans-retinoic acid-induced proteasome activation potentiates the differentiating effect of retinoid in acute myeloid leukemia cells.

    PubMed

    Fang, Yanfen; Zhou, Xinglu; Lin, Meihua; Ying, Meidan; Luo, Peihua; Zhu, Difeng; Lou, Jianshu; Yang, Bo; He, Qiaojun

    2011-01-01

    All-trans retinoic acid (ATRA) is nowadays considered to be the sole efficient agent for differentiation-based therapy in leukemia; however, the mechanisms of ATRA's biological effects remain largely unknown. Here we first reported that ATRA-induced myeloid leukemia differentiation was accompanied with the increased level of ubiquitin-protein conjugates and the upregulation of proteasome activity. To explore the functional role of the activated proteasome in retinoic acid (RA) signaling, the effects of proteasome inhibitors on RA-induced cell differentiation were determined. Our results demonstrated that inhibition of ATRA-elevated proteasome activity obviously promoted the myeloid maturation program triggered by ATRA, suggesting that the overactivated proteasome is not beneficial for ATRA's effects. Further studies demonstrated that the synergistic differentiating effects of ATRA and proteasome inhibitors might be associated with the protection of retinoic acid receptor alpha (RARα) from degradation by the ubiquitin-proteasome pathway (UPP). Moreover, the accumulated RARα was able to enhance the transcription of its target gene, which might also contribute to the enhanced differentiation of leukemia cells. Together, by linking the UPP to ATRA-dependent signaling, our data provide a novel insight into studying the mechanisms of ATRA-elicited cellular effects and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy.

  14. Native structure of rat liver immune proteasomes.

    PubMed

    Stepanova, A A; Lyupina, Yu V; Sharova, N P; Erokhov, P A

    2016-05-01

    Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of "rotated" ubiquitinated proteins. PMID:27417720

  15. Proteasomes and protein conjugation across domains of life

    PubMed Central

    Maupin-Furlow, Julie

    2012-01-01

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes. PMID:22183254

  16. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells

    PubMed Central

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M.; Pyne, Nigel J.; Pyne, Susan

    2016-01-01

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest. PMID:26934645

  17. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

    PubMed

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

    2016-03-29

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

  18. Disruption of NAD(P)H:quinone oxidoreductase 1 gene in mice leads to 20S proteasomal degradation of p63 resulting in thinning of epithelium and chemical-induced skin cancer.

    PubMed

    Patrick, B A; Gong, X; Jaiswal, A K

    2011-03-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic enzyme that protects cells against chemical and radiation-induced oxidative stress and skin cancer. Disruption of NQO1 gene in mice showed thinning of skin epithelium and loss of cytokeratin 14, an early marker of skin differentiation. Immunohistochemistry and western analysis demonstrated downregulation of p63 in NQO1-/- mouse skin, as compared with wild-type (WT) mouse. Further analysis including modulation of NQO1 expression revealed a direct correlation between the levels of NQO1 and p63 in skin-derived keratinocytes and dermal fibroblasts. Modulation of proteasomal activity revealed that p63 is degraded by 20S proteasome and that this degradation is significantly rescued by NQO1. Coimmunoprecipitation studies showed that NQO1 interacts directly with p63 but not 20S to protect against this degradation. In addition, benzo[a]pyrene treatment led to induction of NQO1 and stabilization of p63 in WT but not in NQO1-/- mouse skin and keratinocytes. These data suggest that NQO1 controls stabilization of p63 and progression towards keratinocyte differentiation leading to normal skin development and presumably skin carcinogenesis.

  19. HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest*

    PubMed Central

    Romani, Bizhan; Shaykh Baygloo, Nima; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2015-01-01

    Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G2/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G2/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G2/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G2/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP. PMID:26032416

  20. HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest.

    PubMed

    Romani, Bizhan; Shaykh Baygloo, Nima; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2015-07-10

    Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G2/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G2/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G2/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G2/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP.

  1. Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

    SciTech Connect

    Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung; Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho; Lee, Gye Won; Yun, Mi-Young; Cuong, Nguyen Manh; Shin, Jae-Gook; Song, Gyu-Yong; Oh, Sangtaek

    2010-01-01

    Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

  2. Ell3 stabilizes p53 following CDDP treatment via its effects on ubiquitin-dependent and -independent proteasomal degradation pathways in breast cancer cells

    PubMed Central

    Ahn, Hee-Jin; Kim, Kwang-Soo; Shin, Kyung-Won; Lim, Kee-Hwan; Kim, Jin-Ock; Lee, Je-Yong; Kim, Jiewan; Park, Ji-Hoon; Yang, Kyung-Min; Baek, Kwang-Hyun; Ko, Jeong-Jae; Park, Kyung-Soon

    2015-01-01

    The tumor suppressor protein p53 is unstable in quiescent cells and undergoes proteosomal degradation. Under conditions of cellular stress, p53 is rapidly stabilized by post-translational modification, thereby escaping degradation and translocating to the nucleus where it activates genes related to cell cycle arrest or apoptosis. Here, we report that the transcription elongation factor Ell3 sensitizes luminal type-cancer cell line, MCF7, which have wild-type p53, to the chemotherapeutic agent cis-diamminedichloroplatinum(II) (CDDP) by stabilizing p53. Overexpression of Ell3 in MCF7 cells suppressed the MDM2-mediated ubiquitin-dependent degradation pathway. In addition, Ell3 promoted binding of p53 to NADH quinone oxidoreductase 1, which is linked to the ubiquitin-independent degradation of p53. We found that Ell3 activates interleukin-20 (IL20) expression, which is linked to the ERK1/2 signaling pathway. Chemical inhibition of ERK1/2 signaling or molecular suppression of IL20 revealed that the ERK1/2 signaling pathway and IL20 are the main causes of p53 stabilization in Ell3-overexpressing MCF7 cells. These findings suggest that the ERK1/2 pathway can be targeted in the rational development of therapies to induce chemosensitization of breast cancer cells. PMID:26540344

  3. Phosphorylation of the C-terminal tail of proteasome subunit α7 is required for binding of the proteasome quality control factor Ecm29

    PubMed Central

    Wani, Prashant S.; Suppahia, Anjana; Capalla, Xavier; Ondracek, Alex; Roelofs, Jeroen

    2016-01-01

    The proteasome degrades many short-lived proteins that are labeled with an ubiquitin chain. The identification of phosphorylation sites on the proteasome subunits suggests that degradation of these substrates can also be regulated at the proteasome. In yeast and humans, the unstructured C-terminal region of α7 contains an acidic patch with serine residues that are phosphorylated. Although these were identified more than a decade ago, the molecular implications of α7 phosphorylation have remained unknown. Here, we showed that yeast Ecm29, a protein involved in proteasome quality control, requires the phosphorylated tail of α7 for its association with proteasomes. This is the first example of proteasome phosphorylation dependent binding of a proteasome regulatory factor. Ecm29 is known to inhibit proteasomes and is often found enriched on mutant proteasomes. We showed that the ability of Ecm29 to bind to mutant proteasomes requires the α7 tail binding site, besides a previously characterized Rpt5 binding site. The need for these two binding sites, which are on different proteasome subcomplexes, explains the specificity of Ecm29 for proteasome holoenzymes. We propose that alterations in the relative position of these two sites in different conformations of the proteasome provides Ecm29 the ability to preferentially bind specific proteasome conformations. PMID:27302526

  4. Proteasome Inhibition Increases Recruitment of IκB Kinase β (IKKβ), S536P-p65, and Transcription Factor EGR1 to Interleukin-8 (IL-8) Promoter, Resulting in Increased IL-8 Production in Ovarian Cancer Cells*

    PubMed Central

    Singha, Bipradeb; Gatla, Himavanth Reddy; Manna, Subrata; Chang, Tzu-Pei; Sanacora, Shannon; Poltoratsky, Vladimir; Vancura, Ales; Vancurova, Ivana

    2014-01-01

    Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase β (IKKβ) and an increased recruitment of the nuclear IKKβ, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKβ and with p65, with preferential binding to S536P-p65. Both IKKβ activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKβ and EGR-1 as potential new targets in ovarian cancer combination therapies. PMID:24337575

  5. Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

    SciTech Connect

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji Saeki, Yasushi

    2013-07-05

    Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

  6. Phosphorylation by p38 Mitogen-Activated Protein Kinase Promotes Estrogen Receptor α Turnover and Functional Activity via the SCFSkp2 Proteasomal Complex

    PubMed Central

    Bhatt, Shweta; Xiao, Zhen; Meng, Zhaojing

    2012-01-01

    The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCFSkp2 proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers. PMID:22431515

  7. Proteasome inhibition enhances the killing effect of BikDD gene therapy.

    PubMed

    Sun, Ye; Ponz-Sarvise, Mariano; Chang, Shih-Shin; Chang, Wei-Chao; Chen, Chung-Hsuan; Hsu, Jennifer L; Hung, Mien-Chie

    2015-01-01

    BikDD, a phosphorylation-mimic mutant of pro-apoptotic protein Bik, elicits strong apoptosis in cancer cells when introduced via an expression platform termed VP16-GAL4-WPRE integrated systemic amplifier (VISA) under the control of a cancer-specific promoter both in vitro and in vivo. C-VISA-BikDD expression plasmid encapsulated in liposomes is currently in the process to initiate a phase I clinical trial for pancreatic cancer. In this study, we report a potential combination approach of BikDD with proteasome inhibitors on the basis of our findings that exogenously expressed BikDD protein undergoes proteasome-mediated degradation via both ubiquitin-dependent and -independent pathways. Inhibition of proteasome increases the protein stability of BikDD, enhancing the apoptotic effect of BikDD. Hence, high proteasome activity may be a mechanism by which intrinsic and acquired resistance occurs in BikDD gene therapy, and a combination therapy with current clinically approved proteasome inhibitor may overcome resistance. PMID:25901200

  8. Fast degradable citrate-based bone scaffold promotes spinal fusion

    PubMed Central

    Tang, Jiajun; Guo, Jinshan; Li, Zhen; Yang, Cheng; Xie, Denghui; Chen, Jian; Li, Shengfa; Li, Shaolin; Kim, Gloria B.; Bai, Xiaochun; Zhang, Zhongmin; Yang, Jian

    2015-01-01

    It is well known that high rates of fusion failure and pseudoarthrosis development (5~35%) are concomitant in spinal fusion surgery, which was ascribed to the shortage of suitable materials for bone regeneration. Citrate was recently recognized to play an indispensable role in enhancing osteconductivity and osteoinductivity, and promoting bone formation. To address the material challenges in spinal fusion surgery, we have synthesized mechanically robust and fast degrading citrate-based polymers by incorporating N-methyldiethanolamine (MDEA) into clickable poly(1, 8-octanediol citrates) (POC-click), referred to as POC-M-click. The obtained POC-M-click were fabricated into POC-M-click-HA matchstick scaffolds by compositing with hydroxyapatite (HA) for interbody spinal fusion in a rabbit model. Spinal fusion was analyzed by radiography, manual palpation, biomechanical testing, and histological evaluation. At 4 and 8 weeks post surgery, POC-M-click-HA scaffolds presented optimal degradation rates that facilitated faster new bone formation and higher spinal fusion rates (11.2±3.7, 80±4.5 at week 4 and 8, respectively) than the poly(L-lactic acid)-HA (PLLA-HA) control group (9.3±2.4 and 71.1±4.4) (p<0.05). The POC-M-click-HA scaffold-fused vertebrates possessed a maximum load and stiffness of 880.8±14.5 N and 843.2±22.4 N/mm, respectively, which were also much higher than those of the PLLA-HA group (maximum: 712.0±37.5 N, stiffness: 622.5±28.4 N/mm, p<0.05). Overall, the results suggest that POC-M-click-HA scaffolds could potentially serve as promising bone grafts for spinal fusion applications. PMID:26213625

  9. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  10. Tussilagone suppresses colon cancer cell proliferation by promoting the degradation of β-catenin

    SciTech Connect

    Li, Hua; Lee, Hwa Jin; Ahn, Yeon Hwa; Kwon, Hye Jin; Jang, Chang-Young; Kim, Woo-Young; Ryu, Jae-Ha

    2014-01-03

    Highlights: •Tussilagone (TSL) was purified from plant as an inhibitor of Wnt/β-catenin pathway. •TSL suppressed the β-catenin/T-cell factor transcriptional activity. •The proteasomal degradation of β-catenin was induced by TSL. •TSL suppressed the Wnt/β-catenin target genes, cyclin D1 and c-myc. •TSL inhibit the proliferation of colon cancer cells. -- Abstract: Abnormal activation of the Wnt/β-catenin signaling pathway frequently induces colon cancer progression. In the present study, we identified tussilagone (TSL), a compound isolated from the flower buds of Tussilago farfara, as an inhibitor on β-catenin dependent Wnt pathway. TSL suppressed β-catenin/T-cell factor transcriptional activity and down-regulated β-catenin level both in cytoplasm and nuclei of HEK293 reporter cells when they were stimulated by Wnt3a or activated by an inhibitor of glycogen synthase kinase-3β. Since the mRNA level was not changed by TSL, proteasomal degradation might be responsible for the decreased level of β-catenin. In SW480 and HCT116 colon cancer cell lines, TSL suppressed the β-catenin activity and also decreased the expression of cyclin D1 and c-myc, representative target genes of the Wnt/β-catenin signaling pathway, and consequently inhibited the proliferation of colon cancer cells. Taken together, TSL might be a potential chemotherapeutic agent for the prevention and treatment of human colon cancer.

  11. Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

    PubMed Central

    Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

    2016-01-01

    Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced

  12. The CaVβ Subunit Protects the I-II Loop of the Voltage-gated Calcium Channel CaV2.2 from Proteasomal Degradation but Not Oligoubiquitination.

    PubMed

    Page, Karen M; Rothwell, Simon W; Dolphin, Annette C

    2016-09-23

    CaVβ subunits interact with the voltage-gated calcium channel CaV2.2 on a site in the intracellular loop between domains I and II (the I-II loop). This interaction influences the biophysical properties of the channel and leads to an increase in its trafficking to the plasma membrane. We have shown previously that a mutant CaV2.2 channel that is unable to bind CaVβ subunits (CaV2.2 W391A) was rapidly degraded (Waithe, D., Ferron, L., Page, K. M., Chaggar, K., and Dolphin, A. C. (2011) J. Biol. Chem. 286, 9598-9611). Here we show that, in the absence of CaVβ subunits, a construct consisting of the I-II loop of CaV2.2 was directly ubiquitinated and degraded by the proteasome system. Ubiquitination could be prevented by mutation of all 12 lysine residues in the I-II loop to arginines. Including a palmitoylation motif at the N terminus of CaV2.2 I-II loop was insufficient to target it to the plasma membrane in the absence of CaVβ subunits even when proteasomal degradation was inhibited with MG132 or ubiquitination was prevented by the lysine-to-arginine mutations. In the presence of CaVβ subunit, the palmitoylated CaV2.2 I-II loop was protected from degradation, although oligoubiquitination could still occur, and was efficiently trafficked to the plasma membrane. We propose that targeting to the plasma membrane requires a conformational change in the I-II loop that is induced by binding of the CaVβ subunit. PMID:27489103

  13. The CaVβ Subunit Protects the I-II Loop of the Voltage-gated Calcium Channel CaV2.2 from Proteasomal Degradation but Not Oligoubiquitination*

    PubMed Central

    Page, Karen M.; Rothwell, Simon W.; Dolphin, Annette C.

    2016-01-01

    CaVβ subunits interact with the voltage-gated calcium channel CaV2.2 on a site in the intracellular loop between domains I and II (the I-II loop). This interaction influences the biophysical properties of the channel and leads to an increase in its trafficking to the plasma membrane. We have shown previously that a mutant CaV2.2 channel that is unable to bind CaVβ subunits (CaV2.2 W391A) was rapidly degraded (Waithe, D., Ferron, L., Page, K. M., Chaggar, K., and Dolphin, A. C. (2011) J. Biol. Chem. 286, 9598–9611). Here we show that, in the absence of CaVβ subunits, a construct consisting of the I-II loop of CaV2.2 was directly ubiquitinated and degraded by the proteasome system. Ubiquitination could be prevented by mutation of all 12 lysine residues in the I-II loop to arginines. Including a palmitoylation motif at the N terminus of CaV2.2 I-II loop was insufficient to target it to the plasma membrane in the absence of CaVβ subunits even when proteasomal degradation was inhibited with MG132 or ubiquitination was prevented by the lysine-to-arginine mutations. In the presence of CaVβ subunit, the palmitoylated CaV2.2 I-II loop was protected from degradation, although oligoubiquitination could still occur, and was efficiently trafficked to the plasma membrane. We propose that targeting to the plasma membrane requires a conformational change in the I-II loop that is induced by binding of the CaVβ subunit. PMID:27489103

  14. Ubiquitin-proteasome pathway and cellular responses to oxidative stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Subs...

  15. Ubiquitin-proteasome pathway and cellular responses to oxidative stress

    PubMed Central

    Taylor, Allen

    2011-01-01

    The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in non-canonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. While many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin conjugation enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin conjugation enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells appear to be an indicator of mild oxidative stress. PMID:21530648

  16. Isolation and purification of proteasomes from primary cells.

    PubMed

    Steers, Nicholas J; Peachman, Kristina K; Alving, Carl R; Rao, Mangala

    2014-11-03

    Proteasomes play an important role in cell homeostasis and in orchestrating the immune response by systematically degrading foreign proteins and misfolded or damaged host cell proteins. We describe a protocol to purify functionally active proteasomes from human CD4(+) T cells and dendritic cells derived from peripheral blood mononuclear cells. The purification is a three-step process involving ion-exchange chromatography, ammonium sulfate precipitation, and sucrose density gradient ultracentrifugation. This method can be easily adapted to purify proteasomes from cell lines or from organs. Methods to characterize and visualize the purified proteasomes are also described.

  17. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    PubMed

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy. PMID:27465334

  18. ω-3 PUFAs ameliorate liver fibrosis and inhibit hepatic stellate cells proliferation and activation by promoting YAP/TAZ degradation.

    PubMed

    Zhang, Kun; Chang, Yanan; Shi, Zhemin; Han, Xiaohui; Han, Yawei; Yao, Qingbin; Hu, Zhimei; Cui, Hongmei; Zheng, Lina; Han, Tao; Hong, Wei

    2016-07-20

    Elevated levels of the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivators with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, have been shown to play essential roles in controlling liver cell fate and the activation of hepatic stellate cells (HSCs). The dietary intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been positively associated with a number of health benefits including prevention and reduction of cardiovascular diseases, inflammation and cancers. However, little is known about the impact of ω-3 PUFAs on liver fibrosis. In this study, we used CCl4-induced liver fibrosis mouse model and found that YAP/TAZ is over-expressed in the fibrotic liver and activated HSCs. Fish oil administration to the model mouse attenuates CCl4-induced liver fibrosis. Further study revealed that ω-3 PUFAs down-regulate the expression of pro-fibrogenic genes in activated HSCs and fibrotic liver, and the down-regulation is mediated via YAP, thus identifying YAP as a target of ω-3 PUFAs. Moreover, ω-3 PUFAs promote YAP/TAZ degradation in a proteasome-dependent manner. Our data have identified a mechanism of ω-3 PUFAs in ameliorating liver fibrosis.

  19. ω-3 PUFAs ameliorate liver fibrosis and inhibit hepatic stellate cells proliferation and activation by promoting YAP/TAZ degradation

    PubMed Central

    Zhang, Kun; Chang, Yanan; Shi, Zhemin; Han, Xiaohui; Han, Yawei; Yao, Qingbin; Hu, Zhimei; Cui, Hongmei; Zheng, Lina; Han, Tao; Hong, Wei

    2016-01-01

    Elevated levels of the transcriptional regulators Yes-associated protein (YAP) and transcriptional coactivators with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, have been shown to play essential roles in controlling liver cell fate and the activation of hepatic stellate cells (HSCs). The dietary intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) has been positively associated with a number of health benefits including prevention and reduction of cardiovascular diseases, inflammation and cancers. However, little is known about the impact of ω-3 PUFAs on liver fibrosis. In this study, we used CCl4-induced liver fibrosis mouse model and found that YAP/TAZ is over-expressed in the fibrotic liver and activated HSCs. Fish oil administration to the model mouse attenuates CCl4-induced liver fibrosis. Further study revealed that ω-3 PUFAs down-regulate the expression of pro-fibrogenic genes in activated HSCs and fibrotic liver, and the down-regulation is mediated via YAP, thus identifying YAP as a target of ω-3 PUFAs. Moreover, ω-3 PUFAs promote YAP/TAZ degradation in a proteasome-dependent manner. Our data have identified a mechanism of ω-3 PUFAs in ameliorating liver fibrosis. PMID:27435808

  20. Physiological levels of ATP Negatively Regulate Proteasome Function

    PubMed Central

    Huang, Hongbiao; Zhang, Xiaoyan; Li, Shujue; Liu, Ningning; Lian, Wen; McDowell, Emily; Zhou, Ping; Zhao, Canguo; Guo, Haiping; Zhang, Change; Yang, Changshan; Wen, Guangmei; Dong, Xiaoxian; Lu, Li; Ma, Ningfang; Dong, Weihua; Dou, Q. Ping; Wang, Xuejun; Liu, Jinbao

    2010-01-01

    Intracellular protein degradation by the ubiquitin-proteasome system is ATP-dependent and the optimal ATP concentration to activate proteasome function in vitro is ~100 μM. Intracellular ATP levels are generally in the low millimolar range but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50–100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated, the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly up-regulate proteasome activity upon ATP reduction under stress conditions. PMID:20805844

  1. Proteasome inhibitors suppress the protein expression of mutant p53.

    PubMed

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.

  2. Proteasome inhibitors suppress the protein expression of mutant p53

    PubMed Central

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53. PMID:25485499

  3. Regulation of Cardiac Proteasomes by Ubiquitination, Sumoylation, and Beyond

    PubMed Central

    Cui, Ziyou; Scruggs, Sarah B.; Gilda, Jennifer E.; Ping, Peipei; Gomes, Aldrin V.

    2013-01-01

    The ubiquitin-proteasome system (UPS) is the major intracellular degradation system, and its proper function is critical to the health and function of cardiac cells. Alterations in cardiac proteasomes have been linked to several pathological phenotypes, including cardiomyopathies, ischemia-reperfusion injury, heart failure, and hypertrophy. Defects in proteasome-dependent cellular protein homeostasis can be causal for the initiation and progression of certain cardiovascular diseases. Emerging evidence suggests that the UPS can specifically target proteins that govern pathological signaling pathways for degradation, thus altering downstream effectors and disease outcomes. Alterations in UPS-substrate interactions in disease occur, in part, due to direct modifications of 19S, 11S or 20S proteasome subunits. Post-translational modifications (PTMs) are one facet of this proteasomal regulation, with over 400 known phosphorylation sites, over 500 ubiquitination sites and 83 internal lysine acetylation sites, as well as multiple sites for caspase cleavage, glycosylation (such as O-GlcNAc modification), methylation, nitrosylation, oxidation, and sumoylation. Changes in cardiac proteasome PTMs, which occur in ischemia and cardiomyopathies, are associated with changes in proteasome activity and proteasome assembly; however several features of this regulation remain to be explored. In this review, we focus on how some of the less common PTMs affect proteasome function and alter cellular protein homeostasis. PMID:24140722

  4. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  5. Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation

    PubMed Central

    Elharar, Yifat; Roth, Ziv; Hermelin, Inna; Moon, Alexandra; Peretz, Gabriella; Shenkerman, Yael; Vishkautzan, Marina; Khalaila, Isam; Gur, Eyal

    2014-01-01

    Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S-proteasome, bacterial prokaryotic ubiquitin-like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup-proteasome system (PPS) is important for virulence, yet its physiological role in non-pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto-regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation. PMID:24986881

  6. Cytoplasmic Hsp70 promotes ubiquitination for endoplasmic reticulum-associated degradation of a misfolded mutant of the yeast plasma membrane ATPase, PMA1.

    PubMed

    Han, Sumin; Liu, Yu; Chang, Amy

    2007-09-01

    Cells have a variety of strategies for dealing with misfolded proteins. Heat shock response involves transcriptional induction of chaperones to promote and/or correct folding, and also activation of the ubiquitin/proteasome system to degrade defective proteins. In the secretory pathway, it is primarily luminal misfolded or unassembled proteins that trigger the unfolded protein response which, like heat shock, induces chaperones and components of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. To understand cellular response to a misfolded polytopic membrane protein of the secretory pathway, we studied Pma1-D378S, a model ERAD substrate. Expression of misfolded Pma1 induces heat shock response in the absence of increased temperature. Overexpression of HSF1, the transcription factor that mediates heat shock response, increases degradation of Pma1-D378S without temperature upshift. Nevertheless, efficient Pma1-D378S degradation occurs in an hsf1 mutant that maintains basal transcription levels but cannot mediate transcriptional activation. Thus, heat shock protein induction enhances but is not necessary for ERAD. The Ssa group of cytoplasmic Hsp70 chaperones is required for ERAD of both Pma1-D378S and another transmembrane ERAD substrate, Ste6*. In the absence of Ssa chaperones, ubiquitination of both substrates is impaired, resulting in stabilization. We suggest a role for Hsp70 cytoplasmic chaperones in recognition by the endoplasmic reticulum-associated ubiquitination machinery.

  7. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  8. Evolution of proteasome regulators in eukaryotes.

    PubMed

    Fort, Philippe; Kajava, Andrey V; Delsuc, Fredéric; Coux, Olivier

    2015-05-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles.

  9. Evolution of Proteasome Regulators in Eukaryotes

    PubMed Central

    Fort, Philippe; Kajava, Andrey V.; Delsuc, Fredéric; Coux, Olivier

    2015-01-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles. PMID:25943340

  10. Structural Basis for the Assembly and Gate Closure Mechanisms of the Mycobacterium tuberculosis 20S Proteasome

    SciTech Connect

    Lin, D.; Li, H; Wang, T; Pan, H; Lin, G; Li, H

    2010-01-01

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  11. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

    SciTech Connect

    Li, D.; Li, H.; Li, H.; Wang, T.; Pan, H.; Lin, G.

    2010-06-16

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  12. Multitrait plant growth promoting (PGP) rhizobacterial isolates from Brassica juncea rhizosphere : Keratin degradation and growth promotion.

    PubMed

    Anwar, Mohmmad Shahbaz; Siddique, Mohammad Tahir; Verma, Amit; Rao, Yalaga Rama; Nailwal, Tapan; Ansari, Mohammad; Pande, Veena

    2014-01-01

    Plant growth promoting (PGP) rhizobacteria, a beneficial microbe colonizing plant roots, enhanced crop productivity and offers an attractive way to replace chemical fertilizers, pesticides, and supplements. The keratinous waste which comprises feathers, hairs, nails, skin and wool creates problem of solid waste management due to presence of highly recalcitrant keratin. The multi traits rhizobacteria effective to remove both keratine from the environment by producing keratinase enzyme and to eradicate the chemical fertilizer by providing different PGP activity is novel achievement. In the present study, the effective PM2 strain of PGPR was isolated from rhizospheric soil of mustard (Brassica juncea) field, Pantnagar and they were identified on the basis of different biochemical tests as belonging to Bacillus genera. Different plant growth promoting activity, feather degradation and keratinolytic activity was performed and found very effective toward all the parameters. Furthermore, the efficient strain PM2 was identified on the basis of 16s rRNA sequencing and confirmed as Bacillus cereus. The strain PM2 might be used efficiently for keratinous waste management and PGP activity. Therefore, the present study suggests that Bacillus cereus have multi traits activity which extremely useful for different PGP activity and biotechnological process involving keratin hydrolysis, feather biodegradation or in the leather industry.

  13. CD3ε recruits Numb to promote TCR degradation.

    PubMed

    Martin-Blanco, Nadia; Jiménez Teja, Daniel; Bretones, Gabriel; Borroto, Aldo; Caraballo, Michael; Screpanti, Isabella; León, Javier; Alarcón, Balbino; Canelles, Matilde

    2016-03-01

    Modulation of TCR signaling upon ligand binding is achieved by changes in the equilibrium between TCR degradation, recycling and synthesis; surprisingly, the molecular mechanism of such an important process is not fully understood. Here, we describe the role of a new player in the mediation of TCR degradation: the endocytic adaptor Numb. Our data show that Numb inhibition leads to abnormal intracellular distribution and defective TCR degradation in mature T lymphocytes. In addition, we find that Numb simultaneously binds to both Cbl and a site within CD3ε that overlaps with the Nck binding site. As a result, Cbl couples specifically to the CD3ε chain to mediate TCR degradation. The present study unveils a novel role of Numb that lies at the heart of TCR signaling initiation and termination.

  14. c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine.

    PubMed

    Chaudhary, Pankaj; Vishwanatha, Jamboor K

    2014-10-15

    Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH2-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIPL/S and Bcl2 proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl2 which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl2 at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-l-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death. PMID:25181458

  15. c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine

    PubMed Central

    Chaudhary, Pankaj; Vishwanatha, Jamboor K.

    2014-01-01

    Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH2-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIPL/S and Bcl2 proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl2 which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl2 at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-L-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death. PMID:25181458

  16. Direct Targeting of β-Catenin by a Small Molecule Stimulates Proteasomal Degradation and Suppresses Oncogenic Wnt/β-Catenin Signaling.

    PubMed

    Hwang, So-Young; Deng, Xianming; Byun, Sanguine; Lee, Chan; Lee, Seung-Joo; Suh, Hyunsuk; Zhang, Jianming; Kang, Qiaofeng; Zhang, Ting; Westover, Kenneth D; Mandinova, Anna; Lee, Sam W

    2016-06-28

    The Wnt/β-catenin signaling pathway plays a major role in tissue homeostasis, and its dysregulation can lead to various human diseases. Aberrant activation of β-catenin is oncogenic and is a critical driver in the development and progression of human cancers. Despite the significant potential of targeting the oncogenic β-catenin pathway for cancer therapy, the development of specific inhibitors remains insufficient. Using a T cell factor (TCF)-dependent luciferase-reporter system, we screened for small-molecule compounds that act against Wnt/β-catenin signaling and identified MSAB (methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate) as a selective inhibitor of Wnt/β-catenin signaling. MSAB shows potent anti-tumor effects selectively on Wnt-dependent cancer cells in vitro and in mouse cancer models. MSAB binds to β-catenin, promoting its degradation, and specifically downregulates Wnt/β-catenin target genes. Our findings might represent an effective therapeutic strategy for cancers addicted to the Wnt/β-catenin signaling pathway. PMID:27320923

  17. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.

    PubMed

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

    2015-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.

  18. Pyrrolidine dithiocarbamate and zinc inhibit proteasome-dependent proteolysis.

    PubMed

    Kim, Insook; Kim, Chul Hoon; Kim, Joo Hee; Lee, Jinu; Choi, Jun Jeong; Chen, Zheng Ai; Lee, Min Goo; Chung, Kwang Chul; Hsu, Chung Y; Ahn, Young Soo

    2004-08-01

    Proteasomes play important roles in a variety of cellular processes such as cell cycle progression, signal transduction and immune responses. Proteasome activity is important in maintaining rapid turnover of short-lived proteins, as well as preventing accumulation of misfolded or damaged proteins. Alteration in ubiquitin-proteasome function may be detrimental to its crucial role in maintaining cellular homeostasis. Here, we have found that treatment of pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, resulted in the accumulation of several proteasome substrates including p53 and p21 in HeLa cells. The PDTC effect was due to an extended half-life of these proteins through the mobilization of zinc. PDTC and/or zinc also increased fluorescence intensity of Ub(G76V)-GFP fusion protein that is degraded rapidly by the ubiquitin-proteasome system. Treatment of cells with zinc induced formation of ubiquitinated inclusions in the centrosome, a histological marker of proteasome inhibition. Western blotting showed zinc-induced increase in laddering bands of polyubiquitin-conjugated proteins. In vitro study, zinc inhibited the ubiquitin-independent proteasomal degradations of p21 and alpha-synuclein. These results suggest that zinc may modulate cell functions through its action on the turnover of proteins that are susceptible to proteasome-dependent proteolysis. PMID:15242777

  19. Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication.

    PubMed

    Murphy, Christopher M; Xu, Yanping; Li, Feng; Nio, Kouki; Reszka-Blanco, Natalia; Li, Xiaodong; Wu, Yaxu; Yu, Yanbao; Xiong, Yue; Su, Lishan

    2016-09-13

    The hepatitis B virus (HBV) regulatory protein X (HBx) activates gene expression from the HBV covalently closed circular DNA (cccDNA) genome. Interaction of HBx with the DDB1-CUL4-ROC1 (CRL4) E3 ligase is critical for this function. Using substrate-trapping proteomics, we identified the structural maintenance of chromosomes (SMC) complex proteins SMC5 and SMC6 as CRL4(HBx) substrates. HBx expression and HBV infection degraded the SMC5/6 complex in human hepatocytes in vitro and in humanized mice in vivo. HBx targets SMC5/6 for ubiquitylation by the CRL4(HBx) E3 ligase and subsequent degradation by the proteasome. Using a minicircle HBV (mcHBV) reporter system with HBx-dependent activity, we demonstrate that SMC5/6 knockdown, or inhibition with a dominant-negative SMC6, enhance HBx null mcHBV-Gluc gene expression. Furthermore, SMC5/6 knockdown rescued HBx-deficient HBV replication in human hepatocytes. These results indicate that a primary function of HBx is to degrade SMC5/6, which restricts HBV replication by inhibiting HBV gene expression. PMID:27626656

  20. Proteasome inhibition induces both antioxidant and hb f responses in sickle cell disease via the nrf2 pathway.

    PubMed

    Pullarkat, Vinod; Meng, Zhuo; Tahara, Stanley M; Johnson, Cage S; Kalra, Vijay K

    2014-01-01

    Oxidant stress is implicated in the manifestations of sickle cell disease including hemolysis and vascular occlusion. Strategies to induce antioxidant response as well as Hb F (α2γ2) have the potential to ameliorate the severity of sickle cell disease. Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) is a transcription factor that regulates antioxidant enzymes as well as γ-globin transcription. The Nrf2 in the cytoplasm is bound to its adapter protein Keap-1 that targets Nrf2 for proteasomal degradation, thereby preventing its nuclear translocation. We examined whether inhibiting the 26S proteasome using the clinically applicable proteasome inhibitors bortezomib and MLN 9708 would promote nuclear translocation of Nrf2, and thereby induce an antioxidant response and as well as Hb F in sickle cell disease. Proteasome inhibitors induced reactive oxygen species (ROS) and thereby increased Nrf2-dependent antioxidant enzyme transcripts, elevated cellular glutathione (GSH) levels and γ-globin transcripts as well as Hb F levels in the K562 cell line and also in erythroid burst forming units (BFU-E) generated from peripheral blood mononuclear cells of sickle cell disease patients. These responses were abolished by siRNA-mediated knockdown of Nrf2. Proteasome inhibitors, especially newer oral agents such as MLN9708 have the potential to be readily translated to clinical trials in sickle cell disease with the dual end points of antioxidant response and Hb F induction.

  1. PHF23 (plant homeodomain finger protein 23) negatively regulates cell autophagy by promoting ubiquitination and degradation of E3 ligase LRSAM1

    PubMed Central

    Wang, Zhenda; Hu, Jia; Li, Ge; Qu, Liujing; He, Qihua; Lou, Yaxin; Song, Quansheng; Ma, Dalong; Chen, Yingyu

    2015-01-01

    Autophagy is a multistep process that involves the degradation and digestion of intracellular components by the lysosome. It has been proved that many core autophagy-related molecules participate in this event. However, new component proteins that regulate autophagy are still being discovered. At present, we report PHF23 (PHD finger protein 23) with a PHD-like zinc finger domain that can negatively regulate autophagy. Data from experiments indicated that the overexpression of PHF23 impaired autophagy, as characterized by decreased levels of LC3B-II and weakened degradation of endogenous and exogenous autophagic substrates. Conversely, knockdown of PHF23 resulted in opposite effects. Molecular mechanism studies suggested that PHF23 interacts with LRSAM1, which is an E3 ligase key for ubiquitin-dependent autophagy against invading bacteria. PHF23 promotes the ubiquitination and proteasome degradation of LRSAM1. We also show that the PHD finger of PHF23 is a functional domain needed for the interaction with LRSAM1. Altogether, our results indicate that PHF23 is a negative regulator associated in autophagy via the LRSAM1 signaling pathway. The physical and functional connection between the PHF23 and LRSAM1 needs further investigation. PMID:25484098

  2. Proteasome Inhibitors Block Development of Plasmodium spp.

    PubMed Central

    Gantt, Soren M.; Myung, Joon Mo; Briones, Marcelo R. S.; Li, Wei Dong; Corey, E. J.; Omura, Satoshi; Nussenzweig, Victor; Sinnis, Photini

    1998-01-01

    Proteasomes degrade most of the proteins inside eukaryotic cells, including transcription factors and regulators of cell cycle progression. Here we show that nanomolar concentrations of lactacystin, a specific irreversible inhibitor of the 20S proteasome, inhibit development of the exoerythrocytic and erythrocytic stages of the malaria parasite. Although lactacystin-treated Plasmodium berghei sporozoites are still invasive, their development into exoerythrocytic forms (EEF) is inhibited in vitro and in vivo. Erythrocytic schizogony of P. falciparum in vitro is also profoundly inhibited when drug treatment of the synchronized parasites is prior, but not subsequent, to the initiation of DNA synthesis, suggesting that the inhibitory effect of lactacystin is cell cycle specific. Lactacystin reduces P. berghei parasitemia in rats, but the therapeutic index is very low. Along with other studies showing that lactacystin inhibits stage-specific transformation in Trypanosoma and Entamoeba spp., these findings highlight the potential of proteasome inhibitors as drugs for the treatment of diseases caused by protozoan parasites. PMID:9756786

  3. The proteasome assembly line

    PubMed Central

    Madura, Kiran

    2013-01-01

    The assembly of the proteasome — the cellular machine that eliminates unwanted proteins — is a carefully choreographed affair, involving a complex sequence of steps overseen by dedicated protein chaperones. PMID:19516331

  4. Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

    PubMed Central

    Tsvetkov, Peter; Mendillo, Marc L; Zhao, Jinghui; Carette, Jan E; Merrill, Parker H; Cikes, Domagoj; Varadarajan, Malini; van Diemen, Ferdy R; Penninger, Josef M; Goldberg, Alfred L; Brummelkamp, Thijn R; Santagata, Sandro; Lindquist, Susan

    2015-01-01

    Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans. DOI: http://dx.doi.org/10.7554/eLife.08467.001 PMID:26327695

  5. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling

    PubMed Central

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  6. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

    PubMed

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  7. Long-term incubation with proteasome inhibitors (PIs) induces IκBα degradation via the lysosomal pathway in an IκB kinase (IKK)-dependent and IKK-independent manner.

    PubMed

    Lee, Kyoung-Hee; Jeong, Jiyeong; Yoo, Chul-Gyu

    2013-11-01

    Proteasome inhibitors (PIs) have been reported to induce apoptosis in many types of tumor. Their apoptotic activities have been suggested to be associated with the up-regulation of molecules implicated in pro-apoptotic cascades such as p53, p21(Waf1), and p27(Kip1). Moreover, the blocking of NF-κB nuclear translocation via the stabilization of IκB is an important mechanism of PI-induced apoptosis. However, we found that long-term incubation with PIs (PS-341 or MG132) increased NF-κB-regulated gene expression such as COX-2, cIAP2, XIAP, and IL-8 in a dose- and time-dependent manner, which was mediated by phosphorylation of IκBα and its subsequent degradation via the alternative route, lysosome. Overexpression of the IκBα superrepressor (IκBα-SR) blocked PI-induced NF-κB activation. Treatment with lysosomal inhibitors (ammonium chloride or chloroquine) or inhibitors of cathepsins (Z-FF-FMK or Z-FA-FMK) or knock-down of LC3B expression by siRNAs suppressed PI-induced IκBα degradation. Furthermore, we found that both IKK-dependent and IKK-independent pathways were required for PI-induced IκBα degradation. Pretreatment with IKKβ specific inhibitor, SC-514, partially suppressed IκBα degradation and IL-8 production by PIs. Blockade of IKK activity using insolubilization by heat shock (HS) and knock-down by siRNAs for IKKβ only delayed IκBα degradation up to 8 h after treatment with PIs. In addition, PIs induced Akt-dependent inactivation of GSK-3β. Inactive GSK-3β accelerated PI-induced IκBα degradation. Overexpression of active GSK-3β (S9A) or knock-down of GSK-3β delayed PI-induced IκBα degradation. Collectively, our data demonstrate that long-term incubation with PIs activates NF-κB, which is mediated by IκBα degradation via the lysosome in an IKK-dependent and IKK-independent manner. PMID:24085292

  8. Identification and functional characteristics of chlorpyrifos-degrading and plant growth promoting bacterium Acinetobacter calcoaceticus.

    PubMed

    Zhao, Lei; Wang, Fei; Zhao, Jiao

    2014-05-01

    A bacterial strain D10 with strong ability of degrading chlorpyrifos was isolated from rhizosphere of chives contaminated with pesticide. It was found that it's capable of utilizing chlorpyrifos as the sole source of carbon for growth, and within the first 4 days the extent of degradation at initial concentration of 100 mg L(-1) was 60.0%. It also showed a high ability of degrading chlorpyrifos in sterilized soil, and the degradation reached up to 60.2% after 18 days. In addition, the strain D10 also showed multiple plant growth-promoting traits of phosphate solubilization, indole-3-acetic acid and siderophore production. The results indicate that the strain D10 has potential in the application of pesticide-degrading and plant growth promotion. Strain D10 was identified as Acinetobacter calcoaceticus based on its morphological, physiological-biochemical properties and the 16S rRNA sequence analysis.

  9. mTOR regulates proteasomal degradation and Dp1/E2F1- mediated transcription of KPNA2 in lung cancer cells

    PubMed Central

    Wang, Chun-I; Chen, Yan-Yu; Wang, Chih-Liang; Yu, Jau-Song; Chang, Yu-Sun; Yu, Chia-Jung

    2016-01-01

    Karyopherin subunit alpha-2 (KPNA2) is overexpressed in various human cancers and is associated with cancer invasiveness and poor prognosis in patient. Nevertheless, the regulation of KPNA2 expression in cancers remains unclear. We herein applied epidermal growth factor (EGF) and five EGF receptor (EGFR)-related kinase inhibitors to investigate the role of EGFR signaling in KPNA2 expression in non-small cell lung cancer (NSCLC) cells. We found that EGFR signaling, particularly the mammalian target of rapamycin (mTOR) activity was positively correlated with KPNA2 protein levels in NSCLC cells. The mTOR inhibitors and mTOR knockdown reduced the protein and mRNA levels of KPNA2 in NSCLC and breast cancer cells. Specifically, rapamycin treatment induced proteasome-mediated KPNA2 protein decay and attenuated the transcriptional activation of KPNA2 by decreasing Dp1/E2F1 level in vivo. Immunoprecipitation assay further revealed that KPNA2 physically associated with the phospho-mTOR/mTOR and this association was abolished by rapamycin treatment. Collectively, our results show for the first time that KPNA2 is transcriptionally and post-translationally regulated by the mTOR pathway and provide new insights into targeted therapy for NSCLC. PMID:27009856

  10. Structure of a Proteasome Pba1-Pba2 Complex

    PubMed Central

    Stadtmueller, Beth M.; Kish-Trier, Erik; Ferrell, Katherine; Petersen, Charisse N.; Robinson, Howard; Myszka, David G.; Eckert, Debra M.; Formosa, Tim; Hill, Christopher P.

    2012-01-01

    The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function. PMID:22930756

  11. EBNA3C Augments Pim-1 Mediated Phosphorylation and Degradation of p21 to Promote B-Cell Proliferation

    PubMed Central

    Banerjee, Shuvomoy; Lu, Jie; Cai, Qiliang; Sun, Zhiguo; Jha, Hem Chandra; Robertson, Erle S.

    2014-01-01

    Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr145 residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers. PMID:25121590

  12. Arsenic trioxide disrupts glioma stem cells via promoting PML degradation to inhibit tumor growth

    PubMed Central

    Zhou, Wenchao; Cheng, Lin; Shi, Yu; Ke, Susan Q.; Huang, Zhi; Fang, Xiaoguang; Chu, Cheng-wei; Xie, Qi; Bian, Xiu-wu; Rich, Jeremy N.; Bao, Shideng

    2015-01-01

    Glioblastoma multiforme (GBM) is the most lethal brain tumor. Tumor relapse in GBM is inevitable despite maximal therapeutic interventions. Glioma stem cells (GSCs) have been found to be critical players in therapeutic resistance and tumor recurrence. Therapeutic drugs targeting GSCs may significantly improve GBM treatment. In this study, we demonstrated that arsenic trioxide (As2O3) effectively disrupted GSCs and inhibited tumor growth in the GSC-derived orthotopic xenografts by targeting the promyelocytic leukaemia (PML). As2O3 treatment induced rapid degradation of PML protein along with severe apoptosis in GSCs. Disruption of the endogenous PML recapitulated the inhibitory effects of As2O3 treatment on GSCs both in vitro and in orthotopic tumors. Importantly, As2O3 treatment dramatically reduced GSC population in the intracranial GBM xenografts and increased the survival of mice bearing the tumors. In addition, As2O3 treatment preferentially inhibited cell growth of GSCs but not matched non-stem tumor cells (NSTCs). Furthermore, As2O3 treatment or PML disruption potently diminished c-Myc protein levels through increased poly-ubiquitination and proteasome degradation of c-Myc. Our study indicated a potential implication of As2O3 in GBM treatment and highlighted the important role of PML/c-Myc axis in the maintenance of GSCs. PMID:26510911

  13. Arsenic trioxide disrupts glioma stem cells via promoting PML degradation to inhibit tumor growth.

    PubMed

    Zhou, Wenchao; Cheng, Lin; Shi, Yu; Ke, Susan Q; Huang, Zhi; Fang, Xiaoguang; Chu, Cheng-wei; Xie, Qi; Bian, Xiu-wu; Rich, Jeremy N; Bao, Shideng

    2015-11-10

    Glioblastoma multiforme (GBM) is the most lethal brain tumor. Tumor relapse in GBM is inevitable despite maximal therapeutic interventions. Glioma stem cells (GSCs) have been found to be critical players in therapeutic resistance and tumor recurrence. Therapeutic drugs targeting GSCs may significantly improve GBM treatment. In this study, we demonstrated that arsenic trioxide (As2O3) effectively disrupted GSCs and inhibited tumor growth in the GSC-derived orthotopic xenografts by targeting the promyelocytic leukaemia (PML). As2O3 treatment induced rapid degradation of PML protein along with severe apoptosis in GSCs. Disruption of the endogenous PML recapitulated the inhibitory effects of As2O3 treatment on GSCs both in vitro and in orthotopic tumors. Importantly, As2O3 treatment dramatically reduced GSC population in the intracranial GBM xenografts and increased the survival of mice bearing the tumors. In addition, As2O3 treatment preferentially inhibited cell growth of GSCs but not matched non-stem tumor cells (NSTCs). Furthermore, As2O3 treatment or PML disruption potently diminished c-Myc protein levels through increased poly-ubiquitination and proteasome degradation of c-Myc. Our study indicated a potential implication of As2O3 in GBM treatment and highlighted the important role of PML/c-Myc axis in the maintenance of GSCs. PMID:26510911

  14. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  15. Proteasome-Mediated Processing of Def1, a Critical Step in the Cellular Response to Transcription Stress

    PubMed Central

    Wilson, Marcus D.; Harreman, Michelle; Taschner, Michael; Reid, James; Walker, Jane; Erdjument-Bromage, Hediye; Tempst, Paul; Svejstrup, Jesper Q.

    2013-01-01

    Summary DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a “mechanism of last resort” employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function. PMID:23993092

  16. Dss1 Is a 26S Proteasome Ubiquitin Receptor

    PubMed Central

    Paraskevopoulos, Konstantinos; Kriegenburg, Franziska; Tatham, Michael H.; Rösner, Heike I.; Medina, Bethan; Larsen, Ida B.; Brandstrup, Rikke; Hardwick, Kevin G.; Hay, Ronald T.; Kragelund, Birthe B.; Hartmann-Petersen, Rasmus; Gordon, Colin

    2014-01-01

    Summary The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48. Atomic resolution data show that Dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues. The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions. Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins. PMID:25306921

  17. Regulation of the proteasome by ATP: implications for ischemic myocardial injury and donor heart preservation.

    PubMed

    Majetschak, Matthias

    2013-08-01

    Several lines of evidence suggest that proteasomes are involved in multiple aspects of myocardial physiology and pathology, including myocardial ischemia-reperfusion injury. It is well established that the 26S proteasome is an ATP-dependent enzyme and that ischemic heart disease is associated with changes in the ATP content of the cardiomyocyte. A functional link between the 26S proteasome, myocardial ATP concentrations, and ischemic cardiac injury, however, has been suggested only recently. This review discusses the currently available data on the pathophysiological role of the cardiac proteasome during ischemia and reperfusion in the context of the cellular ATP content. Depletion of the myocardial ATP content during ischemia appears to activate the 26S proteasome via direct regulatory effects of ATP on 26S proteasome stability and activity. This implies pathological degradation of target proteins by the proteasome and could provide a pathophysiological basis for beneficial effects of proteasome inhibitors in various models of myocardial ischemia. In contrast to that in the ischemic heart, reduced and impaired proteasome activity is detectable in the postischemic heart. The paradoxical findings that proteasome inhibitors showed beneficial effects when administered during reperfusion in some studies could be explained by their anti-inflammatory and immune suppressive actions, leading to reduction of leukocyte-mediated myocardial reperfusion injury. The direct regulatory effects of ATP on the 26S proteasome have implications for the understanding of the contribution of the 26S proteasome to the pathophysiology of the ischemic heart and its possible role as a therapeutic target.

  18. Trial Watch: Proteasomal inhibitors for anticancer therapy

    PubMed Central

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called “ubiquitin-proteasome system” (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients. PMID:27308423

  19. ANG II promotes IGF-IIR expression and cardiomyocyte apoptosis by inhibiting HSF1 via JNK activation and SIRT1 degradation

    PubMed Central

    Huang, C-Y; Kuo, W-W; Yeh, Y-L; Ho, T-J; Lin, J-Y; Lin, D-Y; Chu, C-H; Tsai, F-J; Tsai, C-H; Huang, C-Y

    2014-01-01

    Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure. Our previous studies found that the activation of insulin-like growth factor receptor II (IGF-IIR) signaling was critical for hypertensive angiotensin II (ANG II)-induced cardiomyocyte apoptosis. However, the detailed mechanism by which ANG II regulates IGF-IIR in heart cells remains elusive. In this study, we found that ANG II activated its downstream kinase JNK to increase IGF-IIR expression through the ANG II receptor angiotensin type 1 receptor. JNK activation subsequently led to sirtuin 1 (SIRT1) degradation via the proteasome, thus preventing SIRT1 from deacetylating heat-shock transcription factor 1 (HSF1). The resulting increase in the acetylation of HSF1 impaired its ability to bind to the IGF-IIR promoter region (nt −748 to −585). HSF1 protected cardiomyocytes by acting as a repressor of IGF-IIR gene expression, and ANG II diminished this HSF1-mediated repression through enhanced acetylation, thus activating the IGF-IIR apoptosis pathway. Taken together, these results suggest that HSF1 represses IGF-IIR gene expression to protect cardiomyocytes. ANG II activates JNK to degrade SIRT1, resulting in HSF1 acetylation, which induces IGF-IIR expression and eventually results in cardiac hypertrophy and apoptosis. HSF1 could be a valuable target for developing treatments for cardiac diseases in hypertensive patients. PMID:24786827

  20. GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.

    PubMed

    Negahdar, Maria; Aukrust, Ingvild; Molnes, Janne; Solheim, Marie H; Johansson, Bente B; Sagen, Jørn V; Dahl-Jørgensen, Knut; Kulkarni, Rohit N; Søvik, Oddmund; Flatmark, Torgeir; Njølstad, Pål R; Bjørkhaug, Lise

    2014-01-25

    GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 β-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY.

  1. Stimulation of hERG1 channel activity promotes a calcium-dependent degradation of cyclin E2, but not cyclin E1, in breast cancer cells.

    PubMed

    Perez-Neut, Mathew; Shum, Andrew; Cuevas, Bruce D; Miller, Richard; Gentile, Saverio

    2015-01-30

    Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment. Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators.

  2. The 26S proteasome is a multifaceted target for anti-cancer therapies

    PubMed Central

    Grigoreva, Tatyana A; Tribulovich, Vyacheslav G.; Garabadzhiu, Alexander V.; Melino, Gerry; Barlev, Nickolai A.

    2015-01-01

    Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications. PMID:26295307

  3. Galeterone and VNPT55 induce proteasomal degradation of AR/AR-V7, induce significant apoptosis via cytochrome c release and suppress growth of castration resistant prostate cancer xenografts in vivo.

    PubMed

    Kwegyir-Afful, Andrew K; Ramalingam, Senthilmurugan; Purushottamachar, Puranik; Ramamurthy, Vidya P; Njar, Vincent C O

    2015-09-29

    Galeterone (Gal) is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/ARv567es) in LNCaP, CWR22Rv1 and DU145 (transfected with AR-Vs) human PC cells in vitro and CRPC tumor xenografts. Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy.

  4. Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes.

    PubMed

    Liepe, Juliane; Holzhütter, Hermann-Georg; Bellavista, Elena; Kloetzel, Peter M; Stumpf, Michael P H; Mishto, Michele

    2015-01-01

    Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage.

  5. Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes.

    PubMed

    Liepe, Juliane; Holzhütter, Hermann-Georg; Bellavista, Elena; Kloetzel, Peter M; Stumpf, Michael P H; Mishto, Michele

    2015-01-01

    Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage. PMID:26393687

  6. Proteasome proteolysis supports stimulated platelet function and thrombosis

    PubMed Central

    Gupta, Nilaksh; Li, Wei; Willard, Belinda; Silverstein, Roy L.; McIntyre, Thomas M.

    2014-01-01

    Objective Proteasome inhibitors are in use to treat hematologic cancers, but also reduce thrombosis. Whether the proteasome participates in platelet activation or function is opaque since little is known of the proteasome in these terminally differentiated cells. Approach and Results Platelets displayed all three primary proteasome protease activities, which MG132 and bortezomib (Velcade®) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by mono- and poly-ubiquitination. Systemic MG132 strongly suppressed formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed prior to transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the GPIb-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-, ADP-, and LPS-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal GPIbα binding domain. Conclusions Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet-platelet and platelet-wall interactions. PMID:24177323

  7. Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C

    PubMed Central

    Liang, Kai Ling; Paredes, Roberto; Carmody, Ruaidhri; Eyers, Patrick A.; Meyer, Stefan; McCarthy, Tommie V.; Keeshan, Karen

    2016-01-01

    Tribbles homolog 2 (TRIB2) is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Studies of TRIB2 indicate that many of the molecular interactions between the single Drosophila Tribbles (Trbl) protein and interacting partners are evolutionary conserved. In this study, we examined the relationship between TRIB2 and cell division cycle 25 (CDC25) family of dual-specificity protein phosphatases (mammalian homologues of Drosophila String), which are key physiological cell cycle regulators. Using co-immunoprecipitation we demonstrate that TRIB2 interacts with CDC25B and CDC25C selectively. Forced overexpression of TRIB2 caused a marked decrease in total CDC25C protein levels. Following inhibition of the proteasome, CDC25C was stabilized in the nuclear compartment. This implicates TRIB2 as a regulator of nuclear CDC25C turnover. In complementary ubiquitination assays, we show that TRIB2-mediated degradation of CDC25C is associated with lysine-48-linked CDC25C polyubiquitination driven by the TRIB2 kinase-like domain. A cell cycle associated role for TRIB2 is further supported by the cell cycle regulated expression of TRIB2 protein levels. Our findings reveal mitotic CDC25C as a new target of TRIB2 that is degraded via the ubiquitin proteasome system. Inappropriate CDC25C regulation could mechanistically underlie TRIB2 mediated regulation of cellular proliferation in neoplastic cells. PMID:27563873

  8. Human TRIB2 Oscillates during the Cell Cycle and Promotes Ubiquitination and Degradation of CDC25C.

    PubMed

    Liang, Kai Ling; Paredes, Roberto; Carmody, Ruaidhri; Eyers, Patrick A; Meyer, Stefan; McCarthy, Tommie V; Keeshan, Karen

    2016-01-01

    Tribbles homolog 2 (TRIB2) is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Studies of TRIB2 indicate that many of the molecular interactions between the single Drosophila Tribbles (Trbl) protein and interacting partners are evolutionary conserved. In this study, we examined the relationship between TRIB2 and cell division cycle 25 (CDC25) family of dual-specificity protein phosphatases (mammalian homologues of Drosophila String), which are key physiological cell cycle regulators. Using co-immunoprecipitation we demonstrate that TRIB2 interacts with CDC25B and CDC25C selectively. Forced overexpression of TRIB2 caused a marked decrease in total CDC25C protein levels. Following inhibition of the proteasome, CDC25C was stabilized in the nuclear compartment. This implicates TRIB2 as a regulator of nuclear CDC25C turnover. In complementary ubiquitination assays, we show that TRIB2-mediated degradation of CDC25C is associated with lysine-48-linked CDC25C polyubiquitination driven by the TRIB2 kinase-like domain. A cell cycle associated role for TRIB2 is further supported by the cell cycle regulated expression of TRIB2 protein levels. Our findings reveal mitotic CDC25C as a new target of TRIB2 that is degraded via the ubiquitin proteasome system. Inappropriate CDC25C regulation could mechanistically underlie TRIB2 mediated regulation of cellular proliferation in neoplastic cells. PMID:27563873

  9. Proteasomes are tightly associated to myofibrils in mature skeletal muscle.

    PubMed

    Bassaglia, Yann; Cebrian, José; Covan, Silvia; Garcia, Monica; Foucrier, Jean

    2005-01-15

    Proteasomes are the major actors of nonlysosomal cytoplasmic protein degradation. In particular, these large protein complexes (about 2500 kDa) are considered to be responsible for muscular degradation during skeletal muscle atrophy. Despite their unusual and important size, they are widely described as soluble and mobile in the cytoplasm. In mature skeletal muscle, we have previously observed a sarcomeric distribution of proteasomes, as revealed by the distribution of alpha1/p27K, a subunit of the 20S core-particle (prosome) of proteasome. Here, we extend these observations at the electron microscopic level in vivo. We also show that this sarcomeric pattern is dependent of the extension of the sarcomere. Using isolated myofibrils, we demonstrate that proteasomes are still attached to the myofibrils after the isolation procedure, and reproduce the observations made in vivo. In addition, the extraction of actin by gelsolin largely removes proteasomes from isolated myofibrils, but some of them are held in place after this extraction, showing a sarcomeric disposition in the absence of any detectable actin, and suggesting the existence of another molecular partner for these interactions. From these results, we conclude that most of detectable 20S proteasomes in skeletal muscle cells is tightly attached to the myofibrils. PMID:15561103

  10. Assembly, Structure and Function of the 26S proteasome

    PubMed Central

    Bedford, Lynn; Paine, Simon; Sheppard, Paul W.; Mayer, R. John; Roelofs, Jeroen

    2010-01-01

    The 26S proteasome is a large multi-protein complex involved in the regulated degradation of ubiquitinated proteins in the cell. The 26S proteasome has been shown to control an increasing number of essential biochemical mechanisms of the cellular lifecycle including DNA synthesis, repair, transcription, translation and cell signal transduction. Concurrently, it is increasingly seen that malfunction of the ubiquitin proteasome system contributes to the pathogenesis of disease. The recent identification of four molecular chaperones, in addition to five previously identified chaperones, have provided mechanistic insight into how this cellular megastructure is assembled in the cell. These data, together with new insights into the structure and function of the proteasome, provide a much better understanding of this complex protease. PMID:20427185

  11. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  12. Role of the ubiquitin-proteasome system in brain ischemia: friend or foe?

    PubMed

    Caldeira, Margarida V; Salazar, Ivan L; Curcio, Michele; Canzoniero, Lorella M T; Duarte, Carlos B

    2014-01-01

    The ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitin-containing proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review. PMID:24157661

  13. Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit*

    PubMed Central

    Jin, Hongbing; Chiou, Tzu-Ting; Serwanski, David R.; Miralles, Celia P.; Pinal, Noelia; De Blas, Angel L.

    2014-01-01

    We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABAAR subunit promoting GABAAR degradation. PMID:25193658

  14. Characterization of the Brain 26S Proteasome and its Interacting Proteins

    PubMed Central

    Tai, Hwan-Ching; Besche, Henrike; Goldberg, Alfred L.; Schuman, Erin M.

    2010-01-01

    Proteasome-mediated proteolysis is important for synaptic plasticity, neuronal development, protein quality control, and many other processes in neurons. To define proteasome composition in brain, we affinity purified 26S proteasomes from cytosolic and synaptic compartments of the rat cortex. Using tandem mass spectrometry, we identified the standard 26S subunits and a set of 28 proteasome-interacting proteins that associated substoichiometrically and may serve as regulators or cofactors. This set differed from those in other tissues and we also found several proteins that associated only with either the cytosolic or the synaptic proteasome. The latter included the ubiquitin-binding factor TAX1BP1 and synaptic vesicle protein SNAP-25. Native gel electrophoresis revealed a higher proportion of doubly-capped 26S proteasome (19S-20S-19S) in the cortex than in the liver or kidney. To investigate the interplay between proteasome regulation and synaptic plasticity, we exposed cultured neurons to glutamate receptor agonist NMDA. Within 4 h, this agent caused a prolonged decrease in the activity of the ubiquitin-proteasome system as shown by disassembly of 26S proteasomes, decrease in ubiquitin-protein conjugates, and dissociation of the ubiquitin ligases UBE3A (E6-AP) and HUWE1 from the proteasome. Surprisingly, the regulatory 19S particles were rapidly degraded by proteasomal, not lysosomal degradation, and the dissociated E3 enzymes also degraded. Thus the content of proteasomes and their set of associated proteins can be altered by neuronal activity, in a manner likely to influence synaptic plasticity and learning. PMID:20717473

  15. Formation of proteasome-PA700 complexes directly correlates with activation of peptidase activity.

    PubMed

    Adams, G M; Crotchett, B; Slaughter, C A; DeMartino, G N; Gogol, E P

    1998-09-15

    The proteolytic activity of the eukaryotic 20S proteasome is stimulated by a multisubunit activator, PA700, which forms both 1:1 and 2:1 complexes with the proteasome. Formation of the complexes is enhanced by an additional protein assembly called modulator, which also stimulates the enzymatic activity of the proteasome only in the presence of PA700. Here we show that the binding of PA700 to the proteasome is cooperative, as is the activation of the proteasome's intrinsic peptidase activity. Modulator increases the extent of complex formation and peptidase activation, while preserving the cooperative kinetics. Furthermore, the increase in activity is not linear with the number of PA700 assemblies bound to the proteasome, but rather with the number of proteasome-PA700 complexes, regardless of the PA700:proteasome stoichiometry. Hence the stimulation of peptidase activity is fully (or almost fully) effected by the binding of a single PA700 to the 20S proteasome. The stimulation of peptidase by modulator is explained entirely by the increased number of proteasome-PA700 complexes formed in its presence, rather than by any substantial direct stimulation of catalysis. These observations are consistent with a model in which PA700, either alone or assisted by modulator, promotes conformational changes in the proteasome that activate the catalytic sites and/or facilitate access of peptide substrates to these sites. PMID:9737872

  16. Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor.

    PubMed

    Liu, Sen; Zhang, Qiuyang; Chen, Chong; Ge, Dongxia; Qu, Yine; Chen, Rongyi; Fan, Yi-Ming; Li, Nan; Tang, Wendell W; Zhang, Wensheng; Zhang, Kun; Wang, Alun R; Rowan, Brian G; Hill, Steven M; Sartor, Oliver; Abdel-Mageed, Asim B; Myers, Leann; Lin, Qishan; You, Zongbing

    2016-03-22

    Interleukin-17 (IL-17) plays important roles in inflammation, autoimmune diseases, and some cancers. Obese people are in a chronic inflammatory state with increased serum levels of IL-17, insulin, and insulin-like growth factor 1 (IGF1). How these factors contribute to the chronic inflammatory status that promotes development of aggressive prostate cancer in obese men is largely unknown. We found that, in obese mice, hyperinsulinemia enhanced IL-17-induced expression of downstream proinflammatory genes with increased levels of IL-17 receptor A (IL-17RA), resulting in development of more invasive prostate cancer. Glycogen synthase kinase 3 (GSK3) constitutively bound to and phosphorylated IL-17RA at T780, leading to ubiquitination and proteasome-mediated degradation of IL-17RA, thus inhibiting IL-17-mediated inflammation. IL-17RA phosphorylation was reduced, while the IL-17RA levels were increased in the proliferative human prostate cancer cells compared to the normal cells. Insulin and IGF1 enhanced IL-17-induced inflammatory responses through suppressing GSK3, which was shown in the cultured cell lines in vitro and obese mouse models of prostate cancer in vivo. These findings reveal a mechanism underlying the intensified inflammation in obesity and obesity-associated development of aggressive prostate cancer, suggesting that targeting GSK3 may be a potential therapeutic approach to suppress IL-17-mediated inflammation in the prevention and treatment of prostate cancer, particularly in obese men.

  17. Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

    PubMed Central

    Chen, Chong; Ge, Dongxia; Qu, Yine; Chen, Rongyi; Fan, Yi-Ming; Li, Nan; Tang, Wendell W.; Zhang, Wensheng; Zhang, Kun; Wang, Alun R.; Rowan, Brian G.; Hill, Steven M.; Sartor, Oliver; Abdel, Asim B.; Myers, Leann; Lin, Qishan; You, Zongbing

    2016-01-01

    Interleukin-17 (IL-17) plays important roles in inflammation, autoimmune diseases, and some cancers. Obese people are in a chronic inflammatory state with increased serum levels of IL-17, insulin, and insulin-like growth factor 1 (IGF1). How these factors contribute to the chronic inflammatory status that promotes development of aggressive prostate cancer in obese men is largely unknown. We found that, in obese mice, hyperinsulinemia enhanced IL-17-induced expression of downstream proinflammatory genes with increased levels of IL-17 receptor A (IL-17RA), resulting in development of more invasive prostate cancer. Glycogen synthase kinase 3 (GSK3) constitutively bound to and phosphorylated IL-17RA at T780, leading to ubiquitination and proteasome-mediated degradation of IL-17RA, thus inhibiting IL-17-mediated inflammation. IL-17RA phosphorylation was reduced, while the IL-17RA levels were increased in the proliferative human prostate cancer cells compared to the normal cells. Insulin and IGF1 enhanced IL-17-induced inflammatory responses through suppressing GSK3, which was shown in the cultured cell lines in vitro and obese mouse models of prostate cancer in vivo. These findings reveal a mechanism underlying the intensified inflammation in obesity and obesity-associated development of aggressive prostate cancer, suggesting that targeting GSK3 may be a potential therapeutic approach to suppress IL-17-mediated inflammation in the prevention and treatment of prostate cancer, particularly in obese men. PMID:26871944

  18. Role of the Ubiquitin-Proteasome Systems in the Biology and Virulence of Protozoan Parasites.

    PubMed

    Muñoz, Christian; San Francisco, Juan; Gutiérrez, Bessy; González, Jorge

    2015-01-01

    In eukaryotic cells, proteasomes perform crucial roles in many cellular pathways by degrading proteins to enforce quality control and regulate many cellular processes such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein-quality control, and development. The catalytic heart of these complexes, the 20S proteasome, is highly conserved in bacteria, yeast, and humans. However, until a few years ago, the role of proteasomes in parasite biology was completely unknown. Here, we summarize findings about the role of proteasomes in protozoan parasites biology and virulence. Several reports have confirmed the role of proteasomes in parasite biological processes such as cell differentiation, cell cycle, proliferation, and encystation. Proliferation and cell differentiation are key steps in host colonization. Considering the importance of proteasomes in both processes in many different parasites such as Trypanosoma, Leishmania, Toxoplasma, and Entamoeba, parasite proteasomes might serve as virulence factors. Several pieces of evidence strongly suggest that the ubiquitin-proteasome pathway is also a viable parasitic therapeutic target. Research in recent years has shown that the proteasome is a valid drug target for sleeping sickness and malaria. Then, proteasomes are a key organelle in parasite biology and virulence and appear to be an attractive new chemotherapeutic target.

  19. Role of the Ubiquitin-Proteasome Systems in the Biology and Virulence of Protozoan Parasites

    PubMed Central

    Muñoz, Christian; San Francisco, Juan; Gutiérrez, Bessy; González, Jorge

    2015-01-01

    In eukaryotic cells, proteasomes perform crucial roles in many cellular pathways by degrading proteins to enforce quality control and regulate many cellular processes such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein-quality control, and development. The catalytic heart of these complexes, the 20S proteasome, is highly conserved in bacteria, yeast, and humans. However, until a few years ago, the role of proteasomes in parasite biology was completely unknown. Here, we summarize findings about the role of proteasomes in protozoan parasites biology and virulence. Several reports have confirmed the role of proteasomes in parasite biological processes such as cell differentiation, cell cycle, proliferation, and encystation. Proliferation and cell differentiation are key steps in host colonization. Considering the importance of proteasomes in both processes in many different parasites such as Trypanosoma, Leishmania, Toxoplasma, and Entamoeba, parasite proteasomes might serve as virulence factors. Several pieces of evidence strongly suggest that the ubiquitin-proteasome pathway is also a viable parasitic therapeutic target. Research in recent years has shown that the proteasome is a valid drug target for sleeping sickness and malaria. Then, proteasomes are a key organelle in parasite biology and virulence and appear to be an attractive new chemotherapeutic target. PMID:26090380

  20. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    SciTech Connect

    Singh, Chingakham R.; Lovell, Scott; Mehzabeen, Nurjahan; Chowdhury, Wasimul Q.; Geanes, Eric S.; Battaile, Kevin P.; Roelofs, Jeroen

    2014-03-25

    The proteasome-assembly chaperone Nas2 binds to the proteasome subunit Rpt5 using its PDZ domain. The structure of the Nas2 PDZ domain has been determined. The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  1. The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

    PubMed Central

    Lier, Johanna Maria; Burmühl, Stephan; Struchtrup, Andreas; Deutschmann, Kathleen; Vetter, Maik; Leu, Tristan; Reeg, Sandra; Grune, Tilman; Rüther, Ulrich

    2015-01-01

    Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l−/− mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2. PMID:26150391

  2. Computational Approaches for the Discovery of Human Proteasome Inhibitors: An Overview.

    PubMed

    Guedes, Romina A; Serra, Patrícia; Salvador, Jorge A R; Guedes, Rita C

    2016-01-01

    Proteasome emerged as an important target in recent pharmacological research due to its pivotal role in degrading proteins in the cytoplasm and nucleus of eukaryotic cells, regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription, immune response, and signaling processes. The last two decades witnessed intensive efforts to discover 20S proteasome inhibitors with significant chemical diversity and efficacy. To date, the US FDA approved to market three proteasome inhibitors: bortezomib, carfilzomib, and ixazomib. However new, safer and more efficient drugs are still required. Computer-aided drug discovery has long being used in drug discovery campaigns targeting the human proteasome. The aim of this review is to illustrate selected in silico methods like homology modeling, molecular docking, pharmacophore modeling, virtual screening, and combined methods that have been used in proteasome inhibitors discovery. Applications of these methods to proteasome inhibitors discovery will also be presented and discussed to raise improvements in this particular field. PMID:27438821

  3. Determination of Protein Carbonylation and Proteasome Activity in Seeds.

    PubMed

    Xia, Qiong; El-Maarouf-Bouteau, Hayat; Bailly, Christophe; Meimoun, Patrice

    2016-01-01

    Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules in a process called redox signaling. In seeds, ROS are produced at different developmental stages including dormancy release and germination. Main targets of oxidation events by ROS in cell are lipids, nucleic acids, and proteins. Protein oxidation has various effects on their function, stability, location, and degradation. Carbonylation represents an irreversible and unrepairable modification that can lead to protein degradation through the action of the 20S proteasome. Here, we present techniques which allow the quantification of protein carbonyls in complex protein samples after derivatization by 2,4-dinitrophenylhydrazine (DNPH) and the determination proteasome activity by an activity-based protein profiling (ABPP) using the probe MV151. These techniques, routinely easy to handle, allow the rapid assessment of protein carbonyls and proteasome activity in seeds in various physiological conditions where ROS may act as signaling or toxic elements. PMID:27424756

  4. The 26S Proteasome Complex: An Attractive Target for Cancer Therapy

    PubMed Central

    Frankland-Searby, Sarah; Bhaumik, Sukesh R.

    2011-01-01

    The 26S proteasome complex engages in an ATP-dependent proteolytic degradation of a variety of oncoproteins, transcription factors, cell cycle specific cyclins, cyclin-dependent kinase inhibitors, ornithine decarboxylase, and other key regulatory cellular proteins. Thus, the proteasome regulates either directly or indirectly many important cellular processes. Altered regulation of these cellular events is linked to the development of cancer. Therefore, the proteasome has become an attractive target for the treatment of numerous cancers. Several proteasome inhibitors that target the proteolytic active sites of the 26S proteasome complex have been developed and tested for anti-tumor activities. These proteasome inhibitors have displayed impressive anti-tumor functions by inducing apoptosis in different tumor types. Further, the proteasome inhibitors have been shown to induce cell cycle arrest, and inhibit angiogenesis, cell-cell adhesion, cell migration, immune and inflammatory responses, and DNA repair response. A number of proteasome inhibitors are now in clinical trials to treat multiple myeloma and solid tumors. Many other proteasome inhibitors with different efficiencies are being developed and tested for anti-tumor activities. Several proteasome inhibitors currently in clinical trials have shown significantly improved anti-tumor activities when combined with other drugs such as histone deacetylase (HDAC) inhibitors, Akt (protein kinase B) inhibitors, DNA damaging agents, Hsp90 (heat shock protein 90) inhibitors, and lenalidomide. The proteasome inhibitor bortezomib is now in the clinic to treat multiple myeloma and mantle cell lymphoma. Here, we discuss the 26S proteasome complex in carcinogenesis and different proteasome inhibitors with their potential therapeutic applications in treatment of numerous cancers. PMID:22037302

  5. Phosphorylation of ATPase subunits of the 26S proteasome.

    PubMed

    Mason, G G; Murray, R Z; Pappin, D; Rivett, A J

    1998-07-01

    The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.

  6. The cleavage preference of the proteasome governs the yield of antigenic peptides

    PubMed Central

    1995-01-01

    Proteasomes degrade endogenous proteins in the cytosol. The potential contribution of the proteasome to the effect of flanking sequences on the presentation of an antigenic epitope presented by the major histocompatibility complex class I allele Ld was studied. Peptides generated in cells from minigenes coding for peptides of 17- and 19- amino acid length were compared with the in vitro 20S proteasome degradation products of the respective synthetic peptides. The quality of generated peptides was independent of ubiquitination. In vivo and in vitro processing products were indistinguishable with respect to peptide size and abundance. Altering the neighboring sequence substantially improved the yield of the final antigenic nonapeptide by 20S proteasome cleavage. These results suggest that, in addition to the presence of major histocompatibility complex class I allelic motifs, the cleavage preference of the proteasome can define the antigenic potential of a protein. PMID:7500032

  7. Diphenylarsinic acid promotes degradation of glutaminase C by mitochondrial Lon protease.

    PubMed

    Kita, Kayoko; Suzuki, Toshihide; Ochi, Takafumi

    2012-05-25

    Glutaminase C (GAC), a splicing variant of the kidney-type glutaminase (KGA) gene, is a vital mitochondrial enzyme protein that catalyzes glutamine to glutamate. Earlier studies have shown that GAC proteins in the human hepatocarcinoma cell line, HepG2, were down-regulated by diphenylarsinic acid (DPAA), but the mechanism by which DPAA induced GAC protein down-regulation remained poorly understood. Here, we showed that DPAA promoted GAC protein degradation without affecting GAC transcription and translation. Moreover, DPAA-induced GAC proteolysis was mediated by mitochondrial Lon protease. DPAA insolubilized 0.5% Triton X-100-soluble GAC protein and promoted the accumulation of insoluble GAC in Lon protease knockdown cells. DPAA destroyed the native tetrameric GAC conformation and promoted an increase in the unassembled form of GAC when DPAA was incubated with cell extracts. Decreases in the tetrameric form of GAC were observed in cells exposed to DPAA, and decreases occurred prior to a decrease in total GAC protein levels. In addition, decreases in the tetrameric form of GAC were observed independently with Lon protease. Mitochondrial heat shock protein 70 is known to be an indispensable protein that can bind to misfolded proteins, thereby supporting degradation of proteins sensitive to Lon protease. When cells were incubated with DPAA, GAC proteins that can bind with mtHsp70 increased. Interestingly, the association of mtHsp70 with GAC protein increased when the tetrameric form of GAC was reduced. These results suggest that degradation of native tetrameric GAC by DPAA may be a trigger in GAC protein degradation by Lon protease.

  8. Phytoplasma Effector SAP54 Hijacks Plant Reproduction by Degrading MADS-box Proteins and Promotes Insect Colonization in a RAD23-Dependent Manner

    PubMed Central

    MacLean, Allyson M.; Orlovskis, Zigmunds; Kowitwanich, Krissana; Zdziarska, Anna M.; Angenent, Gerco C.; Immink, Richard G. H.; Hogenhout, Saskia A.

    2014-01-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants). PMID:24714165

  9. Phytoplasma effector SAP54 hijacks plant reproduction by degrading MADS-box proteins and promotes insect colonization in a RAD23-dependent manner.

    PubMed

    MacLean, Allyson M; Orlovskis, Zigmunds; Kowitwanich, Krissana; Zdziarska, Anna M; Angenent, Gerco C; Immink, Richard G H; Hogenhout, Saskia A

    2014-04-01

    Pathogens that rely upon multiple hosts to complete their life cycles often modify behavior and development of these hosts to coerce them into improving pathogen fitness. However, few studies describe mechanisms underlying host coercion. In this study, we elucidate the mechanism by which an insect-transmitted pathogen of plants alters floral development to convert flowers into vegetative tissues. We find that phytoplasma produce a novel effector protein (SAP54) that interacts with members of the MADS-domain transcription factor (MTF) family, including key regulators SEPALLATA3 and APETALA1, that occupy central positions in the regulation of floral development. SAP54 mediates degradation of MTFs by interacting with proteins of the RADIATION SENSITIVE23 (RAD23) family, eukaryotic proteins that shuttle substrates to the proteasome. Arabidopsis rad23 mutants do not show conversion of flowers into leaf-like tissues in the presence of SAP54 and during phytoplasma infection, emphasizing the importance of RAD23 to the activity of SAP54. Remarkably, plants with SAP54-induced leaf-like flowers are more attractive for colonization by phytoplasma leafhopper vectors and this colonization preference is dependent on RAD23. An effector that targets and suppresses flowering while simultaneously promoting insect herbivore colonization is unprecedented. Moreover, RAD23 proteins have, to our knowledge, no known roles in flower development, nor plant defence mechanisms against insects. Thus SAP54 generates a short circuit between two key pathways of the host to alter development, resulting in sterile plants, and promotes attractiveness of these plants to leafhopper vectors helping the obligate phytoplasmas reproduce and propagate (zombie plants). PMID:24714165

  10. Functional interactions between mRNA turnover and surveillance and the ubiquitin proteasome system.

    PubMed

    Brooks, Seth A

    2010-01-01

    The proteasome is a critical regulator of protein levels within the cell and is essential for maintaining homeostasis. A functional proteasome is required for effective mRNA surveillance and turnover. During transcription, the proteasome localizes to sites of DNA breaks, degrading RNA polymerase II and terminating transcription. For fully transcribed and processed messages, cytoplasmic surveillance is initiated with the pioneer round of translation. The proteasome is recruited to messages bearing premature termination codons, which trigger nonsense-mediated decay (NMD), as well as messages lacking a termination codon, which trigger nonstop decay, to degrade the aberrant protein produced from these messages. A number of proteins involved in mRNA translation are regulated in part by proteasome-mediated decay, including the initiation factors eIF4G, eIF4E, and eIF3a, and the poly(A)-binding protein (PABP) interacting protein, Paip2. eIF4E-BP (4E-BP) is differentially regulated by the proteasome: truncated to generate a protein with higher eIF4B binding or completely degraded, depending on its phosphorylation status. Finally, a functional proteasome is required for AU-rich-element (ARE)-mediated decay but the specific role the proteasome plays is unclear. There is data indicating the proteasome can bind to AREs, act as an endonuclease, and degrade ARE-binding proteins. How these events interact with the 5'-to-3' and 3'-to-5' decay pathways is unclear at this time; however, data is provided indicating that proteasomes colocalize with Xrn1 and the exosome RNases Rrp44 and Rrp6 in untreated HeLa cells. PMID:21935888

  11. Ubiquitin proteasome system research in gastrointestinal cancer.

    PubMed

    Zhong, Jia-Ling; Huang, Chang-Zhi

    2016-02-15

    The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design. PMID:26909134

  12. Ubiquitin proteasome system research in gastrointestinal cancer

    PubMed Central

    Zhong, Jia-Ling; Huang, Chang-Zhi

    2016-01-01

    The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design. PMID:26909134

  13. Targeted polypeptide degradation

    DOEpatents

    Church, George M.; Janse, Daniel M.

    2008-05-13

    This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

  14. The Role of the Proteasome in Heart Disease

    PubMed Central

    Li, Yi-Fan; Wang, Xuejun

    2010-01-01

    Intensive investigations into the pathophysiological significance of the proteasome in the heart did not start until the beginning of the past decade but exciting progresses have been made and are summarized here as two fronts. First, strong evidence continues to emerge to support a novel hypothesis that proteasome functional insufficiency represents a common pathological phenomenon in a large subset of heart disease, compromises protein quality control in heart muscle cells, and thereby acts as a major pathogenic factor promoting the progression of the subset of heart disease to congestive heart failure. This front is represented by the studies on the ubiquitin-proteasome system (UPS) in cardiac proteinopathy, which have taken advantage of a transgenic mouse model expressing a fluorescence reporter for UPS proteolytic function. Second, pharmacological inhibition of the proteasome has been explored experimentally as a potential therapeutic strategy to intervene some forms of heart disease, such as pressure overload cardiac hypertrophy, viral myocarditis, and myocardial ischemic injury. Not only between the two fronts but also within each one, a multitude of inconsistency and controversy remain to be explained and clarified. At present, the controversy perhaps reflects the sophistication of cardiac proteasomes in terms of the composition, assembly, and regulation, as well as the intricacy and diversity of heart disease in terms of its etiology and pathogenesis. A definitive role of altered proteasome function in the development of various forms of heart disease remains to be established. PMID:20840877

  15. Determination of cypermethrin degradation potential of soil bacteria along with plant growth-promoting characteristics.

    PubMed

    Akbar, Shamsa; Sultan, Sikander; Kertesz, Michael

    2015-01-01

    The pyrethroid insecticide cypermethrin is in extensive use since 1980s for insect control. However, its toxicity toward aquatic animals and humans requires its complete removal from contaminated areas that can be done using indigenous microbes through bioremediation. In this study, three bacterial strains isolated from agricultural soil and identified as Acinetobacter calcoaceticus MCm5, Brevibacillus parabrevis FCm9, and Sphingomonas sp. RCm6 were found highly efficient in degrading cypermethrin and other pyrethroids. These bacterial strains were able to degrade more than 85 % of cypermethrin (100 mg L(-1)) within 10 days. Degradation kinetics of cypermethrin (200 mg kg(-1)) in soils inoculated with isolates MCm5, FCm9, and RCm6 suggested time-dependent disappearance of cypermethrin with rate constants of 0.0406, 0.0722, and 0.0483 d(-1) following first-order rate kinetics. Enzyme assays for Carboxylesterase, 3-PBA dioxygenase, Phenol hydroxylase, and Catechol-1,2 dioxygenase showed higher activities with cypermethrin treated cell-free extracts compared to non-treated cell-free extracts. Meanwhile, SDS-PAGE analysis showed upregulation of some bands in cypermethrin-treated cells. This might suggest that cypermethrin degradation in these strains involves inducible enzymes. Besides, the isolates displayed substantial plant growth-promoting traits such as phosphate solubilization, Indole acetic acid production, and ammonia production. Implying the efficient biodegradation potential along with multiple biological properties, these isolates can be valuable candidates for the development of bioremediation strategies.

  16. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

    PubMed

    Chen, Chao; Cui, Zhenling; Song, Xiangfei; Liu, Ya-Jun; Cui, Qiu; Feng, Yingang

    2016-03-01

    Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes.

  17. Determination of cypermethrin degradation potential of soil bacteria along with plant growth-promoting characteristics.

    PubMed

    Akbar, Shamsa; Sultan, Sikander; Kertesz, Michael

    2015-01-01

    The pyrethroid insecticide cypermethrin is in extensive use since 1980s for insect control. However, its toxicity toward aquatic animals and humans requires its complete removal from contaminated areas that can be done using indigenous microbes through bioremediation. In this study, three bacterial strains isolated from agricultural soil and identified as Acinetobacter calcoaceticus MCm5, Brevibacillus parabrevis FCm9, and Sphingomonas sp. RCm6 were found highly efficient in degrading cypermethrin and other pyrethroids. These bacterial strains were able to degrade more than 85 % of cypermethrin (100 mg L(-1)) within 10 days. Degradation kinetics of cypermethrin (200 mg kg(-1)) in soils inoculated with isolates MCm5, FCm9, and RCm6 suggested time-dependent disappearance of cypermethrin with rate constants of 0.0406, 0.0722, and 0.0483 d(-1) following first-order rate kinetics. Enzyme assays for Carboxylesterase, 3-PBA dioxygenase, Phenol hydroxylase, and Catechol-1,2 dioxygenase showed higher activities with cypermethrin treated cell-free extracts compared to non-treated cell-free extracts. Meanwhile, SDS-PAGE analysis showed upregulation of some bands in cypermethrin-treated cells. This might suggest that cypermethrin degradation in these strains involves inducible enzymes. Besides, the isolates displayed substantial plant growth-promoting traits such as phosphate solubilization, Indole acetic acid production, and ammonia production. Implying the efficient biodegradation potential along with multiple biological properties, these isolates can be valuable candidates for the development of bioremediation strategies. PMID:25194282

  18. Comparative resistance of the 20S and 26S proteasome to oxidative stress.

    PubMed Central

    Reinheckel, T; Sitte, N; Ullrich, O; Kuckelkorn, U; Davies, K J; Grune, T

    1998-01-01

    Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without

  19. Toward an integrated structural model of the 26S proteasome.

    PubMed

    Förster, Friedrich; Lasker, Keren; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2010-08-01

    The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation.

  20. Toward an Integrated Structural Model of the 26S Proteasome*

    PubMed Central

    Förster, Friedrich; Lasker, Keren; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2010-01-01

    The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation. PMID:20467039

  1. Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

    PubMed

    Xia, Hong-Guang; Najafov, Ayaz; Geng, Jiefei; Galan-Acosta, Lorena; Han, Xuemei; Guo, Yuan; Shan, Bing; Zhang, Yaoyang; Norberg, Erik; Zhang, Tao; Pan, Lifeng; Liu, Junli; Coloff, Jonathan L; Ofengeim, Dimitry; Zhu, Hong; Wu, Kejia; Cai, Yu; Yates, John R; Zhu, Zhengjiang; Yuan, Junying; Vakifahmetoglu-Norberg, Helin

    2015-08-31

    Hexokinase II (HK2), a key enzyme involved in glucose metabolism, is regulated by growth factor signaling and is required for initiation and maintenance of tumors. Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non-acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA). Our data demonstrate that FLT3 is an important sensor of cellular nutritional state and elucidate the role and molecular mechanism of CMA in metabolic regulation and mediating cancer cell death. Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability. We reveal a new mechanism by which excessive activation of CMA may be exploited pharmacologically to eliminate cancer cells by inhibiting both FLT3 and autophagy. Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells.

  2. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells.

    PubMed

    Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi; Yamada, Koji; Noguchi, Kohji; Fujimuro, Masahiro

    2011-12-01

    Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-κB inhibitory molecule (IκBα) and suppressed the transcriptional activity of NF-κB in PEL cells. The NF-κB specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-κB signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-κB signaling is upregulated by proteasome-dependent degradation of IκBα. The suppression of NF-κB signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.

  3. Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells

    PubMed Central

    Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark

    2016-01-01

    Summary Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here we report formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, which bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of ‘α4-α4’ proteasomes depends on the relative cellular levels of α4 and α3, and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of a novel mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses. PMID:26997268

  4. Structural analysis of the 26S proteasome by cryoelectron tomography.

    PubMed

    Nickell, Stephan; Mihalache, Oana; Beck, Florian; Hegerl, Reiner; Korinek, Andreas; Baumeister, Wolfgang

    2007-02-01

    The 26S proteasome is the key enzyme of intracellular protein degradation in eukaryotic cells. It is a multisubunit complex of 2.5 MDa confining the proteolytic action to an inner compartment with tightly controlled access. Structural studies of this intriguing molecular machine have been hampered by its intrinsic instability and its dynamics. Here we have used an unconventional approach to obtain a three-dimensional structure of the holocomplex uncompromised by preparation-induced alterations and unbiased by any starting model. We have performed a tomographic reconstruction, followed by averaging over approx. 150 individual reconstructions, of Drosophila 26S proteasomes suspended in a thin layer of amorphous ice.

  5. Mouse zygote-specific proteasome assembly chaperone important for maternal-to-zygotic transition

    PubMed Central

    Shin, Seung-Wook; Shimizu, Natsumi; Tokoro, Mikiko; Nishikawa, Satoshi; Hatanaka, Yuki; Anzai, Masayuki; Hamazaki, Jun; Kishigami, Satoshi; Saeki, Kazuhiro; Hosoi, Yoshihiko; Iritani, Akira; Murata, Shigeo; Matsumoto, Kazuya

    2013-01-01

    Summary During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin–proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT. PMID:23429752

  6. The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction

    PubMed Central

    Koizumi, Shun; Irie, Taro; Hirayama, Shoshiro; Sakurai, Yasuyuki; Yashiroda, Hideki; Naguro, Isao; Ichijo, Hidenori; Hamazaki, Jun; Murata, Shigeo

    2016-01-01

    In response to proteasome dysfunction, mammalian cells upregulate proteasome gene expression by activating Nrf1. Nrf1 is an endoplasmic reticulum-resident transcription factor that is continually retrotranslocated and degraded by the proteasome. Upon proteasome inhibition, Nrf1 escapes degradation and is cleaved to become active. However, the processing enzyme for Nrf1 remains obscure. Here we show that the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is required to cleave and activate Nrf1. Deletion of DDI2 reduced the cleaved form of Nrf1 and increased the full-length cytosolic form of Nrf1, resulting in poor upregulation of proteasomes in response to proteasome inhibition. These defects were restored by adding back wild-type DDI2 but not protease-defective DDI2. Our results provide a clue for blocking compensatory proteasome synthesis to improve cancer therapies targeting proteasomes. DOI: http://dx.doi.org/10.7554/eLife.18357.001 PMID:27528193

  7. The role of allostery in the ubiquitin-proteasome system

    PubMed Central

    Liu, Jin; Nussinov, Ruth

    2012-01-01

    The Ubiquitin-Proteasome System is involved in many cellular processes including protein degradation. Degradation of a protein via this system involves two successive steps: ubiquitination and degradation. Ubiquitination tags the target protein with ubiquitin-like proteins, such as ubiquitin, SUMO and NEDD8, via a cascade involving three enzymes: activating enzyme E1, conjugating enzyme E2, and E3 ubiquitin ligases. The proteasomes recognize the ubiquitin-like protein tagged substrate proteins and degrade them. Accumulating evidence indicates that allostery is a central player in the regulation of ubiquitination, as well as deubiquitination and degradation. Here, we provide an overview of the key mechanistic roles played by allostery in all steps of these processes, and highlight allosteric drugs targeting them. Throughout the review, we emphasize the crucial mechanistic role played by linkers in allosterically controlling the Ubiquitin-Proteasome System action by biasing the sampling of the conformational space, which facilitate the catalytic reactions of the ubiquitination and degradation. Finally, we propose that allostery may similarly play key roles in the regulation of molecular machines in the cell, and as such allosteric drugs can be expected to be increasingly exploited in therapeutic regimes. PMID:23234564

  8. The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells.

    PubMed

    Park, Gwang Hun; Song, Hun Min; Jeong, Jin Boo

    2016-09-01

    Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells. PMID:27424123

  9. The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells.

    PubMed

    Park, Gwang Hun; Song, Hun Min; Jeong, Jin Boo

    2016-09-01

    Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells.

  10. Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells.

    PubMed

    Li, Xu; Zhu, Feng; Jiang, Jianxin; Sun, Chengyi; Zhong, Qing; Shen, Ming; Wang, Xin; Tian, Rui; Shi, Chengjian; Xu, Meng; Peng, Feng; Guo, Xingjun; Hu, Jun; Ye, Dawei; Wang, Min; Qin, Renyi

    2016-09-01

    In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease. PMID:27308733

  11. Potential Roles for Ubiquitin and the Proteasome during Ribosome Biogenesis‡

    PubMed Central

    Stavreva, Diana A.; Kawasaki, Miyuki; Dundr, Miroslav; Koberna, Karel; Müller, Waltraud G.; Tsujimura-Takahashi, Teruko; Komatsu, Wataru; Hayano, Toshiya; Isobe, Toshiaki; Raska, Ivan; Misteli, Tom; Takahashi, Nobuhiro; McNally, James G.

    2006-01-01

    We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome. PMID:16782897

  12. A Non-canonical RNA Silencing Pathway Promotes mRNA Degradation in Basal Fungi

    PubMed Central

    Nicolás, Francisco E.; Vila, Ana; Moxon, Simon; Dalmay, Tamas; Torres-Martínez, Santiago; Garre, Victoriano; Ruiz-Vázquez, Rosa M.

    2015-01-01

    The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post

  13. Structural Insights into the Regulatory Particle of the Proteasome from Methanocaldococcus jannaschii

    SciTech Connect

    Zhang, F.; Hu, M; Tian, G; Zhang, P; Finley, D; Jeffrey, P; Shi, Y

    2009-01-01

    Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding, and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here, we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13 A pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.

  14. Proteomic profiling of expression of proteasomal subunits from livers of mice treated with diethylnitrosamine.

    PubMed

    Yuan, Fuqiang; Lu, Jia; You, Pan; Yang, Zengming; Yang, Pengyuan; Ma, Qiling; Tao, Tao

    2013-01-01

    The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Diethylnitrosamine is metabolized primarily in the liver by cytochrome P-450 and can cause DNA damage. The 26S proteasome is a large proteolytic complex that degrades ubiquitinated proteins, and regulates many physiological processes. We used proteomics-based approaches to examine expressional differences of liver proteasomal subunits from diethylnitrosamine-treated mice. The expression of most proteasomal subunits was observed to be upregulated in the analysis of 2DE and MALDI-TOF MS/MS. Some of these differentially expressed proteasomal subunits were further confirmed by Western blot, RT-PCR, and immunohistochemistry. Our results provided useful information on the relationship between the proteasomal complex and related diseases.

  15. Proteasome Inhibition Enhances the Induction and Impairs the Maintenance of Late-Phase Long-Term Potentiation

    ERIC Educational Resources Information Center

    Dong, Chenghai; Upadhya, Sudarshan C.; Ding, Lan; Smith, Thuy K.; Hegde, Ashok N.

    2008-01-01

    Protein degradation by the ubiquitin-proteasome pathway plays important roles in synaptic plasticity, but the molecular mechanisms by which proteolysis regulates synaptic strength are not well understood. We investigated the role of the proteasome in hippocampal late-phase long-term potentiation (L-LTP), a model for enduring synaptic plasticity.…

  16. Association of metals and proteasome activity in erythrocytes of prostate cancer patients and controls.

    PubMed

    Neslund-Dudas, Christine; Mitra, Bharati; Kandegedara, Ashoka; Chen, Di; Schmitt, Sara; Shen, Min; Cui, Qiuzhi; Rybicki, Benjamin A; Dou, Q Ping

    2012-10-01

    Information is lacking on the effects toxic environmental metals may have on the 26S proteasome. The proteasome is a primary vehicle for selective degradation of damaged proteins in a cell and due to its role in cell proliferation, inhibition of the proteasome has become a target for cancer therapy. Metals are essential to the proteasome's normal function and have been used within proteasome-inhibiting complexes for cancer therapy. This study evaluated the association of erythrocyte metal levels and proteasome chymotrypsin-like (CT-like) activity in age- and race-matched prostate cancer cases (n=61) and controls (n=61). Erythrocyte metals were measured by inductively coupled plasma mass spectrometry (ICP-MS). CT-like activity was measured by proteasome activity assay using a fluorogenic peptide substrate. Among cases, significant correlations between individual toxic metals were observed (r(arsenic-cadmium)=0.49, p<0.001; r(arsenic-lead)=0.26, p=0.04, r(cadmium-lead) 0.53, p<0.001), but there were no significant associations between metals and CT-like activity. In contrast, within controls there were no significant associations between metals, however, copper and lead levels were significantly associated with CT-like activity. The associations between copper and lead and proteasome activity (r(copper-CT-like)=-0.28, p=0.002 ; r(lead-CT-like)=0.23, p=0.011) remained significant in multivariable models that included all of the metals. These findings suggest that biologically essential metals and toxic metals may affect proteasome activity in healthy controls and, further, show that prostate cancer cases and controls differ in associations between metals and proteasome activity in erythrocytes. More research on toxic metals and the proteasome in prostate cancer is warranted.

  17. JAB1/CSN5 inhibits the activity of Luman/CREB3 by promoting its degradation

    PubMed Central

    DenBoer, Lisa M.; Iyer, Aarti; McCluggage, Adam R.R.; Li, Yu; Martyn, Amanda C.; Lu, Ray

    2016-01-01

    Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound transcription factor that has been implicated in the ER stress response. In this study, we used the region of Luman containing the basic DNA-binding domain as bait in a yeast two-hybrid screen and identified the Jun activation domain-binding protein 1 (JAB1) or the COP9 signalosome complex unit 5 (CSN5) as an interacting protein. We confirmed their direct binding by glutathione S-transferase pull-down assays, and verified the existence of such interaction in the cellular environment by mammalian two-hybrid and co-immunoprecipitation assays. Deletion mapping studies revealed that the MPN domain in JAB1 was essential and sufficient for the binding. JAB1 also colocalized with Luman in transfected cells. More interestingly, the nuclear form of Luman was shown to promote the translocation of JAB1 into the nucleus. We found that overexpression of JAB1 shortened the half-life of Luman by 67%, and repressed its transactivation function on GAL4 and unfolded protein response element (UPRE)-containing promoters. We therefore propose that JAB1 is a novel binding partner of Luman, which negatively regulates the activity of Luman by promoting its degradation. PMID:23583719

  18. Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria

    PubMed Central

    Blouin, Jean-Marc; Duchartre, Yann; Costet, Pierre; Lalanne, Magalie; Ged, Cécile; Lain, Ana; Millet, Oscar; de Verneuil, Hubert; Richard, Emmanuel

    2013-01-01

    Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROSC73R mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROSP248Q mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROSC73R and UROSP248Q are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (UrosP248Q/P248Q) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones. PMID:24145442

  19. Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria.

    PubMed

    Blouin, Jean-Marc; Duchartre, Yann; Costet, Pierre; Lalanne, Magalie; Ged, Cécile; Lain, Ana; Millet, Oscar; de Verneuil, Hubert; Richard, Emmanuel

    2013-11-01

    Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROS(C73R) mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROS(P248Q) mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROS(C73R) and UROS(P248Q) are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (Uros(P248Q/P248Q)) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones. PMID:24145442

  20. Quiescent fibroblasts are protected from proteasome inhibition–mediated toxicity

    PubMed Central

    Legesse-Miller, Aster; Raitman, Irene; Haley, Erin M.; Liao, Albert; Sun, Lova L.; Wang, David J.; Krishnan, Nithya; Lemons, Johanna M. S.; Suh, Eric J.; Johnson, Elizabeth L.; Lund, Benjamin A.; Coller, Hilary A.

    2012-01-01

    Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition–mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition–induced cytotoxicity. PMID:22875985

  1. Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

    SciTech Connect

    Zangar, Richard C. ); Kocarek, Thomas A.; Shen, Shang; Bollinger, Nikki ); Dahn, Michael S.; Lee, Donna W.

    2003-06-01

    We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3 A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.

  2. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition

    PubMed Central

    Radhakrishnan, Senthil K; den Besten, Willem; Deshaies, Raymond J

    2014-01-01

    Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., ‘bounce-back’) of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. DOI: http://dx.doi.org/10.7554/eLife.01856.001 PMID:24448410

  3. Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

    PubMed

    Zangar, Richard C; Kocarek, Thomas A; Shen, Shang; Bollinger, Nikki; Dahn, Michael S; Lee, Donna W

    2003-06-01

    We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.

  4. The proteasome is an integral part of solar ultraviolet a radiation-induced gene expression.

    PubMed

    Catalgol, Betul; Ziaja, Isabella; Breusing, Nicolle; Jung, Tobias; Höhn, Annika; Alpertunga, Buket; Schroeder, Peter; Chondrogianni, Niki; Gonos, Efstathios S; Petropoulos, Isabelle; Friguet, Bertrand; Klotz, Lars-Oliver; Krutmann, Jean; Grune, Tilman

    2009-10-30

    Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

  5. [CK2beta promotes Pink1/Parkin-mediated MIRO1 degradation].

    PubMed

    Zhang, Chenliang; Qin, Siyue; Jiang, Chang'an

    2014-12-01

    PTEN-induced putative kinase 1 (PINK1), a Parkinson's disease (PD)-related protein, has two isoforms, the mitochondria-localized full-length isoform PINK1FL and the cytoplasm-localized short isoform PINK1-cyto. Studies have suggested that PINK1FL can selectively accumulate at the surface of damaged mitochondria and cooperate with another Parkinson's Disease-related protein PARKIN to trigger the degradation of MIRO1, a mitochondria trafficking regulator. The functions of PINK1-cyto are, however, not yet clear. To investigate the functions of PINK1-cyto, we expressed different proteins in cultured HEK293 cells by transfecting it with different plasmids, and detected the protein levels by Western blot after expressing for 24 h. We found that in cultured HEK293 cells, PINK1-cyto could also cooperate with PARKIN degrade MIRO1 in the presence of CK23, and the regulatory subunit of Casein Kinase II. Interestingly, this function of CK2P was not dependent on CK2alpha, the catalytic subunit of Casein Kinase II. We also found that CK2P could promote the direct interaction between PINK1-cyto and MIRO1 by immunocoprecipitation analysis. This result suggested that in addition to CK2alpha, CK2beta could also form a kinase complex. PMID:25868250

  6. WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress.

    PubMed

    Leuzzi, Giuseppe; Marabitti, Veronica; Pichierri, Pietro; Franchitto, Annapaola

    2016-07-01

    Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11-mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti-recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1-deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork-protective factor in maintaining genome stability.

  7. Environmental acoustic enrichment promotes recovery from developmentally degraded auditory cortical processing.

    PubMed

    Zhu, Xiaoqing; Wang, Fang; Hu, Huifang; Sun, Xinde; Kilgard, Michael P; Merzenich, Michael M; Zhou, Xiaoming

    2014-04-16

    It has previously been shown that environmental enrichment can enhance structural plasticity in the brain and thereby improve cognitive and behavioral function. In this study, we reared developmentally noise-exposed rats in an acoustic-enriched environment for ∼4 weeks to investigate whether or not enrichment could restore developmentally degraded behavioral and neuronal processing of sound frequency. We found that noise-exposed rats had significantly elevated sound frequency discrimination thresholds compared with age-matched naive rats. Environmental acoustic enrichment nearly restored to normal the behavioral deficit resulting from early disrupted acoustic inputs. Signs of both degraded frequency selectivity of neurons as measured by the bandwidth of frequency tuning curves and decreased long-term potentiation of field potentials recorded in the primary auditory cortex of these noise-exposed rats also were reversed partially. The observed behavioral and physiological effects induced by enrichment were accompanied by recovery of cortical expressions of certain NMDA and GABAA receptor subunits and brain-derived neurotrophic factor. These studies in a rodent model show that environmental acoustic enrichment promotes recovery from early noise-induced auditory cortical dysfunction and indicate a therapeutic potential of this noninvasive approach for normalizing neurological function from pathologies that cause hearing and associated language impairments in older children and adults.

  8. Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death

    PubMed Central

    Xia, Hong-guang; Najafov, Ayaz; Geng, Jiefei; Galan-Acosta, Lorena; Han, Xuemei; Guo, Yuan; Shan, Bing; Zhang, Yaoyang; Norberg, Erik; Zhang, Tao; Pan, Lifeng; Liu, Junli; Coloff, Jonathan L.; Ofengeim, Dimitry; Zhu, Hong; Wu, Kejia; Cai, Yu; Yates, John R.; Zhu, Zhengjiang; Vakifahmetoglu-Norberg, Helin

    2015-01-01

    Hexokinase II (HK2), a key enzyme involved in glucose metabolism, is regulated by growth factor signaling and is required for initiation and maintenance of tumors. Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non–acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA). Our data demonstrate that FLT3 is an important sensor of cellular nutritional state and elucidate the role and molecular mechanism of CMA in metabolic regulation and mediating cancer cell death. Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability. We reveal a new mechanism by which excessive activation of CMA may be exploited pharmacologically to eliminate cancer cells by inhibiting both FLT3 and autophagy. Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells. PMID:26323688

  9. Changes in the Expression and the Enzymic Properties of the 20S Proteasome in Sugar-Starved Maize Roots. Evidence for an in Vivo Oxidation of the Proteasome1

    PubMed Central

    Basset , Gilles; Raymond, Philippe; Malek, Lada; Brouquisse, Renaud

    2002-01-01

    The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of α3 and β6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations. PMID:11891269

  10. Two-substrate association with the 20S proteasome at single-molecule level.

    PubMed

    Hutschenreiter, Silke; Tinazli, Ali; Model, Kirstin; Tampé, Robert

    2004-07-01

    The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal-chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End-on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a 'dead-end' proteasome can process only one substrate at a time. In contrast, the side-on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single-molecule cross-correlation spectroscopy. Thus, the two-stroke engine offers the advantage of speeding up degradation without enhancing complexity. PMID:15175655

  11. Two-substrate association with the 20S proteasome at single-molecule level.

    PubMed

    Hutschenreiter, Silke; Tinazli, Ali; Model, Kirstin; Tampé, Robert

    2004-07-01

    The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal-chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End-on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a 'dead-end' proteasome can process only one substrate at a time. In contrast, the side-on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single-molecule cross-correlation spectroscopy. Thus, the two-stroke engine offers the advantage of speeding up degradation without enhancing complexity.

  12. Two-substrate association with the 20S proteasome at single-molecule level

    PubMed Central

    Hutschenreiter, Silke; Tinazli, Ali; Model, Kirstin; Tampé, Robert

    2004-01-01

    The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal-chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End-on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a ‘dead-end' proteasome can process only one substrate at a time. In contrast, the side-on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single-molecule cross-correlation spectroscopy. Thus, the two-stroke engine offers the advantage of speeding up degradation without enhancing complexity. PMID:15175655

  13. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

    PubMed

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F S; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  14. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form

    PubMed Central

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F. S.; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  15. S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth.

    PubMed

    Albertos, Pablo; Romero-Puertas, María C; Tatematsu, Kiyoshi; Mateos, Isabel; Sánchez-Vicente, Inmaculada; Nambara, Eiji; Lorenzo, Oscar

    2015-01-01

    Plant survival depends on seed germination and progression through post-germinative developmental checkpoints. These processes are controlled by the stress phytohormone abscisic acid (ABA). ABA regulates the basic leucine zipper transcriptional factor ABI5, a central hub of growth repression, while the reactive nitrogen molecule nitric oxide (NO) counteracts ABA during seed germination. However, the molecular mechanisms by which seeds sense more favourable conditions and start germinating have remained elusive. Here we show that ABI5 promotes growth via NO, and that ABI5 accumulation is altered in genetic backgrounds with impaired NO homeostasis. S-nitrosylation of ABI5 at cysteine-153 facilitates its degradation through CULLIN4-based and KEEP ON GOING E3 ligases, and promotes seed germination. Conversely, mutation of ABI5 at cysteine-153 deregulates protein stability and inhibition of seed germination by NO depletion. These findings suggest an inverse molecular link between NO and ABA hormone signalling through distinct posttranslational modifications of ABI5 during early seedling development.

  16. S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth

    PubMed Central

    Albertos, Pablo; Romero-Puertas, María C.; Tatematsu, Kiyoshi; Mateos, Isabel; Sánchez-Vicente, Inmaculada; Nambara, Eiji; Lorenzo, Oscar

    2015-01-01

    Plant survival depends on seed germination and progression through post-germinative developmental checkpoints. These processes are controlled by the stress phytohormone abscisic acid (ABA). ABA regulates the basic leucine zipper transcriptional factor ABI5, a central hub of growth repression, while the reactive nitrogen molecule nitric oxide (NO) counteracts ABA during seed germination. However, the molecular mechanisms by which seeds sense more favourable conditions and start germinating have remained elusive. Here we show that ABI5 promotes growth via NO, and that ABI5 accumulation is altered in genetic backgrounds with impaired NO homeostasis. S-nitrosylation of ABI5 at cysteine-153 facilitates its degradation through CULLIN4-based and KEEP ON GOING E3 ligases, and promotes seed germination. Conversely, mutation of ABI5 at cysteine-153 deregulates protein stability and inhibition of seed germination by NO depletion. These findings suggest an inverse molecular link between NO and ABA hormone signalling through distinct posttranslational modifications of ABI5 during early seedling development. PMID:26493030

  17. Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

    NASA Astrophysics Data System (ADS)

    Anjum, Rana S.; Bray, Sian M.; Blackwood, John K.; Kilkenny, Mairi L.; Coelho, Matthew A.; Foster, Benjamin M.; Li, Shurong; Howard, Julie A.; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J.; Robinson, Nicholas P.

    2015-09-01

    In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

  18. Functions of the Proteasome on Chromatin

    PubMed Central

    McCann, Tyler S.; Tansey, William P.

    2014-01-01

    The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome. PMID:25422899

  19. The ubiquitin-proteasome system regulates plant hormone signaling

    PubMed Central

    Santner, Aaron; Estelle, Mark

    2011-01-01

    SUMMARY Plants utilize the ubiquitin-proteasome system (UPS) to modulate nearly every aspect of growth and development. Ubiquitin is covalently attached to target proteins through the action of three enzymes known as E1, E2, and E3. The ultimate outcome of this post-translational modification depends on the nature of the ubiquitin linkage and the extent of polyubiquitination. In most cases, ubiquitination results in degradation of the target protein in the 26S proteasome. During the last 10 years it has become clear that the UPS plays a prominent regulatory role in hormone biology. E3 ubiquitin ligases in particular actively participate in hormone perception, de-repression of hormone signaling pathways, degradation of hormone specific transcription factors, and regulation of hormone biosynthesis. It is certain that additional functions will be discovered as more of the nearly 1200 potential E3s in plants are elucidated. PMID:20409276

  20. E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair.

    PubMed

    Singh, Randeep K; Dagnino, Lina

    2016-05-01

    Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis.

  1. E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair

    PubMed Central

    Singh, Randeep K.; Dagnino, Lina

    2016-01-01

    Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis. PMID:27028861

  2. Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles*

    PubMed Central

    Waite, Kenrick A.; Mota-Peynado, Alina De-La; Vontz, Gabrielle; Roelofs, Jeroen

    2016-01-01

    The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, although CP largely localized to the cytosol as well as granular structures within the cytosol. In all, our data reveal a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export, and independent vacuolar targeting of CP and RP. Thus, in addition to the well characterized transcriptional up-regulation of genes encoding proteasome subunits, cells are also capable of down-regulating cellular levels of proteasomes through proteaphagy. PMID:26670610

  3. Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

    PubMed Central

    Wang, Feng; Deng, Xing Wang

    2011-01-01

    The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth. PMID:21788985

  4. Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.

    PubMed

    Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R

    2015-05-01

    The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21. PMID:24975575

  5. Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes

    PubMed Central

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Gao, Yuan; Yu, Li-Rong; Alla, Ramani; Shmookler Reis, Robert

    2015-01-01

    Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal Aβ1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10−6). In worms expressing Aβ1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10−6). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly

  6. Molecular Architecture and Assembly of the Eukaryotic Proteasome

    PubMed Central

    Tomko, Robert J.; Hochstrasser, Mark

    2013-01-01

    The eukaryotic ubiquitin-proteasome system is responsible for most cellular quality-control and regulatory protein degradation. Its substrates, which are usually modified by polymers of ubiquitin, are ultimately degraded by the 26S proteasome. This 2.6 MDa protein complex is separated into a barrel-shaped proteolytic 20S core particle (CP) of 28 subunits capped on one or both ends by a 19S regulatory particle (RP) comprising at least 19 subunits. The RP coordinates substrate recognition, removal of substrate polyubiquitin chains, and substrate unfolding and translocation into the CP for degradation. While many atomic structures of the CP have been determined, the RP has resisted high-resolution analysis. Recently, however, a combination of cryo-electron microscopy (cryo-EM), biochemical analysis, and crystal structure determination of several RP subunits has yielded a near-atomic resolution view of much of the complex. Major new insights into chaperone-assisted proteasome assembly have also recently been made. Here we review these novel findings. PMID:23495936

  7. Acute and chronic cadmium exposure promotes E-cadherin degradation in MCF7 breast cancer cells.

    PubMed

    Ponce, Esmeralda; Louie, Maggie C; Sevigny, Mary B

    2015-10-01

    Cadmium is an environmental carcinogen that usually enters the body at minute concentrations through diet or cigarette smoke and bioaccumulates in soft tissues. In past studies, cadmium has been shown to contribute to the development of more aggressive cancer phenotypes including increased cell migration and invasion. This study aims to determine if cadmium exposure-both acute and chronic-contributes to breast cancer progression by interfering with the normal functional relationship between E-cadherin and β-catenin. An MCF7 breast cancer cell line (MCF7-Cd) chronically exposed to 10(-7)  M CdCl2 was previously developed and used as a model system to study chronic exposures, whereas parental MCF7 cells exposed to 10(-6)  M CdCl2 for short periods of time were used to study acute exposures. Cadmium exposure of MCF7 cells led to the degradation of the E-cadherin protein via the ubiquitination pathway. This resulted in fewer E-cadherin/β-catenin complexes and the relocation of active β-catenin to the nucleus, where it interacted with transcription factor TCF-4 to modulate gene expression. Interestingly, only cells chronically exposed to cadmium showed a significant decrease in the localization of β-catenin to the plasma membrane and an increased distance between cells. Our data suggest that cadmium exposure promotes breast cancer progression by (1) down-regulating E-cadherin, thus decreasing the number of E-cadherin/β-catenin adhesion complexes, and (2) enhancing the nuclear translocation of β-catenin to increase expression of cancer-promoting proteins (i.e., c-Jun and cyclin D1).

  8. Targeting Tumor Proteasome with Traditional Chinese Medicine

    PubMed Central

    Yang, Huanjie; Liu, Jinbao; Dou, Q. Ping

    2012-01-01

    The proteasome is a multicatalytic protease complex whose activity is required for the growth of normal or tumor cells. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that the proteasome could be a target of chemotherapy. Studies suggest that traditional Chinese medicine (TCM) is an effective approach for cancer treatment. Here we reviewed several TCMs for their potential in treatment of cancer. This short review focuses mainly on the TCMs that potentially target the tumor cellular proteasome and NF-κB pathway whose activation is dependent on the proteasome activity. PMID:20156140

  9. Purification and characterization of 26S proteasomes from human and mouse spermatozoa.

    PubMed

    Tipler, C P; Hutchon, S P; Hendil, K; Tanaka, K; Fishel, S; Mayer, R J

    1997-12-01

    We purified by fractionation on 10-40% glycerol gradients, 26S proteasomes from normal human spermatozoa. These proteasomes, which participate in the ATP-dependent degradation of ubiquitinated proteins, share a similar sedimentation coefficient to those purified from other human tissues. Fluorogenic peptide assays reveal they have chymotrypsin, trypsin and peptidyl-glutamyl-like peptide hydrolysing activities; the chymotrypsin activity is ablated by the specific 26S proteasome inhibitor MG132. Confirmation that these large proteases are 26S proteasomes is provided by detection of the 20S proteasome subunits HC2, XAPC7, RN3 and Z and regulatory ATPases MSS1, TBP1, SUG1 and SUG2 by Western analyses with monoclonal antisera. These antigens are found only in the gradient fractions enriched in proteolytic activities. We have also shown that, although mature spermatozoa from mice have considerably reduced amounts of a ubiquitin-conjugating enzyme (E2) and ubiquitin-protein conjugates in comparison with less mature germ cells, they retain relatively high values of 26S proteasome activity. This suggests that proteasomes may have further roles to play in normal sperm physiology.

  10. Xilei San Ameliorates Experimental Colitis in Rats by Selectively Degrading Proinflammatory Mediators and Promoting Mucosal Repair

    PubMed Central

    Hori, Kazutoshi; Wang, Shenglan; Kogure, Yoko; Fukunaga, Ken; Kashiwamura, Shinichiro; Yamamoto, Satoshi; Nakamura, Shiro; Li, Junxiang; Miwa, Hiroto; Noguchi, Koichi

    2014-01-01

    Xilei san (XLS), a herbal preparation widely used in China for erosive and ulcerative diseases, has been shown to be effective in ulcerative colitis (UC). The present experiments were conducted to assess its efficacy and determine its mechanism of action in a rat model that resembles human UC. The model was induced by adding 4% dextran sulfate sodium (DSS) to the rats' drinking water for 7 days. XLS was administered daily by retention enema from day 2 to day 7; the rats were sacrificed on day 8. The colon tissues were obtained for further experiments. A histological damage score and the activity of tissue myeloperoxidase were used to evaluate the severity of the colitis. The colonic cytokine levels were detected in a suspension array, and epithelial proliferation was assessed using Ki-67 immunohistochemistry. Intrarectal administration of XLS attenuated the DSS-induced colitis, as evidenced by a reduction in both the histological damage score and myeloperoxidase activity. It also decreased the levels of proinflammatory cytokines, but increased the mucosal repair-related cytokines. In addition, the epithelial Ki-67 expression was upregulated by XLS. These results suggest that XLS attenuates DSS-induced colitis by degrading proinflammatory mediators and promoting mucosal repair. XLS could be a potential topical treatment for human UC. PMID:25120575

  11. Effect on epidermal cell of soybean protein-degraded products and structural determination of the root hair promoting peptide.

    PubMed

    Matsumiya, Yoshiki; Sumiyoshi, Sayoko; Matsukura, Takuma; Kubo, Motoki

    2007-11-01

    Peptide(s) produced from degraded soybean protein by an alkaline protease from Bacillus circulans HA12 (degraded soybean-meal products; DSP) increased the number of both the root hair cells (trichoblasts) and hairless cells (atrichoblasts) of Brassica rapa by about 4.4 times and 1.9 times, respectively. To identify the root hair-promoting peptide(s) in DSP, the origin protein of the root hair-promoting peptide(s) was identified as Kunitz trypsin inhibitor (KTI). The root hair-promoting peptide in the degraded products of KTI was purified and produced a signal of 1,198.2 Da with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A search of the amino acid sequence of KTI located the peptide GGIRAAPTGNER, which had a molecular weight identical to 1,198.2 Da. The peptide GGIRAAPTGNER was chemically synthesized, and the synthetic peptide possessed root hair-promoting activity. Thus, it is concluded that this peptide in DSP is the foreign bioactive peptide promoting the differentiation of root hairs.

  12. Blocking Autophagy Prevents Bortezomib-Induced NF-κB Activation by Reducing I-κBα Degradation in Lymphoma Cells

    PubMed Central

    Jia, Li; Gopinathan, Ganga; Sukumar, Johanna T.; Gribben, John G.

    2012-01-01

    Here we show that bortezomib induces effective proteasome inhibition and accumulation of poly-ubiquitinated proteins in diffuse large B-cell lymphoma (DLBCL) cells. This leads to induction of endoplasmic reticulum (ER) stress as demonstrated by accumulation of the protein CHOP, as well as autophagy, as demonstrated by accumulation of LC3-II proteins. Our data suggest that recruitment of both ubiquitinated proteins and LC3-II by p62 directs ubiquitinated proteins, including I-κBα, to the autophagosome. Degradation of I-κBα results in increased NF-κB nuclear translocation and transcription activity. Since bortezomib treatment promoted I-κBα phosphorylation, ubiquitination and degradation, this suggests that the route of I-κBα degradation was not via the ubiquitin-proteasome degradation system. The autophagy inhibitor chloroquine (CQ) significantly inhibited bortezomib-induced I-κBα degradation, increased complex formation with NF-κB and reduced NF-κB nuclear translocation and DNA binding activity. Importantly, the combination of proteasome and autophagy inhibitors showed synergy in killing DLBCL cells. In summary, bortezomib-induced autophagy confers relative DLBCL cell drug resistance by eliminating I-κBα. Inhibition of both autophagy and the proteasome has great potential to kill apoptosis-resistant lymphoma cells. PMID:22393418

  13. TLR4 mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain

    PubMed Central

    Pla, A; Pascual, M; Renau-Piqueras, J; Guerri, C

    2014-01-01

    New evidence indicates the involvement of protein degradation dysfunctions in neurodegeneration, innate immunity response and alcohol hepatotoxicity. We recently demonstrated that ethanol increases brain proinflammatory mediators and causes brain damage by activating Toll-like receptor 4 (TLR4) signaling in glia. However, it is uncertain if the ubiquitin-proteasome and autophagy-lysosome pathways are involved in ethanol-induced brain damage and whether the TLR4 response is implicated in proteolytic processes. Using the cerebral cortex of WT and TLR4-knockout mice with and without chronic ethanol treatment, we demonstrate that ethanol induces poly-ubiquitinated proteins accumulation and promotes immunoproteasome activation by inducing the expression of β2i, β5i and PA28α, although it decreases the 20S constitutive proteasome subunits (α2, β5). Ethanol also upregulates mTOR phosphorylation, leading to a downregulation of the autophagy-lysosome pathway (ATG12, ATG5, cathepsin B, p62, LC3) and alters the volume of autophagic vacuoles. Notably, mice lacking TLR4 receptors are protected against ethanol-induced alterations in protein degradation pathways. In summary, the present results provide the first evidence demonstrating that chronic ethanol treatment causes proteolysis dysfunctions in the mouse cerebral cortex and that these events are TLR4 dependent. These findings could provide insight into the mechanisms underlying ethanol-induced brain damage. PMID:24556681

  14. Combined use of alkane-degrading and plant growth-promoting bacteria enhanced phytoremediation of diesel contaminated soil.

    PubMed

    Tara, Nain; Afzal, Muhammad; Ansari, Tariq M; Tahseen, Razia; Iqbal, Samina; Khan, Qaiser M

    2014-01-01

    Inoculation of plants with pollutant-degrading and plant growth-promoting microorganisms is a simple strategy to enhance phytoremediation activity. The objective of this study was to determine the effect of inoculation of different bacterial strains, possessing alkane-degradation and 1-amino-cyclopropane-1 -carboxylic acid (ACC) deaminase activity, on plant growth and phytoremediation activity. Carpet grass (Axonopus affinis) was planted in soil spiked with diesel (1% w/w) for 90 days and inoculated with different bacterial strains, Pseudomonas sp. ITRH25, Pantoea sp. BTRH79 and Burkholderia sp. PsJN, individually and in combination. Generally, bacterial application increased total numbers of culturable hydrocarbon-degrading bacteria in the rhizosphere ofcarpet grass, plant biomass production, hydrocarbon degradation and reduced genotoxicity. Bacterial strains possessing different beneficial traits affect plant growth and phytoremediation activity in different ways. Maximum bacterial population, plant biomass production and hydrocarbon degradation were achieved when carpet grass was inoculated with a consortium of three strains. Enhanced plant biomass production and hydrocarbon degradation were associated with increased numbers of culturable hydrocarbon-degrading bacteria in the rhizosphere of carpet grass. The present study revealed that the combined use of different bacterial strains, exhibiting different beneficial traits, is a highly effective strategy to improve plant growth and phytoremediation activity.

  15. The REGγ-proteasome forms a regulatory circuit with IκBɛ and NFκB in experimental colitis

    PubMed Central

    Xu, Jinjin; Zhou, Lei; Ji, Lei; Chen, Fengyuan; Fortmann, Karen; Zhang, Kun; Liu, Qingwu; Li, Ke; Wang, Weicang; Wang, Hao; Xie, Wei; Wang, Qingwei; Liu, Jiang; Zheng, Biao; Zhang, Pei; Huang, Shixia; Shi, Tieliu; Zhang, Biaohong; Dang, Yongyan; Chen, Jiwu; O'Malley, Bert W.; Moses, Robb E.; Wang, Ping; Li, Lei; Xiao, Jianru; Hoffmann, Alexander; Li, Xiaotao

    2016-01-01

    Increasing incidence of inflammatory bowel disorders demands a better understanding of the molecular mechanisms underlying its multifactorial aetiology. Here we demonstrate that mice deficient for REGγ, a proteasome activator, show significantly attenuated intestinal inflammation and colitis-associated cancer in dextran sodium sulfate model. Bone marrow transplantation experiments suggest that REGγ's function in non-haematopoietic cells primarily contributes to the phenotype. Elevated expression of REGγ exacerbates local inflammation and promotes a reciprocal regulatory loop with NFκB involving ubiquitin-independent degradation of IκBɛ. Additional deletion of IκBɛ restored colitis phenotypes and inflammatory gene expression in REGγ-deficient mice. In sum, this study identifies REGγ-mediated control of IκBɛ as a molecular mechanism that contributes to NFκB activation and promotes bowel inflammation and associated tumour formation in response to chronic injury. PMID:26899380

  16. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

    SciTech Connect

    Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi; Yamada, Koji; Noguchi, Kohji; Fujimuro, Masahiro

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors

  17. Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

    2009-09-04

    Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of {sup 35}S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

  18. Structural Determinants Involved in the Regulation of CXCL14/BRAK Expression by the 26S Proteasome

    PubMed Central

    Peterson, Francis C.; Thorpe, Jeffery A.; Harder, Adam; Volkman, Brian F.; Schwarze, Steven R.

    2006-01-01

    The chemokine CXCL14/BRAK participates in immune surveillance by recruiting dendritic cells. CXCL14 gene expression is altered in a number of cancers, but protein expression levels have not been investigated. Here we report that CXCL14 protein can be expressed in primary epithelial cells, however in several immortalized and cancer cell lines this protein is targeted for polyubiquitylation and proteasomal degradation. We determined the NMR structure of CXCL14 to identify motifs controlling its expression. CXCL14 adopts the canonical chemokine tertiary fold but contains a unique five amino acid insertion (41VSRYR45) relative to other CXC chemokines. Deletion or substitution of key residues within this insertion prevented proteasomal degradation. Furthermore, we defined a 15 amino acid fragment of CXCL14 that is sufficient to induce proteasomal degradation. This study elucidates a post-translational mechanism for the loss of CXCL14 in cancer and a novel mode of chemokine regulation. PMID:16987528

  19. Unfolding knots by proteasome-like systems: simulations of the behaviour of folded and neurotoxic proteins.

    PubMed

    Wojciechowski, Michał; Gómez-Sicilia, Àngel; Carrión-Vázquez, Mariano; Cieplak, Marek

    2016-08-16

    Knots in proteins have been proposed to resist proteasomal degradation. Ample evidence associates proteasomal degradation with neurodegeneration. One interesting possibility is that indeed knotted conformers stall this machinery leading to toxicity. However, although the proteasome is known to unfold mechanically its substrates, at present there are no experimental methods to emulate this particular traction geometry. Here, we consider several dynamical models of the proteasome in which the complex is represented by an effective potential with an added pulling force. This force is meant to induce the translocation of a protein or a polypeptide into the catalytic chamber. The force is either constant or applied periodically. The translocated proteins are modelled in a coarse-grained fashion. We do comparative analysis of several knotted globular proteins and the transiently knotted polyglutamine tracts of length 60 alone and fused in exon 1 of the huntingtin protein. Huntingtin is associated with Huntington's disease, a well-known genetically determined neurodegenerative disease. We show that the presence of a knot hinders and sometimes even jams translocation. We demonstrate that the probability to do so depends on the protein, the model of the proteasome, the magnitude of the pulling force, and the choice of the pulled terminus. In any case, the net effect would be a hindrance in the proteasomal degradation process in the cell. This would then yield toxicity via two different mechanisms: one through toxic monomers compromising degradation and another by the formation of toxic oligomers. Our work paves the way for the mechanistic investigation of the mechanical unfolding of knotted structures by the proteasome and its relation to toxicity and disease. PMID:27425826

  20. Proteasome activity is required for the stage-specific transformation of a protozoan parasite

    PubMed Central

    1996-01-01

    A prominent feature of the life cycle of intracellular parasites is the profound morphological changes they undergo during development in the vertebrate and invertebrate hosts. In eukaryotic cells, most cytoplasmic proteins are degraded in proteasomes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, and the intracellular development of the parasite from amastigotes into trypomastigotes, are prevented by lactacystin, or by a peptide aldehyde that inhibits proteasome function. Clasto-lactacystin, an inactive analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S proteasomes from T. cruzi as a target of lactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan. PMID:8920878

  1. LAPTM5 promotes lysosomal degradation of intracellular CD3ζ but not of cell surface CD3ζ.

    PubMed

    Kawai, Yohei; Ouchida, Rika; Yamasaki, Sho; Dragone, Leonard; Tsubata, Takeshi; Wang, Ji-Yang

    2014-07-01

    The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.

  2. Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications

    PubMed Central

    Huang, Qian; Figueiredo-Pereira, Maria E.

    2010-01-01

    The ubiquitin/proteasome pathway is the major proteolytic quality control system in cells. In this review we discuss the impact of a deregulation of this pathway on neuronal function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in pathological inclusion bodies in all the major chronic neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as amyotrophic lateral sclerosis. We describe the intricate nature of the ubiquitin/proteasome pathway and discuss the paradox of protein aggregation, i.e. its potential toxic/protective effect in neurodegeneration. The relations between some of the dysfunctional components of the pathway and neurodegeneration are presented. We highlight possible ubiquitin/proteasome pathway-targeting therapeutic approaches, such as activating the proteasome, enhancing ubiquitination and promoting SUMOylation that might be important to slow/treat the progression of neurodegeneration. Finally, a model time line is presented for neurodegeneration starting at the initial injurious events up to protein aggregation and cell death, with potential time points for therapeutic intervention. PMID:20131003

  3. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death.

    PubMed

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion.

  4. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death

    PubMed Central

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A.; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion. PMID:27529620

  5. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death.

    PubMed

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion. PMID:27529620

  6. Bacterial structure and characterization of plant growth promoting and oil degrading bacteria from the rhizospheres of mangrove plants.

    PubMed

    do Carmo, Flávia Lima; dos Santos, Henrique Fragoso; Martins, Edir Ferreira; van Elsas, Jan Dirk; Rosado, Alexandre Soares; Peixoto, Raquel Silva

    2011-08-01

    Most oil from oceanic spills converges on coastal ecosystems, such as mangrove forests, which are threatened with worldwide disappearance. Particular bacteria that inhabit the rhizosphere of local plant species can stimulate plant development through various mechanisms; it would be advantageous if these would also be capable of degrading oil. Such bacteria may be important in the preservation or recuperation of mangrove forests impacted by oil spills. This study aimed to compare the bacterial structure, isolate and evaluate bacteria able to degrade oil and stimulate plant growth, from the rhizospheres of three mangrove plant species. These features are particularly important taking into account recent policies for mangrove bioreme-diation, implying that oil degradation as well as plant maintenance and health are key targets. Fifty-seven morphotypes were isolated from the mangrove rhizospheres on Bushneil-Haas (BH) medium supplemented with oil as the sole carbon source and tested for plant growth promotion. Of this strains, 60% potentially fixed nitrogen, 16% showed antimicrobial activity, 84% produced siderophores, 51% had the capacity to solubilize phosphate, and 33% produced the indole acetic acid hormone. Using gas chromatography, we evaluated the oil-degrading potential of ten selected strains that had different morphologies and showed Plant Growth Promoting Rhizobacteria (PGPR) features. The ten tested strains showed a promising degradation profile for at least one compound present in the oil. Among degrader strains, 46% had promising PGPR potential, having at least three of the above capacities. These strains might be used as a consortium, allowing the concomitant degradation of oil and stimulation of mangrove plant survival and maintenance. PMID:21887634

  7. Endoplasmic reticulum stress and proteasomal system in amyotrophic lateral sclerosis.

    PubMed

    Karademir, Betul; Corek, Ceyda; Ozer, Nesrin Kartal

    2015-11-01

    Protein processing including folding, unfolding and degradation is involved in the mechanisms of many diseases. Unfolded protein response and/or endoplasmic reticulum stress are accepted to be the first steps which should be completed via protein degradation. In this direction, proteasomal system and autophagy play important role as the degradation pathways and controlled via complex mechanisms. Amyotrophic lateral sclerosis is a multifactorial neurodegenerative disease which is also known as the most catastrophic one. Mutation of many different genes are involved in the pathogenesis such as superoxide dismutase 1, chromosome 9 open reading frame 72 and ubiquilin 2. These genes are mainly related to the antioxidant defense systems, endoplasmic reticulum stress related proteins and also protein aggregation, degradation pathways and therefore mutation of these genes cause related disorders.This review focused on the role of protein processing via endoplasmic reticulum and proteasomal system in amyotrophic lateral sclerosis which are the main players in the pathology. In this direction, dysfunction of endoplasmic reticulum associated degradation and related cell death mechanisms that are autophagy/apoptosis have been detailed.

  8. Colorectal Carcinogenesis, Radiation Quality, and the Ubiquitin-Proteasome Pathway

    PubMed Central

    Datta, Kamal; Suman, Shubhankar; Kumar, Santosh; Fornace, Albert J

    2016-01-01

    Adult colorectal epithelium undergoes continuous renewal and maintains homeostatic balance through regulated cellular proliferation, differentiation, and migration. The canonical Wnt signaling pathway involving the transcriptional co-activator β-catenin is important for colorectal development and normal epithelial maintenance, and deregulated Wnt/β-catenin signaling has been implicated in colorectal carcinogenesis. Colorectal carcinogenesis has been linked to radiation exposure, and radiation has been demonstrated to alter Wnt/β-catenin signaling, as well as the proteasomal pathway involved in the degradation of the signaling components and thus regulation of β-catenin. The current review discusses recent progresses in our understanding of colorectal carcinogenesis in relation to different types of radiation and roles that radiation quality plays in deregulating β-catenin and ubiquitin-proteasome pathway (UPP) for colorectal cancer initiation and progression. PMID:26819641

  9. Does impairment of the ubiquitin-proteasome system or the autophagy-lysosome pathway predispose individuals to neurodegenerative disorders such as Parkinson's disease?

    PubMed

    Matsuda, Noriyuki; Tanaka, Keiji

    2010-01-01

    About twenty years ago, an abnormal enrichment of ubiquitin in the inclusion bodies of various neurodegenerative disorders was reported. To date, this phenotype has been a diagnostic feature of many neurodegenerative disorders including Alzheimer's and Parkinson's diseases (PD). Because ubiquitin tags proteins that must be eliminated from cells, thereby targeting them for proteasomal degradation, many scientists believed that the ubiquitin-proteasome system (UPS) was inactivated in these neurodegenerative disorders. This inactivation would lead to an accumulation of ubiquitylated proteins with their concomitant aggregation into inclusion bodies and subsequent neuronal death. This hypothesis was further fuelled by the discovery that parkin, the causal gene of autosomal recessive juvenile Parkinsonism, functions as a ubiquitin ligase. However, recent findings by several groups demonstrated that ubiquitylation is also relevant to the autophagy system, with parkin promoting autophagy of dysfunctional mitochondria following the loss of mitochondrial membrane potential. These novel topics do not necessarily mean that the proteasome is involved in neurodegeneration of PD. In this review, we describe current evidence and controversies regarding the relationship between UPS and neurodegenerative disorders such as PD, and discuss several scientific discrepancies that await further clarification.

  10. The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells.

    PubMed

    Khan, Saad; Khamis, Imran; Heikkila, John J

    2015-11-01

    In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with the proteasomal inhibitor, MG132, or the environmental toxicants, sodium arsenite or cadmium chloride, induced the accumulation of the small heat shock protein, HSP30, in total and in both soluble and insoluble protein fractions. Immunocytochemical analysis revealed the presence of relatively large HSP30 structures primarily in the perinuclear region of the cytoplasm. All three of the stressors promoted the formation of aggresome-like inclusion bodies as determined by immunocytochemistry and laser scanning confocal microscopy using a ProteoStat aggresome dye and additional aggresomal markers, namely, anti-γ-tubulin and anti-vimentin antibodies. Further analysis revealed that HSP30 co-localized with these aggresome-like inclusion bodies. In most cells, HSP30 was found to envelope or occur within these structures. Finally, we show that treatment of cells with withaferin A, a steroidal lactone with anti-inflammatory, anti-tumor, and proteasomal inhibitor properties, also induced HSP30 accumulation that co-localized with aggresome-like inclusion bodies. It is possible that proteasomal inhibitor or metal/metalloid-induced formation of aggresome-like inclusion bodies may sequester toxic protein aggregates until they can be degraded. While the role of HSP30 in these aggresome-like structures is not known, it is possible that they may be involved in various aspects of aggresome-like inclusion body formation or transport.

  11. [Promotion effects of vitamin B12 on the degradation of 2, 4, 4'-trichlorobiphenyl by Nostoc PD-2].

    PubMed

    Liu, Jia-Yu; Xiao, Wen-Feng; Lu, Li-Ping; Zhang, Hang-Jun

    2014-08-01

    Polychlorinated biphenyls are typical persistent chlorinated organic compounds in the environment. Bioremediation of PCB-contaminated environment has become one of the hot issues. In this study, vitamin B12 (VB12) and chlorine-free culture medium were applied to study the effects of VB12 on the degradation of 2,4,4'-trichlorobiphenyl (PCB28) by Nostoc PD-2 and the gene expression during the PCB-degradation process. Results showed that addition of different concentrations of vitamin B12 could improve the PCB-biodegradation rates by Nostoc PD-2. Compared with the control group, the 7-day degradation rate in 10 microg x L(-1), 100 microg x L(-1), and 1 000 microg x L(-1) VB12-treated groups increased by 11.0%, 19.7%, and 21.9% , respectively. The degradation half-time decreased from 5.53 days (treated with 10 microg x L(-1) VB12) to 3.08 days (treated with 100 microg x L(-1) VB12). The expression of cytochrome b6f complex iron-sulfur protein gene and dioxygenase gene showed significant correlation with PCB28-degradation by Nostoc PD-2. While the expression of iron-sulfur protein gene showed more significant correlation with PCB28-degradation. Results in this study indicated that adding VB12 could promote PCB28-degradation by Nostoc PD-2. Moreover, VB12 addition improved the PCB-degradation activity of Nostoc PD-2 at the gene level. The above conclusions could provide a new choice for developing efficient bioremediation technology for PCB-contaminated environment and a new insight into the PCB-biodegradation mechanism by Nostoc PD-2.

  12. Structure of the human 26S proteasome at a resolution of 3.9 Å

    PubMed Central

    Schweitzer, Andreas; Aufderheide, Antje; Rudack, Till; Beck, Florian; Pfeifer, Günter; Plitzko, Jürgen M.; Sakata, Eri; Schulten, Klaus; Förster, Friedrich; Baumeister, Wolfgang

    2016-01-01

    Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing. PMID:27342858

  13. Hemoglobin fructation promotes heme degradation through the generation of endogenous reactive oxygen species

    NASA Astrophysics Data System (ADS)

    Goodarzi, M.; Moosavi-Movahedi, A. A.; Habibi-Rezaei, M.; Shourian, M.; Ghourchian, H.; Ahmad, F.; Farhadi, M.; Saboury, A. A.; Sheibani, N.

    2014-09-01

    Protein glycation is a cascade of nonenzymatic reactions between reducing sugars and amino groups of proteins. It is referred to as fructation when the reducing monosaccharide is fructose. Some potential mechanisms have been suggested for the generation of reactive oxygen species (ROS) by protein glycation reactions in the presence of glucose. In this state, glucose autoxidation, ketoamine, and oxidative advance glycation end products (AGEs) formation are considered as major sources of ROS and perhaps heme degradation during hemoglobin glycation. However, whether fructose mediated glycation produces ROS and heme degradation is unknown. Here we report that ROS (H2O2) production occurred during hemoglobin fructation in vitro using chemiluminescence methods. The enhanced heme exposure and degradation were determined using UV-Vis and fluorescence spectrophotometry. Following accumulation of ROS, heme degradation products were accumulated reaching a plateau along with the detected ROS. Thus, fructose may make a significant contribution to the production of ROS, glycation of proteins, and heme degradation during diabetes.

  14. Phosphorylation-triggered CUEDC2 degradation promotes UV-induced G1 arrest through APC/C(Cdh1) regulation.

    PubMed

    Zhang, Wei-Na; Zhou, Jie; Zhou, Tao; Li, Ai-Ling; Wang, Na; Xu, Jin-Jing; Chang, Yan; Man, Jiang-Hong; Pan, Xin; Li, Tao; Li, Wei-Hua; Mu, Rui; Liang, Bing; Chen, Liang; Jin, Bao-Feng; Xia, Qing; Gong, Wei-Li; Zhang, Xue-Min; Wang, Li; Li, Hui-Yan

    2013-07-01

    DNA damage triggers cell cycle arrest to provide a time window for DNA repair. Failure of arrest could lead to genomic instability and tumorigenesis. DNA damage-induced G1 arrest is generally achieved by the accumulation of Cyclin-dependent kinase inhibitor 1 (p21). However, p21 is degraded and does not play a role in UV-induced G1 arrest. The mechanism of UV-induced G1 arrest thus remains elusive. Here, we have identified a critical role for CUE domain-containing protein 2 (CUEDC2) in this process. CUEDC2 binds to and inhibits anaphase-promoting complex/cyclosome-Cdh1 (APC/C(Cdh1)), a critical ubiquitin ligase in G1 phase, thereby stabilizing Cyclin A and promoting G1-S transition. In response to UV irradiation, CUEDC2 undergoes ERK1/2-dependent phosphorylation and ubiquitin-dependent degradation, leading to APC/C(Cdh1)-mediated Cyclin A destruction, Cyclin-dependent kinase 2 inactivation, and G1 arrest. A nonphosphorylatable CUEDC2 mutant is resistant to UV-induced degradation. Expression of this stable mutant effectively overrides UV-induced G1-S block. These results establish CUEDC2 as an APC/C(Cdh1) inhibitor and indicate that regulated CUEDC2 degradation is critical for UV-induced G1 arrest.

  15. Isolation and characterization of a bluegill-degrading microorganism, and analysis of the root hair-promoting effect of the degraded products.

    PubMed

    Sanpa, Sirilak; Sumiyoshi, Sayoko; Kujira, Tadakazu; Matsumiya, Yoshiki; Kubo, Motoki

    2006-02-01

    Bluegill-degrading bacteria were isolated from various environmental sources. Brevibacillus sp. BGM1 degraded bluegill efficiently at 50 degrees C, and its culture supernatant showed the highest peptide and amino acid concentrations as trichloroacetic acid (TCA) soluble fraction (ASF) (10.7 mg/ml) of all supernatants obtained with bluegill as a substrate. Strain BGM1 secreted a protease(s) into the medium, and the concentration of peptides and amino acids gradually increased. The fertile effect of the degraded bluegill products (DGP) on Brassica rapa was also investigated. The root hair density of B. rapa grown with DGP at a concentration of 30 mug peptides and amino acids/ml was about 1.7 times higher than when grown with the same concentration of undegraded bluegill. DGP was shown to increase root hair numbers and adventitious root formation. The results of this study suggest that a specific peptide(s) for promotion of root hair is produced from the order Perciformes with a protease(s) from BGM1.

  16. Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors.

    PubMed

    Granot, Zvi; Kobiler, Oren; Melamed-Book, Naomi; Eimerl, Sarah; Bahat, Assaf; Lu, Bin; Braun, Sergei; Maurizi, Michael R; Suzuki, Carolyn K; Oppenheim, Amos B; Orly, Joseph

    2007-09-01

    Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein promoting transfer of cholesterol into steroid making mitochondria in specialized cells of the adrenal cortex and gonads. Our previous work has demonstrated that StAR is rapidly degraded upon import into the mitochondrial matrix. To identify the protease(s) responsible for this rapid turnover, murine StAR was expressed in wild-type Escherichia coli or in mutant strains lacking one of the four ATP-dependent proteolytic systems, three of which are conserved in mammalian mitochondria-ClpP, FtsH, and Lon. StAR was rapidly degraded in wild-type bacteria and stabilized only in lon (-)mutants; in such cells, StAR turnover was fully restored upon coexpression of human mitochondrial Lon. In mammalian cells, the rate of StAR turnover was proportional to the cell content of Lon protease after expression of a Lon-targeted small interfering RNA, or overexpression of the protein. In vitro assays using purified proteins showed that Lon-mediated degradation of StAR was ATP-dependent and blocked by the proteasome inhibitors MG132 (IC(50) = 20 microm) and clasto-lactacystin beta-lactone (cLbetaL, IC(50) = 3 microm); by contrast, epoxomicin, representing a different class of proteasome inhibitors, had no effect. Such inhibition is consistent with results in cultured rat ovarian granulosa cells demonstrating that degradation of StAR in the mitochondrial matrix is blocked by MG132 and cLbetaL but not by epoxomicin. Both inhibitors also blocked Lon-mediated cleavage of the model substrate fluorescein isothiocyanate-casein. Taken together, our former studies and the present results suggest that Lon is the primary ATP-dependent protease responsible for StAR turnover in mitochondria of steroidogenic cells.

  17. Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome.

    PubMed

    Gillette, Thomas G; Kumar, Brajesh; Thompson, David; Slaughter, Clive A; DeMartino, George N

    2008-11-14

    The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.

  18. [BiOBr promoted the photocatalytic degradation of beta-cypermethrin under visible light].

    PubMed

    Peng, Yi-Zhu; Zhao, Xiao-Rong; Jia, Man-Ke; Zhou, Wei; Huang, Ying-Ping

    2014-05-01

    As a visible light photocatalyst, bismuth oxide bromide (BiOBr) was used to catalyze the degradation of beta-cypermethrin (beta-CP). The photocatalytic degradation of beta-CP was studied with gas chromatography. The effects of pH and catalyst dose on the photocatalytic degradation efficiency were discussed. The oxidization and mineralization of beta-CP were detected by chemical oxygen demand (COD) analyzer. The results showed that beta-CP could be effectively degraded under visible light irradiation using BiOBr as the catalyst. At given experimental conditions, the degradation rate of beta-CP reached 94. 68% after 10 h and the COD removal rate reached 67. 99% after 36 h. With the increase of catalyst dose and pH value, the degradation rate was improved. The photocatalytic oxidation species was determined by peroxidase method and terephthalic acid fluorescence method. These results suggested that the photocatalytic degradation process mainly referred to hydroxyl radical ( OH) mechanism. PMID:25055669

  19. The proteasomes of two marine decapod crustaceans, European lobster (Homarus gammarus) and Edible crab (Cancer pagurus), are differently impaired by heavy metals.

    PubMed

    Götze, Sandra; Bose, Aneesh; Sokolova, Inna M; Abele, Doris; Saborowski, Reinhard

    2014-05-01

    The intracellular ubiquitin-proteasome system is a key regulator of cellular processes involved in the controlled degradation of short-living or malfunctioning proteins. Certain diseases and cellular dysfunctions are known to arise from the disruption of proteasome pathways. Trace metals are recognized stressors of the proteasome system in vertebrates and plants, but their effects on the proteasome of invertebrates are not well understood. Since marine invertebrates, and particularly benthic crustaceans, can be exposed to high metal levels, we studied the effects of in vitro exposure to Hg(2+), Zn(2+), Cu(2+), and Cd(2+) on the activities of the proteasome from the claw muscles of lobsters (Homarus gammarus) and crabs (Cancer pagurus). The chymotrypsin like activity of the proteasome of these two species showed different sensitivity to metals. In lobsters the activity was significantly inhibited by all metals to a similar extent. In crabs the activities were severely suppressed only by Hg(2+) and Cu(2+) while Zn(2+) had only a moderate effect and Cd(2+) caused almost no inhibition of the crab proteasome. This indicates that the proteasomes of both species possess structural characteristics that determine different susceptibility to metals. Consequently, the proteasome-mediated protein degradation in crab C. pagurus may be less affected by metal pollution than that of the lobster H. gammarus. PMID:24721378

  20. The proteasomes of two marine decapod crustaceans, European lobster (Homarus gammarus) and Edible crab (Cancer pagurus), are differently impaired by heavy metals.

    PubMed

    Götze, Sandra; Bose, Aneesh; Sokolova, Inna M; Abele, Doris; Saborowski, Reinhard

    2014-05-01

    The intracellular ubiquitin-proteasome system is a key regulator of cellular processes involved in the controlled degradation of short-living or malfunctioning proteins. Certain diseases and cellular dysfunctions are known to arise from the disruption of proteasome pathways. Trace metals are recognized stressors of the proteasome system in vertebrates and plants, but their effects on the proteasome of invertebrates are not well understood. Since marine invertebrates, and particularly benthic crustaceans, can be exposed to high metal levels, we studied the effects of in vitro exposure to Hg(2+), Zn(2+), Cu(2+), and Cd(2+) on the activities of the proteasome from the claw muscles of lobsters (Homarus gammarus) and crabs (Cancer pagurus). The chymotrypsin like activity of the proteasome of these two species showed different sensitivity to metals. In lobsters the activity was significantly inhibited by all metals to a similar extent. In crabs the activities were severely suppressed only by Hg(2+) and Cu(2+) while Zn(2+) had only a moderate effect and Cd(2+) caused almost no inhibition of the crab proteasome. This indicates that the proteasomes of both species possess structural characteristics that determine different susceptibility to metals. Consequently, the proteasome-mediated protein degradation in crab C. pagurus may be less affected by metal pollution than that of the lobster H. gammarus.

  1. Degradation of Jatropha curcas phorbol esters derived from Jatropha oil cake and their tumor-promoting activity.

    PubMed

    Nakao, Motoyuki; Hasegawa, Go; Yasuhara, Tadashi; Ishihara, Yoko

    2015-04-01

    Large amount of oil cake is generated during biodiesel production from Jatropha seeds. Although Jatropha oil cake is rich in plant nutrients, presence of toxic phorbol esters restricts the usage of oil cake as a fertilizer. The objective of this study is to evaluate the components and tumor promoting activity of phorbol esters in Jatropha oil cake-supplemented soil and plants grown in the treated soil. Contents and their biological activity of Jatropha phorbol esters in soil and plants were sequentially analyzed by high-performance liquid chromatography (HPLC) and in vitro cell transformation assay, respectively. Disappearance of Jatropha phorbol-ester-specific peaks were followed with HPLC during incubation of Jatropha oil cake with soil for five weeks. Along with the degradation of Jatropha phorbol ester in soil, tumor-promoting activity in the sample was also attenuated and ultimately disappeared. Jatropha phorbol esters and tumor promoting activity were not detected from mustard spinach grown in the Jatropha oil cake-supplemented soil. In addition, the esterase KM109 degrades DHPB (see definition below; Jatropha phorbol ester) and reduced its tumor-promoting activity. From these data, we conclude: (1) components and tumor promoting activity of Jatropha phorbol esters in the oil cake disappeared completely by incubation with soil for five-week, (2) Jatropha phorbol esters did not transfer into plants grown in the Jatropha oil cake-supplemented soil, and (3) DHPB can be degraded by esterase from soil bacterium. These observations are useful for utilization of Jatropha oil cake as a fertilizer. PMID:25066610

  2. Degradation of Jatropha curcas phorbol esters derived from Jatropha oil cake and their tumor-promoting activity.

    PubMed

    Nakao, Motoyuki; Hasegawa, Go; Yasuhara, Tadashi; Ishihara, Yoko

    2015-04-01

    Large amount of oil cake is generated during biodiesel production from Jatropha seeds. Although Jatropha oil cake is rich in plant nutrients, presence of toxic phorbol esters restricts the usage of oil cake as a fertilizer. The objective of this study is to evaluate the components and tumor promoting activity of phorbol esters in Jatropha oil cake-supplemented soil and plants grown in the treated soil. Contents and their biological activity of Jatropha phorbol esters in soil and plants were sequentially analyzed by high-performance liquid chromatography (HPLC) and in vitro cell transformation assay, respectively. Disappearance of Jatropha phorbol-ester-specific peaks were followed with HPLC during incubation of Jatropha oil cake with soil for five weeks. Along with the degradation of Jatropha phorbol ester in soil, tumor-promoting activity in the sample was also attenuated and ultimately disappeared. Jatropha phorbol esters and tumor promoting activity were not detected from mustard spinach grown in the Jatropha oil cake-supplemented soil. In addition, the esterase KM109 degrades DHPB (see definition below; Jatropha phorbol ester) and reduced its tumor-promoting activity. From these data, we conclude: (1) components and tumor promoting activity of Jatropha phorbol esters in the oil cake disappeared completely by incubation with soil for five-week, (2) Jatropha phorbol esters did not transfer into plants grown in the Jatropha oil cake-supplemented soil, and (3) DHPB can be degraded by esterase from soil bacterium. These observations are useful for utilization of Jatropha oil cake as a fertilizer.

  3. Stimulation of ubiquitin-proteasome pathway through the expression of amidohydrolase for N-terminal asparagine (Ntan1) in cultured rat hippocampal neurons exposed to static magnetism.

    PubMed

    Hirai, Takao; Taniura, Hideo; Goto, Yasuaki; Ogura, Masato; Sng, Judy C G; Yoneda, Yukio

    2006-03-01

    In order to elucidate mechanisms underlying modulation by static magnetism of the cellular functionality and/or integrity in the brain, we screened genes responsive to brief magnetism in cultured rat hippocampal neurons using differential display analysis. We have for the first time cloned and identified Ntan1 (amidohydrolase for N-terminal asparagine) as a magnetism responsive gene in rat brain. Ntan1 is an essential component of a protein degradation signal, which is a destabilizing N-terminal residue of a protein, in the N-end rule. In situ hybridization histochemistry revealed abundant expression of Ntan1 mRNA in hippocampal neurons in vivo. Northern blot analysis showed that Ntan1 mRNA was increased about three-fold after 3 h in response to brief magnetism. Brief magnetism also increased the transcriptional activity of Ntan1 promoter by luciferase reporter assay. Brief magnetism induced degradation of microtubule-associated protein 2 (MAP2) without affecting cell morphology and viability, which was prevented by a selective inhibitor of 26S proteasome in hippocampal neurons. Overexpression of Ntan1 using recombinant Ntan1 adenovirus vector resulted in a marked decrease in the MAP2 protein expression in hippocampal neurons. Our results suggest that brief magnetism leads to the induction of Ntan1 responsible for MAP2 protein degradation through ubiquitin-proteasome pathway in rat hippocampal neurons.

  4. Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination

    NASA Astrophysics Data System (ADS)

    Song, In-Kang; Lee, Jae-Jin; Cho, Jin-Hwan; Jeong, Jihye; Shin, Dong-Hae; Lee, Kong-Joo

    2016-10-01

    Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.

  5. Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination

    PubMed Central

    Song, In-Kang; Lee, Jae-Jin; Cho, Jin-Hwan; Jeong, Jihye; Shin, Dong-Hae; Lee, Kong-Joo

    2016-01-01

    Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1. PMID:27703196

  6. Ubiquitin, Proteasomes and Proteolytic Mechanisms Activated by Kidney Disease

    PubMed Central

    Rajan, Vik; Mitch, William E.

    2008-01-01

    Summary The ubiquitin-proteasome system (UPS) includes 3 enzymes that conjugate ubiquitin to intracellular proteins that are then recognized and degraded in the proteasome. The process participates in the regulation of cell metabolism. In the kidney, the UPS regulates the turnover of transporters and signaling proteins and its activity is down regulated in acidosis-induced proximal tubular cell hypertrophy. In chronic kidney disease (CKD), muscle wasting occurs because complications of CKD including acidosis, insulin resistance, inflammation, and increased angiotensin II levels stimulate the UPS to degrade muscle proteins. This response also includes caspase-3 and calpains which act to cleave muscle proteins to provide substrates for the UPS. For example, caspase-3 degrades actomyosin, leaving a 14kD fragment of actin in muscle. The 14 kD actin fragment is increased in muscle of patient with kidney disease, burn injury and surgery. In addition, acidosis, insulin resistance, inflammation and angiotensin II stimulate glucocorticoid production. Glucocorticoids are also required for the muscle wasting that occurs in CKD. Thus, the UPS is involved in regulating kidney function and participates in highly organized responses that degrade muscle protein in response to loss of kidney function. PMID:18723090

  7. Roles of the ubiquitin proteasome system in the effects of drugs of abuse.

    PubMed

    Massaly, Nicolas; Francès, Bernard; Moulédous, Lionel

    2014-01-01

    Because of its ability to regulate the abundance of selected proteins the ubiquitin proteasome system (UPS) plays an important role in neuronal and synaptic plasticity. As a result various stages of learning and memory depend on UPS activity. Drug addiction, another phenomenon that relies on neuroplasticity, shares molecular substrates with memory processes. However, the necessity of proteasome-dependent protein degradation for the development of addiction has been poorly studied. Here we first review evidences from the literature that drugs of abuse regulate the expression and activity of the UPS system in the brain. We then provide a list of proteins which have been shown to be targeted to the proteasome following drug treatment and could thus be involved in neuronal adaptations underlying behaviors associated with drug use and abuse. Finally we describe the few studies that addressed the need for UPS-dependent protein degradation in animal models of addiction-related behaviors. PMID:25610367

  8. Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System.

    PubMed

    Matilainen, Olli; Jha, Sweta; Holmberg, Carina I

    2016-01-01

    The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions. PMID:27613038

  9. Roles of the ubiquitin proteasome system in the effects of drugs of abuse

    PubMed Central

    Massaly, Nicolas; Francès, Bernard; Moulédous, Lionel

    2015-01-01

    Because of its ability to regulate the abundance of selected proteins the ubiquitin proteasome system (UPS) plays an important role in neuronal and synaptic plasticity. As a result various stages of learning and memory depend on UPS activity. Drug addiction, another phenomenon that relies on neuroplasticity, shares molecular substrates with memory processes. However, the necessity of proteasome-dependent protein degradation for the development of addiction has been poorly studied. Here we first review evidences from the literature that drugs of abuse regulate the expression and activity of the UPS system in the brain. We then provide a list of proteins which have been shown to be targeted to the proteasome following drug treatment and could thus be involved in neuronal adaptations underlying behaviors associated with drug use and abuse. Finally we describe the few studies that addressed the need for UPS-dependent protein degradation in animal models of addiction-related behaviors. PMID:25610367

  10. The ubiquitin-proteasomal system is critical for multiple myeloma: implications in drug discovery

    PubMed Central

    Cao, Biyin; Mao, Xinliang

    2011-01-01

    Bortezomib is a specific inhibitor of proteasomes, the most important protease complexes in protein degradation. Bortezomib can induce apoptosis of a variety of cancer cells, including leukemia, lymphoma, multiple myeloma, breast cancers, prostate cancers, lung cancers, and so on. However, extensive studies and overall evaluation suggested that multiple myeloma is the most sensitive and the best responsive disease which was later approved by Food and Drug Administration for bortezomib treatment. Because proteasomes are an essential component in the ubiquitin-proteasomal protein degradation pathway, the discovery of bortezomib implicates that the UPS is critical for myeloma pathophysiology. The UPS also contains ubiquitin, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), ubiquitin ligases (E3) and deubiquitinases (Dubs). In this review, we examined and analyzed the recent advancements of the UPS components in multiple myeloma and its implications in drug discovery for myeloma treatment. PMID:22432065

  11. Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei Wu Yachun; Yu Le; Li Zhijie; Sung, Joseph Jao Yiu; Cho, C.H.

    2008-09-19

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G{sub 2}/M cell cycle arrest which was associated with the formation of LC3{sup +} autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3{sup +} autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.

  12. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  13. Proteasome Activation is a Mechanism for Pyrazolone Small Molecules Displaying Therapeutic Potential in Amyotrophic Lateral Sclerosis

    PubMed Central

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1G93A cells. PC12-SOD1G93A cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1G93A cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS. PMID:25001311

  14. Resveratrol Enhances Autophagic Flux and Promotes Ox-LDL Degradation in HUVECs via Upregulation of SIRT1

    PubMed Central

    Zhang, Yanlin; Cao, Xueqin; Zhu, Wawa; Liu, Zhihua; Liu, Huihui; Zhou, Yande; Cao, Yongjun; Liu, Chunfeng; Xie, Ying

    2016-01-01

    Oxidized low-density lipoprotein- (Ox-LDL-) induced autophagy dysfunction in human vascular endothelial cells contributes to the development of atherosclerosis (AS). Resveratrol (RSV) protects against Ox-LDL-induced endothelium injury. The objective of this study was to determine the mechanisms underlying Ox-LDL-induced autophagy dysfunction and RSV-mediated protection in human umbilical vein endothelial cells (HUVECs). The results showed that Ox-LDL suppressed the expression of sirtuin 1 (SIRT1) and increased LC3-II and sequestosome 1 (p62) protein levels without altering p62 mRNA levels in HUVECs. Pretreatment with bafilomycin A1 (BafA1) to inhibit lysosomal degradation abrogated the Ox-LDL-induced increase in LC3-II protein level. Ox-LDL increased colocalization of GFP and RFP puncta in mRFP-GFP-tandem fluorescent LC3- (tf-LC3-) transfected cells. Moreover, Ox-LDL decreased the expression of mature cathepsin D and attenuated cathepsin D activity. Pretreatment with RSV increased the expression of SIRT1 and LC3-II and increased p62 protein degradation. RSV induced RFP-LC3 aggregation more than GFP-LC3 aggregation. RSV restored lysosomal function and promoted Ox-LDL degradation in HUVECs. All the protective effects of RSV were blocked after SIRT1 was knocked down. These findings demonstrated that RSV upregulated the expression of SIRT1, restored lysosomal function, enhanced Ox-LDL-induced impaired autophagic flux, and promoted Ox-LDL degradation through the autophagy-lysosome degradation pathway in HUVECs. PMID:27069532

  15. Proteasomes, Sir2, and Hxk2 form an interconnected aging network that impinges on the AMPK/Snf1-regulated transcriptional repressor Mig1.

    PubMed

    Yao, Yanhua; Tsuchiyama, Scott; Yang, Ciyu; Bulteau, Anne Laure; He, Chong; Robison, Brett; Tsuchiya, Mitsuhiro; Miller, Delana; Briones, Valeria; Tar, Krisztina; Potrero, Anahi; Friguet, Bertrand; Kennedy, Brian K; Schmidt, Marion

    2015-01-01

    Elevated proteasome activity extends lifespan in model organisms such as yeast, worms and flies. This pro-longevity effect might be mediated by improved protein homeostasis, as this protease is an integral module of the protein homeostasis network. Proteasomes also regulate cellular processes through temporal and spatial degradation of signaling pathway components. Here we demonstrate that the regulatory function of the proteasome plays an essential role in aging cells and that the beneficial impact of elevated proteasome capacity on lifespan partially originates from deregulation of the AMPK signaling pathway. Proteasome-mediated lifespan extension activity was carbon-source dependent and cells with enhancement proteasome function exhibited increased respiratory activity and oxidative stress response. These findings suggested that the pro-aging impact of proteasome upregulation might be related to changes in the metabolic state through a premature induction of respiration. Deletion of yeast AMPK, SNF1, or its activator SNF4 abrogated proteasome-mediated lifespan extension, supporting this hypothesis as the AMPK pathway regulates metabolism. We found that the premature induction of respiration in cells with increased proteasome activity originates from enhanced turnover of Mig1, an AMPK/Snf1 regulated transcriptional repressor that prevents the induction of genes required for respiration. Increasing proteasome activity also resulted in partial relocation of Mig1 from the nucleus to the mitochondria. Collectively, the results argue for a model in which elevated proteasome activity leads to the uncoupling of Snf1-mediated Mig1 regulation, resulting in a premature activation of respiration and thus the induction of a mitohormetic response, beneficial to lifespan. In addition, we observed incorrect Mig1 localization in two other long-lived yeast aging models: cells that overexpress SIR2 or deleted for the Mig1-regulator HXK2. Finally, compromised proteasome function

  16. Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis

    PubMed Central

    Semren, Nora; Habel-Ungewitter, Nunja C.; Fernandez, Isis E.; Königshoff, Melanie; Eickelberg, Oliver; Stöger, Tobias; Meiners, Silke

    2015-01-01

    Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution. PMID:26340365

  17. Why does threonine, and not serine, function as the active site nucleophile in proteasomes?

    PubMed

    Kisselev, A F; Songyang, Z; Goldberg, A L

    2000-05-19

    Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the