Presence Of Proteasome Inhibitors Research Articles

A nuclear WD40 repeat protein PRL1 regulates stability of MYB4 transcription factor in Arabidopsis

MYB4, a member of the R2R3-type subfamily of MYB transcription factor plays a crucial role in regulating the accumulation of UV-B absorbing phenylpropanoids in plants. UV-B exposure for a longer duration down-regulates the expression of MYB4 gene in Arabidopsis. MYB4 protein represses its own expression by binding to its own promoter. However, at present practically nothing is known about the post-translational regulation of MYB4 protein in vivo. Here, we provide evidence that in Arabidopsis MYB4 protein is phosphorylated in vivo and is targeted by the ubiquitin-26S proteasome-dependent pathway. Immunoprecipitation, immunoblotting, and phosphoprotein staining experiments have revealed that both the accumulation pattern and phosphorylation of MYB4 increase in the light condition during the 24 hours time span under long-day conditions. Yeast two-hybrid and bimolecular fluorescence complementation assays have shown that MYB4 directly interacts with a nuclear WD40 repeat protein, PRL1 in vivo. Cell-free protein degradation assay in the absence and presence of proteasome inhibitor indicates that MYB4 is degraded in a ubiquitin proteasome-dependent manner. Furthermore, analyses of MYB4 protein accumulation levels in transgenic atmyb4–1 mutant line expressing 35 S:AtMYB4 (35 S:AtMYB4-atmyb4–1) and atprl1–1 mutant line indicate that PRL1 regulate stability of MYB4 in Arabidopsis. Overall, our results provide important information on the possible mechanism of post-translational modification and regulation of stability of MYB4 protein in Arabidopsis in vivo.

Sensitization of cancer cells towards Cisplatin and Carboplatin by protein kinase D inhibitors through modulation of ATP7A/B (copper transport ATPases)

Drug resistance of cancer cells is a significant impediment to effective chemotherapy. One primary reason for this is copper exporters - ATPase copper transporting alpha (ATP7A) and ATPase copper transporting beta (ATP7B). These molecular pumps belong to P-type ATPases and dispose off the Platinum (Pt) based anticancer drugs from cancer cells, causing resistance in them. For the disposal of Pt-drugs, copper exporters require phosphorylation mediated by protein kinase D (PKD) for their activation and trafficking. Even though various research works are underway to overcome resistance to anticancer drugs, the role of PKD is mainly ignored. In this study, we have found a significant upregulation of ATP7A and ATP7B in cervical cancer cells (HeLa) and Liver Hepatocellular Carcinoma cells (HepG2) in the presence of Cisplatin or Carboplatin; both at transcriptional as well as translational levels. Interestingly, the expression of ATP7A and ATP7B were significantly downregulated in the presence of a PKD inhibitor (CID2011756), resulting in the reduction of PKD mediated phosphorylation of ATP7A/7B. This causes enhancement of proteasome-mediated degradation of ATP7A/7B and thereby sensitizes the cells towards Cisplatin and Carboplatin. Similarly, the treatment of Cisplatin resistant HepG2 cells with PKD inhibitor causes enhanced sensitivity towards Cisplatin drug. However, the presence of proteasome inhibitor (MG132) reversed the effect of the PKD inhibitor on the expression level of ATP7A/7B, indicating the necessity of phosphorylation for its stability. Hence, we conclude that the combinatorial usage of Cisplatin with drugs targeting PKD can be developed as an effective chemotherapeutic approach to overcome drug resistance.

Selection of CHO host and recombinant cell pools by inhibition of the proteasome results in enhanced product yields and cell specific productivity

Chinese hamster ovary (CHO) cells are the leading mammalian cell expression platform for biotherapeutic recombinant molecules yet some proteins remain difficult to express (DTE) in this, and other, systems. In recombinant cell lines expressing DTE proteins, cellular processes to restore proteostasis can be triggered when the folding and modification capabilities are exceeded, including the unfolded protein response and ER-associated degradation (ERAD) and proteasomal degradation. We therefore investigated whether the proteasome activity of CHO cells was linked to their ability to produce recombinant proteins. We found cell lines with diverse monoclonal antibody (mAb) productivity show different susceptibilities to inhibitors of proteasome activity. Subsequently, we applied selective pressure using proteasome inhibitors on mAb producing cells to determine the impact on cell growth and recombinant protein production, and to apply proteasome selective pressure above that of a metabolic selection marker during recombinant cell pool construction. The presence of proteasome inhibitors during cell pool construction expressing two different model molecules, including a DTE Fc-fusion protein, resulted in the generation of cell pools with enhanced productivity. The increased productivities, and ability to select for higher producing cells, has potential to improve clonal selection during upstream processes of DTE proteins.

Preclinical and Translational Support for Clinical Development of Iberdomide in Combination with Proteasome Inhibitors: Mechanism of Synergy in Clinical Trial CC-220-MM-001

Preclinical and Translational Support for Clinical Development of Iberdomide in Combination with Proteasome Inhibitors: Mechanism of Synergy in Clinical Trial CC-220-MM-001

Nelfinavir inhibits human DDI2 and potentiates cytotoxicity of proteasome inhibitors

Proteasome inhibitors (PIs) are currently used in the clinic to treat cancers such as multiple myeloma (MM). However, cancer cells often rapidly develop drug resistance towards PIs due to a compensatory mechanism mediated by nuclear factor erythroid 2 like 1 (NFE2L1) and aspartic protease DNA damage inducible 1 homolog 2 (DDI2). Following DDI2-mediated cleavage, NFE2L1 is able to induce transcription of virtually all proteasome subunit genes. Under normal condition, cleaved NFE2L1 is constantly degraded by proteasome, whereas in the presence of PIs, it accumulates and induces proteasome synthesis which in turn promotes the development of drug resistance towards PIs. Here, we report that Nelfinavir (NFV), an HIV protease inhibitor, can inhibit DDI2 activity directly. Inhibition of DDI2 by NFV effectively blocks NFE2L1 proteolysis and potentiates cytotoxicity of PIs in cancer cells. Recent clinical evidence indicated that NFV can effectively delay the refractory period of MM patients treated with PI-based therapy. Our finding hence provides a specific molecular mechanism for combinatorial therapy using NFV and PIs for treating MM and probably additional cancers.

Усиленная радиосенсибилизация опухолевых клеток с помощью сочетанного воздействия гипертермии и ингибиторов протеасом

Hyperthermia is used in combination with radiation therapy to enhance the radiation response of the target tumor. However, many tumors are thermoresistant, which makes the thermo-radiosensitization ineffective. In this work, we studied a possibility of enhancing the radiosensitiz-ing effect of hyperthermia on cancer cells in the case of combining heating and treatments with inhibitors of proteasomal protein degradation. The objects of the study were HeLa, MCF-7, and PC-3 cell lines derived from human carcinomas of various localization. Before irradiation (2-6 Gy), cell cultures were subjected to heat stress (42-44 °C for 20-60 min) without or in the pres-ence of proteasome inhibitors (MG132, bortezomib). The death and survival of the treated cells was evaluated by the level of apoptosis/necrosis, by clonogenicity, and in the MTT test. The pro-teotoxicity of the exposures and the subcellular redistribution of heat shock proteins were ana-lyzed using cell fractionation, electrophoresis, and immunoblotting. The results show that, com-pared with hyperthermia, the combined effect (hyperthermia + proteasome inhibitor) significantly increased the radiosensitization of cancer cells, which was manifested in the intensification of their postradiation death and significant suppression of clonogenicity. Thus, the combination of hyperthermia with proteasome inhibitors (for example, with bortezomib, which is clinically ap-proved) can help sensitize radioresistant tumors to radiation therapy.

Compartmentalization of the proteasome-interacting proteins during sperm capacitation

Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

Thymoquinone Selectively Kills Hypoxic Renal Cancer Cells by Suppressing HIF-1α-Mediated Glycolysis.

Several reports have shown that thymoquinone (TQ) effectively attenuates angiogenesis in cancer cells, resulting in suppression of tumor growth. However, it is not yet clear whether TQ reduces hypoxia-inducible factor-1α (HIF-1α) expression in hypoxic cancer cells. Here, we found that TQ was a novel HIF-1α inhibitor through hypoxia response element (HRE)-luciferase assay-based large screening by using 502 natural compounds containing chemical library. TQ reduced HIF-1α protein levels in renal cancer cells; however, it did not affect the HIF-1α protein levels in the presence of proteasome inhibitor, MG132, indicating that the reduction effects of TQ on HIF-1α protein are mediated via the ubiquitination-proteasome dependent pathway. TQ boosted HIF-1α protein degradation, and the mechanism was revealed by inhibiting interaction between HSP90 and HIF-1α. TQ suppressed downstream genes of HIF-1α, indicating negative impact of TQ on HIF-1α transcriptional activities. In addition, TQ altered glucose, lactate, and ATP levels, leading to anaerobic metabolic disturbance. TQ induced apoptosis in hypoxic cancer cells as determined by crystal violet staining and flow cytometry for annexin V-stained cells. Taken together, we suggested that TQ is a potential anticancer agent targeting HIF-1α.

Serum starvation raises turnover of phosphorylated p62/SQSTM1 (Serine 349), reveals expression of proteasome and N-glycanase1 interactive protein RAD23B and sensitizes human synovial fibroblasts to BAY 11-7085-induced cell death.

Phosphorylation of p62/SQSTM1 (p62) on Serine 349 (P-Ser349 p62) as well as proteasome dysfunction have been shown to activate the cell protective Keap1/Nrf2 pathway. We showed previously that BAY 11-7085-induced human synovial fibroblast cell death includes autophagy and p62 downregulation. In this work, we have studied expression of P-Ser349 p62 in human synovial fibroblasts. Results showed that P-Ser349 p62 was not detected in synovial cell extracts unless cells were cultured in the presence of proteasome inhibitor (MG132). MG132 revealed P-Ser349 p62 turnover, that was further increased by concomitant autophagy inhibition and markedly enhanced in serum starved cells. Starvation sensitized synovial fibroblasts to BAY 11-7085 while MG132 protected both non-starved and starved cells from BAY 11-7085-induced cell death. Lentivirus mediated overexpression of phosphorylation-mimetic p62 mutant S349E markedly protected synovial fibroblasts from BAY 11-7085. Inhibitor of Keap1-P-S349 p62 interaction, K67, had synergistic effect with MG132. Starvation increased p62 molecular weight, that was reversed by serum and bovine serum albumin re-feeding. Furthermore, starvation markedly induced RAD23B. Increased endo-β-N-acetylglucosaminidase (ENGase) turnover was detected in starved synovial fibroblasts. PNGase F treatment produced faster migration p62 form in human synovial tissue extracts but starvation-like p62 form of higher molecular weight in synovial cell extracts. Co-transfection of NGLY1, with p62 or p62 mutants S349A and S349E markedly stabilized p62 expressions in HEK293 cells. Tunicamycin upregulated p62 and protected synovial fibroblasts from BAY 11-7085-induced cell death. These results showed that P-Ser349 p62 has pro-survival role in human synovial fibroblasts and that de-glycosylation events are involved in p62 turnover.

Aldehyde-mediated protein degradation is responsible for the inhibition of nucleotide excision repair by cigarette sidestream smoke

We recently reported that cigarette sidestream smoke (CSS) can delay nucleotide excision repair (NER), which was due to the inhibition of repair protein accumulation to DNA damage sites. However, the mechanisms how the protein recruitment was inhibited remains unclear. We hypothesized that aldehydes in CSS could be a candidate taking a role for the inhibition, and tested our hypothesis by removing aldehydes from CSS using cigarette-filters. The NER inhibition potency of CSS or filtered CSS (F-CSS) was compared using human keratinocyte cell line, HaCaT. Cigarette-filters were able to reduce total aldehydes in CSS by half. Pretreating cells with CSS and F-CSS enhanced UVB-induced cell death, with the effect of CSS weakened by filtration.CSS strongly inhibited the repair of UVB-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts (6-4PPs), where the recruitments of repair molecules, TFIIH and XPG, were slowed down. F-CSS showed similar inhibition of NER and accumulation of related proteins, but the effect was weaker than CSS. Semicarbazide (SEM), an aldehyde-trapping agent, alleviated the NER delay induced by both CSS and F-CSS, further confirming that aldehydes in CSS were the main cause for the inhibition of NER and that the different amounts of aldehydes in CSS and F-CSS were responsible for the different inhibition efficiency. Furthermore, TFIIH level was decreased by treatment with CSS and restored in the presence of proteasome inhibitor, indicating that the degradation of NER proteins might be the cause of the inhibition of NER-protein recruitment. These results supported our hypothesis that aldehydes in CSS are the main contributor for the NER inhibition via protein degradation, and reconfirmed that exposure to CSS without filtration could be a severe threat to human health.

Proteasome inhibition protects against diet-induced gallstone formation through modulation of cholesterol and bile acid homeostasis.

Gallstone disease is one of the most prevalent and costly gastrointestinal disorders worldwide. Gallstones are formed in the biliary system by cholesterol secretions in bile, which result from excess cholesterol, a deficiency in bile salts or a combination of the two. The present study examined the effects of proteasome inhibition on gallstone formation using the proteasome inhibitors bortezomib(BT) and carfilzomib(CF). C57BL/6Jmice were fed a lithogenic diet to generate gallstones and injected with BT or CF for 12weeks. After 12weeks of the lithogenic diet, 8out of the 10mice in the control group had developed gallstones, whereas none of the mice who received proteasome inhibitors had developed gallstones. Notably, the expression of genes associated with cholesterol synthesis (sterol regulatory element‑binding protein‑2 and 3‑hydroxy‑3‑methylglutaryl‑CoA reductase), cholesterol secretion [ATP‑binding cassette subfamilyG member5 (ABCG5) and ABCG8] and bile acid synthesis [cytochrome P450 family 7 subfamily A member 1 (Cyp7a1), Cyp7b1, Cyp27a1 and Cyp8b1] was reduced in the livers of mice injected with BT or CF. Cyp7a1 encodes cholesterol 7α‑hydroxylase, the rate‑limiting enzyme in the synthesis of bile acid from cholesterol. The present study therefore measured the expression levels of transcription factors that are known to inhibit Cyp7a1 expression, namely farnesoid X receptor (FXR), pregnaneX receptor(PXR) and small heterodimer partner(SHP). Although FXR, PXR andSHP expression was predicted to increase in the presence of proteasome inhibitors, the expression levels were actually reduced; thus, it was concluded that they were not involved in the proteasome inhibition‑induced regulation of Cyp7a1. Further investigation of the mitogen‑activated protein kinase and protein kinaseA (PKA) signaling pathways in human hepatoma cells revealed that proteasome inhibition‑induced c‑Jun N‑terminal kinase (JNK) phosphorylation reduced CYP7A1 andCYP27A1 expression. Inaddition, reduced PKA phosphorylation as a result of proteasome inhibition regulated ABCG5 andABCG8 expression. Inconclusion, these findings suggest that proteasome inhibition regulates cholesterol and biliary metabolism via the JNK and PKA pathways, and is a promising therapeutic strategy to prevent gallstone disease.

Abstract 4490: Post-transcriptional regulation on PIAS3 expression in malignant mesothelioma

Abstract Protein Inhibitor of Activated STAT3 (PIAS3) was originally identified as an endogenous suppressor of STAT3 activity which can specifically interact with activated STAT3 and block its downstream oncogenic effects. Previous studies from our lab have shown that low PIAS3 protein expression in mesothelioma tumors was associated with poor patient survival. Furthermore, low PIAS3 expression in mesothelioma cell lines correlated with higher STAT3 activity. Over-expression of PIAS3 decreased STAT3 activity and cell growth. These findings suggest that low PIAS3 expression may play an important role in mesothelioma tumorigenesis, thus understanding the regulatory mechanism(s) of PIAS3 expression may provide new therapeutic strategies in mesothelioma treatment. Initially, we examined PIAS3 expression by western blotting and RT-qPCR among six mesothelioma cell lines (H28, 211H, H2052, H2452, HAY, YOU) and found that while PIAS3 mRNA levels were relatively constant, PIAS3 protein expression was highly variable. This finding indicated a potential post-transcriptional regulation of PIAS3 expression in mesothelioma. To investigate this further, we first examined PIAS3 protein stability in the presence of proteasome inhibitors and cycloheximide (CHX). We observed no increase in PIAS3 protein levels during proteasome inhibition using either MG132 (10 μM) or bortezemib (1 μM) for up to 8 h in 211H, H2052 and H2452 cells, whereas TP53 protein expression was increased. Blocking translational protein synthesis with 50 or 100 μg/ml CHX for up to 24 h also produced little change in PIAS3 protein expression in mesothelioma cells, indicating that PIAS3 is a very stable protein. We next explored the PIAS3 3’UTR as a potential site for translational regulation. We initially utilized a miR microarray and computational analysis, followed by a PIAS3 3’UTR luciferase reporter assay, to screen miRNAs that are both increased in cells with low PIAS3 and have seed sequences targeting the PIAS3 3’UTR and identified miR-18a as our first candidate. Taken together, our study suggests that variable expression of PIAS3 protein in mesothelioma is regulated by translational control and not by protein stability. Citation Format: Tian He, Karen McColl, Nneha Sakre, Gary Wildey, Afshin Dowlati. Post-transcriptional regulation on PIAS3 expression in malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4490. doi:10.1158/1538-7445.AM2017-4490

THE ROLE OF NUB1 IN THE CLEARANCE OF TAU AGGREGATES IN ALZHEIMER'S DISEASE

BACKGROUND: Intraneuronal aggregations of abnormal hyperphosphorylated tau are a hallmark of Alzheimer’s disease. Previous data revealed that NEDD8 ultimate buster 1 (NUB1) reduces the aggregation and phosphorylation of tau in an in vitro model. NUB1 is a ubiquitin-like (UBL)/ ubiquitin-associated (UBA) protein that targets the down regulation of ubiquitin-like modifiers (NEDD8, FAT10), and aggregation-prone proteins leading to neurodegenerative disease (synphilin-1, huntingtin). The aim of the study is to understand how NUB1 affects tau aggregation and phosphorylation. METHODS: A GFP-tau inducible neuroblastoma cell line was generated and treated with proteasome inhibitor to induce the formation of aggresomes. GFP-tau cells were transduced with NUB1 lentiviral vectors both in the absence and presence of proteasome inhibitor, followed by western blotting, filter trap assays, immunoprecipitation and immunofluorescence. RESULTS: Inhibition of the proteasome induced the formation of GFP-tau aggresomes that were positive for phospho-tau (p-tau) markers (AT8, pS396). Filter trap assays revealed that NUB1 significantly decreased the ratio of p-tau to tau in the total cellular fraction (both soluble and insoluble), as well as in only the detergent insoluble fraction, both with and without proteasome inhibition. However, the ratio of tau and p-tau in the insoluble fraction to that in the total fraction revealed that the NUB1-mediated decrease in the levels of detergent insoluble tau and p-tau exceeded that of the overall decline in total levels, suggesting NUB1 can target aggregation-prone tau and p-tau. This effect of NUB1 was more pronounced for p-tau following proteasome inhibition. Blocking the proteasome induced the upregulation of p62 and increased the ratio of LC3B-II to LC3B-I. NUB1 induced further upregulation of autophagy following proteasome inhibition as seen by an increase in the LC3B-II/LC3B-I ratio and changes in LC3B positive puncta in cells. Finally, GFP-tau aggresomes were positive for p62 and NUB1 overexpression promoted the interaction between GFP-tau and p62. CONCLUSIONS: These data suggest that NUB1 may play a role in the clearance of aggregation-prone tau and p-tau following inhibition of the proteasome via autophagy.

H2S regulates endothelial nitric oxide synthase protein stability by promoting microRNA-455-3p expression

The aims of the present study are to determine whether hydrogen sulfide (H2S) is involved in the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, and to identify the role of microRNA-455-3p (miR-455-3p) during those processes. In cultured human umbilical vein endothelial cells (HUVECs), the expression of miR-455-3p, eNOS protein and the NO production was detected after administration with 50 μM NaHS. The results indicated that H2S could augment the expression of miR-455-3p and eNOS protein, leading to the increase of NO level. We also found that overexpression of miR-455-3p in HUVECs increased the protein levels of eNOS whereas inhibition of miR-455-3p decreased it. Moreover, H2S and miR-455-3p could no longer increase the protein level of eNOS in the presence of proteasome inhibitor, MG-132. In vivo, miR-455-3p and eNOS expression were considerably increased in C57BL/6 mouse aorta, muscle and heart after administration with 50 μmol/kg/day NaHS for 7 days. We also identified that H2S levels and miR-455-3p expression increased in human atherosclerosis plaque while H2S levels decreased in plasma of atherosclerosis patients. Our data suggest that the stability of eNOS protein and the NO production could be regulated by H2S through miR-455-3p.

Human promyelocytic leukemia protein is targeted to distinct subnuclear domains in plant nuclei and colocalizes with nucleolar constituents in a SUMO-dependent manner.

Eukaryotic nuclei are subdivided into subnuclear structures. Among the most prominent of these structures are the nucleolus and the PML nuclear bodies (PML‐NBs). PML‐NBs are spherical multiprotein aggregates of varying size localized in the interchromosomal area. PML‐NB formation is dependent on the presence of the promyelocytic leukemia protein (PML) as well as on post‐translational modification of core components by covalent attachment of the small ubiquitin‐like modifier (SUMO). So far, PML‐NBs as well as PML have been described in mammalian cells only, whereas no orthologs are known in the plant kingdom. In order to investigate conserved mechanisms in PML targeting, we expressed human PML (hPML) fused to the GFP in Nicotiana benthamiana. Using confocal laser scanning microscopy and coimmunoprecipitation followed by mass spectrometric analysis, we found the fusion protein in association with nucleolar constituents. Importantly, mutants of hPML, which are no longer SUMOylated, showed altered localizations, implying SUMO‐dependent targeting of hPML in plants as has previously been shown for mammalian cells. Interestingly, in the presence of proteasome inhibitors, hPML could also be found in the nucleolus of mammalian cells suggesting conserved targeting mechanisms of PML across kingdoms. Finally, Solanum tuberosum COP1, a proposed PML‐like protein from plants, was fused to the red fluorescent protein (RFP) and coexpressed with hPML::eGFP. Microscopic analysis confirmed the localization of COP1::RFP in nuclear speckles. However, hPML::eGFP did not colocalize with COP1::RFP. Hence, we conclude that plants do not possess specialized PML‐NBs, but that their functions may be covered by other subnuclear structures like the nucleolus. Database Proteomics data have been deposited to the ProteomeXchange Consortium with the identifier PXD004254.

Abstract 2869: FLASH-dependent post-translational regulation of the EMT activator ZEB1

Abstract Understanding the mechanisms of metastasis initiation and tumor persistence despite aggressive treatments is paramount to developing new viable therapeutic targets. Critical to tumor progression is initiation of epithelial-to-mesenchymal transition (EMT), a cellular process characterized by loss of E-cadherin, a critical cell-cell adhesion molecule, gain of migratory properties and chemoresistance. Transcriptional repression of E-cadherin and other genes responsible for maintaining the epithelial phenotype by ZEB1/ZEB2 family is one of the most significant events occurring at the interface between non-metastatic/metastatic cancer and chemo-sensitive/chemo-resistant cancer. Using a novel interdisciplinary high-throughput screen methodology we uncovered novel regulators of E-cadherin. Caspase 8 - associated protein 2 (CASP8AP2 or FLASH) a protein involved in apoptosis and transcriptional control was identified through this screen and we discovered that FLASH depletion resulted in increased E-cadherin levels at the plasma membrane through loss of ZEB1 expression. Subsequent examination of ZEB1 protein expression revealed that ZEB1 is rapidly degraded in FLASH-depleted cells. This process is blocked by proteasome inhibitors suggesting that post-translational modifications (PTMs) and in particular ubiquitination of ZEB1 is responsible for its fast turnover and decreased protein expression in the absence of FLASH. Indeed, ubiquitinated ZEB1 was detected in FLASH-depleted cells in the presence of proteasome inhibitors. Although ZEB1 is degraded through the ubiquitin pathway, its rapid turnover in the absence of FLASH suggests that protective mechanisms are in place to delay and/or prevent this process. Even more, we determined that the atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 is involved in ZEB1 degradation in the absence of FLASH and is upregulated in cells depleted for FLASH. Altogether our data suggests that FLASH can stabilizes ZEB1 indirectly by transcriptional repression of the ubiquitin ligase responsible for its degradation and directly by binding to ZEB1 and masking the recognition site of the ubiquitin ligase. Even more, absence of FLASH reprograms the cells into a more epithelial-like phenotype which is less invasive and is resistant to TGFβ-induced loss of E-cadherin. Not only is FLASH-dependent post-translational regulation of ZEB1 novel, but is also conserved in multiple cells lines from various tissues, making this mechanism an original therapeutic target for cancer metastasis. Citation Format: Camille Abshire, Ana Maria Dragoi. FLASH-dependent post-translational regulation of the EMT activator ZEB1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2869.

Gain-of-function profilin 1 mutations linked to familial amyotrophic lateral sclerosis cause seed-dependent intracellular TDP-43 aggregation

Profilin 1 (PFN1) is an actin monomer-binding protein essential for regulating cytoskeletal dynamics in all cell types. Recently, mutations in the PFN1 gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS). The co-aggregation of PFN1 bearing mutations that cause ALS with TDP-43 (a key molecule in both sporadic and some familial forms of ALS), together with the classical TDP-43 pathology detected in post-mortem tissues of patients with autosomal dominant PFN1 mutation, imply that gain-of-toxic-function of PFN1 mutants is associated with the onset of ALS. However, it remains unknown how PFN1 mutants cause ALS. We found mutant PFN1 that causes ALS formed cytoplasmic aggregates positive for ubiquitin and p62, and these aggregates sequestered endogenous TDP-43. In cells harboring PFN1 aggregates, formation of aggresome-like structures was inhibited in the presence of proteasome inhibitor, and conversion of LC3-I to LC3-II was suppressed in the presence of lysosome inhibitor. Further, insoluble TDP-43 was increased in both cases. Co-expression of ALS-linked mutant PFN1 and TDP-43 increased insoluble and phosphorylated TDP-43 levels. The C-terminal region of TDP-43, essential for aggregation of TDP-43, was also indispensable for the interaction with PFN1. Interestingly, insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 functioned as a seed to induce accumulation and phosphorylation of TDP-43, indicating that TDP-43 accumulated in the presence of the PFN1 mutants is converted to prion-like species. These findings provide new insight into the mechanisms of neurodegeneration in ALS, suggesting that gain-of-toxic-function PFN1 gene mutation leads to conformational change of TDP-43.

Characterization of TcCYC6 from Trypanosoma cruzi, a gene with homology to mitotic cyclins

Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin–CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin–proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.

Abstract 2255: DHA may enhance proteasome activity in human cervical cancer cells: Indirect modulation of proteasome by DHA

Abstract The omega-3 fatty acid, docosahexaenoic acid (DHA), has been shown to induce the proteasomal degradation of various cancer-associated molecules such as beta-catenin and estrogen receptor, implying its regulatory effect on proteasome. To verify this possible link between DHA and proteasome function, we explored the impacts of DHA on human papillomavirus-positive HeLa cervical cancer cells that constitutively express two known endogenous proteasome substrates E6 and E7 viral proteins. Exposure HeLa cells to DHA resulted in elevated proteasome activities along with decreased levels of E6/E7 viral proteins, and these effects of DHA were remarkably reversed in the presence of proteasome inhibitors, suggesting that DHA improves proteasome function in the cells and thereby accelerates the degradation of proteasome substrates. The DHA-induced increase in cellular proteasome activity was preceded by reactive oxygen species (ROS) overproduction and could be blocked by preincubation of the cells with ROS scavengers. Likewise, HeLa cells treated with exogenous ROS, H2O2, also exhibited significantly improved proteasome function. Unexpectedly, when the purified proteasome and whole-cell lysates isolated from HeLa cells were directly exposed to DHA or H2O2, such marked increases in proteasome activity were not seen. Taken together, these findings indicate that DHA may induce proteasome activation in cervical cancer cells mainly by indirect regulation of the proteasome. Citation Format: Kyu Lim, Kaipeng Jing, Soyeon Shin, Soyeon Jeong, Soyeon Kim, Gi-Ryang Kweon, Seung-Kiel Park, Tong Wu, Jong-Il Park. DHA may enhance proteasome activity in human cervical cancer cells: Indirect modulation of proteasome by DHA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2255. doi:10.1158/1538-7445.AM2014-2255

Localization and sub-cellular shuttling of HTLV-1 Tax with the microRNA machinery

The innate ability of the human cell to silence endogenous retroviruses through RNA sequences encoding microRNAs suggests that the cellular RNAi machinery is a major means by which the host mounts a defense response against retroviruses. Indeed, cellular miRNAs target and hybridize to specific sequences of both HTLV-1 and HIV-1 viral transcripts. However, the virus itself contains various mechanisms that assist in the evasion of viral inhibition through control of the cellular RNAi pathway. Retroviruses can hijack components of the RNAi pathway, in some cases to produce novel viral miRNAs that can either assist in active infection or promote a latent state. Here, we show that HTLV-1 Tax contributes to the dysregulation of the RNAi pathway by altering the expression of key components. A survey of uninfected and HTLV-1 infected cells revealed that Drosha is present at lower levels in all HTLV-1 infected cell lines and infected primary cells, while other components such as DGCR8 were not dramatically altered. We show co-localization of Tax and Drosha in the nucleus in vitro as well as co-immunoprecipitation in the presence of proteasome inhibitors, indicating that Tax interacts with Drosha and may target it to specific areas of the cell, namely, the proteasome. In the presence of Tax we observed a prevention of primary miRNA cleavage by Drosha. Finally, the changes in cellular miRNA expression in HTLV-1 infected cells can be mimicked by the add back of Drosha or the addition of antagomiRs against the cellular miRNAs which are downregulated by the virus.