Walter D. Funk

A transcriptionally active DNA-binding site for human p53 protein complexes.

Recent studies have demonstrated transcriptional activation domains within the tumor suppressor protein p53, while others have described specific DNA-binding sites for p53, implying that the protein may act as a transcriptional regulatory factor. We have used a reiterative selection procedure (CASTing: cyclic amplification and selection of targets) to identify new specific binding sites for p53, using nuclear extracts from normal human fibroblasts as the source of p53 protein. The preferred consensus is the palindrome GGACATGCCCGGGCATGTCC. In vitro-translated p53 binds to this sequence only when mixed with nuclear extracts, suggesting that p53 may bind DNA after posttranslational modification or as a complex with other protein partners. When placed upstream of a reporter construct, this sequence promotes p53-dependent transcription in transient transfection assays.

Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site.

The consensus binding site for the muscle regulatory factor myogenin was determined from an unbiased set of degenerate oligonucleotides using CASTing (cyclic amplification and selection of targets). Stretches of totally random sequence flanked by polymerase chain reaction priming sequences were mixed with purified myogenin or myotube nuclear extracts, DNA-protein complexes were immunoprecipitated with an antimyogenin antibody, and the DNA was amplified by polymerase chain reaction. Specific binding was obtained after four to six cycles of CASTing. The population of selected binding sites was then cloned, and a consensus was determined from sequencing individual isolates. Starting from a pool with 14 random bases, purified myogenin yielded a consensus binding site of AACAG[T/C]TGTT, while nuclear extracts retrieved the sequence TTGCACCTGTTNNTT from a pool containing 35 random bases. The latter sequence is consistent with that predicted from combining an E12/E47 half-site (N[not T]CAC) with the purified myogenin half-site ([T/C] TGTT). The presence of paired E boxes in many of the sequences isolated following CASTing with nuclear extracts proves that myogenin can bind cooperatively with other E-box-binding factors.

R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6

The R-Spondin (RSpo) family of secreted proteins act as potent activators of the Wnt/β-catenin signaling pathway. We have previously shown that RSpo proteins can induce proliferative effects on the gastrointestinal epithelium in mice. Here we provide a mechanism whereby RSpo1 regulates cellular responsiveness to Wnt ligands by modulating the cell-surface levels of the coreceptor LRP6. We show that RSpo1 activity critically depends on the presence of canonical Wnt ligands and LRP6. Although RSpo1 does not directly activate LRP6, it interferes with DKK1/Kremen-mediated internalization of LRP6 through an interaction with Kremen, resulting in increased LRP6 levels on the cell surface. Our results support a model in which RSpo1 relieves the inhibition DKK1 imposes on the Wnt pathway.

PAQR proteins: a novel membrane receptor family defined by an ancient 7-transmembrane pass motif.

An emerging series of papers has identified new receptor proteins that predict seven-transmembrane pass topologies. We have consolidated this family to 11 human genes and have named the family PAQR, after two of the initially described ligands (progestin and adipoQ receptors). This protein family has ancient evolutionary roots, with identified homologs found in eubacteria. To date, published data indicate that the prokaryotic members of this family appear to encode hemolysin-type proteins, while in eukaryotes, PAQR proteins encode functional receptors with a broad range of apparent ligand specificities. We provide the complete human and mouse complement of this family, suggest a conserved structure/topology with invariant intracellular amino acid residues, and have measured mRNA expression levels for these genes across a range of human tissues.

R-Spondin Proteins: A Novel Link to β-catenin Activation

The RSpondin (Rspo) protein family is a recently described group of 4 distinct human secreted proteins. Reported activities for RSpo proteins include essential roles in vertebrate development and their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both RSpo and canonical Wnt signaling result in the activation of β-catenin. In a general functional screen for human secreted proteins using transgenic mouse models, we identified human Rspondin 1 (hRSpo1) protein as a potent and specific mitogen for the gastrointestinal epithelium and demonstrated potential therapeutic applications for the protein in mouse models of cancer therapy-induced mucositis. In contrast to previous studies, our data indicated only partial overlap between Wnt and RSpo ligand activities, suggesting that there may be independent receptor/signaling pathways for RSpo proteins that intersect those of Wnt at the level of β-catenin. Here we summarize the current reported data on the RSpo family and discuss these results in terms of alternate mechanisms of action. We have extended our observations on the potential therapeutic application of RSpo proteins by showing that all 4 human Rspo family members are capable of inducing epithelial proliferation and report the first non-vertebrate RSpo family member.

Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins.

Tumor-specific activation of the TAL1 gene is the most common genetic alteration seen in patients with T-cell acute lymphoblastic leukemia. The TAL1 gene products contain the basic helix-loop-helix (bHLH) domain, a protein dimerization and DNA-binding motif common to several known transcription factors. A binding-site selection procedure has now been used to evaluate the DNA recognition properties of TAL1. These studies demonstrate that TAL1 polypeptides do not have intrinsic DNA-binding activity, presumably because of their inability to form bHLH homodimers. However, TAL1 readily interacts with any of the known class A bHLH proteins (E12, E47, E2-2, and HEB) to form heterodimers that bind DNA in a sequence-specific manner. The TAL1 heterodimers preferentially recognize a subset of E-box elements (CANNTG) that can be represented by the consensus sequence AACAGATGGT. This consensus is composed of half-sites for recognition by the participating class A bHLH polypeptide (AACAG) and the TAL1 polypeptide (ATGGT). TAL1 heterodimers with DNA-binding activity are readily detected in nuclear extracts of Jurkat, a leukemic cell line derived from a patient with T-cell acute lymphoblastic leukemia. Hence, TAL1 is likely to bind and regulate the transcription of a unique subset of subordinate target genes, some of which may mediate the malignant function of TAL1 during T-cell leukemogenesis.

CASTing for multicomponent DNA-binding complexes

Degenerate oligonucleotides and polymerase chain reaction-based reiterative selection techniques have been used to define the consensus binding sites for an increasing number of transcription factors. The use of crude nuclear extracts rather than purified proteins permits multicomponent complexes to form, and allows the technique to generate information about the combinatorial interactions involved in gene regulation.

Alfimeprase: a novel recombinant direct-acting fibrinolytic

Alfimeprase is a recombinant, direct-acting fibrinolytic zinc metalloprotease. Alfimeprase has direct proteolytic activity primarily against the fibrin(ogen) Aα chain. Alfimeprase is covalently bound and neutralised by serum α2-macroglobulin, a prevalent mammalian protease inhibitor. Preclinical pharmacology studies have shown that fibrinolysis with alfimeprase is up to sixfold more rapid than with select plasminogen activators, such as tissue-type plasminogen activator and urokinase. Alfimeprase directly delivered to a site of thrombosis has the potential to be a fast and effective fibrinolytic, which does not generate the systemic lytic state seen with plasminogen activators that is associated with major bleeding, including intracerebral haemorrhage. Phase I and II studies in individuals with arterial or venous thrombotic events indicate that alfimeprase is active and generally well tolerated.

Cellular and molecular advances in elucidating p53 function

The finding that in many human tumors there is allelic loss and/or mutations in p53, in combination with recognition that these events may play a role in multi-stage carcinogenesis, has focused considerable interest on this gene. To help keep abreast of this rapidly expanding field, recent experiments on the role and potential regulation of p53 are described: these include discussions of p53 as an anti-proliferative agent, the p53 mutations found in human tumors and tumor cell lines, the conformational states of p53, phosphorylation of p53 by p34cdc2, and signals for the nuclear localization of p53. p53 may act as a transcriptional activator and the specific DNA sequences to which p53 protein binds are also discussed as is the importance of abrogation of p53 function in overcoming cellular senescence.

The lymphoid cell surface receptor NTB-A: a novel monoclonal antibody target for leukaemia and lymphoma therapeutics

NTB‐A is a CD2‐related cell surface protein expressed primarily on lymphoid cells including B‐lymphocytes from chronic lymphocytic leukaemia (CLL) and lymphoma patients. We have generated a series of monoclonal antibodies (mAbs) against NTB‐A and assessed their therapeutic potential for CLL. Selective mAbs to NTB‐A were further tested in functional complement‐dependent cytotoxicity (CDC) and antibody‐dependent cellular cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL patients. While lower levels of NTB‐A were detected in T and natural killer (NK) cells, CDC activity was demonstrated primarily in B cells isolated from CLL patients and B lymphoma cell lines. Knockdown of NTB‐A by small interfering RNA in target cells results in lower cytotoxicity, demonstrating the specificity of the mAbs. Furthermore, anti NTB‐A mAbs demonstrated anti‐tumour activity against CA46 human lymphoma xenografts in nude mice and against systemically disseminated Raji human lymphoma cells in severe combined immunodeficient mice. Taken together, these results demonstrate NTB‐A as a potential new target for immunotherapy of leukaemia and lymphomas.