Structural and functional analysis of peptidyl oligosaccharyl transferase inhibitors

The peptide cyclo(hex-Amb(1)-Cys(2))-Thr(3)-Val(4)-Thr(5)-Nph(6)-NH2 was previously shown to be a slow, tight-binding inhibitor (Ki = 37 nM) of the yeast oligosaccharyl transferase (OT) [Hendrickson et al. (1996) J. Am. Chem. Soc. 118, 7636-7637]. This enzyme catalyzes the transfer of a carbohydrate moiety to an asparagine residue in the consensus sequence Asn-Xaa-Thr/Ser. Herein we present a study of the contribution of the residues in positions 1, 3, 4, and 5 to OT binding. Replacement of the threonine (residue 3) by valine or (S)-2-aminobutyric acid dramatically reduced the potency of the inhibitor while, surprisingly, the incorporation of an additional methylene into the side chain of residue 1 [(S)-2,3-diaminobutyric acid changed to ornithine] had very little effect. Variants with acidic, basic, hydrophilic/polar, and hydrophobic side chains in positions 4 and 5 were also evaluated for both yeast and porcine liver OT inhibition. This aspect of the study reveals that basic (lysine) and acidic (glutamic acid) residues are detrimental to the binding, whereas hydrophobic (valine) and polar/hydrophilic (threonine) residues are both well tolerated. The kinetic behavior of substrate analogs [cyclo(hex-Asn(1)-Cys(2))-Thr(3)-Xaa(4)-Yaa(5)-Nph-NH2] corresponding to inhibitors of weak, medium, and strong potency was also examined in order to provide insight into the nature of these inhibitors.     

Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins

Topoisomerases are enzymes that catalyse the transient breakage and rejoining of either one (topo I) or two (topo II) DNA strands, to allow one strand to pass through another and prevent unresolvable tangles during processes such as DNA replication. A number of important clinical antitumour agents act through inhibition of topo II enzymes, while some topo I inhibitors appear likely to enter clinical use. Although these chemicals do not covalently interact with DNA, they have strong mutagenic potential, generally causing events at the level of the chromosome rather than that of the gene. Most are recombinogens, may affect gene expression and can also lead to aneuploidy through effects on chromosome segregation. Most topo I and topo II inhibitors primarily cause mutagenic events associated with the replication fork. However, at least in mitotic chromosomes, topo II enzymes are located at the base of chromosome loops, and topo II inhibitors may facilitate subunit exchanges, leading to major deletions and illegitimate recombinational events. There is evidence that programmed cell death provides an alternative pathway to mutagenesis following treatment by either topo I or topo II inhibitors. The final fate of the cell will result from a balance between these two processes.     

Structural and functional analysis of SFT, a stimulator of Fe Transport

Previous studies demonstrated that SFT (Stimulator of Fe Transport) facilitates both transferrin and nontransferrin-bound iron uptake in HeLa cells (Yu, J., and Wessling-Resnick, M. (1998) J. Biol. Chem. 273, 6909-6915). To further characterize the structure and function of SFT, we studied this human factor in rodent BHK cells. Kyte-Doolittle analysis suggests that SFT has six transmembrane-spanning segments. This transport protein also displays an REXXE motif resembling domains involved in iron binding by ferritin and in iron uptake mediated by the yeast transporter Ftr1. Using N- and C-terminal epitope tags, we have identified that modification of either protein terminus does not interfere with SFT function in nontransferrin-bound iron uptake. The N- and C-terminal domains are intracellularly disposed since antibodies against these epitopes fail to recognize expressed proteins unless BHK cells are solubilized with detergents. To define the topology of two large extramembranous loop domains, anti-peptide antibodies were employed; anti-loop 4 antibodies show no immunoreactivity unless cells are permeabilized but anti-loop 5 antibodies recognize and bind surface SFT. Thus, loop 4 must be intracellular while loop 5 is extracellular. These topological studies situate the putative iron-binding REXXE domain on the cytosolic face of the plasma membrane. However, 55Fe-binding studies reveal that the ability of SFT to bind and mediate transport of extracellular iron is defective in mutants with Glu --> Ala conversions in this motif. Curiously, we also find that depletion of intracellular iron by desferrioxamine impairs SFT transport and iron-binding functions. These observations lead to the speculation that the REXXE motif may play an important role in regulating SFT activity through interaction with intracellular iron and demonstrate that iron transport mediated by SFT is itself an iron-dependent process.     

Structural and functional analysis of the metal-binding sites of Clostridium thermocellum endoglucanase CelD

Crystallographic analysis indicated that Clostridium thermocellum endoglucanase CelD contained three Ca(2+)-binding sites, termed A, B, and C, and one Zn(2+)-binding site. The protein contributed five, six, and three of the coordinating oxygen atoms present at sites A, B, and C, respectively. Proteins altered by mutation in site A (CelDD246A), B (CelDD361A), or C (CelDD523A) were compared with wild type CelD. The Ca(2+)-binding isotherm of wild type CelD was compatible with two high affinity sites (Ka = 2 x 10(6) M-1) and one low affinity site (Ka < 10(5) M-1). The Ca(2+)-binding isotherms of the mutated proteins showed that sites A and B were the two high affinity sites and that site C was the low affinity site. Atomic absorption spectrometry confirmed the presence of one tightly bound Zn2+ atom per CelD molecule. The inactivation rate of CelD at 75 degrees C was decreased 1.9-fold upon increasing the Ca2+ concentration from 2 x 10(-5) to 10(-3) M. The Km of CelD was decreased 1.8-fold upon increasing the Ca2+ concentration from 5 x 10(-6) to 10(-4) M. Over similar ranges of concentration, Ca2+ did not affect the thermostability nor the kinetic properties of CelDD523A. These findings suggest that Ca2+ binding to site C stabilizes the active conformation of CelD in agreement with the close vicinity of site C to the catalytic center.     

Structural analysis of Drosophila merlin reveals functional domains important for growth control and subcellular localization

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila. As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane- associated NH2-terminal 350 amino acids of Merlin. Removal of a seven-amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene.     

Structural and functional analysis of the cytidine deaminase gene in patients with acute myeloid leukaemia

Gene transfer of the cytidine deaminase (CDD) cDNA has recently been shown to induce cellular resistance to cytarabine (AraC) in vitro. To investigate the role for CDD in acute myeloid leukaemia (AML) we analysed the CDD activity and CDD gene structure in blast material from well-defined patients with untreated and AraC refractory (RF) AML. Median CDD activity in previously untreated AML was significantly lower than in RF-AML blasts (P=0.015) and was significantly lower in patients with complete remission than with blast persistence following induction chemotherapy (P=0.043). Structural investigation of the CDD gene by Southern analyses and RT-PCR showed no detectable aberrations. Sequence analysis of the CDD cDNA from nine RF-AML patients showed inconsistent aberrations in three patients. Semiquantitative assessment of CDD mRNA expression revealed a significant correlation with CDD activity. In conclusion, concordant with another recent study our data suggest a correlation of pretherapeutic CDD activity with induction treatment response. Besides the previously described prognostic impact of mdrl expression, this result could be useful for the development of risk-adapted AML treatment strategies and warrants further studies of CDD activity in well-defined cohorts of AML patients and of the mechanisms involved in the regulation of CDD activity.     

Structural and functional analysis of the Pig-a protein that is mutated in paroxysmal nocturnal hemoglobinuria

There is now convincing evidence that the Pig-a gene is mutated in patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease in which one or more clones of hematopoietic cells have incomplete assembly of glycosylphosphatidylinositol (GPI) anchors and absence of GPI-linked protein expression on the cell surface. Little is known, however, about the Pig-a protein product that is necessary for GPI anchor bioassembly. Relatively few substitution (missense) Pig-a gene mutations have been identified, but we noted two apparent clusters at codons 128-129 and 151-156 and hypothesized that these might represent critical regions of the Pig-a protein. We therefore used site-directed mutagenesis to create conservative mutations in the Pig-a protein, then performed structural and functional analysis. Each Pig-a mutation generated a Pig-a protein of normal size and stability, but certain mutations had substantial deleterious effects on protein function. Conservative mutation of codons histidine 128 (H128), serine 129 (S129), and serine 155 (S155) had greatly diminished function, while mutations of flanking residues had no effect on function. Our results represent the first structure/function analysis of the Pig-a protein, and suggest that codons H128, S129, and S155 represent critical regions of the Pig-a protein. Our results also suggest a means by which transgenic mice with a "partial knock-out" of Pig-a function could be generated, which would allow investigation of PNH in an animal model.     

Structural and functional characterisation of hFSH and hLH isoforms

Human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) are gonadotropins which are secreted as multiple forms by the pituitary. Evidence supporting the structural and functional heterogeneity of 15 purified hFSH isoforms and 20 purified hLH isoforms from pituitary extracts will be presented. Gonadotropin isoforms were purified by a combination of preparative isoelectric focusing and ion-exchange chromatography. The protein mass of each isoform was determined by amino acid analysis, which also correlated (data for hLH) (r = 0.999, P < 0.001, n = 15) with the UV area under the curve at 280 nm of the isoforms following gel-filtration HPLC. The alpha and beta subunits of FSH and LH were shown to be intact by SDS-PAGE under reducing condition, with no evidence of proteolytic nicking or presence of contaminating proteins. hFSH radioreceptor activity varied over a seven-fold range, and a positive correlation (r = 0.85, P < 0.001, n = 9) was observed between FSH receptor activity and the sialic acid (SA) content (1.5-13.7 mol SA/mol hFSH) of the isoforms, as determined by an HPLC-based microfluorometric assay. FSH in vitro activities varied over a similar range with a high correlation (r = 0.82, n = 15) with receptor activities, suggesting that the initial association of the hormone with the receptor is the key interaction with less differences attributed to subsequent effects in the signaling pathway. A similar result was seen with the hLH isoforms. To explore FSH/LH in vivo, the circulating half-life (LH/FSH) and the in vivo bioactivity (LH) using an acute in vivo assay was investigated. The clearance of hLH and hFSH showed a bi-exponential pattern for all isoform preparations with the proportion of the slower dissociating component (t 1/2 50-60 min) increasing three-fold with increasing sialic acid content of the isoform. The more rapidly cleared component (t 1/2 approx 10 min) is attributed to hepatically cleared gonadotropin, rather than gonadotropin equilibration between body compartments. The in vivo assay procedure for LH was based on the 24 h integrated plasma testosterone levels in rats following administration of graded doses of hLH isoform or standard. A 16-fold range in vivo activities between LH isoforms (n = 14) was observed. A comparison between hLH in vitro and in vivo activities showed a good correlation (r = 0.75) with the slope of the regression line (1.39) not significantly different from unity. These results suggest that in this acute in vivo assay method, the differences in circulating half-lives between hLH isoforms although large is not a key factor in their in vivo activity. However, in chronic in vivo assay systems the differences in clearance rates between isoforms may be important in their subsequent biological response. It is concluded that structural heterogeneity of FSH and LH contributes to functional differences, with a key interaction occurring at the receptor level. The contribution of sialic acid to these activities was also investigated.     

Structural and functional analysis of gp64, a membrane protein of the cellular slime mold Polysphondylium pallidum

We examined 38 patients with an arthroscopic bioabsorbable tack repair for anterior shoulder instability in a prospective evaluation. The mean follow-up was 22 months (range 12 to 33). The average age was 28.4 years (range 15 to 57), the operation was performed at average of 50 months (3 to 244 months) after injury. Assessment using the Rowe score revealed excellent results in 33 and good results in 3 patients. 1 patient had a fair result and 1 had a poor result. 26 should obtained full range of motion, 11 had minor (< 10 degrees) loss of external rotation, 1 experienced greater (< 20 degrees) loss of external rotation. 3 of the 38 patients (8%) had recurrent instability, 1 patient with 2 preceding operations and atraumatic and voluntary dislocation, respectively. The recurrence rate of arthroscopic Bankart repair with bioabsorbable tacks are comparable to open Bankart procedures. Success of the procedure depends on appropriate surgical technique and suitable selection of patients with unidirectional, posttraumatic, anterior instability who are found to have well-developed ligamentous tissue.     

Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.     

Structural, functional, and genetic characterization of Gastrophilus hemoglobin

Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified and characterized. At least two isoforms have been identified by isoelectrofocusing, mass spectrometry, and genomic Southern blotting. Functional studies show a high oxygen affinity due to a low ligand dissociation rate (koff = 2.4 s-1) and a relatively high autoxidation rate (t1/2 = 1.6/h). The globins were separated under denaturing conditions, and the sequence of Hb1 (Mr = 17,965 +/- 2) was determined at the protein and DNA level. The open reading frame codes for a polypeptide of 150 amino acids. Although the globin is distantly related to globins from other species, it has a low penalty score against globin templates. Freshly isolated hemoglobin was crystallized from polyethylene glycol. Crystals contain two hemoglobin molecules per asymmetric unit. Solution of the three-dimensional structure by molecular replacement could not be achieved, possibly due to the presence of three protein isoforms in the crystals. In order to determine its three-dimensional structure, G. intestinalis Hb1 was overexpressed in Escherichia coli, resulting in a fully functional molecule as confirmed by ligand binding affinity. The globin gene contains two introns at positions D7.0 and G7.0. The D7.0 intron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought.     

Pallidal lesions. Structural and functional magnetic resonance imaging

OBJECTIVE: To study noninvasively the functional anatomy and pathophysiologic characteristics of the globus pallidus external (GPe) and internal (GPi) divisions. DESIGN: Structural and functional neuroimaging using high-resolution magnetic resonance imaging. SETTING: University medical center research facility. SUBJECTS. Seven patients with pallidal lesions, 4 with an akinetic-rigid syndrome and 3 with a dystonic syndrome, and 15 age-matched volunteers. MAIN OUTCOME MEASURES: T2-weighted anatomical magnetic resonance imaging and number of activated voxels in the GP during rapid supination and pronation of the hand. RESULTS: T2-weighted images showed hyperintense bilateral lesions in the GP of all patients. Patients with dystonic syndromes had isolated lesions in the GPi. Patients with signs of akinetic-rigid syndromes showed abnormalities in the GPe or in central portions of the GP (GPc). Patients with lesions in both parts of the GP had akinetic-rigid or dystonic syndromes. All patients showed activation in the areas of the lesions. The number of activated voxels in the GP was significantly smaller (P < .005, Wilcoxon signed rank test) in patients than in control subjects. Activation of the GP was predominantly contralateral to the moving hand. CONCLUSIONS: Lesions in the GPi result in a loss of inhibitory pallidal projections to the thalamus, which may explain the hyperkinetic signs. Lesions in the GPe lead to an increased inhibition of the thalamus, which may explain the hypokinetic signs. Neuronal activation in lesion sites suggests the presence of remaining functionally vital tissue.     

Structural and functional characterization of IS1358 from Vibrio cholerae

The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374-10378, 1995) and designated IS1358O139. We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 x 10(-8). Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.     

Structural and functional characterization of the human brain D-aspartate oxidase

The most common commercially available test measuring activated protein C (APC) resistance relies on the the anticoagulant response to added APC in an activated partial thromboplastin time (APTT) based method. Another method is a Russell Viper venom time (RVVT) based system. To improve the specificity for factor V Leiden of the APTT based method, pre-dilution of test plasma in FV-deficient plasma has recently been recommended. In this study we tested the relative suitabilities of the APTT-based system, the RVVT-based system and their corresponding assays modified by pre-dilution in FV-deficient plasma, for screening asymptomatic subjects, a group of thrombophilic patients (in particular those with low APC ratios), patients on oral anticoagulants, and patients with lupus anticoagulant (LAC). We found the RVVT-based assay to be superior to the APTT-based method in the separation of normals from those with FV Leiden mutation both in asymptomatic subjects and in the thrombophilic patient group. Both modified assays demonstrated a sensitivity and specificity of 100% for FV Leiden, as verified by genotyping in asymptomatic subjects, thrombophilic patients and patients on oral anticoagulants, with the modified RVVT-based assay giving better separation between normals and FV Leiden. Inhibition of phospholipid-dependent coagulation by LAC antibodies rendered the APTT-based system less suitable than the phospholipid-rich RVVT-based one, and as nine of the 20 LAC-positive patients were on warfarin, we showed only the modified RVVT assay to be a reliable predictor of factor V Leiden in this patient group.     

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.     

Structural and functional effects of PA700 and modulator protein on proteasomes

Control and targeting of the proteolytic activity of the major intracellular protease, the proteasome, is accomplished by various regulatory protein complexes that may form higher-order assemblies with the proteasome. An activator of proteolytic activity, PA700, has been shown to have an ATP-dependent stimulatory effect on the peptidase activities of the proteasome, and another protein factor, the modulator, further enhances the effect of PA700. Here we show that the addition of PA700 endows the proteasome with the ability to cleave ubiquitinated proteins, a property associated with the previously isolated 26 S form of the proteasome. The modulator further stimulates this specific activity, without having any such effect on the proteasome alone. Using electron microscopy, we show that addition of PA700 causes the appearance of protein "caps" at one or both ends of proteasomes, forming structures that are indistinguishable from 26 S proteasomes. Quantitation of the numbers of uncapped, singly capped and doubly capped complexes indicates cooperativity in the association of PA700 with the two ends of the proteasome. Addition of modulator protein makes no further structural modification that is detectable by electron microscopy, but does cause an increase in the number of capped complexes visible at subsaturating concentrations of PA700. Hence PA700 converts the proteasome both functionally and structurally to the 26 S form, and the modulator promotes this transformation, apparently without stable association with the resulting complex.