Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

Molecular heterogeneity of myophosphorylase deficiency (McArdle's disease): a genotype-phenotype correlation study.

We report on 54 Spanish patients with McArdle's disease from 40 unrelated families. Molecular analysis revealed that the most common R49X mutation was present in 70% of patients and 55% of alleles. The G204S mutation was less frequent and found in 14.8% of patients and 9% of mutant alleles. The W797R mutation was observed in 16.5% of patients, accounting for 13.7% of mutant alleles. Moreover, 78% of mutant alleles among Spanish patients can be identified by using polymerase chain reaction-restriction fragment length polymorphism analysis for the R49X, G204S, and W797R mutations, which makes noninvasive diagnosis possible through molecular genetic analysis of blood DNA. Six novel mutations were found. Three were missense mutations, E348K, R601W, and A703V; two nonsense mutations, E124X and Q754X; and one single base pair deletion, 533 delA. No clear genotype-phenotype correlation emerges from our study. Most of the mutations of uncharged and solvent inaccessible residues and the truncations must disrupt the basic structure of the protein. The mutations of charged residues would be expected to interfere with internal hydrogen bonding networks, introducing severe incompatible partnering that is caused by poor packing or electrostatic repulsions.

Structural relationships among regulated and unregulated phosphorylases.

Species and tissue-specific isozymes of phosphorylase display differences in regulatory properties consistent with their distinct roles in particular organisms and tissues. In this review, we compare crystallographic structures of regulated and unregulated phosphorylases, including maltodextrin phosphorylase (MalP) from Escherichia coli, glycogen phosphorylase from yeast, and mammalian isozymes from muscle and liver tissues. Mutagenesis and functional studies supplement the structural work and provide insights into the structural basis for allosteric control mechanisms. MalP, a simple, unregulated enzyme, is contrasted with the more complicated yeast and mammalian phosphorylases that have evolved regulatory sites onto the basic catalytic architecture. The human liver and muscle isozymes show differences structurally in their means of invoking allosteric activation. Phosphorylation, though common to both the yeast and mammalian enzymes, occurs at different sites and activates the enzymes by surprisingly different mechanisms.

A comparison of the crystallographic structures of two catalytic antibodies with esterase activity.

The crystallographic structure of the Fab fragment of the catalytic antibody, 29G11, complexed with an (S)-norleucine phenyl phosphonate transition state analog was determined at 2.2 A resolution. The antibody catalyzes the hydrolysis of norleucine phenyl ester with (S)-enantioselectivity. The shape and charge complementarity of the binding pocket for the hapten account for the preferential binding of the (S)-enantiomer of the substrate. The structure is compared to that of the more catalytically efficient antibody, 17E8, induced by the same hapten transition state analog. 29G11 has different residues from 17E8 at eight positions in the heavy chain, including four substitutions in the hapten-binding pocket: A33V, S95G, S99R and Y100AN, and four substitutions at positions remote from the catalytic site, I28T, R40K, V65G and F91L. The two antibodies show large differences in the orientations of their variable and constant domains, reflected by a 32 degrees difference in their elbow angles. The VL and VH domains in the two antibodies differ by a rotation of 8.8 degrees. The hapten binds in similar orientations and locations in 29G11 and 17E8, which appear to have catalytic groups in common, though the changes in the association of the variable domains affect the precise positioning of residues in the hapten-binding pocket.

Biochemical characterization and crystallographic structure of an Escherichia coli protein from the phosphotriesterase gene family.

Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase, a hydrolytic, bacterial enzyme with an unusual substrate specificity for synthetic organophosphate triesters and phosphorofluoridates, which are common constituents of chemical warfare agents and agricultural pesticides. No natural substrate has been identified for phosphotriesterase, and it has been suggested that the enzyme may have evolved the ability to hydrolyze synthetic compounds in bacteria under selective pressure to meet nutritional needs. PHP, which has 28% sequence identity with phosphotriesterase, may belong to the family of proteins from which phosphotriesterase evolved. Here we report the cloning, expression, initial characterization, and high-resolution X-ray crystallographic structure of PHP. Biochemical analysis shows that PHP is monomeric and binds two zinc ions per monomer. Unlike phosphotriesterase, PHP does not catalyze the hydrolysis of nonspecific phosphotriesters. The structure, similar to that of phosphotriesterase, consists of a long, elliptical alpha/beta barrel and has a binuclear zinc center in a cleft at the carboxy end of the barrel at the location of the presumptive active site.

Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation.

Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 310 helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in Vmax, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in Vmax, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S0.5 for glucose 1-phosphate. The ability of the mutations to enhance binding of AMP and glucose 1-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.

Ethnic background and antecedents of religious conversion among Israeli Jewish outpatients.

This study explored the association of ethnocultural background (Ashkenazi vs Sephardi origin) with antecedents of religious conversion among Israeli Jewish penitents who applied for psychiatric help in an outpatient clinic. A basic assumption underlying the comparison was that Sephardic Jews in Israel are more inclined toward Jewish tradition and collectivistic than Ashkenazim. The interview data indicated that for both groups emotional factors were more dominant in the conversion process than cognitive ones; however, cognitive factors were more strongly present in the conversion process of the Ashkenazim whose prepenitence cultural orientation had been more secularized and individualistic. In both groups a high prevalence of problematic relations with the father (but not with the mother) during childhood was noticed. Over-all, conversion tended to be gradual rather than abrupt and devoid of mystical experiences.

Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding.

The functional role in allosteric regulation of a flexible loop (residues 280-288) located near the active site of muscle glycogen phosphorylase was investigated. Mutations were made in residues 283-285 based on crystallographic studies that indicate that the loop functions as a gate controlling access of substrates to the active site and that these specific residues play distinct roles in mimicking the substrate and binding inhibitors when the enzyme is in an inactive conformation. Substitution of Ala or Asn for Asp-283, the putative substrate mimic, results in a 15-fold decrease in Vmax, a 10-fold decrease in the S0.5 for glucose 1-phosphate, a 10-fold increase in the Ka for AMP, and a 10-20-fold increase in the Ki for glucose. Substitution of Ala for Asn-284, which normally forms a hydrogen bond with the inhibitor glucose, reduces Vmax 3-fold, increases the Ki for glucose 2-fold, but has little effect on AMP or glucose 1-phosphate binding or cooperativity. Substitution of Asp at 284, on the other hand, reduces Vmax 10-fold, elevates the Ki for glucose 10-fold, decreases AMP cooperativity, but has little effect on the affinity of AMP or the cooperativity and binding of glucose 1-phosphate. Substitution of Leu for Phe-285, which forms aromatic stacking interactions with purine inhibitors, reduces Vmax 2-fold, decreases the affinity for caffeine at least 10-fold, raises the Ka for AMP 3-fold, and decreases AMP cooperativity but has little effect on glucose 1-phosphate binding or cooperativity. The results of the mutagenesis studies show the importance of the 280's loop for inhibitor binding and modulation of substrate affinity and suggest a role for the loop in allosteric activation. The propagation of allosteric effects across the domain interface may depend upon specific contacts between residues of the 280's loop and the C-terminal domain.

Mutations in paired alpha-helices at the subunit interface of glycogen phosphorylase alter homotropic and heterotropic cooperativity.

Allosteric switching between inactive and active conformational states in muscle glycogen phosphorylase alters the pattern of van der Waals contacts and hydrogen bonds between two helices located at the dimer interface of the enzyme. Alanine was substituted for residues N270, N274, and R277 to perturb helix interactions, which differ in inactive and active conformations. In addition, the entire alpha-helix in each subunit was exchanged with the analogous region from yeast phosphorylase. The N274A mutant shows increased affinity and reduced cooperativity for the activator, AMP, and reduced cooperativity for the substrate, glucose 1-phosphate. The N270A and R277A mutants, in contrast, show reduced binding and cooperativity for AMP and relatively little change in binding or cooperativity for glucose 1-phosphate. The substitution of the helix from the yeast enzyme results in an 8-fold reduction in Vmax, a loss in cooperativity for both AMP and glucose 1-phosphate, but little change in the affinities of either ligand. Crystallographic analyses of the N274A and R277A mutants show that these substitutions cause only small changes in the structure of the unliganded, inactive form of phosphorylase. The substitution at N274 eliminates intersubunit interactions which selectively stabilize the enzyme in an inactive conformation. The kinetic results indicate that the mutations at N270 and R277, on the contrary, perturb packing interactions at the dimer interface of the activated enzyme and weaken binding of AMP. The relatively modest effects of the replacement with the helices from the yeast enzyme indicate that the helices are not crucial for catalytic function.

Crystallization and preliminary diffraction studies of thioesterase II from rat mammary gland.

Thioesterase II from rat mammary gland has been crystallized in the presence of decanoic acid by the vapor diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), and have cell dimensions, a = 52.7 A, b = 78.0 A, and c = 133.6 A. The asymmetric unit likely consists of two protein monomers based on predictions from its calculated Matthews coefficient. Crystals typically diffract to at least 2.5 A resolution and are suitable for X-ray crystallographic analysis.

The professional credo of an ultra-orthodox psychotherapist.

The somewhat unorthodox working guidelines and professional attitudes of an ultra-orthodox (UO) psychotherapist are presented. Despite relatively low status in the UO society, the UO therapist can contribute much to UO society: by enhancing individuals' religious, as well as, general functioning, and by serving as a consultant to rabbinical and community leaders. Ensuing from the centrality of religion in the therapist's life, usual therapy norms and parameters may be abrogated or modified in the direction of increased stringency (eg, in avoiding sexual misconduct and subtle forms of "robbery") or leniency (eg, extra-therapeutic helpfulness). It is noted that secular professionals who are knowledgable and respectful of religion are at no disadvantage in working with the UO. A culturally-sensitive approach is suggested when treating this population.

Stereochemistry of metal ion coordination to the terminal thiophosphoryl group of adenosine 5'-O-(3-thiotriphosphate) at the active site of pyruvate kinase.

Epimers of [gamma-17O]adenosine 5'-O-(3-thiotriphosphate) ([gamma-17O]ATP gamma S) have been used to determine the stereochemistry of Mn2+ coordination to the terminal thiophosphoryl group in complexes of pyruvate kinase, oxalate, ATP gamma S, and Mg2+, Zn2+, Co2+, or Cd2+. The complex of pyruvate kinase with oxalate and ATP binds 2 equiv of divalent cation per active site. The terminal phosphoryl group of ATP in this enzymic complex becomes a chiral center as a result of coordination to both divalent metal ions. Electron paramagnetic resonance (EPR) data for complexes of pyruvate kinase with Rp- or Sp-[gamma-17O]-ATP gamma S, [17O]oxalate, and mixtures of Mn2+ with Mg2+, Zn2+, or Co2+ show that Mn2+ binds selectively at the site defined by coordination to oxalate and the pro-R oxygen of the thiophosphoryl group of ATP gamma S. In mixtures containing Mn2+ and Cd2+ with Tl+ as the monovalent cation, two hybrid complexes form, enzyme-oxalate-MnII-ATP gamma S-CdII and enzyme-oxalate-CdII-ATP gamma S-MnII, as in the analogous complexes with ATP and K+ or Tl+ (Buchbinder, J. L., & Reed, G. H. (1990) Biochemistry 29, 1799-1806). In the enzyme-oxalate-MnII-ATP gamma S-CdII species, Mn2+ binds exclusively to the pro-R oxygen of the thiophosphoryl group. In the enzyme-oxalate-CdII-ATP gamma S-MnII species, Mn2+ binds to the pro-R oxygen (60%) and to the pro-S oxygen (40%).(ABSTRACT TRUNCATED AT 250 WORDS)

Mysticism and psychosis: the fate of Ben Zoma.

This paper examines the link between psychosis and mystical study through the cases of four young men who 'entered the garden' of Jewish mystical speculation and subsequently became psychotic. The role of such study as a precipitating factor is suggested, as three had no signs of disturbance prior to their mystical studies. All had suffered personal losses, and their choice of mystical texts and rites showed that their attraction to mysticism included a search for atonement for guilt they felt over their loss. The features of normative mysticism are presented with each case and it is apparent that hallucinations, grandiose and paranoid delusions, and social withdrawal, are phenomena that do not distinguish the psychotic from the mystic. Diagnosis of psychosis is made on the basis of duration of the state, ability to control entry into the state and the associated deterioration of habits, particularly the neglect of daily religious duties. These findings emphasize the need for the examining psychiatrist to be aware of the cultural background, despite the presence of seemingly florid psychopathology. Four eminent Rabbis entered the garden of mystical speculation. Ben Azzai glimpsed and died. Ben Zoma glimpsed and was damaged (lost his sanity). Elisha ben Avuyah lost his faith. Rabbi Akiva departed in peace.

Crystallization and preliminary analysis of enzyme-substrate complexes of pyruvate kinase from rabbit muscle.

Pyruvate kinase from rabbit muscle has been crystallized in a form suitable for high resolution X-ray analysis. Complexes of the enzyme with Mn2+ and either pyruvate or oxalate crystallize from solutions of polyethyl-eneglycol 8000 at pH 6.0. Crystals obtained from solutions of the complexes with pyruvate or oxalate appear isomorphous and belong to the triclinic space group P1. The crystals have unit cell dimensions a = 83.3(4) A, b = 109.4(6) A, c = 145.7 (7) A, alpha = 94.9 degrees, beta = 93.6 degrees, gamma = 112.2 degrees. These crystals diffract to better than 2.4 A resolution and are stable in the X-ray beam for at least 20 hr. Electron paramagnetic resonance measurements on a single crystal show that Mn2+ is bound to the crystalline protein.

Electron paramagnetic resonance studies of the coordination schemes and site selectivities for divalent metal ions in complexes with pyruvate kinase.

Electron paramagnetic resonance (EPR) spectroscopy has been used to investigate the properties of the binuclear divalent metal center at the active site of pyruvate kinase. The preferred binding sites for different types of divalent cation in complexes of the enzyme with ATP and oxalate were determined in hybrid metal complexes with Mn(II). Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17O in isotopically enriched forms of oxalate and ATP was used to determine the position of Mn(II) at the binuclear metal center. When Mn(II) is present in combination with Zn(II), Ni(II), or Co(II), Mn(II) binds predominantly at the site defined by ligands from the protein, oxalate, and the gamma-phosphate of ATP. In contrast, EPR data of samples with mixtures of Mn(II)/Ca(II) or Mn(II)/Cd(II) reveal signals of two distinct hybrid-metal complexes. In one species, Mn(II) binds at the oxalate/gamma-phosphate site, and Ca(II) or Cd(II) binds at the ATP site. In the other species, the positions of Mn(II) and the second metal ion are reversed. The results indicate that, in enzymic complexes with ATP and oxalate, the relative size of the cation is a major factor controlling site selectivity. Metal ions that have ionic radii smaller than Mn(II) bind preferentially at the site occupied by ATP whereas metal ions that have ionic radii larger than Mn(II) bind preferentially at the site occupied by oxalate. EPR data of one of the hybrid complexes formed by Cd(II) and Mn(II) show that an alpha,beta,gamma-tridentate species of MnIIATP binds to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Summoning a punishing angel: treatment of a depressed patient with dissociative features.

The authors describe their treatment of a 24-year-old repentant, extremely observant Jewish man with major depressive disorder who complained of persecution by a personal angel. The therapists initiated a culturally sensitive psychotherapy of the patient, enacting a ritual summoning of the angel that resulted in the angel's transformation into an ally. The authors discuss the relationship of the patient's symptomatology to pathological mourning, trance, and dissociation. They advocate the use of a strategic combination of culture-specific concepts with modern psychiatric approaches in similar cases.