Short C-terminal Peptide Research Articles

Inhibitory Potency and Specificity of Subtilase-like Pro-protein Convertase (SPC) Prodomains

The SPCs (subtilisin-like pro-protein convertases) are a family of enzymes responsible for the proteolytic processing of numerous precursor proteins of the constitutive and regulated secretory pathways. SPCs are themselves synthesized as inactive zymogens. Activation of SPCs occurs via the intramolecular autocatalytic removal of the prodomain. SPC prodomains have been proposed as templates in the development of potent and specific SPC inhibitors. In this study, we investigated the specificity and potency of complete prodomains and short C-terminal prodomain peptides of each SPC on highly purified, soluble enzyme preparations of human SPC1, SPC6, and SPC7. Progress curve kinetic analysis of prodomain peptides and complete prodomains showed competitive inhibitory profiles in the low nanomolar range. Complete prodomains were 5-100 times more potent than C-terminal prodomain peptides, suggesting that N-terminal determinants are involved in the recognition process. However, complete prodomains and prodomain peptides exhibit only a partial specificity toward their cognate enzyme. Ala-scan structure activity studies indicated the importance of basic residues in the P(4), P(5), and P(6) positions for inhibition of SPC1. In contrast, hydrophobic residues in P(6) and P(7), as well as basic residues in P(4) and P(5), were critical for inhibition of SPC7. Our data demonstrated that the use of prodomains as specific inhibitors acting in trans would be of limited usefulness, unless modified into more specific compounds.

Mechanism and role of PDZ domains in signaling complex assembly.

PDZ domains are protein-protein recognition modules that play a central role in organizing diverse cell signaling assemblies. These domains specifically recognize short C-terminal peptide motifs, but can also recognize internal sequences that structurally mimic a terminus. PDZ domains can therefore be used in combination to bind an array of target proteins or to oligomerize into branched networks. Several PDZ-domain-containing proteins play an important role in the transport, localization and assembly of supramolecular signaling complexes. Examples of such PDZ-mediated assemblies exist in Drosophila photoreceptor cells and at mammalian synapses. The predominance of PDZ domains in metazoans indicates that this highly specialized scaffolding module probably evolved in response to the increased signaling needs of multicellular organisms.

PDZ domains and the organization of supramolecular complexes.

PDZ domains are modular protein interaction domains that bind in a sequence-specific fashion to short C-terminal peptides or internal peptides that fold in a beta-finger. The diversity of PDZ binding specificities can be explained by variable amino acids lining the peptide-binding groove of the PDZ domain. Abundantly represented in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes, PDZ domains are frequently found in multiple copies or are associated with other protein-binding motifs in multidomain scaffold proteins. PDZ-containing proteins are typically involved in the assembly of supramolecular complexes that perform localized signaling functions at particular subcellular locations. Organization around a PDZ-based scaffold allows the stable localization of interacting proteins and enhances the rate and fidelity of signal transduction within the complex. Some PDZ-containing proteins are more dynamically regulated in distribution and may also be involved in the trafficking of interacting proteins within the cell.

Heat stress response and heat stress transcription factors

Expression of heat shock protein (HSP)-coding genes is controlled by heat stress transcription factors (Hsfs). They are structurally and functionally conserved throughout the eukaryotic kingdom. In addition to the DNA-binding domain with the helix-turn-helix motif essential for DNA recognition, three functional parts in the C-terminal activator domain were characterized: (i) the HR-A/B region is responsible for oligomerization and activity control, (ii) the nuclear localizing signal (NLS) formed by a cluster of basic amino acid residues which is required and sufficient for nuclear import and (iii) short C-terminal peptide motifs with a central Trp residue (AHA elements). These three parts are indispensible for the activator function. A peculiaritiy of plants is the heat shock-inducible new synthesis of Hsfs. In tomato HsfA1 is constitutively expressed, whereas Hsfs A2 and B1 are heat shock-inducible proteins themselves. We used Hsf knock-out strains of yeast and transient reporter assays in tobacco protoplasts for functional analysis of Hsf-coding cDNA clones and mutants derived from them. HsfA2, which in tomato cell cultures is expressed only after heat shock induction, tends to form large cytoplasmic aggregates together with other HSPs (heat stress granules). In the transient expression assay its relatively low activator potential is evidently due to the inefficient nuclear import. However, the intramolecular shielding of the NLS can be released either by deletion of a short C-terminal fragment or by coexpression with HsfA1, which forms hetero-oligomers with HsfA2.

Acetylcholinesterase: C-terminal domains, molecular forms and functional localization

Acetylcholinesterase (AChE) possesses short C-terminal peptides that are not necessary for catalytic activity. These peptides belong to different classes (R, H, T, S) and define the post-translational processing and targeting of the enzyme. In vertebrates, subunits of type H (AChE H) and of type T (AChE T) are the most important: AChE H subunits produced glycolipid (GPI)-anchored dimers and AChE T subunits produce hetero-oligometric forms such as membrane-bound tetramer in the mammalian brain (containing a 20 kDa hydrophobic protein) and asymmetric collagen-tailed forms in neuromuscular junctions (containing a specific collagen, ColQ). The T peptide allows the formation of tetrameric assemblies with a proline-rich attachment domain (PRAD) of collagen ColQ. These complex molecular structures condition the functional localization of the enzyme in the supramolecular architecture of cholinegic synapses.

Sorting of Phaseolin to the Vacuole Is Saturable and Requires a Short C-Terminal Peptide

Sorting of Phaseolin to the Vacuole Is Saturable and Requires a Short C-Terminal Peptide

Interaction of proteolytic fragments of calmodulin with caldesmon and calponin.

Interaction of five tryptic fragments of calmodulin with caldesmon and calponin was analysed by native gel electrophoresis. In the presence of Ca2+ intact calmodulin interacts with caldesmon and calponin with apparent Kd values equal to 0.23 and 1.3 microM respectively. The interaction was abolished in the absence of Ca2+. Two large tryptic fragments of calmodulin obtained in the presence of Ca2+ (TR1C, residues 1-77, and TR2C, residues 78-148) interact with caldesmon with apparent Kd values of 11.9 and 4.6 microM. Affinity of TR2C to calponin (Kd 3.8 microM) was comparable with that of native calmodulin and was much higher than the corresponding value for TR1C (Kd 41 microM). The short C-terminal tryptic peptide of calmodulin obtained in the presence of EGTA (TR3E, residues 107-148) interacts with caldesmon and calponin with Kd values of 23.9 and 12.1 microM, whereas the large N-terminal peptide TR1E (residues 1-106) interacts with both caldesmon and calponin with a very low affinity (Kd 60 microM). Thus although both N- and C-terminal domains of calmodulin are involved in the interaction with caldesmon and calponin, the C-terminal part of calmodulin (residues 78-148) is of special importance and has the highest contribution for caldesmon and calponin binding.

Structure-related effects of CRF and CRF-derived peptides: dissociation of behavioral, endocrine and autonomic activity.

Two C-terminal and one N-terminal peptide fragments derived from corticotropin-releasing factor (CRF-1-41), CRF-28-41, CRF-34-41, and CRF-1-8, and the CRF receptor antagonist alpha-helical CRF-9-41 (alpha hCRF) were evaluated for their behavioral, endocrine and autonomic nervous effects in rats. To this purpose, three different approaches were used. First, rats were tested in a passive avoidance behavioral (PAB) task after intracerebroventricular (i.c.v.) injection the peptides at five different doses. I.c.v. CRF-1-41 was found to attenuate PAB. The effects of CRF-1-41 on PAB were completely antagonized by i.c.v. pretreatment with alpha hCRF. When given as a sole treatment, the antagonist produced a bimodal effect on PAB. At low doses, alpha hCRF tended to facilitate PAB, while high doses of the antagonist significantly attenuated PAB. Both CRF fragments showed behavioral effects similar to CRF-1-41. Of all peptides tested, CRF-34-41 was found to most attenuate PAB at both retention tests. In a second experiment, the behaviorally most potent fragment CRF-34-41 was compared to CRF-1-41 for its adrenocorticotropic activity after iv injection in pentobarbital-anesthetized rats. Treatment with CRF-1-41, in a dose-related fashion, produced a significant rise in plasma ACTH, whereas CRF-34-41 was without effect. Finally, the effect of i.c.v. injected CRF-1-41 and CRF fragments on heart rate (HR) and gross activity was measured in conscious rats in their home cages during a 60-min period, using a wireless telemetry system. Concomitantly, the occurrence of grooming behavior was recorded. During the first 10 min after i.c.v. treatment, of all peptides tested, CRF-34-41 produced the most marked increase in HR, which remained significant only during the first 30 min of recording. CRF-28-41 induced a non significant transient tachycardia. The parent molecule CRF-1-41 also induced an immediate, significant tachycardia but this effect lasted longer than 60 min. The N-terminal CRF-1-8 remained without effect. No significant effects on gross activity were observed with the two short C-terminal peptides, whereas i.c.v. injected CRF-1-41 induced excessive grooming behavior. A significant grooming response was also recorded in rats given CRF-34-41 fragment, but not in those treated with CRF-28-41 or CRF-1-8.(ABSTRACT TRUNCATED AT 400 WORDS)

Limited proteolysis of tetanus toxin

Tetanus toxin is synthesized by Clostridium tetani as a 151-kDa peptide chain. The primary gene product is processed post-translationally by removal of the initiating methionine residue, formation of disulfide bridges and limited proteolysis by bacterial or exogenous proteinases. The mature toxins consist of a 52-kDa light chain and a 98-kDa heavy chain, linked together by a disulfide bond. Proteolytic nicking is accompanied by increased pharmacological potency. To identify the structural alterations involved, single-chain toxin has been subjected to limited proteolysis with various enzymes. The new N-termini have been determined by Edman degradation and the C-termini by isolation of short C-terminal peptide fragments and subsequent analysis of the sequence and composition. All two-chain toxins result from proteolytic nicking within the 17-residue segment of residues 445-461. Thus, the protease(s) of the culture broth cleave on the C-terminal side of Glu449 and partially Ala456, giving rise to two heavy chain N-termini. Trypsin and clostripain first attack the C-terminal of Arg454 and later Arg448, whereas endoproteinase Arg-C cleaves the former bond only. Chymotrypsin and endoproteinase Glu-C each split a single peptide bond, i.e. that located after Tyr452 and Glu449, respectively. Papain gives rise to a large number of cleavages within the 17-residue segment, the new C-terminus being Thr445 or Asn446 and the new N-terminus being Asp460 or Leu461. Further papain digestion leads to an additional cleavage within the heavy chain between Ser863 and Lys864. The original N-terminal Pro1 and C-terminal Asp1314, predicted from the nucleotide sequence, are conserved in all proteolytic digests. The pharmacological activity of the various two-chain toxins was 5-11 times that of the single-chain toxin, as estimated from the inhibition of [3H]noradrenaline release from rat-brain homogenate. The present data on the processing and activation by limited proteolysis prove the existence of several active tetanus isotoxins. These data, together with our previous data on the localization of disulfide bridges and sulfhydryl groups (Krieglstein, K., Henschen, A., Weller, U. & Habermann, E. (1990) Eur. J. Biochem. 188, 39-45), provide the detailed protein chemical characterization of the tetanus isotoxins.

Some properties of duck gizzard caldesmon

The domain structure of duck gizzard caldesmon was investigated. A single thiol group is located in the vicinity of the C-terminus of the protein. A simple method for the purification of a short (21 kDa) C-terminal peptide formed after chemical cleavage of caldesmon at cysteine residues was evolved. The C-terminal peptide of caldesmon interacts with calmodulin with an affinity one order of magnitude higher than that of native caldesmon. The Ca2+/phospholipid-dependent protein kinase (protein kinase C) transfers about 2 mol of phosphate per mol of caldesmon. All sites phosphorylated by protein kinase C are located in the short (21 kDa) C-terminal peptide of caldesmon. Phosphorylation does not affect the interaction of caldesmon with calmodulin.

Degradation of atrial natriuretic factor in the rat.

The biologically active circulating form of atrial natriuretic factor (ANF) in the rat is the 28-amino-acid peptide ANF-(Ser-99-Tyr-126). Degradation of this peptide in vivo as well as in vitro, in whole blood, in plasma and by the isolated mesenteric artery was investigated. Studies in vivo in the rat demonstrated that the elimination and degradation of ANF was extremely fast: within 3 min more than 95% of the injected immunoreactive material was eliminated from circulation. The production of a short C-terminal peptide was detected on injection of 125I-ANF-(Ser-99-Tyr-126) into the rat. This peptide increased proportionately with incubation time. Experiments in vitro in the presence of whole blood or plasma did not cause any major destruction of ANF even after incubation for 60 min. After this prolonged incubation in plasma, ANF-(Ser-99-Tyr-126) was partially converted into ANF-(Ser-103-Tyr-126), a less potent peptide. Isolated mesenteric-artery preparation appeared to degrade ANF in a manner very similar to the system in vivo. These results suggest that degradation of ANF may occur either after internalization in the vascular cells or by a membrane-bound enzyme in the vasculature.