Preferentially expressed antigen in melanoma (PRAME) has been described as a cancer-testis antigen and is associated with leukaemias and solid tumours. Here we show that PRAME gene transcription in leukaemic cell lines is rapidly induced by exposure of cells to bacterial PAMPs (pathogen associated molecular patterns) in combination with type 2 interferon (IFNγ). Treatment of HL60 cells with lipopolysaccharide or peptidoglycan in combination with IFNγ resulted in a rapid and transient induction of PRAME transcription, and increased association of PRAME transcripts with polysomes. Moreover, treatment with PAMPs/IFNγ also modulated the subcellular localisation of PRAME proteins in HL60 and U937 cells, resulting in targeting of cytoplasmic PRAME to the Golgi. Affinity purification studies revealed that PRAME associates with Elongin B and Elongin C, components of Cullin E3 ubiquitin ligase complexes. This occurs via direct interaction of PRAME with Elongin C, and PRAME colocalises with Elongins in the Golgi after PAMP/IFNγ treatment. PRAME was also found to co-immunoprecipitate core histones, consistent with its partial localisation to the nucleus, and was found to bind directly to histone H3 in vitro. Thus, PRAME is upregulated by signalling pathways that are activated in response to infection/inflammation, and its product may have dual functions as a histone-binding protein, and in directing ubiquitylation of target proteins for processing in the Golgi.
As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8+ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide in vitro. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201+ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME100–108 or PRAME300–309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.
The tumour antigen PReferentially expressed Antigen of MElanoma (PRAME) is expressed in a variety of malignancies, including breast cancer. We have analysed PRAME gene expression in relation to clinical outcome for 295 primary breast cancer patients. Kaplan–Meier survival curves show a correlation of PRAME expression levels with increased rates of distant metastases and decreased overall patient survival. This correlation existed both for the entire patient group (n=295) and for the subgroup of patients (n=185) who did not receive adjuvant chemotherapy. Multivariable analysis indicated that PRAME is an independent marker of shortened metastasis-free interval in patients who did not receive adjuvant chemotherapy. PRAME expression was associated with tumour grade and negative oestrogen receptor status. We conclude that PRAME expression is a prognostic marker for clinical outcome of breast cancer, independent of traditional clinicopathological markers.
PRAME; tumour antigen; prognosis; metastasis; survival; microarray
The physiological and phenotypic differences between human and chimpanzee are largely specified by our genomic differences. We have been particularly interested in recent duplications in the human genome as examples of relatively large-scale changes to our genome. We performed an in-depth evolutionary analysis of a region of chromosome 1, which is copy number polymorphic among humans, and that contains at least 32 PRAME (Preferentially expressed antigen of melanoma) genes and pseudogenes. PRAME-like genes are expressed in the testis and in a large number of tumours, and are thought to possess roles in spermatogenesis and oogenesis.
Using nucleotide substitution rate estimates for exons and introns, we show that two large segmental duplications, of six and seven human PRAME genes respectively, occurred in the last 3 million years. These duplicated genes are thus hominin-specific, having arisen in our genome since the divergence from chimpanzee. This cluster of PRAME genes appears to have arisen initially from a translocation approximately 95–85 million years ago. We identified multiple sites within human or mouse PRAME sequences which exhibit strong evidence of positive selection. These form a pronounced cluster on one face of the predicted PRAME protein structure.
We predict that PRAME genes evolved adaptively due to strong competition between rapidly-dividing cells during spermatogenesis and oogenesis. We suggest that as PRAME gene copy number is polymorphic among individuals, positive selection of PRAME alleles may still prevail within the human population.
The potential for cancer-testis (CT) antigens as targets for immunotherapy in cancer patients has been heavily investigated, and currently cancer vaccine trials based on the CT antigens, MAGE-A3 and NY-ESO-1, are being carried out.
We used specific q-RT-PCR assays to analyse the expression of the CT genes MAGE-A3, NY-ESO-1 (CTAG1B), LAGE-1 (CTAG2) and PRAME in a panel of bladder tumours from 350 patients with long-term follow-up and detailed treatment information.
Overall, 43% of the tumours expressed MAGE-A3, 35% expressed NY-ESO-1, 27% expressed LAGE-1 and 20% expressed PRAME. In all, 56% of the tumours expressed at least one of the CT genes analysed. Univariate Cox regression analysis of CT gene expression in non-muscle-invasive tumours showed that expression of MAGE-A3 (P=0.026), LAGE-1 (P=0.001) and NY-ESO-1 (P=0.040) was significantly associated with a shorter progression-free survival. In addition, we found that patients with tumours expressing PRAME responded poorly to chemotherapy (P=0.02, χ2-test).
Cancer-testis genes are frequently expressed in bladder cancer and especially in tumours of high stage and grade. In addition, the CT gene expression may have both prognostic and predictive value. Development of specific immunotherapy against the CT antigens in bladder cancer may ultimately increase patient survival.
bladder cancer; CT genes; immunotherapy; PCR; prognosis
PRAME belongs to a group of cancer/testis antigens (CTAs) that are characterized by their restricted expression in normal gametogenic tissues and a variety of tumors. The PRAME family is one of the most amplified gene families in the mouse and other mammalian genomes. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins functioning as transcription regulators in cancer cells. However, the role of PRAME in normal gonads is unknown. The objective of this study is to characterize the temporal and spatial expression of the mouse Pramel1 gene, and to determine the cellular localization of the PRAMEL1 protein during the mouse spermatogenesis. Our results indicated that the mouse Pramel1 was expressed in testis only. The mRNA and protein expression level was low in the newborn testes, and gradually increased from 1- to 3-week-old testes, and then remained constant after three weeks of age. Immunofluorescent staining on testis sections with the mouse PRAMEL1 antibody revealed that PRAMEL1 was localized in the cytoplasm of spermatocytes and the acrosomal region of round, elongating and elongated spermatids. Further analyses on the testis squash preparation and spermatozoa at a subcellular level indicated that the protein localization patterns of PRAMEL1 were coordinated with morphological alterations during acrosome formation in spermatids, and were significantly different in connecting piece, middle piece and principal piece of the flagellum between testicular and epididymal spermatozoa. Collectively, our results suggest that PRAMEL1 may play a role in acrosome biogenesis and sperm motility.
Preferentially expressed antigen in melanoma (PRAME) and prostate-specific membrane antigen (PSMA) are tumor-associated antigens implicated in cellular differentiation, genetic stability, and angiogenesis. MKC1106-PP is an immunotherapeutic regimen cotargeting PRAME and PSMA, comprised of a recombinant plasmid (pPRA-PSM encoding fragments derived from both antigens) and 2 peptides (E-PRA and E-PSM derived from PRAME and PSMA, respectively). This multicenter study evaluated MKC1106-PP with a fixed plasmid dose and 2 different peptide doses, administered by intralymph node injection in a prime-boost sequence in human leukocyte antigen-A*0201 and tumor-antigen-positive patients with progressing metastatic solid tumors who had failed standard therapy. Immune monitoring was done by tetramer and enzymatic-linked immune spot analysis. The treatment was well tolerated, with no significant differences in safety, immune response, and clinical outcome relative to peptide doses. Fifteen of 24 evaluable patients showed an immune response, as defined by the expansion of PRAME-specific or PSMA-specific T cells in the blood. There were no partial or complete responses by the Response Evaluation Criteria in Solid Tumors. Seven patients showed stable disease (SD) for 6 months or longer, or prostate specific antigen decline: 4 of 10 with prostate carcinoma, 2 of 2 with renal clear cell carcinoma, and 1 of 10 with metastatic melanoma. In addition, there was an association between the induction and persistence of antigen-specific T cells in blood above baseline levels and disease control, defined as SD for 6 months or longer. These results support further development of MKC1106-PP in specific clinical indications.
active immunotherapy; PRAME; PSMA; solid tumor; intralymph node vaccination
Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA) family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157–165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTA's have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC) in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression.
We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTA's with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment.
A minority (36%) of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment.
These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic chondrosarcoma patients where no proven systemic therapies exist.
The prognosis for patients with Hodgkin lymphoma (HL) has improved in recent decades. On the other hand, not all patients can be cured with the currently established therapy regimes and this therapy is associated with several adverse late effects. Therefore it is necessary to develop new therapy strategies. After treatment of L-540 HL cells with 5′-azacytidine (5AC), we observed increased expression of the preferentially expressed antigen in melanoma (PRAME). In addition, we detected an increased resistance of 5AC-treated cells against cytotoxic drugs. We analyzed the influence of PRAME on cell survival of HL cells by knocking down PRAME in the chemotherapy resistant cell line L-428, a cell line that express PRAME at a high level. After knock-down of PRAME using vector based RNA interference we observed increased sensitivity for cisplatin, etoposide and retinoic acid. DNA microarray analysis of HL cells after PRAME knock-down indicated regulation of several genes including down-regulation of known anti-apoptotic factors. Increased retinoic acid signaling in these cells was revealed by increased expression of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic acid signaling. Our data suggest that PRAME inhibits retinoic acid signaling in HL cells and that the knock-down of PRAME might be an interesting option for the development of new therapy strategies for patients with chemo-resistant HL.
To investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance.
The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia (AL) and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics (gender, age, white blood count, immunophenotype of leukemia, percentage of blast cells, and karyotype) of the patients were performed.
The PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia (AML, n=27), and in 28.6% of the patients with acute lymphoblastic leukemia (ALL, n=7), but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M3, 33.3% in M2, and 28.6% in M5. Gene expression was also found to be correlated with CD15 and CD33 expression and abnormal karyotype, but not with age, gender, white blood count or percentage of blast cells.
The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia.
preferentially expressed antigen of melanoma; gene; acute leukemia; minimal residual disease; immunotherapy
The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t6A37 formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.
Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2−/− mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.
ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11Cul). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11Cul fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11Cul mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.
PRAME/MAPE/OIP4 is a germinal tissue-specific gene that is also expressed at high levels in haematological malignancies and solid tumours. The physiological functions of PRAME in normal and tumour cells are unknown, although a role in the regulation of retinoic acid signalling has been proposed. Sequence homology and structural predictions suggest that PRAME is related to the leucine-rich repeat (LRR) family of proteins, which have diverse functions. Here we review the current knowledge of the structure/function of PRAME and its relevance in leukaemia.
Cullin 4 (Cul4), a member of the evolutionally conserved cullin protein family, serves as a scaffold to assemble multisubunit ubiquitin E3 ligase complexes. Cul4 interacts with the Ring finger-containing protein ROC1 through its C-terminal cullin domain and with substrate recruiting subunit(s) through its N-terminus. Previous studies have demonstrated that Cul4 E3 ligase ubiquitylates key regulators in cell cycle control and mediates their degradation through the proteasomal pathway, thus contributing to genome stability. Recent studies from several groups have revealed that Cul4 E3 ligase can target histones for ubiquitylation, and importantly, ubiquitylation of histones may facilitate the cellular response to DNA damage. Therefore, histone ubiquitylation by Cul4 E3 ligase constitutes a novel mechanism through which Cul4 regulates chromatin function and maintains genomic integrity. We outline these studies and suggest that histone ubiquitylation might play important roles in Cul4-regualted chromatin function including the cellular response to DNA damage and heterochromatin gene silencing.
Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.
HSF2; Cul3; PEST; Ubiquitin; Protein turnover
Cullin proteins are molecular scaffolds that have crucial roles in the post-translational modification of cellular proteins involving ubiquitin. The mammalian cullin protein family comprises eight members (CUL1 to CUL7 and PARC), which are characterized by a cullin homology domain. CUL1 to CUL7 assemble multi-subunit Cullin-RING E3 ubiquitin ligase (CRL) complexes, the largest family of E3 ligases with more than 200 members. Although CUL7 and PARC are present only in chordates, other members of the cullin protein family are found in Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and yeast. A cullin protein tethers both a substrate-targeting unit, often through an adaptor protein, and the RING finger component in a CRL. The cullin-organized CRL thus positions a substrate close to the RING-bound E2 ubiquitin-conjugating enzyme, which catalyzes the transfer of ubiquitin to the substrate. In addition, conjugation of cullins with the ubiquitin-like molecule Nedd8 modulates activation of the corresponding CRL complex, probably through conformational regulation of the interactions between cullin's carboxy-terminal tail and CRL's RING subunit. Genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective CRLs. CRLs target numerous substrates and thus have an impact on a range of biological processes, including cell growth, development, signal transduction, transcriptional control, genomic integrity and tumor suppression. Moreover, mutations in CUL7 and CUL4B genes have been linked to hereditary human diseases.
The Elongin complex was originally identified as a positive regulator of RNA polymerase II and is composed of a transcriptionally active subunit (A) and two regulatory subunits (B and C). The Elongin BC complex enhances the transcriptional activity of Elongin A. “Classical” SOCS box-containing proteins interact with the Elongin BC complex and have ubiquitin ligase activity. They also interact with the scaffold protein Cullin (Cul) and the RING domain protein Rbx and thereby are members of the Cullin RING ligase (CRL) superfamily. The Elongin BC complex acts as an adaptor connecting Cul and SOCS box proteins. Recently, it was demonstrated that classical SOCS box proteins can be further divided into two groups, Cul2- and Cul5-type proteins. The classical SOCS box-containing protein pVHL is now classified as a Cul2-type protein. The Elongin BC complex containing CRL family is now considered two distinct protein assemblies, which play an important role in regulating a variety of cellular processes such as tumorigenesis, signal transduction, cell motility, and differentiation.
ubiquitin; Cullin; Elongin; ECS complex; SCF complex
Active immunotherapy may prevent the relapse of acute myeloid leukemia (AML) by inducing leukemia-specific T cells. Here, we investigated whether Wilms’ tumor 1 (WT1) and preferentially expressed antigen in melanoma (PRAME)-specific T cells could be induced upon the priming of healthy donor- and AML patient-derived T cells with HLA-A2-matched, peptide-loaded allogeneic dendritic cells. AML-reactive, tetramer (Tm)-binding and interferon-producing, cytotoxic T lymphocytes specific for PRAME could readily be isolated from healthy individuals and maintained in culture. In this setting, priming efficacy was significantly higher for PRAME than for WT1. The priming of T cells from patient-derived material proved to be near-to-impossible: No leukemia-associated antigen (LAA)-specific T cell could be primed in 4 patients that had recently achieved a complete response (CR), and in only 1 out of 3 patients exhibiting a sustained CR we did observe WT1-specific T cells, though with a low frequency. These findings suggest that the functionality and/or repertoire of T cells differ in healthy subjects and AML patients in CR, and may have repercussions for the implementation of active vaccination approaches against AML.
acute myeloid leukemia; dendritic cell-based vaccination; immunotherapy; PRAME; T- cell priming; WT1
The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.
Background: BTB-Kelch proteins, including KLHL11, are proposed to bind Cul3 through a “3-box” motif to form E3 ubiquitin ligases.
Results: We solved crystal structures of the KLHL11-Cul3 complex and four Kelch domains.
Conclusion: The 3-box forms a hydrophobic groove that binds a specific N-terminal extension of Cul3.
Significance: Dimeric BTB-Kelch proteins bind two Cul3 molecules and support a two-site model for substrate recognition.
Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.
Proteasome; Protein-Protein Interactions; Signaling; Ubiquitination; X-ray Crystallography; β-Propeller; Degradation
The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.
The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation.
This study aims to analyze the expression of 14 cancer/testis
(CT) antigens in multiple myeloma (MM) to identify possible prognostic markers
and therapeutic targets. The expression of MAGEA1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA10, MAGEA12, BAGE1, MAGEC1/CT7,
the GAGE family, LAGE-1, PRAME, NY-ESO-1, SPA17 and SSX1 was
studied by RT-PCR in 15 normal tissues, a pool of 10 normal bone
marrow samples, 3 normal tonsils and bone marrow aspirates from
6 normal donors, 3 monoclonal gammopathies of undetermined significance
(MGUS), 5 solitary plasmacytomas, 39 MM samples (95% advanced
stage) and the MM cell line U266. MAGEC1/CT7 was
expressed in bone marrow aspirates from one MGUS and one plasmacytoma.
The frequencies at which CT antigen were found to be expressed in
MM patients were MAGEC1/CT7 77%, LAGE-1 49%, MAGEA3/6 41%, MAGEA2 36%, GAGE family
33%, NY-ESO-1 33%, BAGE-1 28%, MAGEA1 26%, PRAME 23%, SSX-1 26%, MAGEA12 20.5%, MAGEA4 0%,
and MAGEA10 0%. Cox's regression model
showed that GAGE family expression and having >6
CT antigens expressed were independent prognostic factors when all
patients were analyzed. However, MAGEC1/CT7 expression
was the only independent prognostic factor when non-transplanted
patients where analyzed. Based on our findings, MAGEC1/CT7,
MAGEA3/6 and LAGE-1 are good candidates for immunotherapy,
since together they cover 85% of our MM cases. Furthermore,
expression of the GAGE family, >6 CT antigens and MAGEC1/CT7 seem
to have impact on MM prognosis.
myeloma; CT antigens; mRNA; tissue distribution; prognosis
Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.