Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP 4 ), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-␣. Binding specificity was determined using displacement of 1-O- Receptor-stimulated phosphoinositide (PI) 1 metabolism generates numerous inositol polyphosphates (InsP n s) and inositol phospholipids, many of which may function as potential second messengers (1). Of the possible PI metabolites, Ins(1,4,5)P 3 (InsP 3 ) and diacylglycerol (DAG) are the best characterized second messengers. Generated by receptor-stimulated phospholipase C, hydrolysis of PtdIns(4,5)P 2 (2), Ins(1,4,5)P 3 binds to and gates an InsP 3 receptor calcium channel on the endoplasmic reticulum (2, 3). The lipid DAG remains in the membrane where it activates several protein kinase C isoforms and may regulate other targets (4, 5). In the membrane, DAG is metabolized rapidly to monoacylglycerol and to several phospholipids. In the cytoplasm, Ins(1,4,5)P 3 can be phosphorylated to Ins(1,3,4,5)P 4 by an InsP 3 3-kinase. Other isomers of InsP 4 , InsP 5 , and InsP 6 , some of which are synthesized independently of Ins(1,4,5)P 3 , have been identified (for review, see Ref. 6), and their production may also be regulated by receptors or during cell growth. Information from receptor binding studies, using radioactive InsP 4 and InsP 6 , have demonstrated that a number of important regulatory proteins contain high affinity InsP n binding sites. InsP n s have been implicated in the regulation of clathrin assembly proteins AP-2 (7, 8), AP-3 (9), the non-clathrin-associated coatomer proteins (10), synaptotagmin (11), and the regulation of the small GTPases ras and/or rap via a specific GTPase-activating protein (GAP) activity (12). Inositol phospholipids have also been postulated as messenger molecules. From in vivo, genetic, and permeabilized cell studies, evidence for critical roles for the inositol phospholipids PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P 2 as regulators of membrane vesicle trafficking and cytoskeletal rearrangements is accumulating rapidly (5, 13-15). One inositol phospholipid, PtdIns(3,4,5)P 3 , has emerged as a potential messenger molecule in receptor-stimulated cells (16 -18). Synthesized by receptor-stimulated PI 3-kinase phosphorylation of PtdIns(4,5)P 2 (18), PtdInsP 3 is not a substrate for PI-specific phospholipase * This work was supported in part by National Institutes of Mental Health Grants R29MH50102 and DDRC P50HD32901 (to A. B. T.). Work at Stony Brook was supported by National Institutes of Health Grant NS29632 (to G. D. P.). The first two authors contributed equally to this study. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank § §...
We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 binding sites in four phospholipase C (PLC) isozymes (delta1, beta1, beta2, and beta3), by probing these proteins with analogs of inositol phosphates, D-Ins(1,4,5)P3, D-Ins(1,3,4,5)P4, and InsP6, and polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-delta1 was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated delta1-PH domain was also specifically labeled. Equilibrium binding of D-Ins(1,4,5)P3 to PLC-delta1 indicated the presence of a single, high-affinity binding site; binding of D-Ins(1,4,5)P3 to PLC-beta1, -beta2, or -beta3 was not detected. The catalytic activity of PLC-delta1 was inhibited by the product D-Ins(1,4,5)P3, whereas no inhibition of PLC-beta1, -beta2, or -beta3 activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P2 binding site of PLC-delta1 and that a similar site is not present in PLC-beta1, -beta2, or -beta3. The data are consistent with the idea that the PH domain of PLC-delta1, but not the beta isozymes, directs the catalytic core to membranes enriched in PI(4,5)P2 and is subject to product inhibition.
The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of hIAPP22–29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman, and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22–29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β-sheet secondary structure. TEM images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the -C=C- ring mode (ca. 1600 cm−1) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22–29.
Recent cloning of a rat brain phosphatidylinositol 3,4,5-trisphosphate binding protein, centaurin α, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin α is Gcs1p, the product of theGCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin α, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novelGCS1 disruption strain (gcs1Δ) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Δ was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 andSLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 andSAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.
The majority of protein kinase inhibitors described to date are ATP analogues. However, the selectivity of these species is highly suspect, given the enormous number of ATP-dependent processes that transpire in living cells. Inhibitors that target the protein binding site do not suffer from this disadvantage but exhibit comparatively low inhibitory activity. An alternative approach for the design of protein tyrosine kinase inhibitors is described herein. We have constructed species that simultaneously bind to the active site and the SH2 domain of the Src kinase. Since the region of the inhibitor that associates with the SH2 domain coordinates with relatively high affinity, the overall effect is a substantial enhancement in inhibitory potency (230-fold). This design element offers a strategy to overcome the otherwise poor efficacy of peptide-based protein tyrosine kinase inhibitors.
AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K i of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/ K)X n (R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.Phosphoinositides and inositol polyphosphates (referred to collectively as inositides) are components of many pathways in eukaryotic cells, functioning in second messenger cascades, acting as regulators of many proteins, and operating as membrane localization signals (1-3). Numerous protein and lipid kinases, adaptor proteins, ion channels, phospholipases, modulators of small GTPases, and actin-binding proteins are regulated by inositides (1-3). To identify novel targets for inositides, our laboratories and others have used purification schemes employing affinity resins that contain tethered inositol polyphosphate head groups (3-9). These affinity purifications were successful in the identification of inositide binding in the clathrin adaptor/assembly protein AP-2 (6), centaurin ␣ (7), a centaurin ␣ orthologue (8), and a phospholipase C (PLC) 1 -related protein (9). In addition to AP-2 and centaurin ␣, we isolated several other proteins from rat brain, one of which was approximately 80 kDa (5). Published reports show that inositol polyphosphates modulate the activity of the enzyme AMP deaminase (EC 3.5.4.6) (AMPD) (10, 11), an enzyme family whose endogenous, purified subunit molecular masses are between 66 and 88 kDa. Therefore, we hypothesized that AMPD could be the 80-kDa protein isolated using the inositide affinity resin.AMPD is a diverse and highly regulated enzyme located at a branchpoint in the adenine nucleotide catabolic pathway and is important in regulating nucleotide pools. AMPD is also a component of the purine nucleotide cycle, an energy-generating pathway reportedly operative in many animal tissues (reviewed in Ref. 12). The AMPD1 gene encodes human isoform M and rat iso...
. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP 6 appeared to induce a binding-compatible structure in the short peptide.The synaptotagmins (Syts) 1 are synaptic vesicle proteins that play essential roles in nucleating the clathrin coat during endocytosis and in acting as Ca 2ϩ sensors (1-3) and phosphoinositide sensors (4) during exocytosis. They are a critical part of a complex machinery of intracellular protein transport (5, 6) and the synaptic vesicle cycle (7). In addition to a short Nterminal intravesicular region and single transmembrane domain, Syts have two copies of highly conserved repeats, known as the C2A and C2B domains, which are homologous to the C2 regulatory region of protein kinase C (8). In particular, the C2B domain appears to play several roles. First, the C2B domain of mouse Syt II shows specific binding to high polyphosphate inositols (IP n s) (9), a feature that is not shared by the highly homologous C2A domain nor with the C2 domains of other proteins such as rabphilin. Moreover, the C2B domain is necessary but not sufficient for IP n binding. Although Syt II and IV show high affinity binding of IP 4 and IP 6 , the Syt III-C2B domain shows negligible binding, despite a high sequence identity with the Syt II-C2B domain, including the 32-residue region examined by mutational analysis (10). Inhibition of C2B function in the squid giant axon disrupts synaptic vesicle release and recycling (11). Similarly, Caenorhabditis elegans mutants lacking Syt or simply the C2B domain cannot recycle synaptic vesicles (12), and the presence of Syt I in cerebrospinal fluid of Alzheimer's patients (13) may provide clues to the disruption of synaptic function in humans. The C2B domain acts as a high affinity receptor for clathrin assembly protein AP-2 (14), and Ca 2ϩ appears to mediate Syt dimerization via the C2B domain (15).Synaptotagmin II isolated from mouse cerebellum shows high affinity binding to Ins(1,3,4,5)P 4 with a K D of 30 M (16) and 117 nM for the GST-Syt II-C2B construct (9). Curiously, the rank order of IP n s binding for native Syt II was Ins-(1,3,4,5,6)-P 5 Ͼ (1,3,4,5)-P 4 Ͼ Ins(1,2,3,4,5,6)-P 6 Ͼ Ins(1,4,5)-P 3 , whereas for the GST-Syt II-C2B domain the order was IP 6 Ͼ IP 5 Ͼ IP 4 Ͼ IP 3 . Deletion mutants allowed mapping of the IP n binding site to the central region of the mouse Syt II-C2B domain, specifically residues 315-346 (IHLMQNGKRLKKKK-TTVKKKTLNPYFNESFSF) (9). In order to obtain direct evidence for the binding site of IP n s in the C2B domain, to examine the InsP n selectivity of the domains, to explore the Ca 2ϩ dependence of binding, to determine the role of the central 315-346 peptide, and to evaluate the reason for the failure of Syt III-C2B domain to bind IP n s, we undertook a series of photoaffinity labeling experiments using four tritium-labeled, benzophenone-containing derivatives of IP 3 , IP 4 , and IP 6 (17). EXPERIMENTAL PROCEDURES Chemicals-P-1-Tethered
A comprehensive investigation of peptides derived from the 22–29 region of human islet amyloid polypeptide (hIAPP) that contain phenylalanine analogs at position 23 with a variety of electron donating and withdrawing groups, along with heteroaromatic surrogates, has been employed to interrogate how π-electron distribution effects amyloid formation. Kinetic aggregation studies using turbidity measurements indicate that electron rich aromatic ring systems consistently abolish the amyloidogenic propensity of hIAPP22–29. Electron poor systems modulate the rate of aggregation. Raman and Fourier transform infrared spectroscopy confirm the parallel β-sheet secondary structure of aggregates derived from peptides containing electron poor phenylalanine analogs and provide direct evidence of ring stacking. Transmission electron microscopy confirms the presence of amyloid fibrils. The effect of aryl substituent geometry on peptide self-assembly reveals that the electronic nature of substituents and not their steric profile is responsible for failure of the electron donating group peptides to aggregate. Non-aggregating hIAPP22–29 peptides were found to inhibit the self-assembly of full-length hIAPP1–37. The most potent inhibitory peptides contain phenylalanine with the p-amino and p-formamido functionalities. These novel peptides may serve as leads for the development of future aggregation inhibitors. A potential mechanism for inhibition of amylin self-assembly by electron rich hIAPP22–29 peptides is proposed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.