Biomedical subjects
Publications and source records attributed to Aphrodite Kapurniotu.
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OBJECTIVES: Novel stents loaded with an integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) were analyzed for their potential to limit coronary neointima formation and to accelerate endothelialization by attracting endothelial progenitor cells (EPCs). BACKGROUND: Re-endothelialization is important for healing after arterial injury. METHODS: Effects of cRGD on EPC number, recruitment in flow, and invasion were analyzed in vitro. A durable polymer coating containing 67 microg cRGD per stent was developed for Guidant Tetra stents. Twelve cRGD-loaded polymer, 12 unloaded polymer, and 12 bare metal stents were deployed in porcine coronary arteries. Quantification of cRGD in peri-stent tissue was established by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Histomorphometry and immunostaining were performed after 4 and 12 weeks. Recruitment of labeled porcine EPCs was assessed 7 days after intracoronary infusion. RESULTS: The cRGD clearly supported the outgrowth, recruitment, and migration of EPCs in vitro. At 4 weeks, there was no difference for mean neointimal area and percent area stenosis in the cRGD-loaded, polymer, or bare metal stent group. At 12 weeks, neointimal area (2.2 +/- 0.3 mm2) and percent area stenosis (33 +/- 5%) were significantly reduced compared with polymer stents (3.8 +/- 0.4 mm2, 54 +/- 6%; p = 0.010) or bare metal stents (3.8 +/- 0.3 mm2, 53 +/- 3%; p < 0.001). The HPLC/MS confirmed cRGD tissue levels of 1 to 3 mug/stent at 4 weeks, whereas cRGD was not detectable at 12 weeks. Staining for CD34 and scanning electron microscopy indicated enhanced endothelial coverage on cRGD-loaded stents at 4 weeks associated with a significant increase in the early recruitment of infused EPCs. CONCLUSIONS: Stent coating with cRGD may be useful for reducing in-stent restenosis by accelerating endothelialization.
Protein aggregation into cytotoxic oligomers and fibrils in vivo is linked to cell degeneration and the pathogenesis of >25 uncurable diseases, whereas the high aggregation propensity and insolubility of several bioactive polypeptides and proteins in vitro prevent their therapeutic use. Aggregation of human islet amyloid polypeptide (IAPP) into pancreatic amyloid is strongly associated with the pathogenesis of type II diabetes. IAPP is a 37-residue polypeptide that acts as a neuroendocrine regulator of glucose homeostasis. However, IAPP misfolds and self-associates into cytotoxic aggregates and fibrils even at nanomolar concentrations. Because IAPP aggregation causes beta-cell death and prohibits therapeutic application of IAPP in diabetes, we pursued a minimalistic chemical design approach to generate a molecular mimic of a nonamyloidogenic and bioactive IAPP conformation that would still be able to associate with IAPP and thus inhibit its fibrillogenesis and cytotoxicity. We show that the double N-methylated full length IAPP analog [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI) is a highly soluble, nonamyloidogenic, and noncytotoxic IAPP molecular mimic and an IAPP receptor agonist. Moreover, IAPP-GI binds IAPP with low nanomolar affinity and completely blocks IAPP cytotoxic self-assembly and fibrillogenesis with activity in the low nanomolar concentration range. Importantly, IAPP-GI dissociates cytotoxic IAPP oligomers and fibrils and is able to reverse their cytotoxicity. Bifunctional soluble IAPP mimics that combine bioactivity with the ability to block and reverse IAPP cytotoxic self-assembly are promising candidates for the treatment of diabetes. Moreover, our amyloid disease inhibitor design concept may be applicable to other protein aggregation diseases.
Macrophage migration inhibitory factor (MIF) has widespread actions in the immune, endocrine, and nervous systems. Previously, we reported that increases in the intracellular levels of MIF depress the firing of hypothalamus/brain stem neurons in culture, including the chronotropic actions of angiotensin II. The objective of this study was to investigate the effects of MIF on delayed rectifier K+ current (I(Kv)), one of the component currents whose activity contributes to neuronal firing. Intracellular perfusion of MIF (80 nM) into Sprague-Dawley rat neuronal cultures caused a significant increase in I(Kv), as measured by patch-clamp recordings. This effect was apparent by 3 min, and was maximal after 20-30 min. I(Kv) current density (pA/pF) increased from 31.58 +/- 2.36 in controls to 41.88 +/- 3.76 in MIF-treated neurons (mean +/- SE; n = 9; P < 0.01). MIF that had been inactivated by boiling did not alter I(Kv), and MIF-neutralizing antibodies abolished the action of recombinant MIF (rMIF). The stimulatory effect of MIF on I(Kv) current density was mimicked by intracellular application of either P1S-MIF (80 nM) or the peptide MIF-(50-65) (0.8-8 microM), both of which harbor the thiol-protein oxidoreductase (TPOR) activity of the MIF molecule. Conversely, neither C60S-MIF (80 nM) nor the MIF homologue D-dopachrome tautomerase (80 nM), both of which lack TPOR activity, altered I(Kv). Finally, the increase in I(Kv) produced by rMIF was abolished by the superoxide scavenger Tiron (1 mM). These studies indicate that the neuronal action of MIF includes a stimulatory action on I(Kv) that may be mediated by a TPOR/superoxide-scavenging mechanism.
Macrophage migration inhibitory factor (MIF) is a 12.5 kD polypeptide that serves as a critical regulator of cell functions such as gene expression, proliferation or apoptosis. However, the signal transduction pathways through which MIF takes part in cellular regulation are only incompletely understood. MIF leads to CD74-dependent "sustained" activation of ERK1/2 MAPK, but MIF's role in "transient" ERK activation and the involved upstream pathways are unknown. Here we report that the transient ERK pathway was markedly activated by MIF. This effect involved the phosphorylation and activation of Raf-1, MEK, ERK, and Elk-1. Of note, rapid and transient ERK phosphorylation by MIF was measurable in MIF-deficient cells, suggesting that MIF acted in a non-autocrine fashion. Applying the inhibitor genistein, a tyrosine kinase (TPK) activity was identified as a critical upstream signalling event in MIF-induced transient ERK signalling. Experiments using the Src kinase inhibitor PP2 indicated that the involved TPK was a Src-type tyrosine kinase. A role for an upstream Src kinase was proven by applying Src-deficient cells which did not exhibit transient ERK activation upon treatment with MIF, but in which MIF-induced ERK signalling could be restored by re-expressing Src. Intriguingly, JAB1/CSN5, a signalosome component, cellular binding protein of MIF and regulator of cell proliferation and survival, had a marked, yet dual, effect on MIF-induced ERK signalling. JAB1 overexpression inhibited sustained, but not transient, ERK phosphorylation. By contrast, JAB1-knock-down by siRNA revealed that minimum JAB1 levels were necessary for transient activation of ERK by MIF. In conclusion, MIF rapidly and transiently activates the ERK pathway, an effect that has not been recognized previously. This signalling pathway involves the upstream activation of a Src-type kinase and is co-regulated by the cellular MIF binding protein JAB1/CSN5. Our study thus has unravelled a novel MIF-driven signalling pathway and an intricate regulatory system involving extra- and possibly intracellular MIF, and which likely critically participates in controlling cell proliferation and survival.
The pathogenesis of type II diabetes is associated with the aggregation of the 37-residue human islet amyloid polypeptide (hIAPP) into cytotoxic beta sheet aggregates and fibrils. We have recently shown that introduction of two N-methyl rests in the beta sheet- and amyloid-core-containing sequence hIAPP(22-27), or NFGAIL converted this amyloidogenic and cytotoxic sequence into nonamyloidogenic and noncytotoxic NF(N-Me)GA(N-Me)IL. Here, we show that NF(N-Me)GA(N-Me)IL is able to bind with high-affinity full-length hIAPP and to inhibit its fibrillogenesis. NF(N-Me)GA(N-Me)IL also inhibits hIAPP-mediated apoptotic beta cell death. By contrast, unmodified NFGAIL does not inhibit hIAPP amyloidogenesis and cytotoxicity, suggesting that N-methylation conferred on NFGAIL the properties of NF(N-Me)GA(N-Me)IL. These results support the concept that rational N-methylation of hIAPP amyloid-core sequences may be a valuable strategy to design pancreatic-amyloid diagnostics and therapeutics for type II diabetes.
alpha-Synuclein aggregation into fibrils is associated with the pathogenesis of Parkinson's disease (PD). Li et al. provide strong evidence that rifampicin interacts with alpha-synuclein and inhibits its fibrillization. Rifampicin could be a promising candidate for therapeutic application for PD.
Restricting the conformational flexibility of medium-sized linear polypeptides is a valuable approach to identify and characterize the structural and conformational features that define their biological activities and to design analogs with enhanced agonistic or antagonistic properties and with potential therapeutic applications. The calcium-regulating and bone resorption-inhibiting hormone calcitonin (Ct) is a conformationally flexible polypeptide of 32 amino acid residues that has long been applied therapeutically for the treatment of osteoporosis and other bone disorder diseases. This review describes studies on the structural and conformational features of the Ct sequence that are relevant for Ct bioactivity and focuses on research work performed on rationally designed conformationally constrained Ct analogs as tools for the understanding of the molecular basis of Ct bioactivity and as potential candidates or lead structures for novel Ct-based bone disorder therapeutics.
Macrophage migration inhibitory factor (MIF) is a cytokine that participates in the host inflammatory response. A Cys-Xaa-Xaa-Cys (CXXC)-based thiol-protein oxidoreductase activity of MIF is associated with certain biological functions. Peptides spanning the CXXC region of thiol-protein oxidoreductases retain some biochemical properties of the full-length protein. We report on the characterization of CXXC-spanning MIF-(50-65) and its serine variant, C57S/C60S-MIF-(50-65). Following disulfide-mediated cyclization, MIF-(50-65) adapted a beta-turn conformation comparable with that of beta-turn-containing cyclo-57,60-[Asp57,Dap60]MIF-(50-65). MIF-(50-65) had a redox potential E'0 of -0.258 V and formed mixed disulfides with glutathione and cysteine. MIF-(50-65) but not C57S/C60S-MIF-(50-65) had oxidoreductase activity in vitro. Intriguingly, MIF-(50-65) exhibited MIF-like cellular activities. The peptide but not its variant had glucocorticoid overriding and proliferation-enhancing activity and stimulated ERK1/2 phosphorylation. MIF-(50-65) and its variant bound to the MIF-binding protein JAB1 and enhanced cellular levels of p27Kip1. As the peptide and its variant were endocytosed at similar efficiency, sequence 50-65 appears sufficient for the JAB1-related effects of MIF, whereas other activities require CXXC. Cyclo-57,60-[Asp57,Dap60]MIF-(50-65) activated ERK1/2, indicating that CXXC-dependent disulfide and beta-turn formation is associated with an activity-inducing conformation. We conclude that CXXC and sequence 50-65 are critical for the activities of MIF. MIF-(50-65) is a surprisingly short sequence with MIF-like functions that could be an excellent molecular template for MIF therapeutics.
The aggregation process of beta-amyloid peptide Abeta into amyloid is strongly associated with the pathology of Alzheimer's disease (AD). Aggregation may involve a transition of an alpha helix in Abeta(1-28) into beta sheets and interactions between residues 18-20 of the "Abeta amyloid core." We applied an i, i+4 cyclic conformational constraint to the Abeta amyloid core and devised side chain-to-side chain lactam-bridged cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28). In contrast to Abeta(1-28) and [Lys(17), Asp(21)]Abeta(1-28), cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was not able to form beta sheets and cytotoxic amyloid aggregates. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was able to interact with Abeta(1-28) and to inhibit amyloid formation and cytotoxicity. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) also interacted with Abeta(1-40) and interfered with its amyloidogenesis. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) or similarly constrained Abeta sequences may find therapeutic and diagnostic applications in AD.
We have recently reported the potent hypocalcemic effects of side-chain lactam-bridged analogues of human calcitonin (hCT) (Kapurniotu, A.; et al. Eur. J. Biochem. 1999, 265, 606-618). To extend these studies, we have now synthesized a new series of (Asp(17), Lys(21)) and (Asp(17), Orn(21)) side-chain bridged salmon calcitonin (sCT) and hCT analogues. The affinities of these analogues for the human calcitonin receptor, hCTR(I1)(-), and for rat-brain membrane receptors were assayed in competitive binding assays, and agonist potencies at the hCTR(I1)(-) receptors were assessed, using a cAMP-responsive gene-reporter assay. The bridged sCT analogues had activities similar to sCT itself. In contrast, an (Asp(17), Orn(21)) side-chain bridged hCT analogue, cyclo(17-21)-[Nle(8), Phe(12), Asp(17), Orn,(21) Tyr(22))-hCT, was 80 and 450 times more active than hCT in the hCTR(I1)(-) and rat-brain receptor binding assays, respectively, and was 90 times more potent than hCT and 16 times more potent than sCT in initiating receptor signaling. An uncyclized, isosteric analogue of this peptide was also more potent than hCT, demonstrating that the cyclization constraint and these single-residue substitutions enhance the activities of hCT in an additive fashion. This study demonstrates that the potency-enhancing effects of lactam-bridge constraints at hCT residues 17-21 are not transferable to sCT. We also show that, in comparison to the hCT analogues, sCT and its analogues are less potent agonists than expected from their hCTR(I1)(-) affinities. This suggests that it may be possible to preserve the efficient signal transduction of hCT while introducing additional receptor affinity-enhancing elements from sCT into our potent lactam-bridged hCT analogue, thereby creating new super-potent, hCT-based agonists.
Pancreatic amyloid is formed by the aggregation of the 37-residue islet amyloid polypeptide (IAPP) in type II diabetes patients and is cytotoxic. Pancreatic amyloid deposits are found in more than 95 % of type II diabetes patients and their formation is strongly associated with disease progression. IAPP amyloid forms via a conformational transition of soluble IAPP into aggregated beta-sheets. We recently identified IAPP(22-27) (NFGAIL) as a minimum length sequence sufficient to self-associate into beta-sheet-containing amyloid fibrils. Here, we have used the NFGAIL model of the IAPP amyloid core as a structural template to design non-amyloidogenic derivatives of amyloidogenic sequences of IAPP that are able to interact with the native sequences and inhibit amyloid formation. The design of the derivatives was based on a simple, structure-based minimalistic and selective N-methylation approach. Accordingly, a minimum number of two amide bonds on the same side of the beta-strand of the amyloid core was N-methylated. This was expected to eliminate the two intermolecular backbone NH to CO hydrogen bonds which are critical for the extension of the beta-sheet dimers into multimers and amyloid. Other beta-strand "contact sides" remained intact allowing for the derivatives to interact with the native sequences. Double N-methylated derivatives of amyloidogenic and cytotoxic partial IAPP sequences generated included F(N-Me)GA(N-Me)IL, NF(N-Me)GA(N-Me)IL, SNNF(N-Me)GA(N-Me)IL, and SNNF(N-Me)GA(N-Me)ILSS and were found to be devoid of beta-sheet structure, amyloidogenicity and cytotoxicity according to Fourier transform-infrared spectroscopy (FT-IR), Congo red (CR) staining, electron microscopy (EM), and cell viability tests. The derivatives were able to interact with the native sequences and inhibit amyloid formation as shown by circular dichroism spectroscopy (CD), FT-IR and EM. Moreover, SNNF(N-Me)GA(N-Me)ILSS inhibited cytotoxicity of SNNFGAILSS and is thus the first reported inhibitor of IAPP amyloid formation and cytotoxicity. Our results demonstrate the validity of the design approach for IAPP and suggest that it may find application in understanding the structural features of amyloid formation and in the development of inhibitors of amyloid formation and cytotoxicity of other amyloidogenic polypeptides as well.
Calcitonin (Ct) is a 32-residue peptide hormone that is mainly known for its hypocalcemic effect and the inhibition of bone resorption. Our previous studies have led to potent, side-chain lactam-bridged human Ct (hCt) analogues [Kapurniotu, A. Kayed, R., Taylor, J.W. & Voelter W. (1999) Eur. J. Biochem. 265, 606-618; Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38, 836-847]. We have hypothesized that a possibly type I beta turn/beta sheet conformation in the region 17-21 may play an important role in hCt bioactivity. To investigate this hypothesis, analogues of the potent hCt agonist cyclo17,21-[Asp17,Lys21]hCt (1) bearing type I (and II') or II beta turn-promoting substituents at positions 18 and 19 were designed, synthesized and their solution conformations, human Ct receptor binding affinities and in vivo hypocalcemic potencies were assessed. The novel analogues include cyclo17,21-[Asp17,D-Phe19, Lys21]hCt (2), cyclo17,21-[Asp17,Aib18,Lys21]hCt (3), cyclo17,21-[Asp17,D-Lys18,Lys21]hCt (4), corresponding partial sequence peptides containing the lactam-bridged region 16-22, and nonbridged control peptides. Only 1 showed a higher Ct receptor binding affinity than hCt, whereas analogues 2-4 had similar receptor affinities to hCt. In the in vivo hypocalcemic assay, 3 and 4 were as potent as 1, whereas 2 completely lost the high potency of 1, suggesting that type I (and II') beta turn-promoting substituents are fully compatible with in vivo bioactivity. CD spectroscopy showed that analogues 1-4 were markedly beta sheet-stabilized compared to hCt and indicated the presence of distinct beta turn conformeric populations in each of the analogues. Unexpectedly, the D-amino acid- or Aib-containing cyclic analogues 2-4 but not 1 or hCt self-associated into SDS denaturation-stable dimers. Our results demonstrate a crucial role of the conformational and topological features of the residues in sequence 17-21 and in particular of residues 18 and 19 for human Ct receptor binding and in vivo bioactivity and also for the self association state of hCt. These results may assist to delineate the structure-function relationships of hCt and to design novel hCt agonists for the treatment of osteoporosis and other bone-disorder-related diseases.