Multidimensional HPLC purification of proteins.
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Animals↗
Biomedical subjects
Marie-Isabel Aguilar
Publications and source records attributed to Marie-Isabel Aguilar.
HPLC of peptides and proteins: basic theory and methodology.
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Chromatography, High Pressure Liquid↗
Reversed-phase high-performance liquid chromatography.
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Animals↗
Surface plasmon resonance spectroscopy in the study of membrane-mediated cell signalling.
Peptide-membrane interactions contribute to many important biological processes such as cellular signaling, protein trafficking and ion-channel formation. During receptor-mediated signalling, activated intracellular signalling molecules are often recruited into receptor-induced signaling complexes at the cytoplasmic surface of the cell membrane. Such recruitment can depend upon protein-protein and protein-lipid interactions as well as protein acylation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study these interactions and have provided important information on the relationship between the structure of these proteins involved in cell signalling and their biological function. More recently, surface plasmon resonance (SPR) spectroscopy has also been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications, which demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated cellular signalling.
Cell Membrane↗
Isolation and pharmacological characterization of a phospholipase A2 myotoxin from the venom of the Irian Jayan death adder (Acanthophis rugosus).
1. It has long been thought that death adder venoms are devoid of myotoxic activity based on studies done on Acanthophis antarcticus (Common death adder) venom. However, a recent clinical study reported rhabdomyolysis in patients following death adder envenomations, in Papua New Guinea, by a species thought to be different to A. antarcticus. Consequently, the present study examined A. rugosus (Irian Jayan death adder) venom for myotoxicity, and isolated the first myotoxin (acanmyotoxin-1) from a death adder venom. 2. A. rugosus (10-50 micro g ml(-1)) and acanmyotoxin-1 (MW 13811; 0.1-1 micro M) were screened for myotoxicity using the chick directly (0.1 Hz, 2 ms, supramaximal V) stimulated biventer cervicis nerve-muscle (CBCNM) preparation. A significant contracture of skeletal muscle and/or inhibition of direct twitches were considered signs of myotoxicity. This was confirmed by histological examination. 3. High phospholipase A(2) (PLA(2)) activity was detected in both A. rugosus venom (140.2+/-10.4 micro mol min(-1) mg(-1); n=6) and acanmyotoxin-1 (153.4+/-11 micro mol min(-1) mg(-1); n=6). Both A. rugosus venom (10-50 micro g ml(-1)) and acanmyotoxin-1 (0.1-1 micro M) caused dose-dependent inhibition of direct twitches and increase in baseline tension (n=4-6). In addition, dose-dependent morphological changes in skeletal muscle were observed. 4. Prior incubation (10 min) of CSL death adder antivenom (5 units ml(-1); n=4) or inactivation of PLA(2) activity with 4-bromophenacyl bromide (1.8 mM; n=4) prevented the myotoxicity caused by acanmyotoxin-1 (1 micro M). 5. Acanmyotoxin-1 (0.1 micro M; n=4) displayed no significant neurotoxicity when it was examined using the indirectly (0.1 Hz, 0.2 ms, supramaximal V) stimulated CBCNM preparation. 6. In conclusion, clinicians may need to be mindful of possible myotoxicity following death adder envenomation in Irian Jaya.
Amino Acid Sequence↗
Beta-amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH.
Amyloid protein (Abeta1-40) aggregation and conformation was examined using native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the results compared with those obtained by atomic force microscopy, and with Congo red binding, sedimentation and turbidity assays. The amount of Abeta aggregation measured was different, depending upon the method used. Incubation for 15 min at pH 5.0 or in the presence of Fe2+, Cu2+ or Zn2+ did not alter the level of Abeta oligomers observed on SDS and native gels. However, the slow aggregation of Abeta to form high molecular mass species over 5 days was inhibited. In contrast, when Abeta aggregation was monitored using a Congo red binding assay or sedimentation assay, a rapid increase in Abeta aggregation was observed after incubation for 15 min at pH 5.0, or in the presence of Fe2+, Cu2+ or Zn2+. The low pH-, Zn2+- or Cu2+-induced Abeta aggregation measured in a turbidity assay was reversible. In contrast, a considerable proportion of the Abeta aggregation measured by native and SDS/PAGE was stable. Atomic force microscopy studies showed that Abeta aged at pH 5.0 or in the presence of Zn2+ produced larger looser rod-shaped aggregates than at pH 7.4. Abeta that had been aged at pH 7.4 was more cytotoxic than Abeta aged at pH 5.0. Taken together, the results suggest that Abeta oligomerizes via two mutually exclusive mechanisms to form two different types of aggregates, which differ in their cytotoxic properties.
Amyloid beta-Peptides↗
Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes.
Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.
Amyloid beta-Peptides↗
Inhibitors of metalloendopeptidase EC 3.4.24.15 and EC 3.4.24.16 stabilized against proteolysis by the incorporation of beta-amino acids.
The enzyme EC 3.4.24.15 (EP 24.15) is a zinc metalloendopeptidase whose precise function in vivo remains unknown but is thought to participate in the regulated metabolism of a number of specific neuropeptides. The lack of stable and selective inhibitors has hindered the determination of the exact function of EP 24.15. Of the limited number of EP 24.15 inhibitors that have been developed, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but it is unstable in vivo due to cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). This cleavage by EP 24.11 generates a potent inhibitor of angiotensin converting enzyme, thereby limiting the use of CFP for in vivo studies. To develop specific inhibitors of EP 24.15 that are resistant to in vitro and potentially in vivo proteolysis by EP 24.11, this study incorporated beta-amino acids replacing the Ala-Tyr scissile alpha-amino acids of CFP. Both C2 and C3 substituted beta-amino acids were synthesized and substituted at the EP 24.11 scissile Ala-Tyr bond. Significant EP 24.15 inhibitory activity was observed with some of the beta-amino acid containing analogues. Moreover, binding to EP 24.11 was eliminated, thus rendering all analogues containing beta-amino acids resistant to degradation by EP 24.11. Selective inhibition of either EP 24.15 or EP 24.16 was also observed with some analogues. The results demonstrated the use of beta-amino acids in the design of inhibitors of EP 24.15 and EP 24.16 with K(i)'s in the low micromolar range. At the same time, these analogues were resistant to cleavage by the related metalloendopeptidase EP 24.11, in contrast to the alpha-amino acid based parent peptide. This study has therefore clearly shown the potential of beta-amino acids in the design of stable enzyme inhibitors and their use in generating molecules with selectivity between closely related enzymes.
Amino Acid Substitution↗
Electrostatic and hydrophobic forces tether the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus to anionic lipids.
The carboxyl terminus of the type-1 angiotensin II receptor (AT(1A)) is a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal, first 20 amino acids of the carboxyl terminus adopt an alpha-helical conformation in organic solvents, suggesting that the secondary structure of this region may be sensitive to hydrophobic environments. Using surface plasmon resonance, immobilized lipid chromatography, and circular dichroism, we examined whether this positively charged, amphipathic alpha-helical region of the AT(1A) receptor can interact with lipid components in the cell membrane and thereby modulate local receptor attachment and structure. A synthetic peptide corresponding to the proximal region of the AT(1A) receptor carboxyl terminus (Leu(305) to Lys(325)) was shown by surface plasmon resonance to bind with high affinity to the negatively charged lipid, dimyristoyl L-alpha-phosphatidyl-DL-glycerol (DMPG), but poorly to the zwitterionic lipid, dimyristoyl L-alpha-phosphatidylcholine (DMPC). In contrast, a peptide analogue possessing substitutions at four lysine residues (corresponding to Lys(307,308,310,311)) displayed poor association with either lipid, indicating a crucial anionic component to the interaction. Circular dichroism analysis revealed that both the wild-type and substituted peptides possessed alpha-helical propensity in methanol and trifluoroethanol, while the wild-type peptide also adopted partially inserted helical structure in DMPG and DMPC liposomes. In contrast, the substituted peptide exhibited spectra that suggested the presence of beta-sheet and alpha-helical structure in both liposomes. Immobilized lipid chromatography was used to characterize the hydrophobic component of the membrane interaction, and the results demonstrated that hydrophobic and electrostatic interactions mediated the binding of the wild-type peptide but that the substituted peptide bound to the model membranes mainly via hydrophobic forces. We propose that, in intact AT(1A) receptors, the proximal carboxyl terminus associates with the cytoplasmic face of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the amphipathic helicity of this region. Such associations may be important for receptor function and common for G protein-coupled receptors.
Amino Acid Sequence↗
Surface plasmon resonance spectroscopy: an emerging tool for the study of peptide-membrane interactions.
The interactions between peptides and membranes mediate a wide variety of biological processes, and characterization of the molecular details of these interactions is central to our understanding of cellular events such as protein trafficking, cellular signaling and ion-channel formation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study peptide-membrane interactions, and have provided important information on the relationship between membrane-active peptide structure and their biological function. However, what has generally not been reported is a detailed analysis of the affinity of peptide for different membrane systems, which has largely been due to the difficulty in obtaining this information. To address this issue, surface plasmon resonance (SPR) spectroscopy has recently been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications that demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated peptide function.
Amino Acid Sequence↗
Beta-amino acids: versatile peptidomimetics.
The use of peptidomimetics has emerged as a powerful means for overcoming the limitations inherent in the physical characteristics of peptides thus improving their therapeutic potential. A peptidomimetic approach that has emerged in recent years with significant potential, is the use of beta-amino acids. Beta-amino acids are similar to alpha-amino acids in that they contain an amino terminus and a carboxyl terminus. However, in beta-amino acids two carbon atoms separate these functional termini. beta-amino acids, with a specific side chain, can exist as the R or S isomers at either the alpha (C2) carbon or the beta (C3) carbon. This results in a total of 4 possible diastereoisomers for any given side chain. The flexibility to generate a vast range of stereo- and regioisomers, together with the possibility of disubstitution, significantly expands the structural diversity of beta-amino acids thereby providing enormous scope for molecular design. The incorporation of beta-amino acids has been successful in creating peptidomimetics that not only have potent biological activity, but are also resistant to proteolysis. This article reviews the rapidly expanding applications of beta-amino acids in the design of bioactive peptide analogues ranging from receptor agonists and antagonists, MHC-binding peptides, antimicrobial peptides and peptidase inhibitors. Given their structural diversity taken together with the ease of synthesis and incorporation into peptide sequences using standard solid-phase peptide synthesis techniques, beta-amino acids have the potential to form a new platform technology for peptidomimetic design and synthesis.
Amino Acids↗