Ankyrin Motifs Research Articles

Abstract 880: Identifying ANKRD22 and fostamatinib as a theragnostic target and drug candidate for metastatic pancreatic cancer through in silico analysis

Abstract Introduction: Pancreatic cancer (PC) presents significant clinical challenges, marked by a low 5-year survival rate and frequent distant metastasis. When PC metastasizes to other organs, it leads to a notable decrease in the 5-year survival rate for PC in metastasis, dropping from 11% to 3%. Despite the effectiveness demonstrated by advanced anti-cancer therapies targeting these mutated genes, the improvement in survival rates remains modest. Another critical consideration is the rising resistance to Gemcitabine, a standard drug used for treating PC. Hence, it is imperative to gain a more profound understanding of the comprehensive mechanisms underlying metastatic pancreatic cancer (mPC) and actively explore novel drugs for its treatment. Methods: First, we reanalyzed the RNA-seq of three of PC's most common metastatic sites, including liver, lung, and peritoneum. Using the Galaxy platform, we identified DEGs (differentially expressed genes) between metastatic sites. After that, we further utilized other web-based tools and databases, such as GEPIA, TIMER 2.0, Alphafold, PubChem, Drugbank, and Chembl, to identify ANKRD22 and perform molecular docking of Fostamatinib binds to this gene. Results: Our research focused on exploring the role of ANKRD22, which contains four copies of L-shaped ankyrin motifs, an emerging oncogenic molecule associated with various cancers. In the current study, we identified that ANKRD22 exhibited significant expression in the pancreatic adenocarcinoma (PAAD) cohort, correlating with deteriorating OS rates in PC patients. Moreover, it displayed distinct expression patterns in advanced stages of the disease compared to early stages, Furthermore, we uncovered a noteworthy correlation between ANKRD22 and the Kras, TP53, CDKN2A, and SMAD4 genetic anomaly, linked to diminished survival rates, as widely recognized, in the initiation and progression of PC, these four genes stand out as the most frequently mutated. Mechanistically, ANKRD22 exhibited associations with KRAS dependency signature, U12 type spliceosome assembly, multivesicular body routing, cell invasion, tumor cell line invasion, and tumor cell line migration, all indicative of PC progression and dissemination. Expanding our investigation through the DrugBank and ChEMBL databases, we delved into fostamatinib, an FDA-approved drug known as a spleen tyrosine kinase (SYK) inhibitor. Molecular docking experiments revealed the potential interaction between ANKRD22 and fostamatinib, suggesting its candidacy to treat mPC. Conclusion: Our findings identify ANKRD22 as a pivotal oncogenic factor promoting metastasis in PC and propose fostamatinib as a potential inhibitory treatment. Citation Format: Huong Thi Luu Kim Huynh, Hendrick Gao-Min Lim, Yuan-Chii Gladys Lee, Chien-Hsin Chen, Alexander T.H. Wu. Identifying ANKRD22 and fostamatinib as a theragnostic target and drug candidate for metastatic pancreatic cancer through in silico analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 880.

Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.

Antagonistic interactions among structured domains in the multivalent Bicc1-ANKS3-ANKS6 protein network govern phase transitioning of target mRNAs

The growing number of diseases linked to aberrant phase transitioning of ribonucleoproteins highlights the need to uncover how the interplay between multivalent protein and RNA interactions is regulated. Cytoplasmic granules of the RNA binding protein Bicaudal-C (Bicc1) are regulated by the ciliopathy proteins ankyrin (ANK) and sterile alpha motif (SAM) domain-containing ANKS3 and ANKS6, but whether and how target mRNAs are affected is unknown. Here, we show that head-to-tail polymers of Bicc1 nucleated by its SAM domain are interconnected by K homology (KH) domains in a protein meshwork that mediates liquid-to-gel transitioning of client transcripts. Moreover, while the dispersion of these granules by ANKS3 concomitantly released bound mRNAs, co-recruitment of ANKS6 by ANKS3 reinstated Bicc1 condensation and ribonucleoparticle assembly. RNA-independent Bicc1 polymerization and its dual regulation by ANKS3 and ANKS6 represent a new mechanism to couple the reversible immobilization of client mRNAs to controlled protein phase transitioning between distinct metastable states.

Sequence and Structure-Based Analyses of Human Ankyrin Repeats

Ankyrin is one of the most abundant protein repeat families found across all forms of life. It is found in a variety of multi-domain and single domain proteins in humans with diverse number of repeating units. They are observed to occur in several functionally diverse proteins, such as transcriptional initiators, cell cycle regulators, cytoskeletal organizers, ion transporters, signal transducers, developmental regulators, and toxins, and, consequently, defects in ankyrin repeat proteins have been associated with a number of human diseases. In this study, we have classified the human ankyrin proteins into clusters based on the sequence similarity in their ankyrin repeat domains. We analyzed the amino acid compositional bias and consensus ankyrin motif sequence of the clusters to understand the diversity of the human ankyrin proteins. We carried out network-based structural analysis of human ankyrin proteins across different clusters and showed the association of conserved residues with topologically important residues identified by network centrality measures. The analysis of conserved and structurally important residues helps in understanding their role in structural stability and function of these proteins. In this paper, we also discuss the significance of these conserved residues in disease association across the human ankyrin protein clusters.

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA 2 expression.

Phospholipase A2 (PLA2 ) hydrolyzes fatty acids from phospholipids at the sn-2 position. Two intracellular PLA2 s, iPLA2 A and iPLA2 B, have been found in Spodoptera exigua. Both are calcium-independent cellular PLA2 . Their orthologs have been found in other insects. These two iPLA2 s are different in ankyrin motif of N terminal region. The objective of this study was to determine whether Toll/immune deficiency (IMD) signal pathways could mediate cellular immune responses via induction of iPLA2 expression. Both iPLA 2 s were expressed in all developmental stages of S. exigua, showing the highest expression in the adult stage. During larval stage, hemocyte is the main tissue showing expression of these iPLA2 s. Both iPLA2 s exhibited similar expression patterns after immune challenge with different microbial pathogens such as virus, bacteria, and fungi. Promoter component analysis of orthologs encoded in S. frugiperda indicated nuclear factor-κB- and Relish-responsible elements on their promoters, suggesting their expression in S. exigua under Toll/IMD immune signaling pathways. RNA interference (RNAi) of MyD88 or Pelle under Toll pathway suppressed inducible expression levels of both iPLA2 s in response to Gram-positive bacteria containing Lys-type peptidoglycan or fungal infection. In contrast, RNAi against Relish under IMD pathway suppressed both iPLA2 s in response to infection with Gram-negative bacteria. Under RNAi conditions, hemocytes significantly lost cellular immune response measured by nodule formation. However, addition of arachidonic acid (a catalytic product of PLA2 ) rescued such immunosuppression. These results suggest that Toll/IMD signal pathways can mediate cellular immune responses via eicosanoid signaling by inducing iPLA2 expression.

Characterization of putative proteins encoded by variable ORFs in white spot syndrome virus genome

BackgroundWhite Spot Syndrome Virus (WSSV) is an enveloped double-stranded DNA virus which causes mortality of several species of shrimp, being considered one of the main pathogens that affects global shrimp farming. This virus presents a complex genome of ~ 300 kb and viral isolates that present genomes with great identity. Despite this conservation, some variable regions in the WSSV genome occur in coding regions, and these putative proteins may have some relationship with viral adaptation and virulence mechanisms. Until now, the functions of these proteins were little studied. In this work, sequences and putative proteins encoded by WSSV variable regions were characterized in silico.ResultsThe in silico approach enabled determining the variability of some sequences, as well as the identification of some domains resembling the Formin homology 2, RNA recognition motif, Xeroderma pigmentosum group D repair helicase, Hemagglutinin and Ankyrin motif. The information obtained from the sequences and the analysis of secondary and tertiary structure models allow to infer that some of these proteins possibly have functions related to protein modulation/degradation, intracellular transport, recombination and endosome fusion events.ConclusionsThe bioinformatics approaches were efficient in generating three-dimensional models and to identify domains, thereby enabling to propose possible functions for the putative polypeptides produced by the ORFs wsv129, wsv178, wsv249, wsv463a, wsv477, wsv479, wsv492, and wsv497.

The multifunctional polydnavirus TnBVANK1 protein: impact on host apoptotic pathway

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.

Poxviral ankyrin proteins.

Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

Abstract 2393: The BARD1 BRCT domains are essential for maintenance of telomere integrity

Abstract BARD1 has tumor suppressor functions by binding to BRCA1 and to p53 via RING finger and ankyrin repeats, respectively. The BARD1-BRCA1 dimer has E3 ligase activity, and BARD1-p53 interaction induces apoptosis. The C-terminus of BARD1 is binding to many proteins that are targets of the BRCA1-BARD1 ubiquitin ligase, and expression of N-terminally truncated RING-less BARD1 isoforms was correlated with poor prognosis in various cancers, consistent with their oncogenic functions by antagonistic binding to target proteins. Expression and interaction assays showed that the BARD1 BRCT domains were critically involved in telomere maintenance and interacted with the telomere binding protein TRF2. Overexpression of BARD1 or BRCA1 lead to TRF2 degradation, suggesting that TRF2 presents a novel target of the BARD1-BRCA1 E3 ubiquitination ligase. Repression of BARD1 or BRCA1 by siRNA leads to genetic instability, as shown before, and telomere alterations. We observed distinct telomeric alterations in blood cells of patients with a BARD1 germline mutation that causes a translation stop and translation of a truncated protein lacking the BRCT domains. Cells of carriers of a BRCA1 mutation that disrupts the BARD1-BRCA1 interaction show similar telomere alterations. These in vitro and in vivo observations suggest that BARD1 and BRCA1 play distinct roles in maintaining telomere integrity in a dosage dependent manner. BARD1δ, an isoform lacking RING and ankyrin motifs, but retaining the BRCT domains, is abundantly expressed in all cancers investigated so far. Overexpression of BARD1δ in vitro blocked cell cycle progression and induced chromosomal and telomere abnormalities, suggesting that BARD1δ also affected telomere integrity. We conclude that the correct dose of BARD1 and/ or its BRCT domains is critically important for maintaining telomere integrity. Expression of BARD1δ in cancer, or reduced expression of BARD1 in mutation carriers causes chromosomal instability and promotes carcinogenesis and tumor progression. Citation Format: Irmgard Irminger-Finger, Maxim Pilyugin, Magdalena Ratajska. The BARD1 BRCT domains are essential for maintenance of telomere integrity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2393. doi:10.1158/1538-7445.AM2014-2393

The expression of one ankyrin pk2 allele of the WO prophage is correlated with the Wolbachia feminizing effect in isopods

BackgroundThe maternally inherited α-Proteobacteria Wolbachia pipientis is an obligate endosymbiont of nematodes and arthropods, in which they induce a variety of reproductive alterations, including Cytoplasmic Incompatibility (CI) and feminization. The genome of the feminizing wVulC Wolbachia strain harboured by the isopod Armadillidium vulgare has been sequenced and is now at the final assembly step. It contains an unusually high number of ankyrin motif-containing genes, two of which are homologous to the phage-related pk1 and pk2 genes thought to contribute to the CI phenotype in Culex pipiens. These genes encode putative bacterial effectors mediating Wolbachia-host protein-protein interactions via their ankyrin motifs.ResultsTo test whether these Wolbachia homologs are potentially involved in altering terrestrial isopod reproduction, we determined the distribution and expression of both pk1 and pk2 genes in the 3 Wolbachia strains that induce CI and in 5 inducing feminization of their isopod hosts. Aside from the genes being highly conserved, we found a substantial copy number variation among strains, and that is linked to prophage diversity. Transcriptional analyses revealed expression of one pk2 allele (pk2b2) only in the feminizing Wolbachia strains of isopods.ConclusionsThese results reveal the need to investigate the functions of Wolbachia ankyrin gene products, in particular those of Pk2, and their host targets with respect to host sex manipulation.

Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector.

Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

Fetal Hemoglobin in Sickle Cell Anemia: A Novel Method for High-Resolution Discovery of Associated Genomic Copy Number Variations

Fetal hemoglobin (HbF) can inhibit the polymerization of sickle hemoglobin, and the HbF level is an important modulator of the severity and course of sickle cell anemia. Genetic regulation of HbF levels is complex and under active investigation. Although multiple quantitative trait loci have been discovered, it is estimated that half of the genetic variance of HbF levels remains unaccounted for. Genomic copy number variations (CNVs), defined as inherited duplications or deletions of kilo-to mega-base lengths of DNA, represent a significant source of genetic heterogeneity among humans that might be involved in HbF regulation. Additionally, CNVs can significantly alter assumptions about genotype frequencies in their genomic region, and are therefore important to locate for multiple types of genetic association studies. Here, we present a novel method for the high-resolution discovery of CVNs related to HbF levels in sickle cell anemia, using genome-wide association study (GWAS) data. We used the Illumina 610K single nucleotide polymorphism (SNP) genotyping array to examine 727 adult subjects with sickle cell anemia, with or without a thalassemia, who were enrolled in the Cooperative Study of Sickle Cell Disease (CSSCD; aged 18 to 69 years, mean age 31 years; 44% male; not on hydroxyurea therapy). The Illumina array consisted of ~610K probes spread across the entire genome. At each locus, the relative amount of DNA detected was compared to a reference and expressed as the log R ratio score (LRR). Normal diploid regions of DNA have LRRs close to zero, whereas regions with CNVs have LRRs that are either higher for areas of duplication or lower for areas of deletion. Using LRR information in the context of a GWAS, we developed a novel, two-step signal-processing technique that combines CNV discovery with subsequent phenotypical association analysis. First, the distribution of LRR values at each locus is stratified using a +/− 1.5 standard deviation band-pass filter. This created three groups: a central major group comprised of people with diploid amounts of DNA, and two minor variant groups, one composed of people with elevated LRRs, suggesting >2 DNA copies, and one of people with decreased LRRs, suggesting <2 DNA copies at that locus. To reduce noise, loci without at least one minor group containing >5% of the sample were excluded from further analysis. In the second step, a two-sample Student's t-test was used at each locus to examine the variation in distributions of HbF between the major, diploid group and any variant groups with >5% of the population. Using this method, we examined chromosomes 2, 6, and 11, which include regions known to modulate HbF in patients with sickle cell anemia, individuals with β thalassemia, and in the normal population. We successfully detected multiple clear duplications and deletions (approx. 1 per 6–22 mbp, depending on the chromosome) that showed typical CNV LRR distributions with >10% of the population exhibiting the polymorphism. Several of these were mildly related to HbF levels (p<0.05), including deletions in ASB1 on chromosome 2, and HACE1 on chromosome 6, both ankyrin motif containing proteins involved in the ubiquitin ligase system, as well as an upstream duplication and intragenic deletion involving HLA-DRB5 on chromosome 6. None of these clear CNVs, however, overlapped regions known to affect HbF concentration. Additional potential CNVs were detected throughout each chromosome, many exhibiting atypical LRR distributions not easily classified as either a normal diploid or clear CNV region. Further studies are required to confirm the presence of a CNV at these atypical loci. With this method, we were able to detect CNVs and CNV breakpoints across a population with a single-probe resolution, to within <1kb in some cases. This resolution offers a distinct advantage over other detection methods that utilize a multiple-probe, sliding-window approach to detect LRR deviations in an individual sample. In conclusion, this two-step method of high-resolution detection of CNVs followed by analysis of phenotypical associations shows promise for explaining variations in expressed protein levels, such as those typical of HbF in sickle cell anemia, and possibly for future exploration of differences in HbF responses to therapeutics in sickle cell anemia.

Shifting transition states in the unfolding of a large ankyrin repeat protein.

The 33-amino-acid ankyrin motif comprises a beta-turn followed by two anti-parallel alpha-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a "seed" and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain ankyrin repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it.

Crystal Structure of the BARD1 Ankyrin Repeat Domain and Its Functional Consequences

BARD1 is the constitutive nuclear partner to the breast and ovarian cancer-specific tumor suppressor BRCA1. Together, they form a heterodimeric complex responsible for maintaining genomic stability through nuclear functions involving DNA damage signaling and repair, transcriptional regulation, and cell cycle control. We report the 2.0A structure of the BARD1 ankyrin repeat domain. The structure includes four ankyrin repeats with a non-canonical C-terminal capping ankyrin repeat and a well ordered extended loop preceding the first repeat. Conserved surface features show an acidic patch and an acidic pocket along the surface typically used by ankyrin repeat domains for binding cognate proteins. We also demonstrate that two reported mutations, N470S and V507M, in the ankyrin repeat domain do not result in observable structural defects. These results provide a structural basis for exploring the biological function of the ankyrin repeat domain and for modeling BARD1 isoforms.

Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells

Human acyl-coenzyme A binding domain-containing member 6 (ACBD6) is a modular protein that carries an acyl-CoA binding domain at its N terminus and two ankyrin motifs at its C terminus. ACBD6 binds long-chain acyl-CoAs with a strong preference for unsaturated, C18:1-CoA and C20:4-CoA, over saturated, C16:0-CoA, acyl species. Deletion of the C terminus, which is not conserved among the members of this family, did not affect the binding capacity or the substrate specificity of the protein. ACBD6 is not a ubiquitous protein, and its expression is restricted to tissues and progenitor cells with functions in blood and vessel development. ACBD6 was detected in bone marrow, spleen, placenta, cord blood, circulating CD34+ progenitors, and embryonic-like stem cells derived from placenta. In placenta, the protein was only detected in CD34+ progenitor cells present in blood and in CD31+ endothelial cells surrounding the blood vessels. These cells were also positive for the marker CD133, and they probably constitute hemangiogenic stem cells, precursors of both blood and vessels. We propose that human ACBD6 represents a cellular marker for primitive progenitor cells with functions in hematopoiesis and vascular endothelium development.

Ubiquitination and Degradation of Homeodomain-interacting Protein Kinase 2 by WD40 Repeat/SOCS Box Protein WSB-1

Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53- and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses.

Structure of a BCOR Corepressor Peptide in Complex with the BCL6 BTB Domain Dimer

The transcriptional corepressors BCOR, SMRT, and NCoR are known to bind competitively to the BCL6 BTB domain despite the fact that BCOR has no detectable sequence similarity to the other two corepressors. We have identified a 17 residue motif from BCOR that binds directly to the BCL6 BTB domain and determined the crystal structure of the complex to a resolution of 2.6 A. Remarkably, the BCOR BCL6 binding domain (BCOR(BBD)) peptide binds in the same BCL6 binding site as the SMRT(BBD) peptide despite the lack of any significant sequence similarity between the two peptides. Mutations of critical BCOR(BBD) residues cause the disruption of the BCL6 corepression activities of BCOR, and a BCOR(BBD) peptide blocks BCL6-mediated transcriptional repression and kills lymphoma cells.

Structural Requirements for the BARD1 Tumor Suppressor in Chromosomal Stability and Homology-directed DNA Repair

The BRCA1 tumor suppressor exists as a heterodimeric complex with BARD1, and this complex is thought to mediate many of the functions ascribed to BRCA1, including its role in tumor suppression. The two proteins share a common structural organization that features an N-terminal RING domain and two C-terminal BRCT motifs, whereas BARD1 alone also contains three tandem ankyrin repeats. In normal cells, the BRCA1/BARD1 heterodimer is believed to enhance chromosome stability by promoting homology-directed repair (HDR) of double strand DNA breaks. Here we have investigated the structural requirements for BARD1 in this process by complementation of Bard1-null mouse mammary carcinoma cells. Our results demonstrate that the ankyrin and BRCT motifs of BARD1 are each essential for both chromosome stability and HDR. Tandem BRCT motifs, including those found at the C terminus of BARD1, are known to form a phosphoprotein recognition module. Nonetheless, the HDR function of BARD1 was not perturbed by synthetic mutations predicted to ablate the phospho-recognition activity of its BRCT sequences, suggesting that some functions of the BRCT domains are not dependent on their ability to bind phosphorylated ligands. Also, cancer-associated missense mutations in the BRCT domains of BARD1 (e.g. C557S, Q564H, V695L, and S761N) have been observed in patients with breast, ovarian, and endometrial tumors. However, none of these was found to affect the HDR activity of BARD1, suggesting that any increased cancer risk conferred by these mutations is not because of defects in this repair mechanism.