B-box Domain Research Articles

Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

Many tripartite motif (TRIM) proteins self-associate, forming dimers and higher order complexes. For example, dimers of TRIM5α, a host factor that restricts retrovirus infection, assemble into higher order arrays on the surface of the viral capsid, resulting in an increase in avidity. Here we show that the higher order association of different TRIM proteins exhibits a wide range of efficiencies. Homologous association (self-association) was more efficient than the heterologous association of different TRIM proteins, indicating that specificity determinants of higher order self-association exist. To investigate the structural determinants of higher order self-association, we studied TRIM mutants and chimeras. These studies revealed the following: 1) the RING domain contributes to the efficiency of higher order self-association, which enhances the binding of TRIM5α to the human immunodeficiency virus (HIV-1) capsid; 2) the RING and B-box 2 domains work together as a homologous unit to promote higher order association of dimers; 3) dimerization is probably required for efficient higher order self-association; 4) the Linker 2 region contributes to higher order self-association, independently of effects of Linker 2 changes on TRIM dimerization; and 5) for efficiently self-associating TRIM proteins, the B30.2(SPRY) domain is not required for higher order self-association. These results support a model in which both ends of the core TRIM dimer (RING-B-box 2 at one end and Linker 2 at the other) contribute to the formation of higher order arrays.

TRIM32 Protein Sensitizes Cells to Tumor Necrosis Factor (TNFα)-induced Apoptosis via Its RING Domain-dependent E3 Ligase Activity against X-linked Inhibitor of Apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

The cellular level of TRIM31, an RBCC protein overexpressed in gastric cancer, is regulated by multiple mechanisms including the ubiquitin—proteasome system

TRIM (tripartite motif) family proteins, comprising RING finger, B-box and coiled-coil domains, are involved in various cellular processes including tumour development and antiviral response. One of the family proteins, TRIM31, was originally identified as a gene induced by growth-suppressive retinoid. Our previous study showed that TRIM31 is up-regulated in stomach cancer and that TRIM31 protein possesses the common features of the TRIM protein family, for example, ubiquitin ligase activity and homo-oligomerization tendency. Interestingly, TRIM31 negatively regulates growth of certain cell types despite its overexpression in gastric cancer tissues. We herein demonstrated that upon exogenous expression in 293 cells, TRIM31 is polyubiquitylated, which promotes its degradation in the proteasome pathway. The proteasome-mediated degradation of endogenous TRIM31 was further confirmed in AsPC-1 pancreatic cancer cells. Thus, this posttranslational modification governs the intracellular abundance of TRIM31, which is also dependent on inducible transcription as well as alternative splicing. The complicated control of the intracellular TRIM31 protein level may relate to its seemingly contradictory behaviours in the cancer pathology or the urgent response to viral infection.

TRIM8 regulates Nanog via Hsp90β-mediated nuclear translocation of STAT3 in embryonic stem cells

TRIM8 is a member of a protein family defined by the presence of a common domain structure composed of a tripartite motif including a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with Hsp90β, which interacts with STAT3 and selectively downregulates transcription of Nanog in embryonic stem cells. Knock-down of TRIM8 increased phosphorylated STAT3 in the nucleus and also enhanced transcription of Nanog. These findings suggest that TRIM8 modulates translocation of phosphorylated STAT3 into the nucleus through interaction with Hsp90β and consequently regulates transcription of Nanog in embryonic stem cells.

Ret finger protein 2 enhances ionizing radiation-induced apoptosis via degradation of AKT and MDM2

Ret finger protein 2 ( RFP2), a gene frequently deleted in multiple tumor types, encodes a protein with a RING finger, B-box, and coiled-coil domain that belongs to the RBCC/TRIM protein family. Although RBCC proteins are involved in diverse cellular processes such as apoptosis, proliferation, differentiation, and transcriptional regulation, the biological function of RFP2 has not been well defined. Here, we demonstrate that overexpression of RFP2 in cells induced apoptosis through proteasomal degradation of MDM2 and AKT. The expression of RFP2, which possesses RING domain-dependent E3 ubiquitin ligase activity, was increased by ionizing radiation dose- and time-dependently, and RFP2 overexpression induced cell death with increased expression of apoptotic molecules (p53, p21, and Bax). These results depended on the E3 ubiquitin ligase activity of RFP2 because mutant RFP2, which contains a mutated RING domain, failed to drive apoptosis compared with wild-type RFP2. We observed that RFP2 formed a complex with MDM2, a negative regulator of the p53 tumor suppressor, and AKT, a regulator of apoptosis inhibition at the cellular level. Additionally, we found that the interaction of RFP2 with MDM2 and AKT resulted in ubiquitination and proteasomal degradation of MDM2 and AKT in vivo and in vitro. Thus, these data suggest that irradiation causes RFP2 overexpression, which enhances ionizing radiation-induced apoptosis by increasing p53 stability and decreasing AKT kinase activity through MDM2 and AKT degradation.

Functional interactions between ubiquitin E2 enzymes and TRIM proteins

The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM-UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.

Enterovirus 71 induces degradation of TRIM38, a potential E3 ubiquitin ligase

BackgroundThe tripartite motif (TRIM) proteins are a family of more than 70 members in human. However, only a few of them have been well studied. The TRIM proteins contain the conserved RING, B-box, coiled-coil, and SPRY domains, most of which are involved in protein ubiquitination. TRIM38 is a member of the TRIM protein family, which we studied in more detail here as its functions are largely unknown.ResultsOur study shows that, similar to other TRIM family members, TRIM38 is localized in the cytoplasm. TRIM38 increases ubiquitination of other cellular proteins and catalyzes self-ubiquitination. TRIM38 also promotes K63- and K48-linked ubiquitination of cellular proteins. An intact RING domain is important for the functions of TRIM38. In addition, enterovirus 71 infection induces TRIM38 degradation.ConclusionsOur observations demonstrate that TRIM38 has E3 ubiquitin ligase activity and can be degraded during virus infection. These findings may provide insight into innate immune signaling pathways.

Detection and Characterization of the In Vitro E3 Ligase Activity of the Human MID1 Protein

Human MID1 ( midline- 1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM ( tripartite motif) family, MID1 has three consecutive zinc-binding domains—RING ( really interesting new gene), Bbox1, and Bbox2—that have similar ββα-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING–Bbox1 and RING–Bbox1–Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1–Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING–Bbox1 and RING–Bbox1–Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MID1 E3 ligase activity and show how its three zinc-binding domains can contribute to MID1's overall function.

Functional diversification of the RING finger and other binuclear treble clef domains in prokaryotes and the early evolution of the ubiquitin system.

Recent studies point to a diverse assemblage of prokaryotic cognates of the eukaryotic ubiquitin (Ub) system. These systems span an entire spectrum, ranging from those catalyzing cofactor and amino acid biosynthesis, with only adenylating E1-like enzymes and ubiquitin-like proteins (Ubls), to those that are closer to eukaryotic systems by virtue of possessing E2 enzymes. Until recently E3 enzymes were unknown in such prokaryotic systems. Using contextual information from comparative genomics, we uncover a diverse group of RING finger E3s in prokaryotes that are likely to function with E1s, E2s, JAB domain peptidases and Ubls. These E1s, E2s and RING fingers suggest that features hitherto believed to be unique to eukaryotic versions of these proteins emerged progressively in such prokaryotic systems. These include the specific configuration of residues associated with oxyanion-hole formation in E2s and the C-terminal UFD in the E1 enzyme, which presents the E2 to its active site. Our study suggests for the first time that YukD-like Ubls might be conjugated by some of these systems in a manner similar to eukaryotic Ubls. We also show that prokaryotic RING fingers possess considerable functional diversity and that not all of them are involved in Ub-related functions. In eukaryotes, other than RING fingers, a number of distinct binuclear (chelating two Zn atoms) and mononuclear (chelating one zinc atom) treble clef domains are involved in Ub-related functions. Through detailed structural analysis we delineated the higher order relationships and interaction modes of binuclear treble clef domains. This indicated that the FYVE domain acquired the binuclear state independently of the other binuclear forms and that different treble clef domains have convergently acquired Ub-related functions independently of the RING finger. Among these, we uncover evidence for notable prokaryotic radiations of the ZF-UBP, B-box, AN1 and LIM clades of treble clef domains and present contextual evidence to support their role in functions unrelated to the Ub-system in prokaryotes. In particular, we show that bacterial ZF-UBP domains are part of a novel cyclic nucleotide-dependent redox signaling system, whereas prokaryotic B-box, AN1 and LIM domains have related functions as partners of diverse membrane-associated peptidases in processing proteins. This information, in conjunction with structural analysis, suggests that these treble clef domains might have been independently recruited to the eukaryotic Ub-system due to an ancient conserved mode of interaction with peptides.

Function of B-BOX under shade

Plants are capable of perceiving changes in the light environment and finely adjust their growth and development. Reductions of red to far-red ratio (R:FR) generated by an increase of the plant canopy above the plant are sensed by the phytochrome system triggering the shade-avoidance syndrome (SAS) that includes elongation of vegetative structures, reduction of branching and acceleration of flowering. Albeit the SAS is a strategy of major adaptative significance in plant communities, involving massive changes in gene expression, our knowledge of the SAS signaling network is still fragmented. By a selection and characterization of a T-DNA mutant with a long hypocotyl under shade, we identified BBX21, a protein with two B-box domains involved in the SAS. BBX21 belongs to a small eight member family of B-box containing proteins with both opposite and additive functions in the SAS signaling. BBX21 down-regulates the gene expression of auxin, brassinosteroid and ethylene signaling pathway components under shade. Furthermore BBX21 is a transcription factor that interacts genetically with COP1. We propose a model in which a dynamic balance of positive and negative B-box transcriptional regulators acts as a gas-and-brake mechanism into the COP1 signaling to regulate the expression of SAS.

Whole-Transcriptome Sequencing In Chronic Myeloid Leukemia Reveals Novel Gene Mutations That May Be Associated with Disease Pathogenesis and Progression

AbstractAbstract 885Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) has always been regarded as a genetically homogeneous disease. However, the fact that a proportion of patients (pts), especially in the high Sokal risk setting, fail tyrosine kinase inhibitor therapy and progress to blast crisis (BC) suggests that a certain degree of heterogeneity exists. It can be hypothesized that genetic factors additional to the Ph+ chromosome may be present in these pts. To address this issue, we are currently using massively parallel sequencing to perform a qualitative and quantitative survey of the whole transcriptome of Ph+ CML cells at diagnosis and at progression to BC. Results are being integrated with genome-wide search for copy number alterations by Affymetrix SNP 6.0 arrays. We used a Solexa Illumina Genome Analyzer to scan the transcriptome of a CML patient at the time of diagnosis, at the time of remission (major molecular response) and at the time of progression from chronic phase (CP) to lymphoid blast crisis (BC). Both custom scripts and published algorithms were used for read alignment against the human reference genome, for single nucleotide variant (SNV) calling, for identification of alternative splicings and fusion transcripts, and for digital gene expression profiling. Comparison of the SNVs identified in the diagnosis and relapse samples with the SNVs detected in the remission sample – representing inherited sequence variants not specific for the Ph+ clone – allowed the identification of eight missense mutations at diagnosis affecting the coding sequences of AMPD3 (encoding adenosine monophosphate deaminase 3), SUCNR1 (succinate receptor 1), FANCD2 (Fanconi anemia, complementation group D2), INCENP (inner centromere protein), BSPRY (B-box and SPRY domain containing), HEXDC (hexosaminidase containing), NUDT9 (ADP-ribose diphosphatase) and KIAA2018 (encoding a protein with predicted DNA binding and transcriptional regulation activity) genes. Six of these mutations (FANCD2, INCENP, BSPRY, HEXDC, NUDT9) were also detected in the Ph+ clone re-emerged at the time of disease progression, together with seven additional missense mutations affecting the coding sequences of IDH2 (isocitrate dehydrogenase isoform 2), DECR1 (2,4-dienoyl CoA reductase 1), C4Orf14 (mitochondrial nitric oxide synthase), MRM1 (mitochondrial rRNA methyltransferase 1), PRKD2 (protein kinase D2), TCHP (mitostatin) and ABL1 genes. Digital gene expression analysis showed downregulation of SUCNR1, that might be a consequence of the P292A mutation we detected. IDH2, MRM1, AMPD3, and KIAA2018 mutations were found in additional pts. The IDH2 R140Q mutation was detected in 3/75 (4%) myeloid BC, 1/31 (3.2%) lymphoid BC, 0/34 Ph+ ALL and 0/23 Philadelphia-negative (Ph-) ALL pts. The MRM1 C120S mutation was found in 6/70 (9%) additional BC pts (2 lymphoid and 4 myeloid). AMPD3 and KIAA2018 genes were found to harbour the same point mutations (N334S and S1818G, respectively) in 1 out of 20 additional CP patients analyzed. Massively parallel sequencing of the sample collected at diagnosis also revealed that the Bcr-Abl kinase domain was already harbouring point mutations at low levels (E308D, A344G, R386S) but not the T315I that was selected at the time of disease progression. Point mutations in untraslated regions where miRNAs are known to bind were also detected, and are currently under validation. Digital gene expression profiling comparing progression to diagnosis showed significant expression changes including upregulation of 134 genes and downregulation of 88 genes. In particular, we observed an upregulation of the B-cell developmental factor PAX5, its interactor Lef-1 and its targets IRF4, BLNK, Bik, EBF1, CD79A, CD79B, CD19, VpreB1, VpreB3, BOB1, RAG1 and RAG2; upregulation of PAX9; upregulation of WNT3A, WNT9A, GLI3 and downregulation of SFRP1, resulting in aberrant activation of the Wnt signalling pathway. In summary, our preliminary data highlighted putative key genes whose deregulation may be recurrent in a subset of CML patients and may be linked to disease pathogenesis or progression. Their actual role in CML is currently being exlored. Massively parallel sequencing of additional patients is ongoing. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants. Disclosures:Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Direct Suppression of Fas-Mediated Apoptosis by PMLRARa through Forming An Apoptotic Inhibitory Complex with c-FLIP In Acute Promyelocytic Leukemia

AbstractAbstract 3148Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML).PML enhances pro-apoptotic signaling, while its dominant negative form, promyelocytic leukemia–retinoic acid receptor α (PMLRARα) fusion protein, activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα blocks Fas-mediated apoptosis.Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937/PR9 cells and APL primary cells isolated from patients. The PMLRARα-Fas binding was mapped to the PML B-box domain of PMLRARα and death domain of Fas. Flow cytometry analysis of propidium iodide- and Annexin V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody CH-11 indicated that PMLRARα blocks Fas-mediated apoptosis at early and late stages. In line with this finding, knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Detailed analysis showed that expression of PMLRARα prevents procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11) and thus, also prevents cleavage/activation of procaspase-8. Further analysis indicated that PMLRARα recruits caspase-8 inhibitor c-FLIPL/S to Fas to suppress Fas signaling. A significantly higher number of mice transfected with PMLRARα-expressing plasmid than mice transfected with empty vector survived the treatment with the mouse agonistic anti-Fas antibody Jo2 (11 of 12 vs. 0 of 12; P < 0.001). Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with vector-transfected mice.These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which, as we have shown, can occur by regulation via direct interaction. The newly-discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptossis. By neutralizing the effect of death receptor inhibitors such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Disclosures:No relevant conflicts of interest to declare.

P05 B-cell epitope mapping of anti-ro-52 responses in patients with primary biliary cirrhosis

IntroductionUp to 40% of patients with primary biliary cirrhosis (PBC) show reactivity against Ro-52 (Hepatology 2009), a Sjögren's syndrome (SS)-associated autoantibody. Previous studies have shown that anti-Ro-52 in SS targets...

Novel potato C2H2-type zinc finger protein gene, StZFP1, which responds to biotic and abiotic stress, plays a role in salt tolerance

Many TFIIIA-type zinc finger proteins (ZFPs) play important roles in stress responses in plants. In the present study, a novel zinc finger protein gene, StZFP1, was cloned from potato. StZFP1 is a typical TFIIIA-type two-finger zinc finger gene with one B-box domain, one L-box domain and a DLN-box/EAR motif. The StZFP1 genes belong to a small gene family with an estimated copy number of four or five, located on chromosome I. StZFP1 is constitutively expressed in leaves, stems, roots, tubers and flowers of adult plants. Expression of StZFP1 can be induced by salt, dehydration and exogenously applied ABA. StZFP1 expression is also responsive to infection by the late blight pathogen Phytophthora infestans. Transient expression analysis of StZFP1:GFP fusion protein revealed that StZFP1 is preferentially localised in the nucleus. Ectopic expression of StZFP1, driven by the Arabidopsis rd29A promoter in transgenic tobacco, increased plant tolerance to salt stress. These results demonstrate that StZFP1 might be involved in potato responses to salt and dehydration stresses through an ABA-dependent pathway.

TRIM8 modulates STAT3 activity through negative regulation of PIAS3

TRIM8 is a member of the protein family defined by the presence of a common domain structure composed of a tripartite motif: a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with protein inhibitor of activated STAT3 (PIAS3), which inhibits IL-6-dependent activation of STAT3. Ectopic expression of TRIM8 cancels the negative effect of PIAS3 on STAT3, either by degradation of PIAS3 through the ubiquitin-proteasome pathway or exclusion of PIAS3 from the nucleus. Furthermore, expression of TRIM8 in NIH3T3 cells enhances Src-dependent tumorigenesis. These findings indicate that TRIM8 enhances the STAT3-dependent signal pathway by inhibiting the function of PIAS3.

Abstract 2908: Promyelocytic leukemia-retinoic acid receptor α forms complexes with Fas and FLIP and impairs Fas-mediated apoptosis

Abstract Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we have screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while the promyelocytic leukemia-retinoic acid receptor α (PMLRARα) activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα, which typically operates in a dominant-negative manner, blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937-PR9 cells and in APL primary cells. The binding of PMLRARα to Fas was mapped to the B-box domain of PMLRARα. Flow cytometry analysis of propidium iodide-stained and Annexin-V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody (CH-11) indicated that the presence of PMLRARα was associated with blocked Fas-mediated apoptosis at early and late stages. The knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Expression of PMLRARα in U937-PR9 cells prevented Fas-mediated cleavage of procaspase-8 and also prevented procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11). Further analysis indicated that PMLRARα bound to FLIPL/S and forms a complex with Fas associated with suppression of Fas signaling. These data suggest that cancer-specific inhibitors of Fas such as PMLRARα block Fas-mediated apoptosis and thus can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which we have shown to occur by direct regulation. We have identified an attractive potential target to the regulation of apoptosis at the PMLRARα-Fas and PML-Fas interfaces. By neutralizing the effect of death receptor inhibitors such as PMLRARα and other potential inhibitors, we can improve on the success of the many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2908.

Clinical features and functional significance of the P369S/R408Q variant in pyrin, the familial Mediterranean fever protein

ObjectivesFamilial Mediterranean fever (FMF) is caused by mutations in MEFV, which encodes pyrin. The nature of substitutions P369S and R408Q in exon 3 remains unclear. Exon 3 encoding pyrin's B-box...

Hls5, a Novel Ubiquitin E3 Ligase, Modulates Levels of Sumoylated GATA-1.

Abstract 253Hemopoietic lineage commitment is controlled, in part, by transcription factors that regulate specific genes required for the formation of mature blood cells. Differentiation along particular hemopoietic lineages is dependant not only on the presence of particular transcription factors, but also on appropriate concentrations - altering transcription factor levels can force cells into different hemopoietic pathways. Transcription factors undergo numerous post-translational modifications and are controlled spatially via sub-cellular localisation. De-regulation of transcription factors can result in leukemias, or other blood disorders. GATA-1 is an example of a key lineage-determining gene, essential for erythropoiesis. Increasing GATA-1 levels promotes maturation along the erythroid pathway, whereas reducing GATA-1 concentrations favours myelopoiesis. GATA-1 regulation occurs at multiple levels including transcription, translation and post-translational modifications such as phosphorylation, acetylation, ubiquitination and sumoylation. Although GATA-1 ubiquitination modifies the protein for proteasomal degradation, the effect of adding small ubiquitin-like modier (Sumo) to GATA-1 is unclear. Several examples of hemopoietic differentiation plasticity have been observed. We reported a lineage switch by erythroleukemic J2E cells which spontaneously developed a monoblastoid phenotype. Two genes (Hls5 and Hls7/Mlf1) were isolated from this lineage switch with potential lineage-determining features. Hls5 is a member of the RBCC (Ring finger, B-box, Coiled-coil) family of proteins, which includes PML. Ectopic expression of Hls5 impedes erythroid differentiation by reducing GATA-1 levels, and suppressing hemoglobin synthesis. Significantly, Hls5 relocates from the cytoplasm to associate with GATA-1 in the nucleus, where it interferes with DNA binding and transactivation of GATA-1. Several members of the RBCC family are ubiquitin E3 ligases, catalysing the final step in the ubiquitination process - these molecules play a vital role in regulating the levels of target proteins. Here we show that Hls5 is a bona fide ubiquitin E3 ligase, in partnership with several ubiquitin E2 enzymes. The Ring finger is critical for Hls5 ligase activity as mutation of key residues within the Ring finger ablates catalytic activity. Interestingly, a yeast 2 hybrid screen for Hls5 interactors identified Ubc9 and Pias1, which act as E2 and E3 enzymes in the sumoylation cascade. Co-immunoprecipitation, BRET and co-localization experiments confirmed the Hls5 association with Ubc9 and Pias1. Moreover, Hls5 binds Sumo-1 (but not Sumo-2 or 3), and co-localizes with Sumo-1 in discrete nuclear bodies. Thus, Hls5 interacts with several components of the intracellular sumoylation machinery. Hls5 can also reduce sumoylated proteins globally, indicating it may target these modified proteins for degradation. Recently, a new family of ubiquitin E3 ligases has been described which specifically mark sumoylated proteins for degradation. These Sumo-targeted ubiquitin ligases (STUbL) are found primarily in yeast, and only one mammalian STUbL has been identified. We postulated that Hls5 may be a STUbL, capable of regulating sumoylated GATA-1. Our data demonstrate that while Hls5 is able to bind GATA-1 via the B-box and Coiled-coil domains, it preferentially associates with sumoylated GATA-1 through a canonical Sumo interacting motif (SIM). This results in increased GATA-1 ubiquitination and, as a consequence, levels of sumoylated GATA-1 are reduced substantially. Since mutation of the lysine necessary for Sumo attachment does not affect GATA-1 transactivation, sumoylation may act as a prelude to ubiquitination and protein turn-over. We propose, therefore, that GATA-1 mediates transcription of target genes, and is subsequently sumoylated by Pias1 and Ubc9 – addition of Sumo moieties to GATA-1 enhance binding to Hls5, which in turn impedes GATA-1 DNA binding, and promotes ubiquitination for proteasomal degradation. This model is consistent with decreased levels of GATA-1 in erythroid cells ectopically expressing Hls5, and with the original isolation of Hls5 as a potential lineage-determining gene involved with the erythroid to monoblastoid lineage switch. Thus, Hls5 is a novel STUbL which plays a role in hemopoietic lineage commitment by modulating GATA-1 activity and content. Disclosures:No relevant conflicts of interest to declare.

Promyelocytic Leukemia-Retinoic Acid Receptor α Binds and Prevents Activation of the Fas Receptor and Suppresses Fas-Mediated Apoptosis.

Abstract 3976Poster Board III-912Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas killing observed in some normal and cancer cells, we have screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified a group of peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while the promyelocytic leukemia–retinoic acid receptor α (PMLRARα) activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα, which typically operates in a dominant-negative manner, blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937-PR9 cells and in APL primary cells. The binding of PMLRARα to Fas was mapped to the B-box domain of PMLRARα. Flow cytometry analysis of propidium iodide-stained and Annexin-V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody (CH-11) indicated that the presence of PMLRARα was associated with blocked Fas-mediated apoptosis at early and late stages. The knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Expression of PMLRARα in U937-PR9 cells prevented Fas-mediated cleavage of procaspase-8 and also prevented procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11). Further analysis indicated that PMLRARα bound to FLIPL/S and forms an apoptotic inhibitory complex with Fas, which prevents Fas activation. The data suggest that tissue-specific inhibitors of Fas such as PMLRARα block Fas-mediated apoptosis and thus can contribute to cancer development. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which we have shown to occur by direct regulation. We have identified an attractive potential target to the regulation of apoptosis at the PMLRARα-Fas and PML-Fas interfaces. By neutralizing the effect of death receptor inhibitors such as PMLRARα and other potential inhibitors, we can improve on the success of the many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Disclosures:No relevant conflicts of interest to declare.

Searching for Genes for Cleft Lip and/or Palate Based on Breakpoint Analysis of a Balanced Translocation t(9;17)(q32;q12)

Identification of the breakpoints of disease-associated chromosome rearrangements can provide informative clues to a positional cloning approach for genes responsible for inherited diseases. Recently, we found a three-generation Japanese family segregating balanced chromosome translocation t(9;17)(q32;q12). One of the subjects had cleft lip and palate. We examined whether regions near the breakpoint could be associated with cleft lip and/or palate. We determined the breakpoints involved in the translocation by fluorescence in situ hybridization analysis and subsequent long-range polymerase chain reaction. In order to study the role of these disrupted regions in nonsyndromic cleft lip and/or palate, we performed mutation analysis and a haplotype-based transmission disequilibrium test using tagging single-nucleotide polymorphisms in the flanking regions of the breakpoints in white and Filipino nonsyndromic cleft lip and/or palate populations. Sequence analysis demonstrated that two genes, SLC31A1 (solute carrier family 31 member 1) on chromosome 9 and CCL2 (chemokine ligand 2) on chromosome 17, were rearranged with the breaks occurring within their introns. It is interesting that SLC31A1 lies closed to BSPRY (B-box and SPRY domain), which is a candidate for involvement with cleft lip and/or palate. Some of the variants in BSPRY and CCL2 showed significant p values in the cleft lip and/or palate population compared with the control population. There was also statistically significant evidence of transmission distortion for haplotypes on both chromosomes 9 and 17. The data support previous reports that genes on chromosomal regions of 9q and 17q play an important role in facial development.