Proteasome-dependent Protein Degradation Research Articles

C. elegans spermatocyte divisions show a weak spindle checkpoint response.

Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies have shown that spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in Caenorhabditis elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillates between the two divisions. However, the SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.

Targeting Proteasomes and the MHC Class I Antigen Presentation Machinery to Treat Cancer, Infections and Age-Related Diseases

The majority of T-cell responses involve proteasome-dependent protein degradation and the downstream presentation of oligopeptide products complexed with major histocompatibility complex (MHC) class I (MHC-I) molecules to peptide-restricted CD8+ T-cells. However, evasion of host immunity is a cancer hallmark that is achieved by disruption of host antigen processing and presentation machinery (APM). Consequently, mechanisms of immune evasion promote cancer growth and survival as well as de novo and acquired resistance to immunotherapy. A multitude of cell signaling pathways modulate the APM and MHC-I-dependent antigen presentation. Pharmacologics that specifically target and modulate proteasome structure and activity represent a novel emerging strategy to improve the treatment of cancers and other diseases characterized by aberrant protein accumulation. FDA-approved pharmacologics that selectively activate proteasomes and/or immunoproteasomes can be repositioned to overcome the current bottlenecks that hinder drug development to enhance antigen presentation, modulate the immunopeptidome, and enhance the cytotoxic activity of endogenous or engineered T-cells. Strategies to enhance antigen presentation may also improve the antitumor activity of T-cell immunotherapies, checkpoint inhibitors, and cancer vaccines. Proteasomes represent actionable therapeutic targets to treat difficult-to-treat infectious processes and neurodegenerative diseases that are characterized by the unwanted accrual of insoluble, deleterious, and potentially toxic proteins. Taken together, we highlight the breadth and magnitude of the proteasome and the immense potential to amplify and unmask the immunopeptidomic landscape to improve the treatment of a spectrum of human diseases.

CBX7 reprograms metabolic flux to protect against meningioma progression by modulating the USP44/c-MYC/LDHA axis.

Meningioma is one of the most common primary neoplasms in the central nervous system, but no specific molecularly targeted therapy has been approved for the clinical treatment of aggressive meningiomas. There is hence an urgent demand to decrypt the biological and molecular landscape of malignant meningioma. Here, through the in-silica prescreening and 10-year follow-up studies of 445 meningioma patients, we uncovered that CBX7 expression progressively decreases with malignancy grade and neoplasia stage in meningioma, and a high CBX7 expression level predicts a favorable prognosis in meningioma patients. CBX7 restoration significantly induces cell cycle arrest and inhibits meningioma cell proliferation. iTRAQ-based proteomics analysis indicated that CBX7 restoration triggers the metabolic shift from glycolysis to oxidative phosphorylation. The mechanistic study demonstrated that CBX7 promotes the proteasome-dependent degradation of c-MYC protein by transcriptionally inhibiting the expression of a c-MYC deubiquitinase, USP44, consequently attenuates c-MYC-mediated transactivation of LDHA transcripts, and further inhibits glycolysis and subsequent cell proliferation. More importantly, the functional role of CBX7 was further confirmed in subcutaneous and orthotopic meningioma xenograft mouse models and meningioma patients. Altogether, our results shed light on the critical role of CBX7 in meningioma malignancy progression and identify the CBX7/USP44/c-MYC/LDHA axis as a promising therapeutic target against meningioma progression.

A ubiquitin-proteasome pathway degrades the inner nuclear membrane protein Bqt4 to maintain nuclear membrane homeostasis.

Aberrant accumulation of inner nuclear membrane (INM) proteins is associated with deformed nuclear morphology and mammalian diseases. However, the mechanisms underlying the maintenance of INM homeostasis remain poorly understood. In this study, we explored the degradation mechanisms of the INM protein Bqt4 in the fission yeast Schizosaccharomyces pombe. We have previously shown that Bqt4 interacts with the transmembrane protein Bqt3 at the INM and is degraded in the absence of Bqt3. Here, we reveal that excess Bqt4, unassociated with Bqt3, is targeted for degradation by the ubiquitin-proteasome system localized in the nucleus and Bqt3 antagonizes this process. The degradation process involves the Doa10 E3 ligase complex at the INM. Bqt4 is a tail-anchored protein and the Cdc48 complex is required for its degradation. The C-terminal transmembrane domain of Bqt4 was necessary and sufficient for proteasome-dependent protein degradation. Accumulation of Bqt4 at the INM impaired cell viability with nuclear envelope deformation, suggesting that quantity control of Bqt4 plays an important role in nuclear membrane homeostasis.

An ORFeome of rice E3 ubiquitin ligases for global analysis of the ubiquitination interactome

BackgroundUbiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown.ResultsHere, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice (Oryza sativa L.) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1–OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity.ConclusionsThe rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3–substrate interactome in plants.

P865: BIOMARKER ANALYSIS TO SUPPORT DOSE OPTIMIZATION OF IBERDOMIDE AS MONOTHERAPY AND IN COMBINATION WITH STANDARD OF CARE TREATMENTS FOR MULTIPLE MYELOMA FROM A PHASE 1/2 TRIAL

Background: Iberdomide (IBER), a novel cereblon E3 ligase modulator (CELMoD®) compound, induces ubiquitination and proteasome-dependent degradation of Ikaros and Aiolos proteins, resulting in tumoricidal and immunomodulatory activity in multiple myeloma (MM). IBER is being investigated for the treatment of relapsed/refractory MM (RRMM) in a phase 1/2 study (NCT02773030). Due to the recent focus by health authorities on dose selection for oncology products other than the maximum tolerated dose (MTD), a comprehensive analysis of pharmacodynamics (PD) and pharmacokinetics (PK) was implemented to support dose optimization of IBER. Aims: To inform dose selection of IBER as monotherapy, and in combination with dexamethasone (DEX; Iber+d) and with DEX and daratumumab (DARA; IberDd). Methods: PK samples were collected on treatment of cycles (C) 1–4 to estimate IBER exposure using population PK (area under the concentration curve over 24-h dosing interval [AUCτ]). Biomarkers included analysis of peripheral blood samples on C1 day (D)1 and mid-cycle through C4 for immunophenotyping, absolute neutrophil counts (ANC), and assessment of serum free light chain (sFLC), as a biomarker of tumor burden. Bone marrow samples were collected at screening and C2D15 for immunohistochemistry. Results: IBER AUCτ increased in a dose-proportional manner between 0.3 and 1.0 mg as monotherapy and 0.3 and 1.6 mg in combination with DEX, with moderate variability. Median time to maximum serum concentration of IBER was 2–4 h post dose with an elimination half-life of ~12 h. Comparable IBER exposure was observed between monotherapy and Iber+d. Reductions in Ikaros/Aiolos protein levels in tumor cells were observed at all dose levels in both cohorts with a >90% reduction at the 0.45-mg dose. However, a >50% decrease in sFLCs was observed only at doses ≥0.9 mg, and doses of 1.6 mg induced faster and deeper decreases vs lower doses. In the immune compartment, IBER treatment induced similar dose-/exposure-dependent PD changes as monotherapy and Iber+d, which appeared to saturate at higher doses. A >80% reduction in mature B cells and ~2-fold increase in proliferating/activated T and NK cells were observed at doses ≥1.0 mg. PD activity of IBER was not attenuated by prior refractoriness to immunomodulatory drugs (IMiD® agents) or anti-CD38 therapy. Based on these results, doses between 1.0 and 1.6 mg were tested in the IberDd cohort. PK and PD of IBER were similar with concomitant administration of DARA. In this cohort, a reduction in mature B cells and sFLCs was more consistently observed at 1.6 mg vs lower doses; however, higher IBER exposures were associated with more pronounced decreases in ANC when compared with Iber+d. This was clinically manageable and the MTD of IBER was not determined in any cohort. Summary/Conclusion: Similar PK and PD results were observed in patients treated with IBER monotherapy, Iber+d, and IberDd. Across all cohorts, a >20% difference in dose was needed for meaningful change in IBER exposure, immune PD began to saturate at doses ≥1.0 mg, and decreases in tumor burden were greatest at the 1.6-mg dose. Based on these results, IBER doses of 1.0, 1.3, and 1.6 mg were chosen for dose optimization of IberDd for RRMM. Taken together with data suggesting immune surveillance is a driver of maintenance therapy efficacy and a desire to increase IBER tolerability with long-term treatment, doses of 0.75, 1.0, and 1.3 mg were selected for dose optimization of IBER monotherapy in the newly diagnosed MM maintenance setting.

Prefoldin Function in Cellular Protein Homeostasis and Human Diseases.

Cellular functions are largely performed by proteins. Defects in the production, folding, or removal of proteins from the cell lead to perturbations in cellular functions that can result in pathological conditions for the organism. In cells, molecular chaperones are part of a network of surveillance mechanisms that maintains a functional proteome. Chaperones are involved in the folding of newly synthesized polypeptides and assist in refolding misfolded proteins and guiding proteins for degradation. The present review focuses on the molecular co-chaperone prefoldin. Its canonical function in eukaryotes involves the transfer of newly synthesized polypeptides of cytoskeletal proteins to the tailless complex polypeptide 1 ring complex (TRiC/CCT) chaperonin which assists folding of the polypeptide chain in an energy-dependent manner. The canonical function of prefoldin is well established, but recent research suggests its broader function in the maintenance of protein homeostasis under physiological and pathological conditions. Interestingly, non-canonical functions were identified for the prefoldin complex and also for its individual subunits. We discuss the latest findings on the prefoldin complex and its subunits in the regulation of transcription and proteasome-dependent protein degradation and its role in neurological diseases, cancer, viral infections and rare anomalies.

The multiomics landscape of serum exosomes during the development of sepsis

IntroductionSepsis is an infection-induced severe inflammatory disorder leading to multiple organ dysfunction. It remains a highly lethal condition for which early diagnosis and therapy achieve unsatisfactory results. Circulating exosomes containing biomarkers and mediators of sepsis have recently received attention, but the progress has been far from optimal. ObjectivesThe present study focuses on the profiles of molecular dynamics in serum exosomes and explores the potential molecular mechanisms on serum exosomes during the process of sepsis. MethodsWe used high-performance liquid chromatography-tandem mass spectrometry and RNA-seq to detect the dynamic profiles of exosome proteins and RNAs (including mRNAs, lncRNAs and miRNAs) in serum exosomes from 3 healthy individuals and 9 septic patients at the different stages. Then integrative multiomics analyses were performed and the results were validated by qRT-PCR, LiquiChip assay and metabolomics analysis on mice subjected to cecal ligation and puncture (CLP) modeling. ResultsA total of 354 proteins, 195 mRNAs, 82 lncRNAs and 55 miRNAs were identified as differentially expressed molecules in serum exosomes from septic patients. Integrative multiomics analysis showed that exosome components were associated with cytokine storm, complement and clotting cascades, the endothelial barrier, 20S proteasome-dependent protein degradation and vitamin metabolism. Importantly, pretreatment with serum exosomes derived from mice subjected to CLP significantly restrained proinflammatory cytokine expression and alleviated tissue injury in septic mice. Further metabolomics analysis demonstrated that pretreatment with septic serum exosomes significantly affected the metabolites associated with vitamin digestion and absorption in CLP mice. ConclusionOur study for the first time describes the landscape of the molecular dynamics of serum exosomes during the development of sepsis and proposes some hypothetical molecular mechanisms by integrative multiomics analysis, which may provide helpful diagnostic and therapeutic insights for the ongoing battle against sepsis.

Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae.

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

Potential involvement of DSCAML1 mutations in neurodevelopmental disorders.

The molecular mechanisms underlying neurodevelopmental disorders (NDDs) remain unclear. We previously identified Down syndrome cell adhesion molecule like 1 (Dscaml1) as a responsible gene for Ihara epileptic rat (IER), a rat model for human NDDs with epilepsy. However, the relationship between NDDs and DSCAML1 in humans is still elusive. In this study, we screened databases of autism spectrum disorders (ASD), intellectual disability (ID)/developmental disorders (DD) and schizophrenia for genomic mutations in human DSCAML1. We then performed in silico analyses to estimate the potential damage to the mutated DSCAML1 proteins and chose three representative mutations (DSCAML1C729R , DSCAML1R1685* and DSCAML1K2108Nfs*37 ), which lacked a cysteine residue in the seventh Ig domain, the intracellular region and the C-terminal PDZ-binding motif, respectively. In overexpression experiments in a cell line, DSCAML1C729R lost its mature N-glycosylation, whereas DSCAML1K2108Nfs*37 was abnormally degraded via proteasome-dependent protein degradation. Furthermore, in primary hippocampal neurons, the ability of the wild-type DSCAML1 to regulate the number of synapses was lost with all mutant proteins. These results provide insight into understanding the roles of the domains in the DSCAML1 protein and further suggest that these mutations cause functional changes, albeit through different mechanisms, that likely affect the pathophysiology of NDDs.

MPL Overexpression Induces a High Level of Mutant-Calr/MPL Complex: A Novel Mechanism of Ruxolitinib Resistance in Myeloproliferative Neoplasms with Calr Mutations

【Introduction】 Although Ruxolitinib (RUX), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients, causing poor prognosis of MPN patients. Until now, various studies have elucidated the mechanisms of RUX-resistance in JAK2V617F-mutant MPN cells, including 1) increased JAK2 heterodimerization leading to sustained JAK2 activation, 2) JAK2 kinase domain mutations, and 3) JAK1/JAK3 activations. However, mechanisms of RUX-resistance in MPN cells with CALR mutations have not been fully characterized to date. In this study, we have clarified a mechanism of RUX-resistance in MPN tumor cells with CALR mutations. 【Materials and Methods】 At first, we have created several human cell lines with exogenous MPL expressions (exMPL) and CALR +1 frameshift mutations (CALR-fs) by introductions of V5-tagged MPL and/or FLAG-tagged CALR Del52/Ins5 expressing vectors or the CRISPR/Cas9 technology. We have confirmed that these cell lines had increased JAK/STAT signaling and respond to RUX-treatment. To establish RUX-resistant cell lines, we have cultured these cell lines with low-dose RUX (0.2μM), and gradually increased the concentrations of RUX by 0.1μM every week. We have successfully established RUX-resistant cells that proliferated in the presence of RUX at 0.8μM. Then, we characterized the RUX-resistant cells with CALR-fs/exMPL. To examine whether RUX-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted RUX-resistant cells as well as RUX-sensitive cells into immunocompromised mice. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors pathologically examined. MPL knockdown experiments showed that high levels of MPL were indispensable in the resistant cell lines. Co-immunoprecipitation assaies showed the interactions of mutant CALR and MPL proteins in RUX-resistant cells. To examine reversibility of RUX-resistance, RUX-resistant cells were cultured without RUX for three months. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We have found that RUX-resistant cells had high MPL transcripts, overexpression of both immature and mature MPL, and JAK2. We also found that RUX-resistant cells had increased phosphorylations of JAK1, JAK2, JAK3 STAT5, MEK and ERK. In vivo assay using immunocompromised mice showed the immunohistochemical staining of MPL in the tumors from RUX-resistant cells showed high expression of MPL in the tumor cells. We also found that mature MPL proteins were more stable since proteasome-dependent degradation of mature MPL proteins was impaired in RUX-resistant cells. Knockdown of MPL of RUX-resistant cells by shRNAs decreased intensity of phosphorylations of JAK1, JAK2, STAT5, MEK and ERK, suggesting that the high expression of MPL leads to more potent signaling. Notably, when mutant-CALR proteins were immunoprecipitated, both immature and mature MPL proteins were co-immunoprecipitated: more MPL proteins were pulled down in RUX-resistant cells. In a reciprocal experiment, when MPL proteins were immunoprecipitated, mutant-CALR proteins were co-immunoprecipitated: more mutant-CALR proteins were pulled down in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells, suggesting that a high level of mutant-CALR/MPL complex contributed to RUX-resistance in these cells. When the resistant cells were cultured in the absence of RUX, RUX-resistance was reversed with reduction of MPL transcripts, mature MPL and JAK2 proteins and mutant-CALR/MPL complex. Immunohistochemical staining showed that MPL staining intensity of megakaryocytes of MF patients with CALR mutations were higher than those of patients with JAK2V617F or normal individuals. MPL expressions were higher after RUX-treatment in MF patients with both JAK2V617F mutations and CALR mutations. 【Discussion】 Overexpression of MPL is a common mechanism after RUX-treatment in MPN cells with both JAK1V617F mutations and CALR mutations. In RUX-resistant cells with CALR mutations, MPL overexpression leads to the high level of mutant-CALR/MPL complex, causing resistance to RUX. This novel mechanism could be a new therapeutic target to overcome RUX-resistance in MPN cells with CALR mutations. Disclosures Komatsu: Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

Abstract 1472: Paraoxonase 3 is a novel tumor suppressor protein in xenograft mouse model

Abstract Introduction: Dysfunction of mitochondrial respiratory chains or its associated machinery favors the aerobic glycolysis leading to upregulation of genes related to cell proliferation, metastasis, drug resistant and cancer cell stemness, i.e., the Warburg effect, which has a major role in the pathogenesis of tumor formation. Various studies have documented that perturbation of mitochondria leads to apoptosis suggesting that the precise spatiotemporal regulation of mitochondrial function is a critical regulator of both cell survival and death, whcih determine tumorigenesis. Paraoxonase 3 (PON3) is an intracellular protein localized to the mitochondrial associated membrane. PON3 binds coenzymes Q10 and regulates the respiratory complex activity and prevents the ubisemiquinone-mediated mitochondrial superoxide levels in response to stress conditions. PON3 deficiency leads to dysfunction of mitochondria, which along with an increase in mitochondrial oxidative stress causes systemic inflammation, atherosclerosis, obesity and gall stone formation. PON3 is upregulated in ovarian cancer tissues. However, it's expression in various stages of ovarian cancer and its role on cancer development have not been studied. Hypothesis: PON3 expression may regulate the ovarian cancer development by enhancing the mitochondrial function. Methods and Results: Using human ovarian carcinoma tissue array, we identified that PON3 expression was higher in both early and late stages of ovarian cancer when compared to normal tissue. Interestingly, we demonstrated that overexpression of PON3 prevented tumor formation in a mouse ovarian cancer xenograft model. Overexpression of PON3, (i) reduced the tumor angiogenesis marker, CD31 levels in the tumor microenvironment and (ii) inhibited ovarian cancer cell proliferation in both normoxic and hypoxic condition in vitro. Moreover, PON3 reduced VEGF levels (but not IGF-1 levels) under hypoxic conditions. Furthermore, mechanistically, overexpression of PON3 decreased the VEGF level by accelerating the proteasome-dependent protein degradation of hypoxia inducible factor-1 α (HIF-1α) under hypoxic conditions. Finally, we demonstrate that the inhibitory effect of PON3 on HIF-1α levels is, in part, mediated by mitochondrial function. Conclusion: We report for the first time that PON3 functions as a tumor suppressor in ovarian cancer possibly by suppressing VEGF signaling via HIF-1α by enhancing the mitochondrial function suggesting that PON3 might be a successful strategy to inhibit the ovarian cancer development. Citation Format: Asokan Devarajan, Feng Su, Dawoud Sulaiman, Dorothy Nguyen, Robin Farias-Eisner, Srinivasa T. Reddy. Paraoxonase 3 is a novel tumor suppressor protein in xenograft mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1472.

Novel small-molecule LG1836 inhibits the in vivo growth of castration-resistant prostate cancer.

Androgen deprivation therapy (ADT) is the mainstay of treatment for castration-resistant prostate cancer (CRPC). Unfortunately, although ADT initially prolongs survival, most patients relapse and develop resistance. Clinical failure of these treatments in CRPC highlights the urgent need to develop novel strategies to more effectively block androgen receptor (AR) signaling and target other oncogenic factors responsible for ADT resistance. We developed a small-molecule compound LG1836 and investigated the in vitro and in vivo activity of LG1836 against CRPC in cellular and animal models. LG1836 exhibits potent in vitro cytotoxicity in CRPC cells. Mechanistic studies demonstrated that LG1836 inhibits the expression of AR and AR variant 7, partially mediated via proteasome-dependent protein degradation. LG1836 also suppresses survivin expression and effectively induces apoptosis in CRPC cells. Significantly, as a single agent, LG1836 is therapeutically efficacious in suppressing the in vivo growth of CRPC in the subcutaneous and intraosseous models and extends the survival of tumor-bearing mice. These preclinical studies indicate that LG1836 is a promising lead compound for the treatment of CRPC.

Inhibition of jasmonate-mediated plant defences by the fungal metabolite higginsianin B.

Infection of Arabidopsis thaliana by the ascomycete fungus Colletotrichum higginsianum is characterized by an early symptomless biotrophic phase followed by a destructive necrotrophic phase. The fungal genome contains 77 secondary metabolism-related biosynthetic gene clusters, whose expression during the infection process is tightly regulated. Deleting CclA, a chromatin regulator involved in the repression of some biosynthetic gene clusters through H3K4 trimethylation, allowed overproduction of three families of terpenoids and isolation of 12 different molecules. These natural products were tested in combination with methyl jasmonate, an elicitor of jasmonate responses, for their capacity to alter defence gene induction in Arabidopsis. Higginsianin B inhibited methyl jasmonate-triggered expression of the defence reporter VSP1p:GUS, suggesting it may block bioactive jasmonoyl isoleucine (JA-Ile) synthesis or signalling in planta. Using the JA-Ile sensor Jas9-VENUS, we found that higginsianin B, but not three other structurally related molecules, suppressed JA-Ile signalling by preventing the degradation of JAZ proteins, the repressors of jasmonate responses. Higginsianin B likely blocks the 26S proteasome-dependent degradation of JAZ proteins because it inhibited chymotrypsin- and caspase-like protease activities. The inhibition of target degradation by higginsianin B also extended to auxin signalling, as higginsianin B treatment reduced auxin-dependent expression of DR5p:GUS. Overall, our data indicate that specific fungal secondary metabolites can act similarly to protein effectors to subvert plant immune and developmental responses.

A novel function of AAA-ATPase p97/VCP in the regulation of cell motility.

High level of the multifunctional AAA-ATPase p97/VCP is often correlated to the development of cancer; however, the underlying mechanism is not understood completely. Here, we report a novel function of p97/VCP in actin regulation and cell motility. We found that loss of p97/VCP promotes stabilization of F-actin, which cannot be reversed by actin-destabilizing agent, Cytochalasin D. Live-cell imaging demonstrated reduced actin dynamics in p97/VCP-knockdown cells, leading to compromised cell motility. We further examined the underlying mechanism and found elevated RhoA protein levels along with increased phosphorylation of its downstream effectors, ROCK, LIMK, and MLC upon the knockdown of p97/VCP. Since p97/VCP is indispensable in the ubiquitination-dependent protein degradation pathway, we investigated if the loss of p97/VCP hinders the protein degradation of RhoA. Knockdown of p97/VCP resulted in a higher amount of ubiquitinated RhoA, suggesting p97/VCP involvement in the proteasome-dependent protein degradation pathway. Finally, we found that p97/VCP interacts with FBXL19, a molecular chaperone known to guide ubiquitinated RhoA for proteasomal degradation. Reduction of p97/VCP may result in the accumulation of RhoA which, in turn, enhances cytoplasmic F-actin formation. In summary, our study uncovered a novel function of p97/VCP in actin regulation and cell motility via the Rho-ROCK dependent pathway which provides fundamental insights into how p97/VCP is involved in cancer development.

PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia.

GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) are sequentially responsible for de novo synthesis of tetrahydrobiopterin (BH4), a known co-factor for nitric oxide synthase (NOS). The implication of BH4-biosynthesis process in tumorigenesis remains to be investigated. Here, we show that PTPS, which is highly expressed in early-stage colorectal cancer, is phosphorylated at Thr 58 by AMPK under hypoxia; this phosphorylation promotes PTPS binding to LTBP1 and subsequently drives iNOS-mediated LTBP1 S-nitrosylation through proximal-coupling BH4 production within the PTPS/iNOS/LTBP1 complex. In turn, LTBP1 S-nitrosylation results in proteasome-dependent LTBP1 protein degradation, revealing an inverse relationship between PTPS pT58 and LTBP1 stability. Physiologically, the repressive effect of PTPS on LTBP1 leads to impaired transforming growth factor β (TGF-β) secretion and thereby maintains tumor cell growth under hypoxia. Our findings illustrate a molecular mechanism underlying the regulation of LTBP1-TGF-β signaling by the BH4-biosynthesis pathway and highlight the specific requirement of PTPS for tumor growth.

Proteasome-dependent degradation of intracellular carbamylated proteins.

Carbamylation, which corresponds to the binding of isocyanic acid to the amino groups of proteins, is a nonenzymatic post-translational modification responsible for alterations of protein structural and functional properties. Tissue accumulation of carbamylation-derived products and their role in pathological processes such as atherosclerosis or chronic renal failure have been previously documented. However, few studies have focused on the carbamylation of intracellular proteins and their subsequent role in cellular aging. This study aimed to determine the extent of intracellular protein carbamylation, its impact on cell functions and the ability of cells to degrade these modified proteins. Fibroblasts were incubated with cyanate or urea and the carbamylation level was evaluated by immunostaining and homocitrulline quantification. The results showed that carbamylated proteins accumulated intracellularly and that all proteins were susceptible. The presence of intracellular carbamylated proteins did not modify cell proliferation or type I collagen synthesis nor did it induce cell senescence, but it significantly decreased cell motility. Fibroblasts were able to degrade carbamylated proteins through the ubiquitin-proteasome system. In conclusion, intracellular proteins are susceptible to carbamylation but their accumulation does not seem to deeply affect cell function, owing largely to their elimination by the ubiquitin-proteasome system.

Suppression of PAMP-triggered immunity (PTI) by effector proteins synthesized by phytopathogens and delivered into cells of infected plant

Plant immunity is constituted by multilayered system involving two intertwined lines of defence: a first level of immunity termed PAMP-triggered immunity (PTI) or basal resistance, and a second layer of plant defence, called effector-triggered immunity (ETI). The second line of defence depends on the ability of the plant to recognize phytopathogen-synthesized effector proteins delivered into host plant cells. The effector proteins employ common as well as pathogen-specific strategies to disturb plant immunity and to promote pathogen survival and favor their multiplication. They target pattern-recognition receptors (PRRs) and key components in the PTI signaling pathways, as well as, they interfere with many cellular processes including vesicle transport, cytoskeleton reorganization, proteasome-dependent protein degradation, phytohormone biosynthesis and signaling, and gene expression. This results in effector-triggered susceptibility (ETS). However, in some cases, pathogen effectors are recognized by plant intracellular immune receptors NB-LRR/NLR that identify effector proteins. Conformational changes in the NB-LRR/NLR immune receptors accompanying the recognition of the effector proteins activate intracellular signaling pathways initiating a whole range of defence responses that form the second line of local defence.

Moderate Exercise Improves Experimental Cancer Cachexia by Modulating the Redox Homeostasis.

Cachexia is a debilitating syndrome that complicates the management of cancer patients. Muscle wasting, one of the main features of cachexia, is associated with hyper-activation of protein degradative pathways and altered mitochondrial function that could both result from impaired redox homeostasis. This study aimed to investigate the contribution of oxidative stress to cancer-induced cachexia in the presence or in the absence of moderate exercise training. Mice bearing the colon C26 carcinoma, either sedentary or exercised, were used. The former showed muscle wasting and redox imbalance, with the activation of an antioxidant response and with upregulation of markers of proteasome-dependent protein degradation and autophagy. Moderate exercise was able to relieve muscle wasting and prevented the loss of muscle strength; such a pattern was associated with reduced levels of Reactive Oxygen Species (ROS), carbonylated proteins and markers of autophagy and with improved antioxidant capacity. The muscle of sedentary tumor hosts also showed increased levels of molecular markers of mitophagy and reduced mitochondrial mass. Conversely, exercise in the C26 hosts led to increased mitochondrial mass. In conclusion, moderate exercise could be an effective non-pharmacological approach to prevent muscle wasting in cancer patients, decreasing muscle protein catabolism and oxidative stress and preserving mitochondria.

Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation

Anthrax is a life-threatening disease caused by infection with Bacillus anthracis, which expresses lethal factor and the receptor-binding protective antigen. These two proteins combine to form anthrax lethal toxin (LT), whose proximal targets are mitogen-activated kinase kinases (MKKs). However, the downstream mediators of LT toxicity remain elusive. Here we report that LT exposure rapidly reduces the levels of c-Jun, a key regulator of cell proliferation and survival. Blockade of proteasome-dependent protein degradation with the 26S proteasome inhibitor MG132 largely restored c-Jun protein levels, suggesting that LT promotes degradation of c-Jun protein. Using the MKK1/2 inhibitor U0126, we further show that MKK1/2-Erk1/2 pathway inactivation similarly reduces c-Jun protein, which was also restored by MG132 pre-exposure. Interestingly, c-Jun protein rebounded to normal levels 4 h following U0126 exposure but not after LT exposure. The restoration of c-Jun in U0126-exposed cells was associated with increased c-Jun mRNA levels and was blocked by inactivation of the JNK1/2 signaling pathway. These results indicate that LT reduces c-Jun both by promoting c-Jun protein degradation via inactivation of MKK1/2-Erk1/2 signaling and by blocking c-Jun gene transcription via inactivation of MKK4-JNK1/2 signaling. In line with the known functions of c-Jun, LT also inhibited cell proliferation. Ectopic expression of LT-resistant MKK2 and MKK4 variants partially restored Erk1/2 and JNK1/2 signaling in LT-exposed cells, enabling the cells to maintain relatively normal c-Jun protein levels and cell proliferation. Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation.