Proteomic Analysis Reveals that Proteasome Subunit Beta 6 Is Involved in Hypoxia-Induced Pulmonary Vascular Remodeling in Rats

Background

Chronic hypoxia (CH) is known to be one of the major causes of pulmonary hypertension (PH), which is characterized by sustained elevation of pulmonary vascular resistance resulting from vascular remodeling. In this study, we investigated whether the ubiquitin proteasome system (UPS) was involved in the mechanism of hypoxia-induced pulmonary vascular remodeling. We isolated the distal pulmonary artery (PA) from a previously defined chronic hypoxic pulmonary hypertension (CHPH) rat model, performed proteomic analyses in search of differentially expressed proteins belonging to the UPS, and subsequently identified their roles in arterial remodeling.

Results

Twenty-two proteins were differently expressed between the CH and normoxic group. Among them, the expression of proteasome subunit beta (PSMB) 1 and PSMB6 increased after CH exposure. Given that PSMB1 is a well-known structural subunit and PSMB6 is a functional subunit, we sought to assess whether PSMB6 could be related to the multiple functional changes during the CHPH process. We confirmed the proteomic results by real-time PCR and Western blot. With the increase in quantity of the active subunit, proteasome activity in both cultured pulmonary artery smooth muscle cells (PASMCs) and isolated PA from the hypoxic group increased. An MTT assay revealed that the proteasome inhibitor MG132 was able to attenuate the hypoxia-induced proliferation of PASMC in a dose-dependent manner. Knockdown of PSMB6 using siRNA also prevented hypoxia-induced proliferation.

Conclusion

The present study revealed the association between increased PSMB6 and CHPH. CH up-regulated proteasome activity and the proliferation of PASMCs, which may have been related to increased PSMB6 expression and the subsequently enhanced functional catalytic sites of the proteasome. These results suggested an essential role of the proteasome during CHPH development, a novel finding requiring further study.     

IDENTIFICATION OF PROTEASOME ALPHA6 SUBUNIT ASSOCIATED WITH DELTAMETHRIN RESISTANCE IN Drosophila melanogaster Kc CELLS

Differential expression of the proteasome alpha6 (prosalpha6) was previously reported between Plutella xylostella strains that are resistant or susceptible to the pesticide deltamethrin (DM). This finding indicated that the prosalpha6 may be involved in DM resistance. In this article, qPCR analysis revealed that the prosalpha6 was also significantly upregulated in Drosophila Kc cells treated with DM. To better understand the contribution of prosalpha6 in DM resistance, RNA interference, heterologous expression, and a proteasome inhibitor (MG‐132) were used. MG‐132 was used to suppress proteasomal activity, and the dsRNA was designed to block the function of prosalpha6. The results indicated that both MG‐132 and prosalpha6 knockdown decreased the cellular viability following DM treatment. Prosalpha6 was cloned and transfected into Drosophila Kc cells. The result showed that overexpression of prosalpha6 in Drosophila Kc cells conferred some protection against DM. Taken together, our results indicate that prosalpha6 is involved in Drosophila cells DM resistance.     

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3): POTENTIAL ROLE IN BORTEZOMIB-MEDIATED ANTICANCER THERAPY*

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome.     

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.     

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

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 ³⁵S-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.     

Ribosomal Protein S29 Regulates Metabolic Insecticide Resistance through Binding and Degradation of CYP6N3

Background

Many diseases are transmitted by mosquitoes, including malaria, dengue fever, yellow fever, filariasis, and West Nile fever. Chemical control plays a major role in managing mosquito-borne diseases. However, excessive and continuous application of insecticides has caused the development of insecticide resistance in many species including mosquito, and this has become the major obstacle to controlling mosquito-borne diseases. Insecticide resistance is the result of complex polygenic inheritance, and the mechanisms are not well understood. Ribosomal protein RPS29 was found to be associated with DM resistance in our previous study. In this study, we aim to further investigate the involvement of RPS29 in deltamethrin resistance.

Methodology and Principal Findings

In this study, tandem affinity purification was used to identify proteins that can interact with RPS29. Among the candidate proteins, CYP6N3, a member of the CYP450 superfamily, was identified, and binding to RPS29 was confirmed in vitro and in vivo by GST pull-down and immunofluorescence. CCK-8 assay was used to investigate the RPS29-CTP6N3 interaction in relation to DM resistance. CYP6N3 overexpression significantly enhanced DM resistance and insect cell viability, but this was reversed by RPS29 overexpression. Western blot was used to study the mechanism of interaction between RPS29 and CYP6N3. RPS29 increases CYP6N3 protein degradation through the proteasome.

Conclusions and Significance

These observations indicate that CYP6N3, a novel RPS29-interacting partner, could stimulate deltamethrin resistance in mosquito cells and RPS29 overexpression targeted CYP6N3 for proteosomal degradation, abrogating the CYP6N3-associated resistence to deltamethrin. Our findings provide a novel mechanism associated with CYP450s mediated DM resistance.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

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 G₂/M cell cycle arrest which was associated with the formation of LC3⁺ 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⁺ 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.     

Proteasomal interference prevents zona pellucida penetration and fertilization in mammals

The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al., Proc Natl Acad Sci U S A 2002; 99:1223-1228). The present study provides experimental evidence demonstrating proteasome involvement in the penetration of mammalian zona pellucida (ZP). Using porcine in vitro fertilization as a model, penetration of ZP was completely inhibited by specific proteasomal inhibitors MG-132 and lactacystin. Three commercial rabbit sera recognizing 20S proteasomal core subunits beta-1i, beta-2i, alpha-6, and beta-5 completely blocked fertilization at a very low concentration (i.e., diluted 1/2000 to 1/8000 in fertilization medium). Neither proteasome inhibitors nor antibodies had any effects on sperm-ZP binding and acrosome exocytosis in zona-enclosed oocytes or on fertilization rates in zona-free oocytes, which were highly polyspermic. Consistent with a possible role of ubiquitin-proteasome pathway in ZP penetration, ubiquitin and various alpha and beta type proteasomal subunits were detected in boar sperm acrosome by specific antibodies, immunoprecipitated and microsequenced by MALDI-TOF from boar sperm extracts. Antiubiquitin-immunoreactive substrates were detected on the outer face of ZP by epifluorescence microscopy. This study therefore provides strong evidence implicating the ubiquitin-proteasome pathway in mammalian fertilization and zona penetration. This finding opens a new line of acrosome/ZP research because further studies of the sperm acrosomal proteasome can provide new tools for the management of polyspermia during in vitro fertilization and identify new targets for contraceptive development.     

Identification of collaborative activities with oxidative phosphorylation in bipolar disorder

Bipolar disorder (BD) is a psychiatric disease considered to polygenic with multiple factors in genetics, each of which is not dominant but collaborative during pathogenic progression. We describe a method that estimates the collaborative contribution to the disease between a certain well-studied pathway and the other candidate pathway using Gene Set Enrichment Analysis (GSEA). We describe a modified GSEA (improved derivation) to identify genes that are significantly and differentially expressed between disease and non-disease states and that are consistently co-expressed with a target pathway which is deeply related to disease etiology. The modified GSEA uses available gene expression data to identify molecular mechanism (ubiquitin-proteasome and inflammatory response) associated with the disease. We believe that this approach could reveal hidden relations between a certain well-studied pathway and the other candidate pathway known in literature.

Abbreviations

ATP5I - ATP synthase H+ transporting mitochondrial F0 complex subunit E, ATP5J - ATP synthase H+ transporting mitochondrial F0 complex subunit F6, BAD - Bcl-2-associated death promoter, BAX - Bcl-2-associated x protein, Bcl-2 - B-cell lymphoma 2, BDNF - brain derived neurotrophic factor, COX5B - Cytochrome c oxidase subunit Vb, COX7A2 - cytochrome c oxidase subunit VIIa polypeptide 2, DLK - dual leucine zipper-bearing kinase, GABA - Gamma aminobutyric acid, IL-8 - Interleukin 8, NDUFA1 - NADH dehydrogenase 1 alpha subcomplex 1, NDUFB2 - NADH dehydrogenase1 beta subcomplex 2, NDUFS4 - NADH dehydrogenase Fe-S protein 4, NGF - nerve growth factor, PPP2R5C - protein phosphatase 2 regulatory subunit B gamma, PSMA3 - proteasome subunit alpha type 3, PSMA7 - proteasome subunit alpha type 7, PSMB1 - proteasome subunit beta type 1, PSMB6 - proteasome subunit beta type 6, PSMB7 - proteasome subunit beta type 7, PSMC2 - proteasome 26S subunit ATPase 2, PSMC5 - proteasome 26S subunit ATPase 5, SLC6A4 - solute carrier family 6 member 4, TNFa - tumor necrosis factor a, UBE2A - ubiquitinconjugating enzyme E2A, UCRC - ubiquinol-cytochrome c reductase complex, UFC1 - ubiquitin-fold modifier conjugating enzyme 1, UQCRQ - ubiquinol-cytochrome c reductase complex III subunit VII, USP14 - ubiquitin specific protease 14.     

A novel crosstalk between two major protein degradation systems

Eukaryotes have two major intracellular protein degradation pathways, namely the ubiquitin-proteasome system (UPS) and autophagy. Inhibition of proteasomal activities has been previously shown to induce autophagy, indicating a coordinated and complementary relationship between these two systems. However, little is known about the regulation of the UPS by autophagy. In this study, we showed for the first time that proteasomes were activated in response to pharmacological inhibition of autophagy as well as disruption of autophagy-related genes by RNA interference under nutrient-deficient conditions in cultured human colon cancer cells. The induction was evidenced by the increased proteasomal activities and the upregulation of proteasomal subunits, including the proteasome β5 subunit, PSMB5. Co-inhibition of the proteasome and autophagy also synergistically increased the accumulation of polyubiquitinated proteins. Collectively, our findings suggest that proteasomes are activated in a compensatory manner for protein degradation upon autophagy inhibition. Our studies unveiled a novel regulatory mechanism between the two protein degradation pathways.     

Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.     

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin-like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S-20S-19S proteasomes, and abrogation of expression of beta 1i, beta 2i and beta 5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic beta1, beta2 and beta 5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase-like, trypsin-like and chymotrypsin-like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, gamma-irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti-apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro-apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis-resistant phenotype.     

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.     

Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21^Waf1/Cip1 mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21^Waf1/Cip1 induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.