Structural biology and structural genomics/proteomics.
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Genomics↗
Biomedical subjects
M Cascio
Publications and source records attributed to M Cascio.
Functional reconstitution and characterization of recombinant human alpha 1-glycine receptors.
By utilizing a baculoviral expression system described previously (Cascio, M., Schoppa, N. E., Grodzicki, R. L., Sigworth, F. J., and Fox, R. O. (1993) J. Biol. Chem. 268, 22135-22142), functional recombinant homomeric human alpha(1)-glycine receptors (GlyR) were overexpressed in insect cell culture, solubilized, purified, and reconstituted into lipid vesicles via gel filtration. Reconstituted GlyR channels were observed to retain native-like activity in single channel recordings of planar bilayers and in flux assays of small unilamellar vesicles, providing evidence that the recombinant homomeric receptor may be functionally reconstituted. This reconstitution is significant in that it indicates that the overexpressed homomeric receptor is an appropriate substrate for subsequent biophysical characterization aimed at the general elucidation of structure-function. Circular dichroism spectroscopy of reconstituted GlyR indicated a low alpha-helical content and a significant fraction of polyproline structure. The small fraction of observed alpha-helix is insufficient to accommodate the four helical transmembrane domains proposed in models for this receptor. By inference, other members of the homologous ligand-gated channel superfamily, which include the ionotropic gamma-aminobutyric acid, acetylcholine, and serotonin receptors, may also be erroneously modeled, and alternate models should be considered.
Amino Acid Sequence↗
Structure of ligand-gated ion channels: critical assessment of biochemical data supports novel topology.
Rapid signaling across the synaptic junction is partially mediated by the ligand-gated ion channel superfamily (LGICS), which includes inhibitory glycine and GABA receptors and excitatory acetylcholine and serotonin receptors. The glycine receptor (GlyR) can assemble as homopentamers of alpha subunits, and baculovirus expression systems are capable of overexpressing large quantities of active receptors. Limited proteolysis coupled to mass spectrometry on reconstituted alpha1 GlyR homopentamers identified proteolytic cleavages within proposed transmembrane domains postulated to fold as bilayer-spanning alpha helices in the "classical" model and identified unexpected membrane-associated regions in the N-terminal domain (J. F. Leite et al., 2000, J. Biol. Chem. 275, 13683-13689). In this review, optimized sequence alignments were used to integrate these proteolysis data with biochemical information determined in studies of all the LGICS members in order to construct a novel topological model.
Animals↗
Coupled proteolytic and mass spectrometry studies indicate a novel topology for the glycine receptor.
Members of the heteropentameric ligand-gated ion channel superfamily rapidly mediate signaling across the synaptic cleft. Sequence analysis and limited experimental studies have yielded a topological model containing four transmembrane alpha-helices, labeled M1 to M4, and a large soluble, extracellular N-terminal domain. This model persists to date despite some recent structural studies that suggest it may be inappropriate. In this study, the topology of the glycine receptor was probed by limited proteolysis coupled to mass spectrometry. Of particular note, accessible cleavage sites within the putative M1 and M3 transmembrane helices were identified. Membrane-associated fragments within the postulated globular extracellular N-terminal domain were also observed. This report presents several key details incorporated in a new topological model and is the first direct experimental evidence that a subset of the transmembrane regions are too short to be membrane-spanning alpha-helices; rather, these regions are proposed to be a mix of alpha-helices and beta-sheets. This report is also the first to exploit the capability of mass spectrometry to probe critically the topology of a class of membrane proteins of unknown structure.
Amino Acid Sequence↗
Identification of a novel Stat3 recruitment and activation motif within the granulocyte colony-stimulating factor receptor.
Stat3 is essential for early embryonic development and for myeloid differentiation induced by the cytokines granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). Two isoforms of Stat3 have been identified, (p92) and beta (p83), which have distinct transcriptional and biological functions. Activation of both Stat3 and Stat3beta requires the distal cytoplasmic domain of the G-CSFR, which contains four Tyr at positions 704, 729, 744, and 764. The studies reported here were undertaken to determine which, if any, of these tyrosine residues participated in Stat3/beta recruitment and activation. We showed that Stat3 and Stat3beta were affinity purified using phosphopeptides containing Y704 and Y744 but not by nonphosphorylated peptide analogues or by phosphopeptides containing Y729 and Y764. Complementary results were obtained in studies examining the ability of these peptides to destabilize and inhibit DNA binding of activated Stat3. Both Y704 and Y744 contributed to optimal activation of Stat3/beta in M1 murine myeloid leukemia cells containing wild-type and Y-to-F mutant G-CSFR constructs. Carboxy-terminal to Y704 at the +3 position is Gln; YXXQ represents a consensus Stat3 recruitment and activation motif. Y744 is followed at the +3 position by Cys (C); YXXC, represents a novel motif implicated in the recruitment and activation of Stat3. Modeling of the SH2 domain of Stat3 based on homologous SH2 domains of known structure revealed polar residues whose side chains contact the +3 position. This substitution may confer specificity for the Y704- and Y744-based ligands by allowing H-bond formation between the binding surface and the Gln or Cys found at the respective +3 position.
Amino Acid Sequence↗
Labour analgesia with intrathecal fentanyl decreases maternal stress.
PURPOSE: Lumbar epidural analgesia (LEA) decreases maternal stress as measured by maternal circulating plasma catecholamine concentrations. Intrathecal fentanyl (ITF) provides effective labour analgesia but its effect on maternal epinephrine (Epi) and norepinephrine (NE) concentrations is not known. This study assesses whether ITF reduces maternal stress in the same manner as conventional LEA. METHODS: Twenty-four healthy women in active labour received either 25 micrograms ITF (n = 12) or epidural lidocaine 1.5% (n = 12) for analgesia. Venous blood samples were collected before anaesthesia and at five minute intervals for 30 min following anaesthesia for the measurement of plasma Epi and NE by high performance liquid chromatography. Maternal blood pressure (BP), heart rate (HR), visual analog scores (VAS) to pain and pruritus were recorded at the same time. RESULTS: Both ITF and LEA decreased pain VAS scores, maternal BP, and plasma Epi concentrations with only minimal effects on plasma NE concentrations. Intrathecal fentanyl (ITF) and LEA reduced plasma epi to a similar extent, with ITF reducing the levels slightly faster than LEA. Intrathecal fentanyl(ITF) and LEA reduced plasma Epi concentrations by 52% and 51%, respectively (P value < 0.01). CONCLUSION: We conclude that ITF is as effective as LEA in producing pain relief in the labouring patient. Intrathecal Fentanyl (ITF) is also capable of reducing maternal plasma epinephrine concentration, thus avoiding the possibly deleterious side effects of excess amounts of this catecholamine during labour.
Adult↗
Attachment site of grafted SCN influences precision of restored circadian rhythm.
Fetal hypothalamic grafts containing the suprachiasmatic nucleus (SCN) restore circadian locomotor rhythmicity when implanted into the third ventricle of SCN-lesioned hamsters. However, the quality of restored rhythms is variable, and the locomotor rhythms of grafted animals are generally less robust than those of intact animals. The present study explored whether anatomical features of the graft predict the quality of the recovered rhythm and whether such information might provide insight as to the target of the signal from the SCN that controls locomotor rhythmicity. The following graft parameters were assessed: distance between the attachment site of the graft and potential targets for the output signal from the SCN, number and overall size of SCN clusters, the size of the cluster closest to the SCN lesion site, and extent of vasoactive intestinal polypeptide (VIP) and vasopressin-associated neurophysin (NP) positive fiber outgrowth from the graft. The restored circadian activity rhythm was assessed by quantifying the precision of activity onset and the amount, period, and robustness of rhythmicity. The results indicate a significant positive correlation between the precision of activity onset and the proximity of the closest SCN cluster to the site of the lesioned host SCN. A more detailed analysis of the spatial location of the graft indicates that proximity of the graft in the dorsal and caudal directions, but not the rostral direction, is positively correlated with the precision of the recovered rhythm. This suggests two possibilities: the coupling signal may act on a site very near the SCN and travel preferentially in a rostro-caudal direction. Alternatively, the coupling signal may act on a site rostral to the SCN. That the site is not far rostral to the SCN was suggested by the lack of a correlation between the precision of the restored rhythm and the rostrally lying anterior medial preoptic nucleus. Finally, evaluation of NP- and VIP-ergic fibers in nuclei known to receive input from the SCN indicates that the extent of such innervation by graft efferents does not predict either the occurrence of recovery or the precision of the recovered rhythm. Overall, these results suggest that the target(s) of SCN pacemakers regulating locomotor rhythmicity lie in the hypothalamus, close to or rostral to the SCN.
Animals↗
Meningitis following a combined spinal-epidural technique in a labouring term parturient.
PURPOSE: To report a case of probable bacterial meningitis in a parturient who received a combined spinal-epidural (CSE) technique for labour analgesia. CLINICAL FEATURES: A 28-yr-old GIPO at 40 wk gestational age presented in labour and requested analgesia. A CSE technique was used to inject a mixture of fentanyl 25 micrograms and bupivacaine 2.5 mg into the subarachnoid space following which a catheter was inserted into the epidural space. Sixteen hours after delivery her body temperature increased to 38.7 degrees C and she complained of a non-positional frontal headache. This was associated with chills, photophobia, and mild nuchal rigidity. Diagnostic lumbar puncture revealed cloudy cerebrospinal fluid (CSF) with increased polymorphonuclear (PMN) white cell count and increased protein and decreased glucose concentrations. Peripheral blood cytology also showed an increased leukocyte count with a shift to the left. Immediate treatment with antibiotics was instituted and the patient rapidly recovered without sequelae. CONCLUSIONS: Meningitis may occur, albeit rarely, with any neuraxial blocks including CSE techniques. Early diagnosis and appropriate antibiotic therapy are important in preventing serious neurological sequelae.
Adult↗
Hemodynamic effects of intrathecal fentanyl in nonlaboring term parturients.
STUDY OBJECTIVE: To determine the effect of intrathecal fentanyl on maternal hemodynamics. DESIGN: Prospective. SETTING: Labor and delivery suite of a university medical center. PATIENTS: 23 ASA status I nonlaboring term parturients presenting for elective cesarean section. INTERVENTIONS: Patients were given either 1,200 ml lactated Ringer's Solution (Group 1, n = 12) or no intravenous (i.v.) fluid (Group 2, n = 11). A combined spinal-epidural technique was then performed. Fentanyl 25 micrograms was administered intrathecally through a 24-gauge Sprotte or 25-gauge Whitacre spinal needle. After completion of the hemodynamic study, a catheter was threaded into the epidural space for local anesthetic administration. MEASUREMENTS AND MAIN RESULTS: Baseline hemodynamic data [systolic (SBP), diastolic, and mean arterial pressure, heart rate, stroke volume, cardiac output, end-diastolic volume, and ejection fraction] were obtained in triplicate using noninvasive blood pressure monitoring and impedance cardiography. After administration of intrathecal fentanyl, hemodynamic measurements were recorded at 3-minute intervals for 30 minutes. These values were compared with baseline for both groups. Ten patients in each group completed the study. Intrathecal fentanyl administration did not result in any maternal hemodynamic changes in Group 1, and a few small statistically significant changes in Group 2. Measured SBP was always greater than 100 mmHg in all patients during the study. CONCLUSION: Intrathecal administration of fentanyl 25 micrograms in nonlaboring term parturients does not produce clinically important maternal hemodynamic changes.
Adult↗
Physical characterization of gap junction membrane connexons (hemi-channels) isolated from rat liver.
Enriched subcellular fractions of double membrane gap junctions (plaques) from rat livers were treated under reducing conditions with high salt and non-ionic detergent concentrations at high pH to obtain a preparation of structural 80-90 A complexes of oligomers (connexons). The isolated oligomers were chromatographically purified, and subsequently characterized immunologically, morphologically by electron microscopy, hydrodynamically by gel filtration and ultracentrifugation, spectroscopically by circular dichroism, and chemically via cross-linking studies. The physical characteristics of these isolated gap junction complexes were compared to those of native membrane-bound gap junctions in rat liver. These analyses indicate that the isolated complex (connexon) principally contains a hexameric arrangement of gap junction protein to form a single membrane hemi-channel.
Animals↗
Effects of local environment on the circular dichroism spectra of polypeptides.
The secondary structures of proteins may be estimated by analysis of their circular dichroism spectra using techniques which empirically fit component spectra derived from water-soluble protein databases. However, local environment effects red- or blue-shift peptide energy transitions primarily through solvent polarization effects and may be an especially important factor for proteins in nonaqueous environments. In this study the reference basis spectra were deconvoluted into three component Gaussian curves corresponding to each of the peptide absorbances in the wavelength range from 190 to 240 nm. These Gaussians were then systematically shifted and utilized in the analyses of the spectra of crambin solubilized in a variety of nonaqueous solvents, including that of the polypeptide associated with small unilamellar phospholipid vesicles. Crambin was selected since its well-characterized structure was found to retain native structure in a variety of nonaqueous environments. This method was more successful in estimating the helical content of this protein than conventional methodologies. This type of analysis may prove to be appropriate for estimating the secondary structure of proteins found in nonaqueous environments, such as membrane proteins.
Alcohols↗
Intrathecal administration of liposomal morphine in a mouse model.
The authors determined the duration of analgesia, toxicity, and neuraxial distribution of liposomal morphine after intrathecal administration in the mouse. Analgesic duration was determined using the tail-flick test after intrathecal injection of 12.5, 25, or 50 micrograms of plain or liposomal morphine (n = 6 mice/dose/formulation). Toxicity of the formulations was compared by estimating LD50. Neuraxial morphine distribution was determined after 20 micrograms of plain or liposomal morphine. The excised spinal cord and brain were divided into five segments at 1 min, and at 1, 4, and 8 h after injection for both formulations. In addition, for the liposomal morphine, similar sections were obtained at 24 h (n = 6 mice/formulation/time point). Segmental morphine concentration was quantified using radioimmunoassay. Liposomal encapsulation significantly prolonged duration of analgesia for the 25-micrograms (13.4 +/- 1.64 [SE] vs 4.1 +/- 0.5 h) and 50-micrograms doses (16.8 +/- 4.0 vs 4.6 +/- 1.0 h). The estimated LD50 was 200 (confidence interval 151-257 micrograms) for plain morphine, but was not determinable for the liposomal formulation, since no deaths occurred at the largest dose level which could be tested (371 micrograms). For plain morphine, the drug was not confined to a specific neuraxial segment, and segmental levels declined rapidly. After liposomal morphine, the most morphine was concentrated and persisted in the low spinal cord segment at each time interval. These results show that a single dose of liposomal morphine produces prolonged analgesia with decreased toxicity compared to the plain formulation.
Animals↗
Functional expression and purification of a homomeric human alpha 1 glycine receptor in baculovirus-infected insect cells.
The human alpha 1 glycine receptor (GlyR) was expressed in Sf9 insect cells infected with a recombinant Autographa californica nuclear polyhedrosis baculovirus. Previous studies had indicated that transient expression of this subunit in Xenopus oocytes or human kidney cell lines is sufficient to form active agonist-gated chloride channels. Expression of the alpha 1 GlyR protein resulted in functional channels present on the cell surface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 48-kDa protein could be easily visualized on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha 1 GlyR protein was solubilized from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminostrychnine agarose, yielding 0.33 mg/liter of cells. Given the low natural abundance of the native channel, the development of this expression system now provides sufficient purified channel protein for future biochemical and biophysical characterization. Since the glycine receptor shares sequence and structural homology with other members of a ligand-gated channel superfamily, further characterization may establish general rules governing the structure and mechanism of these membrane protein channels.
Amino Acid Sequence↗
The secondary structure of gap junctions. Influence of isolation methods and proteolysis.
Paired intercellular transmembrane channels, termed connexons, comprised of hexameric assemblies of gap junction protein, were isolated and purified from rat liver by exploiting their resistance to either Sarkosyl detergent solubilization or alkali extraction. The secondary structures of the gap junction proteins prepared by these methods were compared by circular dichroism (CD) spectroscopy. Both the spectra and the calculated net secondary structures of the proteins obtained by the two isolation methods were different. The protein isolated by the Sarkosyl treatment was found to be approximately 50% alpha-helical, while protein isolated by alkali extraction had a lower helix content (approximately 40%). In both types of preparations, however, the helical content of the gap junction protein was sufficiently large to be consistent with an all-helical model for the membrane-spanning parts of the structure. CD spectroscopy was also used to examine the effects of proteolytic digestion of the cytoplasmic domain on the net secondary structure of the detergent-treated gap junction protein. The membrane-bound fragments had a slightly higher proportion of their residues that were alpha-helical in nature, suggesting that the transmembrane and/or intra-gap domains are indeed enriched in this type of secondary structure. This information constrains the range of models which can be realistically proposed for the channel structure.
Animals↗
The secondary structure of human amyloid deposits as determined by circular dichroism spectroscopy.
The deposition of amyloid fibrils has been associated with a diversity of pathologies including plasma cell dyscrasias, chronic inflammatory diseases, and several types of neurological diseases including Alzheimer's disease. Using circular dichroism spectroscopy, the secondary structure of a human amyloid protein deposit from a patient with plasma cell (light chain)-associated amyloidosis (amyloidosis AL) has been determined. The protein contains 52% beta structure, which is consistent with these depositions arising from the aberrant catabolism of immunoglobulin light chains, which are rich in beta sheets. The protein was also found to contain 20% alpha-helix, suggesting that partial refolding may occur during amyloidogenesis.
Amino Acids↗
Conformation of alamethicin in phospholipid vesicles: implications for insertion models.
The secondary structure of alamethicin, a membrane channel-forming polypeptide, has been examined by circular dichroism spectroscopy to determine the relationship of its conformation in organic solution to its conformation in a membrane-bound state. The spectrum of alamethicin in small unilamellar dimyristoyl phosphatidylcholine vesicles is significantly different from its spectrum in 10% methanol/acetonitrile, the solvent from which it was crystallized (Fox and Richards: Nature 300:325-330, 1982), as well as its spectrum in methanol, the solvent in which NMR studies have been done (Banerjee and Chan: Biochemistry 22:3709-3713, 1983). This suggests that structural models based on studies of the molecule in organic solvents may not be entirely appropriate for the membrane-bound state. To distinguish between different models for channel formation and insertion, two different methods were used to associate the alamethicin with vesicles; in addition, the effect of oligomerization on the conformation of the membrane-bound state was investigated. These studies are consistent with a modified insertion model in which alamethicin monomers, dimers, or trimers associate with the bilayer and then spontaneously oligomerize to form a prechannel with a higher helix content. This aggregate could then "open" upon application of an appropriate gating transmembrane potential.
Alamethicin↗
Evaluation of methods for the prediction of membrane protein secondary structures.
With the advent of molecular cloning methods, the amino acid sequences for a number of membrane proteins have been determined. The relative paucity of detailed three-dimensional structural information available for these molecules has led to attempts to predict the secondary structures of membrane proteins based on folding motifs found in soluble proteins of known three-dimensional structure and sequence. In this study, we evaluated the accuracy of several of these methods in predicting the conformation of 15 integral membrane proteins and membrane-spanning polypeptides for which both primary and secondary structural information are available. chi 2 analyses indicated a less than 0.5% correlation between the net predicted secondary structures and the experimental results. A more stringent test of the accuracy of the methods, the index of prediction, was calculated for individual residues in four of the polypeptides for which the crystal structures were known; this criterion also indicated that the predicted assignments for the secondary structures of the residues were inaccurate. Thus, prediction schemes using soluble protein bases appear to be inappropriate for the prediction of membrane protein folding.
Amino Acid Sequence↗