Structural flexibility of multifunctional HABP1 may be important for regulating its binding to different ligands

Hyaluronan-binding protein 1 (HABP1)/p32/gC1qR was characterized as a highly acidic and oligomeric protein, which binds to different ligands like hyaluronan, C1q, and mannosylated albumin. It exists as trimer in high ionic and reducing conditions as shown by crystal structure. In the present study, we have examined the structural changes of HABP1 under a wide range of ionic environments. HABP1 exhibits structural plasticity, which is influenced by the ionic environment under in vitro conditions near physiological pH. At low ionic strength HABP1 exists in a highly expanded and loosely held trimeric structure, similar to that of the molten globule-like state, whereas the presence of salt stabilizes the trimeric structure in a more compact fashion. It is likely that the combination of the high net charge asymmetrically distributed along the faces of the molecule and the relatively low intrinsic hydrophobicity of HABP1 result in its expanded structure at neutral pH. Thus, the addition of counter ions in the molecular environment minimizes the intramolecular electrostatic repulsion in HABP1 leading to its stable and compact conformations, which reflect in its differential binding toward different ligands. Whereas the binding of HABP1 toward HA is enhanced on increasing the ionic strength, no significant effect was observed with the two other ligands, C1q and mannosylated albumin. Thus, although HA interacts only with compact HABP1, C1q and mannosylated albumin can bind to loosely held oligomeric HABP1 as well. In other words, structural changes in HABP1 mediated by changes in the ionic environment are responsible for recognizing different ligands.     

Induction of string rescues the neuroblast proliferation defect in trol mutant animals

In trol mutants, neuroblasts fail to exit G1 for S phase. Increasing string expression in trol mutants rescues the number of S phase neuroblasts without an increase in M phase neuroblasts. Decreasing string expression further decreased the number of S phase neuroblasts. Coexpression of cyclin E and string did not produce additional S phase cells. Unlike cyclin E, cdk2, and cdk2AF, elevated expression of neither cyclin A, cyclin D, nor cdk1AF was able to promote S phase progression in arrested neuroblasts, indicating that String-induced activity of a Cyclin A or Cyclin D complex is unlikely to drive trol neuroblasts into S phase. Biochemical analyses revealed a rapid increase of Cyclin E-Cdk2 kinase activity to wild-type levels upon increased string expression. These results suggest that Drosophila Cdc25 may directly or indirectly increase the kinase activity of Cyclin E-Cdk2 complexes in vivo, thus driving arrested neuroblasts into cell division.     

CEA is the major PHA-L-reactive glycoprotein in colon carcinoma cell lines and tumors: relationship between K-ras activation and beta1-6 branching of N-linked carbohydrate on CEA

Previously we have shown that a positive correlation existed between the presence of beta1-6 branching of N-linked carbohydrate (detected as PHA-L reactivity) and the level of Ras activation in colon carcinoma cell lines. In these cell lines the major PHA-L-reactive species was found to be 180 kDa. Here we identified this species to be carcinoembryonic antigen (CEA) by demonstrating that: (a) CEA immunoreactivity and PHA-L reactivity colocalized on blots of crude cellular membranes from these cell lines, and that (b) immunoprecipitation of CEA resulted in quantitative coprecipitation of PHA-L reactivity at 180 kDa. Metabolic labeling of cell line HTB39 with [(3)H]mannose revealed that CEA was the predominantly labeled glycoprotein. This indicated that CEA was the major PHA-L-reactive species due its high level of expression. The amount of PHA-L reactivity present on CEA, expressed as the PHA-L/CEA ratio, was found to vary between cell lines. This ratio was found to correlate closely with the level of Ras activation in these cells. In cellular membrane isolated from primary colon carcinoma, the major PHA-L-reactive species was also 180 kDa. This reactivity colocalized with CEA immunoreactivity, indicating that the major beta1-6-branching glycoprotein in membranes from primary colon carcinoma was CEA. Similar to that seen in cell lines, the amount of PHA-L reactivity on CEA in human tumor samples varied, suggesting that a similar paradigm of Ras-induced expression of beta1-6 branching may occur in human colon carcinoma.     

The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.     

Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.     

Inhibition of insulin-like growth factor-I-mediated cell signaling by the von Hippel-Lindau gene product in renal cancer

Insulin-like growth factor-I (IGF-I)-mediated signaling is thought to be involved in the regulation of multiple cellular functions in different tumors including renal cell carcinoma (RCC). Blocking IGF-I signaling by any of the several strategies abolishes or delays the progression of a variety of tumors in animal models. Herein, we demonstrate that in RCC cell lines, IGF-I-mediated signaling is found to be inhibited in the presence of wild type von Hippel-Lindau (VHL) tumor suppresser gene. Moreover, molecular modeling and biochemical approaches have revealed that beta-domain of the VHL gene product by interacting directly with protein kinase Cdelta inhibits its association with IGF-IR for downstream signaling. We also demonstrated that RCC has IGF-I-mediated invasive activity where protein kinase Cdelta is an important downstream molecule, and this invasiveness can be blocked by wild type VHL. These experiments thus elucidate a novel tumor suppresser function of VHL with its unique kinase inhibitory domain.     

Phorbol ester-induced generation of reactive oxygen species is protein kinase cbeta -dependent and required for SAPK activation

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with protein kinase C (PKC) betaII-mediated activation of the stress-activated protein kinase (SAPK) pathway. The present studies demonstrate that the TPA response of U-937 cells includes the generation of reactive oxygen species (ROS). By contrast, the TPA-resistant U-937 cell variant (TUR), which is deficient in PKCbetaII expression, failed to respond to TPA with the induction of ROS. Moreover, we show that TPA-induced ROS production is restored in TUR cells stably transfected to express PKCbetaII. The results also demonstrate that TPA-induced ROS production is required for activation of the MEK kinase-1 (MEKK-1)--> SAPK pathway. In concert with this observation, treatment of U-937 with H(2)O(2) as a source of ROS is associated with activation of the MEKK-1-->SAPK cascade. These findings indicate that PKCbetaII is required for TPA-induced ROS production and that the MEKK-1-->SAPK pathway is activated by a ROS-mediated mechanism.     

Proteolytic cleavage and activation of protein kinase C [micro] by caspase-3 in the apoptotic response of cells to 1-beta -D-arabinofuranosylcytosine and other genotoxic agents

Protein kinase C (PKC) mu is a novel member of the PKC family that differs from the other isozymes in structural and biochemical properties. The precise function of PKCmu is not known. The present studies demonstrate that PKCmu is cleaved during apoptosis induced by 1-beta-d-arabinofuranosylcytosine (ara-C) and other genotoxic agents. PKCmu cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-x(L) protein or the baculovirus p35 protein. Our results demonstrate that PKCmu is cleaved by caspase-3 at the CQND(378)S site. Cleavage of PKCmu is associated with release of the catalytic domain and activation of its kinase function. We also show that, unlike the cleaved fragments of PKCdelta and theta, overexpression of the PKCmu catalytic domain is not lethal. Cells stably expressing the catalytic fragment of PKCmu, however, are more sensitive to apoptosis induced by genotoxic stress. In addition, expression of the caspase-resistant PKCmu mutant partially inhibits DNA damage-induced apoptosis. These findings demonstrate that PKCmu is cleaved by caspase-3 and that expression of the catalytic domain sensitizes cells to the cytotoxic effects of ara-C and other anticancer agents.     

The use of enzyme therapy to regulate the metabolic and phenotypic consequences of adenosine deaminase deficiency in mice. Differential impact on pulmonary and immunologic abnormalities

Adenosine deaminase (ADA) deficiency results in a combined immunodeficiency brought about by the immunotoxic properties of elevated ADA substrates. Additional non-lymphoid abnormalities are associated with ADA deficiency, however, little is known about how these relate to the metabolic consequences of ADA deficiency. ADA-deficient mice develop a combined immunodeficiency as well as severe pulmonary insufficiency. ADA enzyme therapy was used to examine the relative impact of ADA substrate elevations on these phenotypes. A "low-dose" enzyme therapy protocol prevented the pulmonary phenotype seen in ADA-deficient mice, but did little to improve their immune status. This treatment protocol reduced metabolic disturbances in the circulation and lung, but not in the thymus and spleen. A "high-dose" enzyme therapy protocol resulted in decreased metabolic disturbances in the thymus and spleen and was associated with improvement in immune status. These findings suggest that the pulmonary and immune phenotypes are separable and are related to the severity of metabolic disturbances in these tissues. This model will be useful in examining the efficacy of ADA enzyme therapy and studying the mechanisms underlying the immunodeficiency and pulmonary phenotypes associated with ADA deficiency.