Structural determinants of the calpain inhibitory activity of calpastatin peptide B27-WT

Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of mu- and m-calpain. In this report, a combination of beta-alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two "hot spots," Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19), in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19) backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.     

Interdependence of inoculum size and trace element supply in growth of Aspergillus oryzae

Summary Further to earlier reports indicating that the effects of inoculum size on growth of Aspergillus oryzae in synthetic media are abolished by certain trace element compositions, it has now been shown that those effects can be made more pronounced, but can also be reversed, by varying the balance of available trace elements.Addition of Ca to substrate A ₁ increases the usual effects of inoculum size whereas Cu abolishes them with a tendency for reversal.Supplementation of substrate A ₁ with suitable amounts of ethylenediaminetetraacetic acid results in a stronger inhibition of small-inoculum cultures than large ones, thus increasing the usual effects of inoculum size. These effects are reversed if the sugar of substrate A ₁ is purified by Al(OH)₃ co-precipitation method, but only if the treatment is carried out at neutral or alkaline reaction. This reversal of the phenomena is due to an almost complete absence of Zn after purification and presence of Fe in substrate A ₁.The Al(OH)₃ treatment of the sugar results in substrates so poor in Zn that addition of as little as 5 g ZnSO₄ · 7 H₂O/l has an effect. This influence, however, results in an inhibition of the growth rate, and higher concentrations of Zn are required in order to obtain stimulation with both large and small-inoculum cultures.The above-mentioned reversal of the phenomena was abolished in a substrate with slightly caramelized glucose.

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Zusammenfassung Diese Arbeit ist eine Erweiterung vorhergehender Berichte über den Einfluß der Größe der Impfmenge auf das Wachstum von Aspergillus oryzae, wo u. a. festgestellt wurde, daß die beobachteten Effekte durch bestimmte Spurenelement-Zusammensetzungen aufgehoben werden.Es wurde nun festgestellt, daß Zugabe von Ca zu Substrat A ₁ die gewohnten Effekte erhöht, während Cu diese verringert mit einer Tendenz zur Umkehrung.Werden geeignete Mengen Äthylendiamintetraessigsäure zu Substrat A ₁ zugegeben, so werden Kulturen, die aus kleiner Impfmenge erwachsen sind, stärker in ihrer Wachstumsgeschwindigkeit und maximaler Ausbeute an Mycel gehemmt als Kulturen von großer Impfdichte, was einer Verstärkung der gewohnten Effekte gleich kommt. Diese Effekte werden umgekehrt, wenn die Glucose des Substrates A ₁ durch Al(OH)₃-Fällung gereinigt wird, jedoch nur wenn diese Behandlung bei neutraler oder alkalischer Reaktion erfolgt. Diese Umkehrung ist auf nahezu vollständige Entfernung von Zn und gleichzeitige Anwesenheit von Fe in Substrat A ₁ zurückzuführen.Schon Zugabe sehr kleiner Mengen Zn (5 g ZnSO₄ · 7 H₂O/l) zum gereinigten Substrat übt eine Wirkung aus, welche aber in einer Hemmung der Wachstumsgeschwindigkeit besteht. Höhere Zn-Konzentrationen sind nötig, um das Wachstum der Kulturen von großen und kleinen Impfmengen zu fördern.Die vorerwähnte Umkehrung der Phänomene wurde aufgehoben, wenn ein Substrat mit leichter Caramelisierung der Glucose angewendet wurde.

     

Structural determinants of binding and specificity in transforming growth factor-receptor interactions.

Transforming growth factor (TGF-beta) protein families are cytokines that occur as a large number of homologous proteins. Three major subgroups of these proteins with marked specificities for their receptors have been found-TGF-beta, activin/inhibin, and bone morphogenic protein. Although structural information is available for some members of the TGF-beta family of ligands and receptors, very little is known about the way these growth factors interact with the extracellular domains of their cell surface receptors, especially the type II receptor. In addition, the elements that are the determinants of binding and specificity of the ligands are poorly understood. The structure of the extracellular domain of the receptor is a three-finger fold similar to some toxin structures. Amino acid exchanges between multiply aligned homologous sequences of type II receptors point to a residue at the surface, specifically finger 1, as the determinant of ligand specificity and complex formation. The "knuckle" epitope of ligands was predicted to be the surface that interacts with the type II receptor. The residues on strands beta2, beta3, beta7, beta8 and the loop region joining beta2 and beta3 and joining beta7 and beta8 of the ligands were identified as determinants of binding and specificity. These results are supported by studies on the docking of the type II receptor to the ligand dimer-type I receptor complex.     

Structural determinants for activity of glucagon-like peptide-2

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide. The majority of position 2 substitutions exhibited normal to enhanced GLP-2 receptor (GLP-2R) binding; in contrast, position 2 substitutions were less well tolerated in studies of receptor activation as only Gly, Ile, Pro, alpha-aminobutyric acid, D-Ala, or nor-Val substitutions exhibited enhanced GLP-2R activation. In contrast, alanine replacement at positions 5,6,17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding. Position 2 substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21 exhibited normal to enhanced GLP-2R binding but greater than 75% reduction in receptor activation. D-Ala(2), Pro(2) and Gly(2), Ala(16) exhibited significantly lower EC(50)s for receptor activation than the parent peptide (p < 0.01-0.001). Circular dichroism analysis indicated that the enhanced activity of these GLP-2 analogues was independent of the alpha-helical content of the peptide. These results indicate that single amino acid substitutions within GLP-2 can confer structural changes to the ligand-receptor interface, allowing the identification of residues important for GLP-2R binding and receptor activation.     

Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

In recent years, orotidine-5′-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.     

Synthesis and inhibitory activity of oligosaccharide thiazolines as a class of mechanism-based inhibitors for endo-beta-N-acetylglucosaminidases

A facile synthesis of oligosaccharide-thiazoline derivatives of N-glycans as a novel class of inhibitors for endo-beta-N-acetylglucosaminidases was described. It was found that the external sugar residues on the N-glycan core could enhance the inhibitory potency. While the Manbeta1,4GlcNAc- and Man3GlcNAc-thiazolines were only moderate inhibitors, the large Man9GlcNAc-thiazoline demonstrated potent inhibitory activity, with an IC(50) of 0.22 and 0.42 microM against the Arthrobacter enzyme (Endo-A) and the human endo-beta-N-acetylglycosaminidase (hENGase), respectively. It was also observed that the oligosaccharide thiazolines could differentially inhibit endo-beta-N-acetylglucosaminidases from different sources. These oligosaccharide thiazolines represent the first class of endo-beta-N-acetylglucosaminidase inhibitors.     

Pore size and negative charge as structural determinants of permeability in the Torpedo nicotinic acetylcholine receptor channel.

To gain an insight into the molecular basis of ion permeation mechanism through the nicotinic acetylcholine receptor (AChR) channel, we have determined permeability ratios of organic cations relative to Na+ of specifically mutated Torpedo californica AChR channels expressed in Xenopus oocytes. The mutations involved mainly the side chains of the amino acid residues in the intermediate ring, where mutations have been found to exert strong effects on single-channel conductance and ion selectivity among alkali metal cations. The results obtained reveal that both the size and the net charge of the side chains of the intermediate ring are involved in determining the permeability, and provide experimental evidence that the pore size at the intermediate ring is a critical determinant of permeability. Our findings further suggest that changes in net charge exert effects on permeability by affecting the pore size of the channel.     

Cell size and growth rate are major determinants of replicative lifespan

Yeast cells, like mammalian cells, enlarge steadily as they age. Unabated cell growth can promote cellular senescence; however, the significance of the relationship between size and cellular lifespan is not well understood. Herein, we report a genetic link between cell size, growth rate and lifespan. Mutations that increase cell size concomitantly increase growth rate and decrease lifespan. As a result, large cells grow, divide and age dramatically faster than small cells. Conversely, small cell mutants age slowly and are long-lived. Investigation of the mechanisms involved suggests that attainment of a maximal size modulates lifespan. Indeed, cumulative results revealed that life expectancy is size-dependent, and that the rate at which cells age is determined in large part by the amount of cell growth per generation.Key words: size, cell growth, longevity, senescence, aging     

Structural determinants of Clostridium difficile toxin A glucosyltransferase activity

The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.     

Structural determinants for the biological activity of Vav proteins

We have used an extensive mutagenesis approach to study the specific role of the eight structural domains of Vav during both the activation and signaling steps of this Rac1 exchange factor. Our results indicate that several Vav domains (Dbl homology, pleckstrin homology, and zinc finger) are essential for all the biological activities tested, whereas others are required for discrete, cell type-specific biological effects. Interestingly, we have found that Vav domains have no unique functions. Thus, the calponin homology domain mediates the inhibition of Vav both in vitro and in vivo but, at the same time, exerts effector functions in lymphocytes upon receptor activation. The Vav SH2 and SH3 regions play regulatory roles in the activation of Vav in fibroblasts, mediating both its phosphorylation and translocation to the plasma membrane. In contrast, the Vav SH2 and SH3 regions act as scaffolding platforms in T-cells, ensuring the proper phosphorylation of Vav and the subsequent engagement of downstream effectors. We also provide evidence indicating that the zinc finger region exerts at least three different functional roles in Vav, aiding in the down-regulation of its basal activity, the engagement of substrates, and the induction of ancillary pathways required for cell transformation. Finally, the results obtained are consistent with a new regulatory model for Vav, in which the calponin homology region inhibits the basal activity of Vav through interactions with the zinc finger region.     

Structural determinants of the eosinophil cationic protein antimicrobial activity

Abstract Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affinity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specific cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be sufficient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological significance.     

Structural studies of migration inhibitory activity induced during murine tumor allograft rejection.

Lymphocytes from mice rejecting a tumor allograft produce a soluble activity that inhibits the migration of murine macrophages. The present studies show that this activity is stable with 56 °C heating for 30 min, is inactivated by trypsin, has a molecular size of about 45,000, and has an isoelectric point of 5.0–5.5. In addition, it does not inhibit the migration of guinea pig macrophages. These results indicate that this lymphokine is similar in its physicochemical properties to murine, guinea pig and human migration inhibitory factors (MIF) studied previously, but they also suggest that there may be structural differences in the functionally active portions of MIF from different species.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.     

Structural effects of carbohydrate-containing polycations on gene delivery. 1. Carbohydrate size and its distance from charge centers

Cationic polymers have the ability to bind plasmid DNA (pDNA) through electrostatic interactions and condense it into particles that can be readily endocytosed by cultured cells. The effects that polycation structure has on toxicity and gene delivery efficiency are investigated here by synthesizing a series of amidine-based polycations that contain the carbohydrates d-trehalose and beta-cyclodextrin (CD) within the polycation backbone. The carbohydrate size (trehalose vs CD) and its distance from the charge centers affect the gene delivery behavior in BHK-21 cells. It is found that as the charge center is further removed from the carbohydrate unit, the toxicity is increased. Also, as the size of the carbohydrate moiety is enlarged from trehalose to beta-cyclodextrin, the toxicity is reduced. The absence of a carbohydrate in the polycation produces high toxicity. All carbohydrate polycations transfect BHK-21 cells to approximately the same level of gene expression.     

Separate oligosaccharide determinants mediate interactions of the low-molecular-weight salivary mucin with neutrophils and bacteria.

The low-molecular-weight human salivary mucin (MG2) coats oral surfaces, where it is in a prime location for governing cell adhesion. Since oligosaccharides form many of the interactive facets on mucin molecules, we examined MG2 glycosylation as it relates to the molecule's adhesive functions. Our previous study of MG2 oligosaccharide structures showed that the termini predominantly carry T, sialyl-T, Lewisx (Lex), sialyl Lex (sLex), lactosamine, and sialyl lactosamine determinants [Prakobphol, A., et al. (1998) Biochemistry 37, 4916-4927]. In addition, we showed that sLex determinants confer L-selectin ligand activity to this molecule. Here we studied adhesive interactions between MG2 and cells that traffic in the oral cavity: neutrophils and bacteria. Under flow conditions, neutrophils tethered to MG2-coated surfaces at forces between 1.25 and 2 dyn/cm2, i.e., comparable to the shear stress generated at the tooth surface by salivary flow ( approximately 0.8 dyn/cm2). MG2 was also found in association with neutrophils isolated from the oral cavity, evidence that the cells interact with this mucin in vivo. Since MG2 serves as an adhesion receptor for bacteria, the MG2 saccharides that serve this function were also identified. Seven of 18 oral bacteria strains that were tested adhered to MG2. Importantly, six of these seven strains adhered via T antigen, sialyl-T antigen, and/or lactosamine sequences. No adherence to Lex and sLex epitopes was detected in all the strains that were tested. Together, these results suggest that distinct subsets of MG2 saccharides function as ligands for neutrophil L-selectin and receptors for bacterial adhesion, a finding with interesting implications for both oral health and mucin function.     

Structural and mutational studies on the importance of oligosaccharide binding for the activity of yeast PNGase

Peptide:N-glycanase (PNGase) is an important component of the endoplasmic reticulum-associated protein degradation pathway in which it de-glycosylates misfolded glycoproteins, thus facilitating their proteasomal degradation. PNGase belongs to the transglutaminase superfamily and features a Cys, His, and Asp catalytic triad, which is essential for its enzymatic activity. An elongated substrate-binding groove centered on the active site Cys191 was visualized in the crystal structure of apo-PNGase, whereas its complex with Z-VAD-fmk, a peptide-based inhibitor of PNGase, revealed that the inhibitor occupied one end of the substrate-binding groove while being covalently linked to the active site Cys. Recently, haloacetamidyl-containing carbohydrate-based inhibitors of PNGase were developed and shown to specifically label the active site Cys. In this study, we describe the crystal structure of yeast PNGase in complex with N,N'-diacetylchitobiose (chitobiose). We found that the chitobiose binds on the side opposite to the peptide binding site with the active site Cys191 being located approximately midway between the carbohydrate and peptide binding sites. Mutagenesis studies confirm the critical role of the chitobiose-interacting residues in substrate binding and suggest that efficient oligosaccharide binding is required for PNGase activity. In addition, the N-terminus of a symmetry-related PNGase was found to bind to the proposed peptide-binding site of PNGase. Together with the bound chitobiose, this enables us to propose a model for glycoprotein binding to PNGase. Finally, deleting the C-terminal residues of yeast PNGase, which are disordered in all structures of this enzyme, results in a significant reduction in enzyme activity, indicating that these residues might be involved in binding of the mannose residues of the glycan chain.     

Structural requirements for IgM assembly and cytolytic activity. Effects of mutations in the oligosaccharide acceptor site at Asn402

Glycosylation of IgG occurs at asparagine 297 of the gamma H chain and is necessary for the normal capacity of IgG to activate the classical pathway of complement-dependent cytolysis. IgM is glycosylated at five sites in the constant region of the mu H chain, of which glycosylation at asparagine 402 seems analogous to the glycosylation of IgG. In order to assess the importance of glycosylation at asparagine 402 for IgM cytolytic activity, we have used site-directed mutagenesis to produce IgM which is not glycosylated at this position. In particular we have tested the effects of substituting Gln for Asn 402 and Thr-Gly for Gly 403-Thr 404 in the third constant region domain. We tested the effects of these substitutions by expressing the mutant mu genes in hybridoma cells which produce the hapten-specific kappa-chain. The normal mu-chain is glycosylated at Asn 402, and, as expected, these mutations appear to abrogate glycosylation of the mutant mu-chains at position 402 and do not affect the hapten affinity of the IgM. However, both of these mutations cause the increased production of monomeric rather than polymeric IgM: the ratio of monomeric to polymeric IgM is 0.21, 3.5, and 10.3 for wild-type IgM, IgM-Gln 402, and IgM-Thr 403-Gly 404, respectively. The wild-type and mutant polymeric IgM preparations were compared for their capacity to promote complement-dependent cytolysis: IgM-Gln 402 and IgM-Thr 403-Gly 404 have approximately 31% and 4%, respectively, of the capacity of wild-type IgM.     

Structural determinants of developmental toxicity in hamsters.

A CASE/MULTICASE structure activity relationship (SAR) model of developmental toxicity of chemicals in hamsters (HaDT) was developed. The model exhibited a predictive performance of 74%. The model's overall predictivity and informational content were similar to those of an SAR model of mutagenicity in Salmonella. However, unlike the Salmonella mutagenicity model, the HaDT model did not identify overtly chemically reactive moieties as associated with activity. Moreover, examination of the number and nature of significant structural determinants suggested that developmental toxicity in hamsters was not the result of a unique mechanism or attack on a specific molecular target. The analysis also indicated that the availability of experimental data on additional chemicals would improve the performance of the SAR model.     

Synthesis of oligosaccharide determinants with amide spacers of T-type antigens

The hapten of the T-antigen was synthesized with a peptide-like amide-spacer as 2-(4-methoxycarbonylbutanecarboxamido)ethyl 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside and coupled with serum albumin to give a synthetic antigen. Other O-beta-D-galactopyranosyl haptens, 2-(4-methoxycarbonylbutanecarboxamido)ethyl 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside, O-beta-D-galactopyranosyl-(1 leads to 3)-O-[beta-D-galactopyranosyl-(1 leads to 4)]-2-acetamido-2-deoxy-alpha-D-galactopyranoside, and 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-glucopyranoside, the last compound being the determinant of the Lewis Lec antigen, were also synthesized.