Identification of (22R)-3 alpha,7 alpha,12 alpha,22- and (23R)-3 alpha,7 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acids in urine from a patient with Zellweger's syndrome

The nature of two novel C27 bile acids present as the taurine conjugates in urine from a patient with Zellweger's syndrome was studied. Bile acids conjugated with taurine were isolated from unconjugated and glycine-conjugated bile acids by means of ion-exchange chromatography. After alkaline hydrolysis of the taurine conjugates, the hydrolysate was acidified and extracted with ether; the extract was again subjected to ion-exchange chromatography to separate neutral from acidic compounds. The neutral fraction, which consisted mainly of two steroidal lactones, was treated with lithium aluminum hydride, and the reduction products were identified as (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,26-pentol and (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,26-pentol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of chemically synthesized authentic samples. Thus, the chemical structure of two native bile acids present in urine from a patient with Zellweger's syndrome should be formulated as (22R)-3 alpha,7 alpha,12 alpha,22-tetrahydroxy-5 beta-cholestanoic acid and (23R)-3 alpha,7 alpha,12 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acid, respectively.     

Improved solid-phase synthesis of alpha,alpha-dialkylated amino acid-rich peptides with antimicrobial activity.

A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid-phase synthetic techniques. Each nonapeptide was rich in alpha,alpha-dialkylated amino acids [one 4-aminopiperidine-4-carboxylic acid (Api) and six alpha-aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9-fluorenylmethyloxycarbonyl (Fmoc)-Aib-Aib-OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3(10)-helical, amphipathic design of these peptides was born out most prominently in the N-terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations < or =100 microM) and the acetylated peptides (concentrations < or = 200 microM) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Nuclear targeting of prothymosin alpha

Prothymosin alpha is a highly acidic protein which lacks an amino-terminal signal peptide, yet was once thought to be a precursor for thymosin alpha 1, a putative peptide hormone secreted by the thymus. Here, two lines of evidence are presented that strongly implicate prothymosin alpha as a nuclear protein: 1) in COS cells transfected with the human prothymosin alpha gene copious amounts of prothymosin alpha were present in sealed nuclei obtained by treating these cells with cytochalasin B and enucleating them centrifugally. 2) Constructs in which human prothymosin alpha nucleic acid sequences were fused in-frame either near the amino terminus of the beta-galactosidase gene in pCH110 or at the carboxyl terminus, when expressed in COS cells, resulted in nuclear localization of the fusion protein; indirect immunofluorescence in situ was used as the assay. The basic cluster of amino acids at the carboxyl terminus of prothymosin alpha, TKKQKT, has been identified as part of the nuclear targeting signal, whereas the basic cluster of amino acids situated within the thymosin alpha 1 sequence at the amino terminus failed to effect nuclear transport.     

N-methylation of N(alpha)-acylated, fully C(alpha)-methylated, linear, folded peptides: synthetic and conformational aspects

Peptides characterized by single or multiple N-methylated, C(alpha)-trisubstituted (e.g., protein) amino acids are of great interest in medicinal chemistry. Several naturally occurring peptides, remarkably stable to enzymatic attacks, are based on N-methylated residues. The classical conditions (CH(3)I/Ag(2)O in DMF, 24 h, room temperature) for N-methylation of the peptide function are useful tools for distinguishing solvent exposed from intramolecularly H-bonded -CO-NH- groups in peptides. In this work we have extended this reaction to N(alpha)-acylated, linear peptides based exclusively on helicogenic C(alpha)-tetrasubstituted alpha-amino acids, e.g., Aib (alpha-aminoisobutyric acid) or (alphaMe)Nva (C(alpha)-methyl norvaline) residues. Under the experimental conditions used, only amide monomethylation (on the N-terminal, acylated, residue) takes place. Methylation of internal peptide groups linking two C(alpha)-tetrasubstituted residues was not observed. Our FT-IR absorption, NMR, and X-ray diffraction investigations support the view that the beta-turn and 3(10)-helical conformations preferred by the original peptides are not dramatically perturbed in the derivatives monomethylated at position 1. In particular, the tertiary amide bonds are trans. Conversely, the packing modes in the crystals are strongly influenced by the reduction of the number of H-bonding donors. The MeXxx-Xxx peptide bond is readily disrupted under mild acidic conditions.     

Nicotine binding to native and substituted peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha1, alpha2, alpha3, alpha4, alpha5, and alpha7 subunits

Structural determinants of L-[(3)H]nicotine binding to synthetic peptides comprising residues 188-207 of nicotinic acetylcholine receptor alpha subunits were invesitigated by equilibrium binding analysis. Two binding components were detected, one of low affinity (K(d) approximately 1.5 microM) that did not differ significantly among peptides and another of high affinity. The high affinity binding component was higher for the neuronal peptides (K(d) = 14-23 nM) than the muscle alpha1 peptides (K(d) = 52 nM). The following nonconservative substitutions in the alpha4 peptide resulted in a significant decrease in nicotine affinity for the peptide: Y190A, Y190D, C192G, E195A, E195-, P199A, P199-, and Y203A. Substitution of alpha4P199 with a leucine which is present in the alpha1 sequence decreased the affinity of the alpha4 peptide for nicotine and substitution of alpha1L199 with a proline (alpha4) or a glutamine (alpha3) increased the affinity of the alpha1 peptide. It is concluded that aromatic residues contribute to the binding site for nicotine on the alpha4 subunit and that the residue present at position 199 partly determines differences in nicotine affinity for different alpha subunits.     

Identification of 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid in Zellweger's syndrome

In order to confirm the occurrence of 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid in Zellweger's syndrome, the nature of tetrahydroxycholestanoic acids present in a patient with this disease was studied. Urinary bile acids were extracted with a Sep-pak C18 cartridge and methylated after alkaline hydrolysis. The methyl esters were purified by silica gel column chromatography, and the methyl tetrahydroxycholestanoate fraction was analyzed by gas liquid chromatography-mass spectrometry. Along with already known side chain hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid, 3 alpha, 7 alpha, 12 alpha, 24- and 3 alpha, 7 alpha, 12 alpha, 26-tetrahydroxy-5 beta-cholestanoic acids, three nuclear hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid were found. One of them was identified as 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholestanoic acid by direct comparison with the authentic standard which was chemically synthesized from 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholanoic acid by side chain elongation.     

Structure of conformationally constrained peptides: from model compounds to bioactive peptides

The use of backbone conformational constraints has acquired increasing importance in the design and synthesis of structurally restricted agonists and antagonists of bioactive peptides. Here I discuss the preferred conformations of four among the most popular types of such peptide surrogates: (a) Peptides from C alpha, alpha-dialkylated residues, (b) tetrazolyl peptides, (c) (gamma- and delta-) lactam-containing peptides, and (d) thiated peptides. Emphasis is given to conformational energy computations and x-ray diffraction analyses of selected model compounds and analogues of small bioactive peptides such as the formylmethionyl tripeptide chemoattractant and MIF.     

The signal transfer regions of G alpha(s)

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.     

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin)

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.     

Slotoxin, alphaKTx1.11, a new scorpion peptide blocker of MaxiK channels that differentiates between alpha and alpha+beta (beta1 or beta4) complexes

A novel peptide from Centruroides noxius Hoffmann scorpion venom was isolated and sequenced. The 37 amino acid peptide belongs to the charybdotoxin sub-family (alphaKTx1) and was numbered member 11. alphaKTx1.11 has 75% sequence identity with iberiotoxin and 54% with charybdotoxin. alphaKTx1.11 revealed specificity for mammalian MaxiK channels (hSlo), thus, was named slotoxin. Slotoxin blocks the MaxiK pore-forming alpha subunit reversibly (K(d)=1.5 nM). Slotoxin association with alpha+beta (beta1 or beta4) channels was approximately 10 times slower than iberiotoxin and charybdotoxin, leading to a lack of effect on alpha+beta4 when tested at 100 nM for 5 min. Thus, slotoxin is a better tool to distinguish MaxiK alpha+beta complexes.     

Identification of novel short peptides derived from the alpha 4, alpha 5, and alpha 6 fibrils of type IV collagen with anti-angiogenic properties

Angiogenesis, or neovascularization, is tightly controlled by positive and negative regulators, many of which reside in the extracellular matrix. We have now identified eight novel 19- to 20-residue peptides derived from the alpha4, alpha5, and alpha6 fibrils of type IV collagen, which we have designated tetrastatins, pentastatins, and hexastatins, respectively. We have shown that these endogenous peptides suppress the proliferation and migration of HUVECs in vitro. By performing clustering analyses of the sequences using sequence similarity criteria and of the experimental results using a hierarchical algorithm, we report that the clusters identified by the experimental results coincide with the sequence-based clusters, indicating a tight relationship between peptide sequence and anti-angiogenic potency. These peptides may have potential as anti-angiogenic therapeutic agents.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.     

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.     

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.     

New tools for the control of peptide conformation and supramolecular chemistry: crown-carrier, C(alpha)-methyl L-DOPA amino acids

The preferred conformation of five, terminally protected, model peptide series to the hexamer level, based on three novel crowned, C(alpha)-methyl L-DOPA amino acids combined with either L-Ala/Aib or Gly/Aib, were assessed in structure supporting solvents using FT-IR absorption, (1)H NMR, and CD techniques. The FT-IR absorption spectra strongly suggest that the contribution of the crowned C(alpha)-tetrasubstituted residue to intramolecular H-bonding is equivalent to that of Aib and is much more significant than that of either L-Ala or Gly. In addition, the (1)H NMR titrations and the CD patterns resemble those typically exhibited by (right-handed) 3(10)-helical structures.     

Peptides with indirect in vivo activity against an intracellular pathogen: selective lysis of infected macrophages.

A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50-80% alpha,alpha-dialkylated glycines (alpha,alpha-Dags) were synthesized on solid-phase resin as the C-terminus amides using N-alpha-fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in alpha,alpha-Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 microm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 microg for melittin, whereas 500 microg doses were nonlethal for many of the other peptides. Several of the alpha,alpha-Dag-rich peptides selectively destroy B. abortus-infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.     

Separation and chemical differentiation of alpha and beta subunits in yeast phosphofructokinase

The two types of subunits alpha and beta constitutive of yeast phosphofructokinase have been separated by ion-exchange chromatography under denaturating conditions. Amino acid analysis and peptide mapping were performed on the isolated subunits. The frequence of most of the amino acids significantly differs between the two types of polypeptide chains. Moreover, tryptic peptide maps of alpha and beta subunits are clearly not superimposable. These chemical differences seem sufficient to account for the distinct catalytic and regulatory functions of beta and alpha subunits in the yeast phosphofructokinase reaction.     

Theoretical prediction of the basic helix types in alpha,beta-hybrid peptides

This study provides a complete overview on all possible helical- folding patterns, their stabilities, and their detailed molecular structure in the novel foldamer class of alpha,beta-hybrid peptides on the basis of ab initio molecular orbital (MO) theory. The results indicate a considerable intrinsic potential of backbone folding. As found for other peptide foldamers, representatives of mixed or beta-helices are most stable in more apolar media, whereas polar environments favor the helices with the hydrogen bonds pointing in only one direction. The theoretical results confirm the hydrogen-bonding patterns found in the first experimental studies on these hybrid peptides. Selecting special backbone substitution patterns, the secondary structure potential of the alpha,beta-hybrid peptides could be of great importance for a rational peptide and protein design.     

Cloning and targeted mutations of G alpha 7 and G alpha 8, two developmentally regulated G protein alpha-subunit genes in Dictyostelium.

GTP-binding protein (G protein)-mediated signal transduction pathways play essential roles during the aggregation and differentiation process of Dictyostelium. In addition to the five known G protein alpha-subunit genes, we recently identified three novel alpha-subunit genes, G alpha 6, G alpha 7, and G alpha 8, using the polymerase chain reaction technique. We present here a more complete analysis of G alpha 7 and G alpha 8. The cDNAs of these two genes were cloned, and their complete nucleotide sequences were determined. Sequence analyses indicate that G alpha 8 possesses some unusual features. It lacks the "TCATDT" motif, a sequence of amino acids highly conserved among G alpha subunits, and has an additional 50 amino acids at its C-terminus consisting of long stretches of asparagine. Moreover, G alpha 8 is unusually resistant to protease digestion, which may indicate a slow GTP hydrolysis rate. The possible functions of these alpha-subunits were assessed by generating mutants lacking G alpha 7 or G alpha 8 by gene targeting through homologous recombination and by overexpressing G alpha 7 or G alpha 8 protein. Overexpression of G alpha 7 resulted in abnormal morphogenesis starting at the slug stage, whereas analysis of the other strains failed to reveal any obvious growth or developmental defects under either normal or stressful conditions. The implications of these results are discussed.