Synthetic peptides based upon a three-dimensional model for the receptor recognition site of follicle-stimulating hormone exhibit antagonistic or agonistic activity at low concentrations.

Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated alpha and beta. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10(-4) to 10(-5) M) relative to the hormone itself (10(-8) to 10(-11) M). This suggests that the peptides represent only a portion of a larger recognition site in the intact hormone that comprises parts of both the beta and the alpha chains. To develop peptides that exhibit FSH-antagonistic activity at low concentrations, we have constructed a three-dimensional model for FSH, which is based on an alignment of both the beta and the alpha chains of glycoprotein hormones with thioredoxin, for which x-ray diffraction data are available. This model resulted in the prediction of a conformational receptor-binding site in FSH, in which (parts of) three earlier proposed binding regions on the FSH molecule [namely, the regions FSH alpha-(34-37), with the amino acid sequence SRAY; FSH beta-(40-43), with the amino acid sequence TRDL; and FSH beta-(87-94), the "determinant loop" with the amino acid sequence CDSDSTDC] are located within 10 A of one another. On the basis of this model, peptides have been synthesized in which two of these binding regions are linked by a synthetic amino acid whose length was derived from the model, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH2 and Ac-SRAY-NH-(CH2)4-CO-TRDL-NH2. Both peptides inhibited FSH-induced cAMP production in Sertoli cells at 1000-fold lower concentrations (10(-7) M) than the peptides Ac-TRDL-NH2, Ac-SRAY-NH2, or Ac-TDSDS-NH2. In another peptide, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH-(CH2)4-CO-TRDL-NH2, all three binding regions have been linked. This peptide appeared to be a strong agonist of FSH action, as measured by the ability to stimulate cAMP production, at concentrations as low as 10(-7) M. The observation that a synthetic peptide, in which (parts of) three earlier described receptor interaction sites are combined according to the three-dimensional model, can mimic the action of FSH, at 10(-7) M, shows that this model is useful to predict a conformational receptor-binding site in FSH and that combination of only a few amino acid residues from the alpha and beta chains of FSH in a small synthetic peptide is sufficient to transduce a signal upon binding to the receptor.     

Conformational epitopes detected by cross-reactive antibodies to envelope 2 glycoprotein of the hepatitis C virus.

The prevalence of anti-E2 antibody in persons chronically infected with hepatitis C virus (HCV) is high irrespective of viral genotype, and this cross-reactive antibody is thought to react with a conformational epitope. To investigate the characteristics of this anti-E2 antibody, the immunoreactivity of sera from HCV-1b-infected patients was measured against various modified forms of E2 glycoprotein derived from HCV-H (genotype 1a) by an immunofluorescence technique. Twelve of 18 patients were positive for anti-E2 antibody, and 10 of the 12 required a minimal amino acid (aa) region including aa 406-644 for strong reactivity, suggesting that the major E2 antibody has a conformational epitope in this region. Subsequent analysis using mutant E2 glycoproteins designed to lose N-glycosylation potential at varying sites revealed seven important N-glycosylation sites in this region. Four of these (aa 423, 430, 448, and 576) are indispensable for an antibody response.     

Plasminogen: a structural review.

Plasminogen is the zymogen form of plasmin, a broad specificity serine protease whose activity contributes to a variety of normal and pathological conditions, including intravascular thrombolysis and extracellular proteolysis. Plasminogen contains seven structural units or 'domains', each of which confer specific properties on the molecule. The kringle domains possess fibrin-binding functions and, together with the N-terminal peptide, regulate the ability of plasminogen to adopt at least three dissimilar conformations. These conformational forms influence the rate of formation, following activation by plasminogen activators, of the plasmin active site within its C-terminal serine protease domain. Structural and functional analogies are postulated between these plasminogen structures and the conformations of other proteins related by sequence homology.     

Structural effects of substitutions on the p21 proteins.

The conformational effects of different amino acid substitutions (lysine, serine, proline, and D-valine) for glycine at position 12 in the p21 oncogene-encoded proteins have been investigated by using conformational energy calculations. The normal cellular gene codes for a glycine at position 12 in the amino acid sequence, in the middle of a hydrophobic p21-(6-15)-decapepetide from Leu-6 to Gly-15. Mutations that cause amino acid substitutions for Gly-12 result in a protein product that produces malignant transformation of cells. We now find that not only are the preferred structures for the lysine- and serine-containing peptides more restricted and more helical than those for the glycine-containing peptide, but the lowest-energy structure for each substituted peptide is exactly the same as that previously found for the peptide with Val-12, suggesting the existence of a "malignancy-causing" conformation. None of the preferred conformations for the valine-, lysine-, and serine-containing peptides contain chain reversals at positions 11 and 12. However, we find that proline, unlike these residues but like glycine, at position 12 causes helix termination at positions 11 and 12, a result that suggests that the p21 protein product with proline at position 12 may exhibit lowered transforming potential, in agreement with the results of recent genetic recombination experiments.     

Amino-acid sequence of equine renal metallothionein-1B.

The amino-acid sequence of a metallothionein is reported. Metallothionein is a widely distributed, extremely cysteine-rich, low-molecular-weight protein containing large amounts of cadmium and/or zinc. Metallothionein-1B is one of the two prinicipal variants occurring in equine kidney cortex. The single-chain protein contains 61 amino acids and has the composition Cys20 Ser8Lys7Arg1Ala7Gly5Val3Asp2Asn1-Glu1Gln2Pro2Thr1Met1(Cd + Zn)7. Its amino-terminal residue is N-acetylmethionine. The sequence shows distinct clustering of the twenty cysteinyl residues into seven groups separated by stretches of at least three other residues. Within these groups the cysteines occur seven times in alternating Cys-X-Cys sequences and three times each in Cys-Cys and Cys-X-X-Cys sequences, where X is an amino acid other than cysteine. Another unique feature is the close association of serine and the basic amino acids with cysteine, as manifested by the occurrence of seven Ser-Cys, four Cys-Lys, one Cys-Arg, and three Lys-Cys sequences. These findings are in agreement with the previous suggestion that metallothionein has structurally defined metal-binding sites, most of which contain three cysteinyl residues as the principal metal-binding ligands. The charge difference between the metal-free and the metal-containing protein is consistent with the formation of negatively charged trimercaptide complexes with cadmium and/or zinc ions. The possible additional involvement of serine and the basic amino acids in metal binding is discussed.     

Designing amino acids to determine the local conformations of peptides.

The local conformations of proteins and peptides are determined by the amino acid sequence. However, the 20 amino acids encoded by the genome allow the peptide backbone to fold into many conformations, so that even for a small peptide it becomes very difficult to predict the three-dimensional structure. By using empirical conformational energy calculations, a set of amino acids has been designed that would be expected to constrain the conformation of a peptide or a protein to one or two local minima. Most of these amino acids are based on asymmetric substitutions at the C alpha atom of each residue. The H alpha atom of alanine was replaced by various groups: -OCH3, -NCH3, -SCH3, -CONH2, -CONHCH3, -CON(CH3)2, -NH.CO.CH3, -phenyl, or -o-(OCH3)phenyl. Several of these new amino acids are predicted to fold into unique peptide conformations such as right-handed alpha-helical, left-handed alpha-helical, or extended. In an attempt to produce an amino acid that favored the C(eq)7 conformation (torsion angles: phi = -70 degrees and psi = +70 degrees), an extra amide group was added to the C beta atom of the asparagine side chain. Conformationally restricted amino acids of this type could prove useful for developing new peptide pharmaceuticals, catalysts, or polymers.     

Phosphorylation controls binding of acidic proteins to the ribosome.

The replacement of each one of the eight serine residues present in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal phosphoprotein YP2 beta (L45) by different amino acids has been performed by heteroduplex site-directed mutagenesis in the cloned gene. The mutated DNA was used to transform a yeast strain previously deprived of the original protein YP2 beta (L45) by gene disruption. The replacement of serine in position 19 by either alanine, aspartic acid, or threonine prevents in vivo phosphorylation of the protein and its interaction with the ribosome. The serine-19 mutated gene is unable to rescue the negative effect on the growth rate caused by elimination of the original protein in YP2 beta (L45) gene disrupted strains. The mutation of any one of the other seven serine residues has no effect on either the phosphorylation or the ribosome binding capacity of the protein, although replacement of serine-72 seems to increase the sensitivity of the polypeptide to degradation. These results provide strong evidence indicating that ribosomal protein phosphorylation plays an important part in the activity of the particle and that it supports the existence of a control mechanism of protein synthesis, which would regulate the level of phosphorylation of acidic proteins.     

Amino acid synthesis deficiencies

In recent years the number of disorders known to affect amino acid synthesis has grown rapidly. Nor is it just the number of disorders that has increased: the associated clinical phenotypes have also expanded spectacularly, primarily due to the advances of next generation sequencing diagnostics. In contrast to the “classical” inborn errors of metabolism in catabolic pathways, in which elevated levels of metabolites are easily detected in body fluids, synthesis defects present with low values of metabolites or, confusingly, even completely normal levels of amino acids. This makes the biochemical diagnosis of this relatively new group of metabolic diseases challenging. Defects in the synthesis pathways of serine metabolism, glutamine, proline and, recently, asparagine have all been reported. Although these amino acid synthesis defects are in unrelated metabolic pathways, they do share many clinical features. In children the central nervous system is primarily affected, giving rise to (congenital) microcephaly, early onset seizures and varying degrees of mental disability. The brain abnormalities are accompanied by skin disorders such as cutis laxa in defects of proline synthesis, collodion-like skin and ichthyosis in serine deficiency, and necrolytic erythema in glutamine deficiency. Hypomyelination with accompanying loss of brain volume and gyration defects can be observed on brain MRI in all synthesis disorders. In adults with defects in serine or proline synthesis, spastic paraplegia and several forms of polyneuropathy with or without intellectual disability appear to be the major symptoms in these late-presenting forms of amino acid disorders. This review provides a comprehensive overview of the disorders in amino acid synthesis.     

Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin).

The amino acid sequence of galactoglycoprotein purified from human plasma was elucidated to 75% completeness by using chemical degradation of peptides and glycopeptides derived from digests of the protein with seven specific proteases. This sequence represents a polypeptide chain of approximately 220 amino acid residues including a high content of serine, threonine, alanine, and proline with one N-linked and multiple O-linked glycans. Comparison of peptide sequences from the native galactoglycoprotein and the deglycosylated derivative demonstrated the locations of 25 sites of O-glycosylation and three serine sites that are not glycosylated. The homogeneous N terminus was established as serine. C-terminal analysis revealed multiple C-terminal residues, suggesting that galactoglycoprotein molecules are of varying lengths. A search of a protein data base revealed that the galactoglycoprotein polypeptide is identical to the N-terminal (extracellular) polypeptide region of the blood-cell surface molecule CD43 (sialophorin, leukosialin). Further support of the relatedness of these molecules was obtained by immunoprecipitation of 125I-labeled galactoglycoprotein by monoclonal anti-CD43 antibodies. The composition and properties of the molecules together with the known structure of the gene encoding CD43 suggest that galactoprotein is derived by proteolytic cleavage from transmembrane "hexasaccharide CD43," known to be expressed on neutrophils, activated T lymphocytes, and platelets.     

A developmentally regulated mRNA from 3T3 adipocytes encodes a novel serine protease homologue.

We previously have isolated cDNA clones for several mRNAs that increase in abundance during the differentiation of 3T3 adipocytes but whose physiological role is unknown. We show here that a mRNA that is complementary to one of these clones and encodes a protein of 28 kDa is expressed abundantly in mouse fat pads but not in several other mouse tissues. Sequence analysis of the corresponding cDNA clone indicated that the encoded protein shows 30% overall amino acid homology to several serine proteases including trypsin, chymotrypsin, and elastase. Homology is much higher (64%) between the 28-kDa protein and regions that are strongly conserved among the members of the serine protease family. The derived protein also has key features characteristic of active serine proteases, including the histidine, aspartic acid, and serine residues, which comprise the charge relay system, and a potential cleavage site for activation of the zymogen. Primer extension analysis performed to obtain the sequence of the 5' end of mRNA that encodes the 28-kDa protein indicates that two forms of this mRNA exist and probably arise through alternative splicing. The two mRNAs encode signal sequences that differ by the deletion of one amino acid near the predicted cleavage site of the signal peptide. These results demonstrate that adipocyte differentiation is accompanied by the expression of mRNA encoding a serine protease homologue that can be synthesized with two different signal peptides.     

Effect of single amino acid substitutions on the formation of the PlA and Bak alloantigenic epitopes.

The subunits that comprise the platelet-specific integrin alpha IIb beta 3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PlA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PlA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PlA2-associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PlA)-specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PlA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PlA1 and PlA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 in equilibrium Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes.     

Effect of IGF-I on serine metabolism in fetal sheep

Acute infusion of IGF-I to the fetus has been shown to inhibit amino acid oxidation and appears to increase fetoplacental amino acid uptake. This study was designed to investigate further the effects of IGF-I on fetal amino acid metabolism. Radiolabeled serine was used to test the hypothesis that fetal IGF-I infusion enhances serine uptake into the fetus and/or placenta and inhibits serine oxidation. Eight fetal sheep were studied at 127 days of gestation before and during a 4-h infusion of IGF-I (50 microg/h per kg). During the infusion there was no change in uptake of serine or its oxidation by fetus or placenta. However, both uptake and oxidation of serine and glycine decreased in the fetal carcass. There was also a decrease in fetal blood serine and glycine concentrations which could indicate a decrease in protein breakdown, although reduced amino acid synthesis cannot be excluded. Thus IGF-I appeared to influence the distribution of these amino acids as oxidative substrates between different fetal tissues. In addition, fetal IGF-I infusion increased the conversion of serine to glycine which is likely to have increased the availability of one-carbon groups for biosynthesis. Our data provide further evidence that IGF-I plays a role in the regulation of fetoplacental amino acid metabolism.     

Functional dissociation between proforms and mature forms of proteinase 3, azurocidin,and granzyme B in regulation of granulopoiesis

OBJECTIVE: We previously demonstrated that secreted proform(s) of the neutrophil serine protease PR3 (proteinase 3) can down-modulate the fraction of normal human colony-forming unit granulocyte-macrophage (CFU-GM) in S-phase, whereas PR3 extracted from mature neutrophils lacks this ability. The objective of this study was to characterize the structural and functional dissociation between secreted proforms and granule-stored mature forms and to extend the investigation to other related hematopoietic serine proteases. MATERIALS AND METHODS: Conditioned media containing secreted proteases from transfectant cell lines with stable expression of human PR3, neutrophil elastase, cathepsin G, azurocidin, and granzymes A, B, H, K, and M were tested for their ability to reduce the fraction of normal human CFU-GM in S phase. Furthermore, recombinant PR3, azurocidin, and granzyme B with defined N-terminal propeptides, and the respective mature forms without propeptide, were functionally characterized. RESULTS: In addition to PR3, secreted proforms of azurocidin and granzymes A, B, H, K, and M, but not cathepsin G or neutrophil elastase, have S-phase reducing activity. This activity is restricted to the dipeptide proforms, whereas mature forms without propeptide have no S-phase reducing activity. On the other hand, only the mature forms of PR3 and granzyme B could bind the serine protease inhibitor diisopropylfluorophosphate (DFP), or aprotinin in the case of azurocidin. We also demonstrate that granulocyte colony-stimulating factor-stimulated CD34+ cells and interleukin-2-stimulated lymphocytes secrete active proforms of PR3 and granzyme B, respectively. CONCLUSION: These results demonstrate distinctive functional and conformational differences between proforms and mature forms of these hematopoietic serine proteases and suggest novel growth regulatory mechanisms in granulopoiesis.     

Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the alpha1 and alpha2 helices (Deltaloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Deltaloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel induced about one-third the level of apoptosis seen with wild-type Bcl-2. To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K-->R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) had an effect on Bcl-2 phosphorylation. Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-70; forms of Bcl-2 lacking the loop region are much more effective at preventing apoptosis than wild-type Bcl-2 because they cannot be phosphorylated. JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.     

Protein balance during very-low-calorie diets for the treatment of severe obesity

Very-low-calorie diets (less than 500 kcal/day; VLCD) are widely used for the treatment of severe obesity. We report the effects of such diets, consisting of proteins only or proteins and carbohydrates (CH), on nitrogen balance and protein nutritional status of morbidly obese patients. Cumulative nitrogen loss, serum albumin, transferrin, prealbumin (PA) and retinol-binding protein (RBP) concentrations, and plasma amino acid profile were determined in two groups of obese patients: 5 subjects (3 women, 2 men: BMI 55.3 +/- 2.2 kg/m2) subjected for 4 weeks to a protein VLCD (40 g protein + 2 g fat) and 7 others (4 women, 3 men: BMI 45.6 +/- 2.8 kg/m2) received for the same length of time a protein + CH VLCD (34 g protein + 26 g CH). Nitrogen balance was determined weekly whilst plasma and serum variables were measured on days 0, 3, 5, 10, 20 and 28 of treatment. Nitrogen balance did not significantly differ between the two groups of patients throughout the treatment. Serum PA and RBP concentrations decreased from day 5 and day 10, respectively, in both groups. Plasma amino acids showed a similar pattern in the protein and protein + CH groups. Alanine gradually decreased below baseline values; after a peak value on day 5, branched-chain amino acids (valine, leucine, isoleucine) returned to baseline values in both groups. In conclusion, in severely obese patients subjected to VLCD, nitrogen balance, labile protein concentrations and plasma amino acid profile are not significantly affected by adding CH to proteins.     

Characterization of a point mutation in the pyruvate dehydrogenase E1α gene from two boys with primary lactic acidaemia

Summary We report here a novel mutation in the codon for amino acid 263 resulting in the change from arginine to glutamine in the pyruvate dehydrogenase (PDH) E1 gene, in two boys with primary lactic acidaemia, from independent families. The mutation changes an amino acid located between the two serine residues which are the sites of phosphorylation of the subunit protein. In one family, the mutation wasde novo and in the other it was transmitted from mother to son. The amino acid substitution may affect function of the PDH complex via phosphorylation and dephosphorylation of the E1 subunit. Derangement in the regulation of activity of the PDH complex may explain the primary lactic acidaemia in the patients.     

Characterization of a cDNA coding for human factor VII.

Factor VII is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it participates in blood coagulation by activating factor X and/or factor IX in the presence of tissue factor and calcium. Clones coding for factor VII were obtained from two cDNA libraries prepared from poly(A) RNA from human liver and Hep G2 cells. The amino acid sequence deduced from the cDNAs indicates that factor VII is synthesized with a prepro-leader sequence of 60 or 38 amino acids. The mature protein that circulates in plasma is a single-chain polypeptide composed of 406 amino acids. The amino acid sequence analysis of the protein and the amino acid sequence deduced from the cDNAs indicate that factor VII is converted to factor VIIa by the cleavage of a single internal bond between arginine and isoleucine. This results in the formation of a light chain (152 amino acids) and a heavy chain (254 amino acids) that are held together by a disulfide bond. The light chain contains a gamma-carboxyglutamic acid (Gla) domain and two potential epidermal growth factor domains, while the heavy chain contains the serine protease portion of the molecule. Factor VII shows a high degree of amino acid sequence homology with the other vitamin K-dependent plasma proteins.     

Diversified sequences of peptide epitope for same-RNA recognition.

We replaced an essential RNA-binding, 30-amino acid helix-loop in an Escherichia coli tRNA synthetase with an inactive and simplified "generic" sequence having 23 of the 30 amino acids as alanine and serine. Wild-type residues were restored in random combinations to generate a library with a sequence complexity of about 1.9 x 10(7). Active molecules were obtained by genetic selection at a frequency of approximately 1% and contained variants with as many as 11 alanine/serine replacements and a total of 17 alanine/serine residues. These variants have activities which are thermodynamically competitive with that of the native protein and therefore are functionally and, most likely, conformationally equivalent.