Comparison of the analgesic properties of lipotropin C-Fragment and stabilized enkephalins in the rat

The C-Fragment of lipotropin (LPH 61–91) was shown to produce strong and long-lasting analgesia on intraventricular infusion in the rat. Its analgesic properties were compared with those of three synthetic derivatives of methionine enkephalin (LPH 61–65) which had been stabilized against enzymic degradation by blocking one or both termini with N-methyl and C-amide groups. C-Fragment was approximately 50 times more potent on a molar basis than N-methyl methionine enkephalin amide. The singly blocked pentapeptides, like methionine enkephalin, produced little more than transient analgesia. It was concluded that the analgesic properties of C-Fragment depend on the length and nature of the peptide chain rather than on the resistance of its N-terminal pentapeptide to degradation.     

Ubiquitination of p53 at multiple sites in the DNA-binding domain

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.     

Isolation of the C-fragment and C''-fragment of lipotropin from pig pituitary and C-fragment from brain.

Three novel peptides derived from lipotropin, the C-Fragment (residues 61-91), C'-Fragment (61-87) and N-Fragment (1-38), were isolated from pig pituitary, and the C-Fragment was shown to be present in brain. The experimental procedures developed for their isolation are described. The formation of each of the fragments involves enzymic cleavage of lipotropin at consecutive basic residues, with specificity identical with that involved in the activation of known prohormones. In brain assays C-Fragment exhibits a range of biological activities related to its ability to act as an inhibitory neurotransmitter.     

Dependence creating properties of lipotropin C-fragment (beta-endorphin): evidence for its internal control of behavior

The C-fragment of lipotropin (β-endorphin) possesses reinforcing properties, in that this peptide, like heroin, induced intraventricular self-administering behavior in drug naive rats. Only mild behavioral signs reminiscent of physical dependence were present. After injection into the nucleus raphé magnus, C-Fragment appeared to act as a discriminative stimulus in rats trained to discriminate fentanyl from saline. These data indicate that naturally occuring C-Fragment exerts powerful control of behavior, which may be of significance for the understanding of the mechanisms underlying opiate dependence.     

Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity

Bacterially expressed fusion proteins containing the COOH-terminal domain of the human poly(ADP-ribose)polymerase were analyzed by means of a novel assay, the "activity blot," which allows the detection of transferred polypeptides involved in poly(ADP-ribose) synthesis. Deletion analysis demonstrated that the 40-kDa COOH-terminal region of the enzyme is an autonomous catalytic domain exhibiting both the polymerizing and branching activities in the absence of DNA. Site-directed mutagenesis demonstrated that lysine 893 is essential for these catalytic processes. In addition, sequence similarities obtained with the NAD(P)+ amino acid dehydrogenases suggest that (i) lysine 893 may interact with the substrates of poly(ADP-ribose)polymerase and (ii) the COOH-terminal part of the 40-kDa fragment may also contain a Rossman fold structure.     

Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.     

Structural studies of bovine heart cytochrome c1

The complete primary structure of bovine heart cytochrome c1 was established by analyses of peptide fragments prepared by digestion using trypsin, staphylococcal protease, and chymotrypsin and by cyanogen bromide cleavage of cytochrome c1 and its derivatives. The total number of amino acid residues is 241, giving a molecular weight of 27,924 including the heme group. The NH2- and COOH-terminal residues are serine and lysine, respectively. One characteristic of the protein is that cytochrome c1 contains 43.6% hydrophobic residues and the polarity is estimated to be 41.1%. No clear homology was found between cytochrome c1 and other membranous proteins such as cytochrome b5 or the subunits of cytochrome oxidase for which sequences have been reported. Cytochrome c1 is predicted to have a high content of alpha-helix (46%). Partial sequence studies were also carried out on cytochrome c1 preparations obtained by different procedures and showed that there is no difference among the sequences of various preparations of cytochrome c1. The presence of a hydrophobic cluster near the COOH-terminal region indicates that the COOH-terminal region of cytochrome C1 associates with, or is buried in, the phospholipid bilayer of the mitochondrial membrane.     

The carboxy-terminal amino acid sequence of rabbit liver fructose 1,6-bisphosphatase

A peptide derived from the COOH-terminus of rabbit liver fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) has been isolated and its amino acid sequence determined. The COOH-terminus is lysine, but some preparations contain COOH-terminal alanine or lysyl lysine. This region of the protein appears to be susceptible to modification by the action of an endogenous peptidyldipeptidase.     

Truncation of the COOH-terminal region of the paramyxovirus SV5 fusion protein leads to hemifusion but not complete fusion

The role of the simian virus 5 (SV5) fusion (F) protein 20 residue COOH- terminal region, thought to represent the cytoplasmic tail, in fusion activity was examined by constructing a series of COOH-terminal truncation mutants. When the altered F proteins were expressed in eukaryotic cells, by using the vaccinia virus-T7 transient expression system, all the F proteins exhibited similar intracellular transport properties and all were expressed abundantly on the cell surface. Quantitative and qualitative cell fusion assays indicated that all of the F protein COOH-terminal truncation mutants mediated lipid mixing with similar kinetics and efficiency as that of wild-type F protein. However, the cytoplasmic content mixing activity decreased in parallel with the extent of the deletion in the F protein COOH-terminal truncation mutants. These data indicate that it is possible to separate the presumptive early step in the fusion reaction, hemifusion, and the final stage of fusion, content mixing, and that the presence of the F protein COOH-terminal region is important for the final steps of fusion.     

The nature of post-translational formation of MM creatine kinase isoforms

Isoforms (derived from the same isoenzyme but distinguished by differences in isoelectric point) of MM creatine kinase appear in plasma after myocardial infarction. They are formed by conversion of the tissue form of creatine kinase (MM-A, pI 7.80) to progressively more acidic species (MM-B, pI 7.50) and MM-C (pI 7.20) after release into the circulation. To define the changes responsible for myocardial MM creatine kinase isoform formation in humans and dogs, purified isoforms were treated with trypsin or cyanogen bromide. The digests were fractionated by reverse-phase high pressure liquid chromatography. Comparison of proteolytic maps showed that MM-A and MM-C were each characterized by a single, unique peptide peak. Maps of MM-B creatine kinase contained both of these peaks. Sequence analysis and comparison with the complete amino acid sequence of MM creatine kinase showed that the peptide unique to MM-A corresponded to the COOH-terminal tryptic or CNBr peptide. The peptide unique to MM-C was shown to have the same amino acid composition except for lysine (the COOH-terminal amino acid). Thus, isoform formation is characterized by the successive removal of the COOH-terminal lysine residue from one M subunit at a time resulting in the conversion of MM-A to isoforms MM-B and MM-C.     

Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence

We have identified a novel peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human catalase. The last four amino acids of this protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter protein, chloramphenicol acetyl transferase. However, this PTS differs from the extensive family of COOH-terminal PTS tripeptides collectively termed PTS1 in two major aspects. First, the presence of the uncharged amino acid, asparagine, at the penultimate residue of the human catalase PTS is highly unusual, in that a basic residue at this position has been previously found to be a common and critical feature of PTS1 signals. Nonetheless, this asparagine residue appears to constitute an important component of the catalase PTS, in that replacement with aspartate abolished peroxisomal targeting (as did deletion of the COOH-terminal four residues). Second, the human catalase PTS comprises more than the COOH-terminal three amino acids, in that COOH-terminal-ANL cannot functionally replace the PTS1 signal-SKL in targeting a chloramphenicol acetyl transferase fusion protein to peroxisomes. The critical nature of the fourth residue from the COOH terminus of the catalase PTS (lysine) is emphasized by the fact that substitution of this residue with a variety of other amino acids abolished or reduced peroxisomal targeting. Targeting was not reduced when this lysine was replaced with arginine, suggesting that a basic amino acid at this position is required for maximal functional activity of this PTS. In spite of these unusual features, human catalase is sorted by the PTS1 pathway, both in yeast and human cells. Disruption of the PAS10 gene encoding the S. cerevisiae PTS1 receptor resulted in a cytosolic location of chloramphenicol acetyl transferase appended with the human catalase PTS, as did expression of this protein in cells from a neonatal adrenoleukodystrophy patient specifically defective in PTS1 import. Furthermore, through the use of the two-hybrid system, it was demonstrated that both the PAS10 gene product (Pas10p) and the human PTS1 receptor can interact with the COOH-terminal region of human catalase, but that this interaction is abolished by substitutions at the penultimate residue (asparagine-to- aspartate) and at the fourth residue from the COOH terminus (lysine-to-glycine) which abolish PTS functionality. We have found no evidence of additional targeting information elsewhere in the human catalase protein. An internal tripeptide (-SHL-, which conforms to the mammalian PTS1 consensus) located nine to eleven residues from the COOH terminus has been excluded as a functional PTS. Additionally, in contrast to the situation for S. cerevisiae catalase A, which contains an internal PTS in addition to a COOH-terminal PTS1, human catalase lacks such a redundant PTS, as evidenced by the exclusive cytosolic location of human catalase mutated in the COOH-terminal PTS. Consistent with this species difference, fusions between catalase A and human catalase which include the catalase A internal PTS are targeted, at least in part, to peroxisomes regardless of whether the COOH-terminal human catalase PTS is intact.     

Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

Characterization of rat heart tropoelastin

Several overlapping rat tropoelastin cDNA clones were isolated from a lambda gt11 rat heart cDNA library and their nucleotide sequence was determined. The corresponding deduced amino acid sequence of rat tropoelastin revealed strong homology to bovine and human tropoelastins although possessing some unique features including greater size (18%) and composition of repetitive units. Comparison of the amino acid sequence of rat tropoelastin to four other tropoelastin species reveals that the hydrophobic peptide repeat regions in the middle of each molecule and the crosslinking areas containing three lysine residues are remarkably conserved. A possible function for the clustering of three lysine residues in providing a mechanism for the in vivo reduction of dehydrolysinonorleucine via a redox shuttle with dihydrodesmosine is proposed. In addition, the COOH-terminal sequence of the rat tropoelastin is virtually identical to tropoelastins of other species in possessing a cysteine/arginine/lysine containing segment. There are no obvious amino acid insertions or substitutions in the COOH-terminal half of the rat tropoelastin molecule which would signal unique cleavage or glycosylation sites. Examination of the steady-state levels of rat tropoelastin mRNA in 8- and 12-day neonatal lung, heart, and aortic tissues showed that the amount of tropoelastin mRNA was abundant and of similar size (3.9 kb) in all three tissues.     

Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay

AIMS: To generate a stable Escherichia coli lysine auxotroph for the lysine bioavailability assay. METHODS AND RESULTS: An E. coli lysine auxotrophic strain was constructed by deleting the entire lysA gene and replacing it with a gene that confers resistance to ampicillin (bla). The linear DNA contained 50 bp homologous sequence of upstream of lysA in one end and 50 bp of downstream of lysA in the other end. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The deltalysA::bla strain exhibited a linear response to lysine supplementation and can be used for quantifying lysine.     

Identification of six phosphorylation sites in the COOH-terminal tail region of the rat neurofilament protein M.

The COOH-terminal tail domain of the neurofilament polypeptide M from rat nervous tissue contains approximately six molecules of phosphate. We report here that protein kinases in a crude cytoskeleton preparation of rat nervous tissue phosphorylated a set of tryptic peptides of M similar (but not identical) to those phosphorylated by living dorsal root ganglion cells in culture. Using these phosphopeptides as markers, we purified these same peptides from rat spinal cord and identified six specific phosphorylation sites in M by enzymatic and chemical criteria. These sites, serines 502, 506, 536, 606, 608, and 666, are all located in the COOH-terminal tail domain. Four are embedded in the repeated motif KSP whereas two are within variants of this motif, KSD and ESP. All of the sites that were preceded by lysine were resistant to alkaline phosphatase prior to modification of the lysine with citraconic anhydride. The identification of these sites should aid in investigations of the function of the phosphorylation of this protein and provides criteria for identifying the relevant kinases.     

A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance

In fungal species, lysine 56 of newly synthesized histone H3 molecules is modified by the acetyltransferase Rtt109, which promotes resistance to genotoxic agents. To further explore how H3 K56ac contributes to genome stability, we conducted screens for suppressors of the DNA damage sensitivity of budding yeast rtt109Δ mutants. We recovered a single extragenic suppressor mutation that efficiently restored damage resistance. The suppressor is a point mutation in the histone H3 gene HHT2, and converts lysine 56 to glutamic acid. In some ways, K56E mimics K56ac, because it suppresses other mutations that interfere with the production of H3 K56ac and restores histone binding to chromatin assembly proteins CAF-1 and Rtt106. Therefore, we demonstrate that enhanced association with chromatin assembly factors can be accomplished not only by acetylation-mediated charge neutralization of H3K56 but also by the replacement of the positively charged lysine with an acidic residue. These data suggest that removal of the positive charge on lysine 56 is the functionally important consequence of H3K56 acetylation. Additionally, the suppressive function of K56E requires the presence of a second H3 allele, because K56E impairs growth when it is the sole source of histones, even more so than does constitutive H3K56 acetylation. Our studies therefore emphasize how H3 K56ac not only promotes chromatin assembly but also leads to chromosomal malfunction if not removed following histone deposition.     

Regulation of dihydrodipicolinate synthase and aspartate kinase in Bacillus subtilis.

The regulation of dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4) was studied in Bacillus subtilis 168. Starvation for lysine gave depression of one aspartate kinase isoenzyme but not of dihydrodipicolinate synthase. Strains resistant to growth inhibition by the lysine analogue thiosine exhibited constitutively derepressed synthesis of one aspartate kinase isoenzyme but had normal levels of dihydrodipicolinate synthase. The data provide strong evidence that lysine is not the signal for derepression of dihydrodipicolinate synthase. Nevertheless, dihydrodipicolinate synthase specific activity increased during sporulation, and it is suggested that this increase may result, in part, from resistance to proteolysis of that enzyme.     

Amino acid sequence of beta-galactosidase. X. Sequence of the COOH-terminal segment, CNBr peptides 18 to 24, residues 654 to 1021.

The sequence of the COOH-terminal third (omega) of beta-galactosidase is presented. The size of the 7 cyanogen bromide peptides of this segment is larger on the average, about 52 amino acid residues as compared to an average size of 42 for cyanogen bromide peptides in the whole molecule. Tyrosine, phenylalanine, and valine are low in this segment whereas alanine and lysine are high. This region has a slight excess of basic groups.     

Salt-dependent structural changes of neurohormones: lithium ions induce conformational rearrangements of ocytocin to a vasopressin-like structure

The preferred average conformation and structural subdomain interactions of the nonapeptide hormones vasopressin and ocytocin have been analyzed through the determination of their hydrodynamic volume and the thermal coefficient of the frictional resistance to rotation of their tyrosine residue. A spherical gross shape and an ellipsoidal gross shape were assessed respectively for ocytocin and vasopressin by fluorescence polarization analysis. Investigation of the thermal coefficient of viscosity and the critical temperature of both hormones and analogues indicated that strong interactions hold together the two structural subdomains of ocytocin (the flexible six-membered ring and the COOH-terminal tripeptide tail). An opposite situation was found in the case of vasopressin where such interactions could not be detected between the rigid ring and the flexible COOH-terminal tail. Lithium ions were shown to promote ocytocin binding to specific neurophysin sites restricted, under standard conditions, to vasopressin. In the presence of lithium, the gross conformational shape of ocytocin becomes similar to that of vasopressin but in the absence of salt. In addition, the ocytocin ring becomes more rigid in the presence of lithium while decreasing interactions between the ring and the COOH-terminal tail were detected. It is proposed that lithium ions induce specific conformational rearrangements of ocytocin toward a vasopressin-like structure, allowing recognition of this hormonal ligand by a specific vasopressin binding domain of neurophysins.     

Molecular genetic and biochemical analyses of FGF23 mutations in familial tumoral calcinosis

Fibroblast growth factor 23 (FGF23) is a hormone required for normal renal phosphate reabsorption. FGF23 gain-of-function mutations result in autosomal dominant hypophosphatemic rickets (ADHR), and FGF23 loss-of-function mutations cause familial hyperphosphatemic tumoral calcinosis (TC). In this study, we identified a novel recessive FGF23 TC mutation, a lysine (K) substitution for glutamine (Q) (160 C>A) at residue 54 (Q54K). To understand the molecular consequences of all known FGF23-TC mutants (H41Q, S71G, M96T, S129F, and Q54K), these proteins were stably expressed in vitro. Western analyses revealed minimal amounts of secreted intact protein for all mutants, and ELISA analyses demonstrated high levels of secreted COOH-terminal FGF23 fragments but low amounts of intact protein, consistent with TC patients' FGF23 serum profiles. Mutant protein function was tested and showed residual, yet decreased, bioactivity compared with wild-type protein. In examining the role of the FGF23 COOH-terminal tail (residues 180-251) in protein processing and activity, truncated mutants revealed that the majority of the residues downstream from the known FGF23 SPC protease site ((176)RXXR(179)/S(180)) were not required for protein secretion. However, residues adjacent to the RXXR site (between residues 188 and 202) were required for full bioactivity. In summary, we report a novel TC mutation and demonstrate a common defect of reduced FGF23 stability for all known FGF23-TC mutants. Finally, the majority of the COOH-terminal tail of FGF23 is not required for protein secretion but is required for full bioactivity.