Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Increased dopamine turnover in the prefrontal cortex impairs spatial working memory performance in rats and monkeys.

The selective activation of the prefrontal cortical dopamine system by mild stress can be mimicked by anxiogenic beta-carbolines such as FG7142. To investigate the functional relevance of elevated levels of dopamine turnover in the prefrontal cortex, the current study examined the effects of FG7142 on the performance of spatial working memory tasks in the rat and monkey. FG7142 selectively increased prefrontal cortical dopamine turnover in rats and significantly impaired performance on spatial working memory tasks in both rats and monkeys. Spatial discrimination, a task with similar motor and motivational demands (rats), or delayed response performance following zero-second delays (monkeys) was unaffected by FG7142. Further, biochemical analysis in rats revealed a significant positive correlation between dopamine turnover in the prefrontal cortex and cognitive impairment on the delayed alternation task. The cognitive deficits in both rats and monkeys were prevented by pretreatment with the benzodiazepine receptor antagonist, RO15-1788, which blocked the increase in dopamine turnover and by the dopamine receptor antagonists, haloperidol, clozapine, and SCH23390. These findings indicate that excessive dopamine activity in the prefrontal cortex is detrimental to cognitive functions mediated by the prefrontal cortex.

The law of mass action governs antigen-stimulated cytolytic activity of CD8+ cytotoxic T lymphocytes.

An analysis of the initial antigen-recognition step in the destruction of target cells by CD8+ cytolytic T lymphocytes (CTLs) shows that a relationship in the form of the law of mass action can be used to describe interactions between antigen-specific receptors on T cells (TCRs) and their natural ligands on target cells (peptide-major histocompatibility protein complexes, termed pepMHC complexes), even though these reactants are confined to their respective cell membranes. For a designated level of lysis and receptor affinities below about 5 X 10(6) M-1, the product of the required number of pepMHC complexes per target cell ("epitope density") and TCR affinity for pepMHC complexes is constant; therefore, over this range TCR affinities can be predicted from epitope densities (or vice versa). At higher receptor affinities ("affinity ceiling") the epitope density required for half-maximal lysis reaches a lower limit of less than 10 complexes per target cell.

Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities.

Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits.

Antagonism of WT1 activity by protein self-association.

Germline loss-of-function mutations at the Wilms tumor (WT) suppressor locus WT1 are associated with a predisposition to WTs and mild genital system anomalies. In contrast, germ-line missense mutations within the WT1 gene encoding the DNA-binding domain often yield a more severe phenotype consisting of WT, sexual ambiguity, and renal nephropathy. In this report, we demonstrate that the products of mutant alleles that impair DNA recognition can antagonize WT1-mediated transcriptional repression. We demonstrate that WT1 can self-associate in vitro and in vivo and that the responsible domain maps to the amino-terminal region of the protein. Oligomers of full-length protein form less efficiently or produce less stable complexes than oligomers between truncated polypeptides and full-length protein. Our data suggest a molecular mechanism to explain how WT1 mutations may act in deregulating cellular proliferation and differentiation.

Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons.

Temporal and spatial changes in the intracellular Ca2+ concentration ([Ca2+]i) were examined in dendrites and somata of rat cerebellar Purkinje neurons by combining whole-cell patch-clamp recording and fast confocal laser-scanning microscopy. In cells loaded via the patch pipette with the high-affinity Ca2+ indicator Calcium Green-1 (Kd approximately 220 nM), a single synaptic climbing fiber response, a so-called complex spike, resulted in a transient elevation of [Ca2+]i that showed distinct differences among various subcellular compartments. With conventional imaging, the Ca2+ signals were prominent in the dendrites and almost absent in the soma. Confocal recordings from the somatic region, however, revealed steep transient increases in [Ca2+]i that were confined to a submembrane shell of 2- to 3-microns thickness. In the central parts of the soma [Ca2+]i increases were much slower and had smaller amplitudes. The kinetics and amplitudes of the changes in [Ca2+]i were analyzed in more detail by using the fast, low-affinity Ca2+ indicator Calcium Green-5N (Kd approximately 17 microM). We found that brief depolarizing pulses produced [Ca2+]i increases in a narrow somatic submembrane shell that resembled those seen in the dendrites. These results provide direct experimental evidence that the surface-to-volume ratio is a critical determinant of the spatiotemporal pattern of Ca2+ signals evoked by synaptic activity in neurons.

The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex.

Signal peptides direct the cotranslational targeting of nascent polypeptides to the endoplasmic reticulum (ER). It is currently believed that the signal recognition particle (SRP) mediates this targeting by first binding to signal peptides and then by directing the ribosome/nascent chain/SRP complex to the SRP receptor at the ER. We show that ribosomes can mediate targeting by directly binding to translocation sites. When purified away from cytosolic factors, including SRP and nascent-polypeptide-associated complex (NAC), in vitro assembled translation intermediates representing ribosome/nascent-chain complexes efficiently bound to microsomal membranes, and their nascent polypeptides could subsequently be efficiently translocated. Because removal of cytosolic factors from the ribosome/nascent-chain complexes also resulted in mistargeting of signalless nascent polypeptides, we previously investigated whether readdition of cytosolic factors, such as NAC and SRP, could restore fidelity to targeting. Without SRP, NAC prevented all nascent-chain-containing ribosomes from binding to the ER membrane. Furthermore, SRP prevented NAC from blocking ribosome-membrane association only when the nascent polypeptide contained a signal. Thus, NAC is a global ribosome-binding prevention factor regulated in activity by signal-peptide-directed SRP binding. A model presents ribosomes as the targeting vectors for delivering nascent polypeptides to translocation sites. In conjunction with signal peptides, SRP and NAC contribute to this specificity of ribosomal function by regulating exposure of a ribosomal membrane attachment site that binds to receptors in the ER membrane.

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

The delta isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-delta associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-delta is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-delta showed that the modified enzyme had diminished activity toward the receptor gamma-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-delta regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.

Mutant rat phosphatidylinositol/phosphatidylcholine transfer proteins specifically defective in phosphatidylinositol transfer: implications for the regulation of phospholipid transfer activity.

The mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs) catalyze exchange of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. We find that Ser-25, Thr-59, Pro-78, and Glu-248 make up a set of rat (r) PI-TP residues, substitution of which effected a dramatic reduction in the relative specific activity for PI transfer activity without significant effect on PC transfer activity. Thr-59 was of particular interest as it is a conserved residue in a highly conserved consensus protein kinase C phosphorylation motif in metazoan PI-TPs. Replacement of Thr-59 with Ser, Gln, Val, Ile, Asn, Asp, or Glu effectively abolished PI transfer capability but was essentially silent with respect to PC transfer activity. These findings identify rPI-TP residues that likely cooperate to form a PI head-group binding/recognition site or that lie adjacent to such a site. Finally, the selective sensitivity of the PI transfer activity of rPI-TP to alteration of Thr-59 suggests a mechanism for in vivo regulation of rPI-TP activity.

Sphingosylphosphocholine, a signaling molecule which accumulates in Niemann-Pick disease type A, stimulates DNA-binding activity of the transcription activator protein AP-1.

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity.

The cleavage specificity of the Pvu II and BamHI restriction endonucleases is found to be dramatically reduced at elevated osmotic pressure. Relaxation in specificity of these otherwise highly accurate and specific enzymes, previously termed "star activity," is uniquely correlated with osmotic pressure between 0 and 100 atmospheres. No other colligative solvent property exhibits a uniform correlation with star activity for all of the compounds tested. Application of hydrostatic pressure counteracts the effects of osmotic pressure and restores the natural selectivity of the enzymes for their canonical recognition sequences. These results indicate that water solvation plays an important role in the site-specific recognition of DNA by many restriction enzymes. Osmotic pressure did not induce an analogous effect on the specificity of the EcoRV endonuclease, implying that selective hydration effects do not participate in DNA recognition in this system. Hydrostatic pressure was found to have little effect on the star activity induced by changes in ionic strength, pH, or divalent cation, suggesting that distinct mechanisms may exist for these observed alterations in specificity. Recent evidence has indicated that BamHI and EcoRI share similar structural motifs, while Pvu II and EcoRV belong to a different structural family. Evidently, the use of hydration water to assist in site-specific recognition is a motif neither limited to nor defined by structural families.

New multivalent calixarene-based ligands for protein recognition and inhibition

One of the challenges that concerns chemistry is the design of molecules able to modulate protein-protein and protein-ligand interactions, since these are involved in many physiological and pathological processes. The interactions occurring between proteins and their natural counterparts can take place through reciprocal recognition of rather large surface areas, through recognition of single contact points and single residues, through inclusion of the substrates in specific, more or less deep binding sites. In many cases, the design of synthetic molecules able to interfere with the processes involving proteins can benefit from the possibility of exploiting the multivalent effect. Multivalency, widely spread in Nature, consists in the simultaneous formation between two entities (cell-cell, cell-protein, protein-protein) of multiple equivalent ligand-recognition site complexes. In this way the whole interaction results particularly strong and specific. Calixarenes furnish a very interesting scaffold for the preparation of multivalent ligands and in the last years calixarene-based ligands demonstrated their remarkable capability to recognize and inhibit or restore the activity of different proteins, with a high efficiency and selectivity in several recognition phenomena. The relevance and versatility of these ligands is due to the different exposition geometries of the binding units that can be explored exploiting the conformational properties of these macrocycles, the wide variety of functionalities that can be linked to their structure at different distances from the aromatic units and to their intrinsic multivalent nature. With the aim of creating new multivalent systems for protein targeting, the work reported in this thesis regards the synthesis and properties of glycocalix[n]arenes and guanidino calix[4]arenes for different purposes. Firstly, a new bolaamphiphile glycocalix[4]arene in 1,3-alternate geometry, bearing cellobiose, was synthesized for the preparation of targeted drug delivery systems based on liposomes. The formed stable mixed liposomes obtained by mixing the macrocycle with DOPC were shown to be able of exploiting the sugar units emerging from the lipid bilayer to agglutinate Concanavalin A, a lectin specific for glucose. Moreover, always thanks to the presence of the glycocalixarene in the layer, the same liposomes demonstrated through preliminary experiments to be uptaken by cancer cells overexpressing glucose receptors on their exterior surface more efficiently respect to simple DOPC liposomes lacking glucose units in their structure. Then a small library of glycocalix[n]arenes having different valency and geometry was prepared, for the creation of potentially active immunostimulants against Streptococcus pneumoniae, particularly the 19F serotype, one of the most virulent. These synthesized glycocalixarenes bearing β-N-acetylmannosamine as antigenic unit were compared with the natural polysaccharide on the binding to the specific anti-19F human polyclonal antibody, to verify their inhibition potency. Among all, the glycocalixarene based on the conformationally mobile calix[4]arene resulted the more efficient ligand, probably due its major possibility to explore the antibody surface and dispose the antigenic units in a proper arrangement for the interaction process. These results pointed out the importance of how the different multivalent presentation in space of the glycosyl units can influence the recognition phenomena. At last, NMR studies, using particularly 1H-15N HSQC experiments, were performed on selected glycocalix[6]arenes and guanidino calix[4]arenes blocked in the cone geometry, in order to better understand protein-ligand interactions. The glycosylated compounds were studied with Ralstonia solanacearum lectin, in order to better understand the nature of the carbohydrate‐lectin interactions in solution. The series of cationic calixarene was employed with three different acidic proteins: GB1, Fld and alpha synuclein. Particularly GB1 and Fld were observed to interact with all five cationic calix[4]arenes but showing different behaviours and affinities.

Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger,with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties.Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA N-terminal β-hairpin reveals differences in terms of length, charge, and spatial distribution. Consequently,four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [Δ(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity,being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells.