Amino Acid C-terminal Peptide Research Articles

The C-terminal tail of CSNAP attenuates the CSN complex.

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitination is the cullin-RING ligases (CRLs). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumor progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises ∼300 complexes, all of which are regulated by the COP9 signalosome complex (CSN). Therefore, CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN-interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Immunological Investigation for the Presence of Lunasin, a Chemopreventive Soybean Peptide, in the Seeds of Diverse Plants.

Lunasin, a 44 amino acid soybean bioactive peptide, exhibits anticancer and anti-inflammatory properties. All soybean varieties that have been examined contain lunasin. It has also been reported in a few other plant species including amaranth, black nightshade, wheat, barley, rye, and triticale. Interestingly, detailed searches of transcriptome and DNA sequence databases of cereals failed to identify lunasin-coding sequences, raising questions about the authenticity of lunasin in cereals. To clarify the presence or absence of lunasin in cereals and other plant species, an immunological investigation was conducted utilizing polyclonal antibodies raised against the first 20 amino acid N-terminal peptide (SKWQHQQDSCRKQLQGVNLT) and a 15 amino acid C-terminal peptide (CEKHIMEKIQGRGDD) of lunasin. Protein blot analyses revealed the presence of proteins from several plants that reacted against the lunasin N-terminal peptide antibodies. However, the same proteins failed to react against the lunasin C-terminal peptide antibodies. These results demonstrate that peptides identical to soybean lunasin are absent in seeds of diverse plants examined in this study.

Guanylin-like peptides, guanylate cyclase and osmoregulation in the European eel ( Anguilla anguilla)

Three guanylin-like peptides, guanylin, uroguanylin and renoguanylin and two guanylate cyclase type C (GC-C) receptor isoforms were cloned and sequenced from the European eel ( Anguilla anguilla). All peptides and both receptors (GC-C1 and GC-C2) were predominantly expressed within the intestine and kidney of both sexually immature yellow, and sexually maturing, migratory silver eels. The derived amino acid sequences for the pre-prohormones and guanylate cyclase isoforms had structural features in common with sequences previously reported for guanylin-like peptides and guanylate cyclases from teleost fish and other species in general. The highest sequence homologies for the prohormones were found within the active, 15–16 amino acid C-terminal peptide domain, whereas the guanylate cyclase receptors exhibited highest homology throughout the transmembrane domain and intracellular region of the protein comprising the kinase homology, oligomerisation/coiled-coil and catalytic domains. In both yellow and silver eels, seawater (SW) acclimation induced sustained increases in the expression of uroguanylin and GC-C1 mRNAs within the intestine but no significant changes were found in the abundance of mRNAs for guanylin, renoguanylin or GC-C2. Likewise there were no significant changes in expression of any of the prohormone or receptor mRNAs within the renal kidney following transfer to SW. The results suggest that uroguanylin and GC-C1 are key components of a cGMP signalling system that may play an important role within intestinal enterocytes for the regulation of salt and water absorption in the SW-acclimated eel.

Goldfish ghrelin: molecular characterization of the complementary deoxyribonucleic acid, partial gene structure and evidence for its stimulatory role in food intake.

Complementary deoxyribonucleic acid (cDNA) encoding goldfish preproghrelin was identified using rapid amplification of the cDNA ends (RACE) and reverse transcription (RT)-polymerase chain reaction (PCR). The 490 bp cDNA encodes a 103 amino acid preproghrelin which has a 26 amino acid signal region, 19 amino acid mature peptide and a 55 amino acid C-terminal peptide region. The mature peptide region of goldfish ghrelin has two putative cleavage sites and amidation signals (GRR); one after 12 amino acids and the other after 19 amino acids. The serine (S) in the second amino acid position in the "active core" of ghrelin is substituted with threonine (T). The goldfish ghrelin gene has four exons and three short introns and resembles the human ghrelin gene. Ghrelin messenger RNA (mRNA) expression was detected in the brain, pituitary, intestine, liver, spleen and gill by RT-PCR followed by Southern blot analysis, and in the intestine by Northern blot. Intracerebroventricular (ICV) injection of n-octanoylated goldfish ghrelin (1-19) stimulates food intake in goldfish.

Crystallization of ClfA and ClfB fragments: the fibrinogen-binding surface proteins of Staphylococcus aureus.

Recombinant constructs encoding the fibrinogen-binding domains of ClfA and ClfB from Staphylococcus aureus have been crystallized. ClfA was crystallized in the orthorhombic space group P212121 with unit-cell parameters a = 39.58, b = 81.39 and c = 112.65 A. A complete data set was recorded to 2.1 A resolution and had a Vm of 2. 3 A3 Da-1 with 46.5% solvent, suggesting one molecule per asymmetric unit. Co-crystals of ClfA with the 17 amino-acid C-terminal peptide of fibrinogen gamma-chain diffracted to 2.1 A resolution and had unit-cell parameters a = 39.11, b = 81.39 and c = 109.51 A in the space group P212121. ClfB was crystallized in the tetragonal space group P41212 or P43212 with unit-cell parameters a = 96.31, b = 96. 31 and c = 84.13 A and diffracted to 2.45 A resolution. The estimated Vm of 2.6 A3 Da-1 with 53% solvent indicated one molecule in the asymmetric unit.

Importance of hydropathic complementarity for the binding of the neuropeptide substance P to a monoclonal antibody: Equilibrium and kinetic studies

In a previous study (Boquet et al., 1995, Molec. Immunol. 32, 303–308) we observed remarkable inversions of hydropathic profiles between complementarity determining regions (CDRs) of an anti-substance P monoclonal antibody (SP31) and the corresponding 5 amino acid C-terminal peptide epitope. Here we demonstrate the importance this hydropathic complementarity by measuring the immunoreactivity of SP-related peptides which have been modified in their C-terminal parts so that hydropathic profile has been conserved (by substituting amino acids in the epitope) or modified (by mixing the sequence of amino acids in the epitope). Experiments performed in equilibrium conditions using a competitive enzyme immunoassay showed that most of the peptides conserving the hydropathic profile of SP epitope were recognized by mAb SP31 (even if marked variations in affinity were observed), while those for which the hydropathic profile was modified exhibited very low or undetectable affinity. The kinetic parameters ( k a and k d) of peptide-antibody interactions were determined using Surface Plasmon Resonance technology (BIACORE 2000). These measurements showed that all peptides recognized by mAb SP31 had similar association rate constants (close to 2 × 10 5 M −1 s −1), differences in binding affinities essentially resulting from differences in dissociation rate constants (ranging from 1.61 × 10 −4 to 1.15 × 10 −1 s −1). From these results, it was concluded that hydropathic complementarity between the epitope and the paratope could be a necessary but not sufficient condition for establishing high-affinity binding. We hypothesize that hydropathic interactions may play a critical role during the first contacts between antibody CDRs and the peptide, possibly by favouring reorganization of water molecules at the antibody-peptide interface.

Characterization of the gene for leucomyosuppressin and its expression in the brain of the cockroach Diploptera punctata

Using HPLC separation, radioimmunoassay, and subsequent bioassay, we have detected the presence of an active peptide, which co-elutes with the insect myoinhibitory peptide leucomyosuppressin, in the brain of the cockroach Diploptera punctata. We have isolated a cDNA encoding the precursor for this peptide from cDNA libraries representing D. punctata brain RNA. The cDNA sequence contains an open reading frame that upon translation would result in a prepropolypeptide of 96 amino acids. Proteolytic cleavage of the predicted precursor could result in several peptides, including a 10 amino acid C-terminal peptide that would, upon modification of the NH 2 and COOH-terminal amino acids, be identical to the insect FLRFamide, leucomyosuppressin. No other RFamide products are predicted to be processed from the precursor. Southern blot analysis indicates that the gene is present in the D. punctata genome in a single copy. Northern blot analysis shows that the gene is predominantly expressed as a 3.8 kb mRNA in cockroach brain. Study of the expression of the leucomyosuppressin gene in D. punctata brain, using in situ hybridization, indicates that expression occurs primarily in the pars intercerebralis of the protocerebrum, a region showing abundant FMRFamide-like immunoreactive neurosecretory cells. Immunohistochemistry and HPLC coupled to radioimmunoassay indicates that leucomyosuppressin represents a significant proportion of FMRFamide-related peptide production in the brain. However, HPLC analysis also indicates the presence of significant levels of other related peptides, demonstrating the presence of more than one FMRFamide-related gene in this insect.

Identification of an IgE-binding determinant of the major allergen Myr pI from the venom of the Australian jumper ant Myrmecia pilosula

The structure of the single allergenic determinant of the major ant venom allergen, Myr, p I from the Australian jumper ant Myrmecia pilosula has been determined by inhibition studies with synthetic peptides. A 14 amino-acid C-terminal peptide sequence has been shown to constitute this determinant. Half-maximal inhibition of binding of ant venom-specific IgE antibodies to the native venom was obtained with this peptide at a concentration of 5 · 10 −8M. This allergenic determinant was invariant for all ant venom-allergic subjects tested whose IgE antibodies recognized this allergen.

Regulation of glucose transport in clone 9 cells by thyroid hormone

Triiodothyronine (T 3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a ‘non-transformed’ rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T 3 is due to an increase in transport V max and occurs in the absence of a change in either the K m for glucose transport (∼ 3 mM) or the K i for inhibition of transport by cytochalasin B ((1–2) · 10 −7 M). Consistent with the observed K i for cytochalasin B, Northern blot analysis of RNA from control and T 3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T 3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T 3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.