Structural requirements of lysophospholipid-regulated mitochondrial Ca2+ transport

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca2+ transport and membrane potential. Lysophospholipids released Ca2+ from mitochondria and reduced the maximal Ca2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C1-position of the glycerol backbone and residues of small size and/or low polarity at the C2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholipase A2 activity and an impairment of mitochondrial function are supposed to play a crucial role.     

Structural requirements of ceramide and sphingosine based inhibitors of mitochondrial ceramidase

The effects of structural analogues of ceramide on rat brain mitochondrial ceramidase (mt-CDase) were investigated. Design of target compounds was mainly based on modifications of the key elements in ceramide and sphingosine, including stereochemistry, the primary and secondary hydroxyl groups, the trans double bond in the sphingosine backbone, and the amide bond. Mt-CDase was inhibited by (1) all stereoisomers of D-erythro-ceramide (D-e-Cer) with an IC50 of 0.11, 0.21, and 0.26 mol % for the L-threo, D-threo, and L-erythro isomers, respectively; (2) all stereoisomers of sphingosine with IC50 ranging from 0.04 to 0.14 mol %, N-methyl-D-erythro-sphingosine (N-Me-Sph, IC50 0.13 mol %); and (3) D-erythro-urea-C16-ceramide (C16-urea-Cer IC50 0.33 mol %). The enzyme was not inhibited by N-methyl ceramide (N-Me-C16-Cer), 1-O-methyl ceramide (1-O-Me-C16-Cer), 3-O-methyl ceramide (3-O-Me-C16-Cer), cis-D-erythro ceramide (cis-D-e-C16-Cer) and 3-O-methyl-D-erythro-sphingosine (3-O-Me-Sph). It was less potently inhibited by D-erythro-sphinganine (D-e-dh-Sph, IC50 0.20 mol %), D-erythro-dehydro sphingosine (D-e-deh-Sph, IC50 0.25 mol %), (2S)-3-keto-sphinganine (3-keto-dh-Sph, IC50 0.34 mol %), (2S) 3-keto-ceramide (3-keto-C16-Cer, IC50 0.60 mol %), and ceramine (C18-ceramine, IC50 0.62 mol %), 1-O-methyl-D-erythro-sphingosine (1-O-Me-Sph), cis-D-erythro-sphingosine (cis-D-e-Sph), (2S)-3-ketosphingosine (3-keto-Sph), (2S)-3-keto-dehyrosphingosine (3-keto-deh-Sph), and N,N-dimethyl-D-erythrosphingosine (N,N-diMe-Sph) were weak inhibitors whereas ceramide-1-phosphate (Cer-1-P) and sphingosine-1-phosphate (Sph-1-P) stimulated the enzyme. Thus, for inhibition, the enzyme requires the primary and secondary hydroxyl groups, the C4-C5 double bond, the trans configuration of this double bond, and the NH-protons from either the amide of ceramide or the amine of sphingosine. Therefore, these results provide important information on the requirements for ceramide-enzyme interaction, and they suggest that ligand interaction with the enzyme occurs in a high affinity low specificity manner, in contrast to catalysis which is highly specific for D-erythro-ceramide (D-e-Cer) but occurs with a lower affinity. In addition, this study identifies two competitive inhibitors of mt-CDase; urea-ceramide (C16-urea-Cer) and ceramine (C18-ceramine) that may be further developed and used to understand the mechanism of mt-CDase in vitro and in biologic responses.     

Structural requirements of mitochondrial D-beta-hydroxybutyrate dehydrogenase with respect to its coenzyme

The purpose of this work was to test structural analogs of NAD+ in order to know enzyme requirements of chemical structure of coenzyme to get catalytic activity and, in a other hand to see which chemical parts of the coenzyme were involved in the coenzyme binding to the active site. The binding of the coenzyme analog at the catalytic site requires an adenosine diphosphoribose structure without any additional phosphate group on the ribose linked to adenine.     

Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis.

The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.     

Structural requirements for heparan sulphate self-association

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.     

Structural requirements for CD43 function

The regulation of T cell activation and adhesion by CD43 (leukosialin, sialophorin) has been thought to be mainly a function of the large size and negative charge of the extracellular domain of the protein. In this work, we demonstrate that the cytoplasmic tail is both necessary and sufficient for the negative regulatory effect of CD43 on cell-cell adhesion. Expression of mutant CD43 proteins in primary T cells from CD43-deficient mice demonstrated that the antiproliferative effect of CD43 is also dependent upon the cytoplasmic tail. In contrast, Ab-mediated costimulation through CD43 does not require the intracellular domain of CD43. These data demonstrate that CD43 primarily serves as a negative regulator of T cell activation and adhesion, and that this is mediated not exclusively by passive effects of the extracellular domain, but requires participation of the cytoplasmic tail, perhaps through interactions with the cytoskeleton, or alternatively, active regulation of intracellular signaling pathways.     

Structural requirements for formyl homooligopeptide chemoattractants

Using solution peptide synthesis, we have made three series of N alpha-formylated homooligopeptides, from the dipeptide to the heptapeptide, derived from L-methionine, L-norleucine, and S-methyl-L-cysteine and related to the chemotactic peptide N alpha-formylmethionylleucylphenylalanine. Compounds were prepared to determine the combined effects of the main-chain length and the presence of a sulfur atom in side-chain gamma- and delta-positions. Each peptide was tested for its ability to induce rabbit peritoneal polymorphonuclear leukocytes in the presence of cytochalasin B to secrete granule enzymes. In parallel, a conformational analysis was carried out in the solid state and in solution, using infrared absorption and circular dichroism. We examined these peptides in solvents of widely different polarities, i.e., chloroform, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoropropan-2-ol, and mixed organic-aqueous media. The tendencies to form antiparallel-chain beta-associated and folded structures were determined. The biological and conformational data are described in terms of a model of the chemotactic peptide receptor of rabbit neutrophils recently proposed by Freer et al. (1982) [Freer, R.J., Day, A.R., Muthukumarswamy, N., Pinon, D., Wu, A., Showell, H.J., & Becker, E.L. (1982) Biochemistry 21, 257-263]. In the three N alpha-formylated C-methoxy homooligopeptide series tested, the highest level of activity attained is at the tetrapeptide or pentapeptide stage, confirming the suggestion that the formylpeptide receptor is large enough to accommodate a peptide with at least four amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

Structural requirements for a primitive adaptor molecule

Adaptor properties of linear hairpin helices have been examined. The analysis suggests that neither right nor left handed hairpin helices can simultaneously read a comma free messenger and align aminoacyl residues for peptide condensation. Comparison of these studies with the model of the present day peptidyl transfer intermediate suggests that the "L" shaped folding of the present day tRNAs may be a prerequisite for adaptor function. Therefore, the three-dimensional organization of the ancestral adaptor molecule must have had structural features similar to its present day counterpart.     

Structural requirements for the binding of prostaglandins

The binding of prostaglandins and analogs to the lipocyte PGE receptor was shown to exhibit a high degree of structural specificity. Small changes, particularly at the 9-keto or 15-hydroxy position, were found to drastically diminish interaction with the receptor. Studies of a rather substantial number of compounds revealed a close relationship between affinity for the lipocyte PGE receptor and the ability to stimulate cyclic AMP synthesis in the isolated mouse ovary. In general, activities in these two parameters follow the biological potencies generally recognized for these compounds.     

Structural requirements for efficient prion protein conversion

To understand why cross species infection of prion disease often results in inefficient transmission and reduced protein conversion, most research has focussed on defining the effect of variations in PrP primary structures, including sequence compatibility of substrate and seed. By contrast, little research has been aimed at investigating structural differences between different variants of PrP^C and secondary structural requirements for efficient conversion. This is despite a clear role for molecular chaperones in formation of prions in non-mammalian systems, indicating the importance of secondary/tertiary structure during the conversion process. Recent data from our laboratory on the cellular location of disease-specific prion cofactors supports the critical role of specific secondary structural motifs and the stability of these motifs in determining the efficiency of disease-specific prion protein conversion. In this paper we summarise our recent results and build on the hypothesis previously suggested by Wuthrich and colleagues, that stability of certain regions of the prion protein is crucial for protein conversion to abnormal isoforms in vivo. It is suggested that one role for molecular co-factors in the conversion process is to stabilise PrP^C structure in a form that is amenable for conversion to PrP^Sc.     

Structural requirements for dermorphin opioid receptor binding

Structural features influencing binding activity of dermorphin to opioid receptors have been investigated in the rat brain through the synthesis and evaluation of binding affinity of a series of synthetic dermorphin analogs. Tritiated dermorphin was used as primary ligand. The single population of high affinity dermorphin binding sites present in the rat brain is clearly of an opioid nature since bound radiolabeled dermorphin was fully displaced with high affinity either by morphine or naloxone. Displacement of tritiated dermorphin by all alkaloid opiates or dermorphin related peptides tested was monophasic, consistent with simple competitive inhibition at a single population of binding sites. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) was the most potent competitor in all experiments. The D-configuration of the amino acid residue in position 2 was found to be of crucial importance for binding. Replacement of D-Ala2 with L-Ala led to a deleterious effect, this analog being 1/5000th as potent as dermorphin in displacing bound tritiated dermorphin from its receptor. Shorter dermorphin homologs, dermorphin-(1-4)-NH2 and dermorphin-(1-3)-NH2, were found to be 20 and 40-fold less potent, respectively, than dermorphin. The C-terminal carboxamide function is of significant importance for manifestation of the full intrinsic binding potency of dermorphin. Deamidated dermorphin had 1/5th the potency of the parent peptide. This suggests that while the whole dermorphin sequence is required for the expression of the full intrinsic binding activity of the molecule, the N-terminal tripeptide is a key structure as it contains the features which allow receptor recognition.     

Biochemical requirements for the maturation of mitochondrial c-type cytochromes

Cytochromes c are metalloproteins that function in electron transfer reactions and contain a heme moiety covalently attached via thioether linkages between the co-factor and a CXXCH motif in the protein. Covalent attachment of the heme group occurs on the positive side of all energy-transducing membranes (bacterial periplasm, mitochondrial intermembrane space and thylakoid lumen) and requires minimally: 1) synthesis and translocation of the apocytochromes c and heme across at least one biological membrane, 2) reduction of apocytochromes c and heme and maintenance under a reduced form prior to 3) catalysis of the heme attachment reaction. Surprisingly, the conversion of apoforms of cytochromes c to their respective holoforms occurs through at least three different pathways (systems I, II and III). In this review, we detail the assembly process of soluble cytochrome c and membrane-bound cytochrome c1, the only two mitochondrial c-type cytochromes that function in respiration. Mitochondrial c-type cytochromes are matured in the intermembrane space via the system I or system III pathway, an intriguing finding considering that the biochemical requirements for cytochrome c maturation are believed to be common regardless of the energy-transducing membrane under study.     

Structural requirements for the antitubercular quaternized triflupromazine pharmacophore

Quaternized triflupromazine derivatives (QTDs) must possess benzyl groups attached to the quaternary nitrogen in order to have significant antitubercular potency. Replacing the quaternary amine with a triazole abolishes antitubercular activity. A modest halogen substitution effect exists, with the 4-bromophenyl QTD 3 having the best selectivity index (>21). All N-benzyl QTDs 1-4 similarly inhibit non-replicating, persistent Mycobacterium tuberculosis with MIC<8μM, and compounds 1-3 were all nontoxic to mammalian cells in vitro (IC(50)>128μM).     

Structural requirements for nucleophilic substrates of carboxypeptidase Y

The composition and structural aspects of the amino and carboxylic acid groups required for incorporation into peptides by transpeptidation and inhibition of hydrolysis in carboxypeptidase Y-catalyzed reactions were studied. Separation of these two groups by even one carbon prevents incorporation by transpeptidation and does not inhibit incorporation of other amino acids into model peptides. Substitution of phosphonic or sulfonic acids for the carboxylic acid group also results in loss of incorporation by transpeptidation. Only the sulfonic acid analog of glycine causes inhibition of hydrolysis and this inhibition is lost when serine is included in the reaction. d-Serine is not incorporated by carboxypeptidase Y, and its presence in the reaction mixture does not inhibit the incorporation of the L-isomer.     

Structural requirements for conserved arginine of parathyroid hormone

Arg-20 is one of two residues conserved in all peptides known to activate the parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclase (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogues with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In descending order of activity, the most active analogues had (S)-4-piperidyl-(N-amidino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (Orn) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N-amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]butanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible activity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. The conformational constraints imposed by the PipGly ring had a negative effect on its ability to substitute for Arg. The side-chain H-bonding potential of the Cit ureimido group was likely an important factor in its mimicry of Arg. The increase in amphiphilicity, as demonstrated by its greater high-performance liquid chromatographic retention, and increased alpha-helix, as shown by circular dichroic spectroscopy, likely contributed to the activity of the Nle-20 analogue. The data demonstrated that specific H-bonding, hydrophobicity of the side chain, stabilization of alpha-helix, and possibly specific cation positioning were all important in the interaction of Arg-20 with receptor groups.