Proximal giant neurofilamentous axonopathy in mice genetically engineered to resist calpain and caspase cleavage of α-II spectrin.

We use 1,2-diacetylbenzene (1,2-DAB) to probe molecular mechanisms of proximal giant neurofilamentous axonopathy (PGNA), a pathological hallmark of amyotrophic lateral sclerosis. The spinal cord proteome of rodents displaying 1,2-DAB PGNA suggests a reduction in the abundance of α-II spectrin (Spna2), a key protein in the maintenance of axonal integrity. Protein immunoblotting indicates that this reduction is due to Spna2 degradation. We investigated the importance of such degradation in 1,2-DAB PGNA. Spna2 mutant mice lacking a calpain- and/or caspase-sensitive domain (CSD), thus hypothetically resistant to 1,2-DAB, and wild-type littermates, were treated with 1,2-DAB, 35 mg/kg/day, or saline control, for 3 weeks. 1,2-DAB induced motor weakness and PGNA, irrespective of the genotype. Spna2-calpain breakdown products were not detected in mutant mice, which displayed a normal structure of the nervous system under saline treatment. Intriguingly, treatment with 1,2-DAB reduced the abundance of the caspase-specific 120-kDa Spna2 breakdown products. Our findings indicate that degradation of Spna2 by calpain- and/or caspase is not central to the pathogenesis of 1,2-DAB axonopathy. In addition, the Spna2-CSD seems to be not required for the maintenance of the cytoskeleton integrity. Our conceptual framework offers opportunities to study the role of calpain-caspase cross talk, including that of the protease degradomics, in models of axonal degeneration.

Spectrin-based skeleton as an actor in cell signaling.

This review focuses on the recent advances in functions of spectrins in non-erythroid cells. We discuss new data concerning the commonly known role of the spectrin-based skeleton in control of membrane organization, stability and shape, and tethering protein mosaics to the cellular motors and to all major filament systems. Particular effort has been undertaken to highlight recent advances linking spectrin to cell signaling phenomena and its participation in signal transduction pathways in many cell types.

Identification of the main ubiquitination site in human erythroid alpha-spectrin.

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.

Identification of ubiquitinated repeats in human erythroid alpha-spectrin.

The spectrin role(s) is (are) very important for the shape and the physical properties of red cells, such as deformability and resistance to mechanical stresses. Moreover a variety of spectrin diseases are known. We have previously demonstrated [Corsi, D., Galluzzi, L., Crinelli, R. & Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935] that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo. In order to define the ubiquitinated repeats of this long protein and find out a possible function, we have produced recombinant peptides encompassing the alphaIII-, alphaIV-, alphaV- and EF hand domains of alpha-spectrin chain. These peptides were tested in in vitro ubiquitin conjugation assays and two regions susceptibles to ubiquitination were found. The first one, in the alphaIV-domain, includes the repeat 17 and the second one, in the alphaV-domain, includes the repeat 20 and a part of repeat 21. We also demonstrated that the susceptibility to ubiquitination of the alphaV-domain is reduced by interaction with the corresponding portion of beta-spectrin chain (betaIV-domain). Thus, at least ubiquitination of alphaV-domain is susceptible to cytoskeleton assembly and spectrin dimerization.

Properties of normal and mutant polypeptide fragments from the dimer self-association sites of human red cell spectrin.

We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the alpha-chain and the C-terminus of the beta-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of two helices from the C-terminal part of the beta-chain and one helix from the N-terminus of the alpha-chain. Three mutant beta-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The alpha- and beta-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold. Unexpectedly, the conformational stability of the folded beta-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible explanations for this phenomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wildtype alpha- and beta-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied by an increase in alpha-helicity. From continuous-variation profiles an association constant in the range 1-2 x 10(6) M-1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus of the spectrin beta-chain. Of the three mutations in the beta-chain fragment, one (an Ala-->Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the alpha-chain fragment, whereas Trp-->Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic conditions associated with the mutations.

Spectrin self-association site: characterization and study of beta-spectrin mutations associated with hereditary elliptocytosis.

Most of hereditary elliptocytosis (HE) cases are related to a spectrin dimer (SpD) self-association defect. The severity of haemolysis is correlated with the extent of the SpD self-association defect, which itself depends on the location of the mutation regarding the tetramerization site. This site is presumed to involve the first C helix of the alpha chain and the last two helices, A and B, of the beta chain to reconstitute a triple helical structure (A, B and C), as observed along spectrin. Using recombinant peptides, we demonstrated that the first C helix of the alpha chain and the last two helices of the beta chain alone are not sufficient to establish interactions, which only occurred when a complete triple-helical repeat was added to each partner. One adjacent repeat is necessary to stabilize the conformation of both N- and C-terminal structures directly involved in the interaction site and is sufficient to generate a binding affinity similar to that observed in the native molecule. Producing peptides carrying a betaHE mutation, we reproduced the tetramerization defect as observed in patients. Therefore, the betaW2024R and betaW2061R mutations, which replace the invariant tryptophan and a residue located in the hydrophobic core, respectively, affect alpha-beta interactions considerably. In contrast, the betaA2013V mutation, which modifies a residue located outside any presumed interacting regions, has a minor effect on the interaction.

Analysis of ETV6 and ETV6-AML1 proteins in acute lymphoblastic leukaemia.

The t(12;21) is a recurring chromosomal abnormality in acute lymphoblastic leukaemias (ALLs) which results in the production of an ETV6-AML1 fusion gene. The association between t(12;21) and the deletion of the untranslocated allele of ETV6 is among the most frequent abnormalities observed in B-lineage ALLs in children. In order to study the proteins encoded by ETV6 and ETV6-AML1, we raised polyclonal antibodies directed against a recombinant peptide corresponding to the junctional region of ETV6-AML1. Cell lysates from various leukaemic cell lines, and from children with B- and T-lineage ALLs, were studied by Western blot. Two isoforms of ETV6 protein were detected in normal bone marrow cells and in leukaemic cells without 12p alteration: a major form (apparent m.w. 63 kD) and a minor one (apparent m.w. 53 kD). In the REH cell line, which expresses the ETV6-AML1 fusion transcript and no normal ETV6 mRNA, the ETV6 isoforms were absent and two new bands were detected corresponding to ETV6-AML1 protein products (apparent m.w. 95 and 105 kD). A similar pattern was obtained with blast cells from patients with a t(12;21) and a deletion of ETV6. In two patients with a t(12;21) but no deletion of ETV6, four bands were detected corresponding to both the normal ETV6 and ETV6-AML1 proteins, suggesting that in these cases the second ETV6 allele was not inactivated. Surprisingly, the expression pattern of ETV6 differed widely from patient to patient. In three out of 13 patients without t(12;21), the relative intensity of the bands corresponding to ETV6 isoforms in blast cells from patients was completely different from normal cells, with a marked predominance of the 53 kD isoform. The pattern of ETV6 expression was normal in bone marrow from the same patients during remission. These finding suggest that ETV6 abnormalities are not restricted to patients with translocations or deletions involving this gene.

Spectrin St Claude, a splicing mutation of the human alpha-spectrin gene associated with severe poikilocytic anemia.

An alpha-spectrin variant with increased susceptibility to tryptic digestion, alpha(II/47), was previously observed in a child with severe, recessively inherited, poikilocytic anemia. The molecular basis of this variant, spectrin St Claude, has now been identified as a splicing mutation of the alpha-spectrin gene due to a T --> G mutation in the 3' acceptor splice site of exon 20. This polypyrimidine tract mutation creates a new acceptor splice site, AT --> AG, and leads to the production of two novel mRNAs. One mRNA contains a 12 intronic nucleotide insertion upstream of exon 20. This insertion introduces a termination codon into the reading frame and is predicted to encode a truncated protein (108 kD) that lacks the nucleation site and thus cannot be assembled in the membrane. In the other mRNA, there is in-frame skipping of exon 20, predicting a truncated (277 kD) alpha-spectrin chain. The homozygous propositus has only truncated 277 kD alpha-spectrin chains in his erythrocyte membranes. His heterozygous parents are clinically and biochemically normal. This allele was identified in 3% of asymptomatic individuals from Benin, Africa.

Human coproporphyrinogen oxidase. Biochemical characterization of recombinant normal and R231W mutated enzymes expressed in E. coli as soluble, catalytically active homodimers.

To obtain recombinant human coproporphyrinogen oxidase (CPX), a cDNA for the coding region of mature human CPX has been expressed in E. coli. CPX was produced as a fusion protein with glutathione S-transferase followed by the hexapeptide recognition site for thrombin cleavage just preceding first amino acid of the CPX protein. The human CPX was found to be in the soluble fraction. This previously unobtainable human heme synthetic enzyme was purified to electrophoretic homogeneity with a specific activity of 4200 nmol/hr./mg of protein using a Glutathione Sepharose 4B column and gel filtration. Recombinant human CPX exhibits homogeneous behavior during high performance liquid chromatography (HPLC) and the N-terminal sequence, confirmed by protein sequencing, revealed a single polypeptide chain. In its active form, human CPX is a homodimer. According to the hydrodynamic properties derived from analytical ultracentrifugation, dimeric CPX has a nearly globular shape. Additionally, naturally occurring Arg to Trp (R231W)-mutated CPX has been also expressed in E. coli and further characterized. The mutated enzyme has a Km value of 0.55 microM as compared to 0.30 microM for the wild type. The catalytic efficiency (specificity constant, kcat/Km) of the mutated CPX was four fold lower than wild-type enzyme. The activity measurement of the mutated enzyme showed higher thermal sensitivity as compared with wild type CPX. The measured pI for mutated CPX is 5.65, compared to 6.40 for wild type. The pH optima for the mutated and wild-type protein are 6.6 and 6.8, respectively. The R231W mutation of CPX does not affect dimer formation and both normal and mutated CPX exhibit identical sedimentation properties. The thermal denaturation of both wild type and mutant CPX was found to be irreversible. The mutated CPX contained a significant amount of tightly bound porphyrin coproporphyrin. No metal association was found either in wild type or in mutated CPX. The availability of the recombinant human CPX will aid in structural and mechanistic studies.

Identification of alpha-spectrin domains susceptible to ubiquitination.

Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study, we mapped the sites of ubiquitination on erythrocyte alpha-spectrin. A peptide map of digested alpha-spectrin, previously submitted to in vitro 125I-ubiquitin conjugation, revealed the presence of four distinct labeled bands with Mr 40,000, 36,000, 29,000, and 25,500. Western blotting experiments using antibodies against each alpha-spectrin domain revealed that only IgG anti-alphaIII domain recognized the 125I-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alphaV domain recognized the Mr 40,000 125I-ubiquitin-labeled peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra and tri multiubiquitin chains. Ubiquitination of the alphaIII and alphaV domains was further confirmed by anti-alpha-spectrin domain immunoaffinity chromatography. Endoprotease Lys C-digested spectrin conjugated previously to 125I-ubiquitin was incubated with antibodies against each trypsin-resistant domain of alpha-spectrin. Gamma counting of the radiolabeled antigen-antibody complexes purified by protein A chromatography showed labeling in the IgG anti-alphaIII and anti-alphaV complexes alone. Domain alphaIII is not associated with any known function, whereas domain alphaV contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiquitin in the modulation of the stability, deformability, and viscoelastic properties of the erythrocyte membrane.

Human erythroid spectrin alpha subunit and its SH3 domain are sensitive to acidic Plasmodium falciparum proteolytic activity.

Many proteases play a crucial role in the Plasmodium intraerythrocytic life cycle. Spectrin depletion, one of the major events involved in parasite release from the red blood cell, results from proteolytic activities associated with the presence of the intracellular parasite. Here, we describe a new acidic proteolytic activity from Plasmodium falciparum, whose target is the alpha-subunit of human spectrin. Immunoblotting experiments with antibodies specific for the tryptic peptides of the alpha-chain and in vitro proteolysis tests on recombinant peptides from different regions of the spectrin alpha subunit demonstrated that cleavage sites for the parasite proteolytic activity were localized within the SH3 motif of the alpha-chain sequence. Remarkably, this Plasmodium protease activity on spectrin SH3 substrate was unable to cleave the SH3 from fodrin, a non-erythroid spectrin.

Epidemiological studies of spectrin mutations related to hereditary elliptocytosis and spectrin polymorphisms in Benin.

We studied an African population in Benin and discovered an unexpectedly high frequency (1.6%) of hereditary elliptocytosis (HE) among the 1447 subjects studied. In approximately two-thirds of HE individuals we identified molecular defects, primarily those in erythrocyte alpha-spectrin (dupL154, L260P and L207P mutations), as well as a novel mutation of erythrocyte beta-spectrin (beta-W2061R mutation). We also identified the genetic basis of a previously identified protein polymorphism of the alpha III domain of spectrin (R1331I mutation). The genetic background of HE in the African population was studied using a number of polymorphisms of the alpha-spectrin gene, including the alpha III domain polymorphism. These studies suggest that the HE mutations appear to have originated from separate genetic backgrounds in this population.

Inhibition of phospholipase D activity by fodrin. An active role for the cytoskeleton.

Phospholipase D (PLD) is a major enzyme implicated in important cellular processes such as secretion and proliferation. The knowledge of its regulation is essential to understand the control of these phenomena. Several proteins activating PLD have been described in the last years. In this report, we chromatographed bovine brain cytosolic proteins to identify fodrin, the non-erythroid spectrin, as the first described inhibitor of PLD. A cytosolic fraction with an inhibitory effect on PLD activity loses its capacity after immunoprecipitation of fodrin. Moreover, at 1 nM, purified fodrin blocks fully and quickly PLD activity, whatever the stimuli used. In contrast, fodrin has no effect on adenylate cyclase activity. Fodrin-analogous proteins like dimeric or tetrameric erythroid spectrin have the same inhibitory effect on PLD, at higher concentrations. Other cytoskeletal proteins, actin and vimentin, are inefficient on PLD inhibition. The mechanisms implicated in PLD modulation such as post-translational modifications of fodrin and the role of small G-proteins on the cytoskeleton regulation are discussed. In conclusion, this study reveals that fodrin is involved in the control of PLD activity, suggesting that the cytoskeleton could have an active role in control of secretion and proliferation.

Location and PCR-based detection of three polymorphisms of the human erythrocyte beta-spectrin gene (SPTB).

Defects of beta spectrin, a structural protein of the erythrocyte membrane skeleton, have been identified in many cases of inherited disorders of red blood cell shape such as hereditary elliptocytosis and spherocytosis. To aid in genetic analyses of families with these disorders, the locations of three beta-spectrin gene (SPTB) polymorphisms were mapped and PCR-based assays designed for their identification. Using these PCR-based assays, the frequencies of these polymorphisms were determined in two populations.

Identification of three novel spectrin alpha I/74 mutations in hereditary elliptocytosis: further support for a triple-stranded folding unit model of the spectrin heterodimer contact site.

Six individuals with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) from three unrelated families were evaluated. Defects in the ability of spectrin (Sp) to undergo self-association were present, and associated with increased recovery of the Sp alpha I 74-kD fragment after limited tryptic digestion (Sp alpha I/74 variant). Because mutations associated with the Sp alpha I/74 variant described to date have been localized to the 5' coding region of the alpha-Sp gene (exon 2) or at the 3' coding end of the beta-Sp gene (exon 30), the polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) method was used to detect mutations in these two regions. In one family with HE, an abnormal pattern of migration of PCR-amplified fragments containing exon 2 was observed, and led to the detection of a new mutation (Ile24Ser) in helix 3 of repeating segment alpha 1. In the two other families, an abnormal pattern of migration of PCR-amplified fragments containing exon 30 was observed in affected individuals, and sequencing led to the identification of two new mutations (Ala2023Val and Trp2024Arg) in helix 1 of repeating segment beta 17. The elliptogenic potential of these mutations emphasizes the importance of the conformational integrity of each of the three helices involved in the formation of the Sp heterodimer contact site, and will help identify critical amino acids involved in this interaction.

[Hospital nuns attending indigent patients in the seventeenth and eighteenth centuries]. / Les soeurs hospitalières au service des pauvres malades aux XVIIe et XVIIIe siècles.

Unacknowledged, the hospital nuns deserve to be studied. Their call (or vocation), tested by the selection during novitiate, sets them into hospital environment of XVIIth and XVIIIth centuries and is rarely refuted. Whatever their own religious families, they represent the majority of nurses. In spite of some conflicts, their increasing success in the service of hospitals depends on cheap, movable, flexible, docile and polyvalent communities.

Molecular basis of clinical and morphological heterogeneity in hereditary elliptocytosis (HE) with spectrin alpha I variants.

The impaired ability of spectrin dimers to self-associate into tetramers is one of the most frequent defects associated with hereditary elliptocytosis (HE) and its more serious form, hereditary pyropoikylocytosis (HPP). We previously described four proteic variants of the spectrin (Sp) alpha I tryptic domain associated with the Sp dimer self-association defect (Sp alpha I/78, Sp alpha I/74, Sp alpha I/65, Sp alpha I/46 variants). Following the characterization of proteic variants, genomic molecular defects were identified and most of the mutations appeared to lie either in or near the self-association site, i.e. in the alpha I tryptic domain or in the beta I tryptic domain. The clinical severity of these different mutations varies considerably and ranges from asymptomatic to severe haemolytic disease such as in heterozygous HPP patients and in some homozygous HE patients. Studies of 113 patients from 61 HE families showed a correlation among parameters and showed which factors modulate the clinical expression of the molecular defect. Our analysis indicated that the clinical expression was directly correlated with the severity of the spectrin dimer self-association defect as evaluated by the increase in the Sp dimer percentage found in the 4 degrees C extract. A critical threshold of 40-50% of unassembled Sp dimer was determined; above that, patients exhibited severe haemolysis requiring splenectomy. The percentage of Sp dimer depends, in turn, on two factors: (i) the nature of the variant in relation to the position of the mutation versus the tetramerization site; (ii) the relative amount of mutant spectrin present in the membrane (ranging from 15% to 80% in heterozygous patients). As for the severity of haemolysis, the ghost mechanical stability to shear stress, as measured by ektacyometer, was also found to depend on the Sp dimer self-association defect. In contrast, the decrease in erythrocyte deformability was not related to the amount of unassembled Sp dimer but appeared to be correlated with the amount of mutant spectrin whatever the variant. Concerning erythrocyte morphology and the number of elliptocytes, the Sp alpha I/65 variant appears to be the most 'elliptocytogenic' variant, indicating that erythrocyte shape abnormality is not linked to the Sp dimer self-association defect.