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At least 1,135 records · Page 63Linked to original sources
PubMed1999

Generation of spectrin breakdown products in peripheral nerves by addition of M-calpain.

Identification of spectrin breakdown products (SBP) in tissues of the central nervous system (CNS) has been used to monitor calpain activity in models of neurodegeneration. We investigated the use of this technique in the peripheral nervous system (PNS) in order to use it as a marker of calpain-mediated proteolysis during axonal degeneration. Using in vitro methods for activation of calpains, we compared brains and sciatic nerves from rats for the presence of calpain-specific SBP. The 150-kDa SBP identified on western blots was demonstrated in brain and nerve homogenates subjected to membrane disruption in the presence of calcium. Incubation of tissues with recombinant m-calpain generated SBP in a dose-dependent fashion, and calpastatin inhibited the generation of SBP by either paradigm. In contrast to brain, sciatic nerves showed the presence of SBP even in noninjured tissues, suggesting a basal level of calpain activity in peripheral nerves. Time-course experiments showed that the generation of SBP in sciatic nerves correlated with the breakdown of axonal neurofilaments. SBP peaked within minutes after addition of m-calpain and disappeared in the homogenates before 1 h, indicating that identification of SBP is a transient phenomenon. These data provide a potential new way for studying axonal degeneration in both experimental and human neuropathies.

Animals↗
PubMed2001

Backbone and side-chain 13C and 15N signal assignments of the alpha-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 Tesla.

The backbone and side-chain 13C and 15N signals of a solid 62-residue (u-13C,15N)-labelled protein containing the alpha-spectrin SH3 domain were assigned by two-dimensional (2D) magic angle spinning (MAS) 15N-13C and 13C-13C dipolar correlation spectroscopy at 17.6 T. The side-chain signal sets of the individual amino acids were identified by 2D 13C-13C proton-driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton-nitrogen and a nitrogen-carbon cross-polarisation step followed by a carbon-carbon homonuclear transfer unit. Interresidue correlations leading to sequence-specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7-61, while the signals of the N- and C-terminal residues 1-6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor-bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three-dimensional versions of the applied or analogous techniques.

Amino Acid Sequence↗
PubMed2001

Assignment of the nonexchanging protons of the alpha-spectrin SH3 domain by two- and three-dimensional 1H-13C solid-state magic-angle spinning NMR and comparison of solution and solid-state proton chemical shifts.

The assignment of nonexchanging protons of a small microcrystalline protein, the alpha-spectrin SH3 domain (7.2 kDa, 62 residues), was achieved by means of three-dimensional (3D) heteronuclear (1H-13C-13C) magic-angle spinning (MAS) NMR dipolar correlation spectroscopy. With the favorable combination of a high B(0)-field, a moderately high spinning frequency, and frequency-switched Lee-Goldburg irradiation applied during 1H evolution, a proton linewidth < or =0.5 ppm at 17.6 Tesla was achieved for the particular protein preparation used. A comparison of the solid-state 1H chemical shifts with the shifts found in solution shows a remarkable similarity, which reflects the identical protein structures in solution and in the solid. Significant differences between the MAS solid- and liquid-state 1H chemical shifts are only observed for residues that are located at the surface of the protein and that exhibit contacts between different SH3 molecules. In two cases, aromatic residues of neighboring SH3 molecules induce pronounced upfield ring-current shifts for protons in the contact area.

Models, Molecular↗
PubMed1989

Severe hemolysis and red cell fragmentation caused by the combination of a spectrin mutation with a thrombotic microangiopathy.

Two patients are described who presented with severe hemolysis and erythrocyte fragmentation. One patient had renal allograft rejection and disseminated intravascular coagulation, and the other had thrombotic thrombocytopenia purpura. The severity of hemolysis and the red cell abnormalities were considerably more profound than usually seen in patients with thrombotic microangiopathies. After evaluation of blood smears prepared before the onset of the disease and biochemical characterization of proteins of the red blood cell skeleton, a mutation of the skeletal protein spectrin, designated Sp alpha l/65, was identified. In the heterozygous form, this mutation manifests as mild, often asymptomatic, hereditary elliptocytosis. We conclude that in these two patients with thrombotic microangiopathy, the intrinsic red cell membrane instability resulting from the underlying skeletal defect aggravated the mechanical red cell fragmentation, producing morphological features similar to the severe hemolytic form of hereditary elliptocytosis or hereditary pyropoikilocytosis.

Adolescent↗
PubMed2006

Spectrin mutations in spinocerebellar ataxia (SCA).

Recently, betaIII spectrins have been recognized as ataxia disease genes, with the identification by Ikeda and co-workers of pathogenic mutations in the SPTBN2 gene in three large (and mapped) SCA5 families of American and European origin.((1)) With their discovery, the large "Lincoln" family has been traced back to the underlying genetic defect for the slowly progressive cerebellar ataxia. In addition, the involvement of this component of the cytoskeleton directs attention towards the possible role of organelle stability during neurodegeneration. The findings suggest that the mechanical properties of neurons and their dynamics may be as important as altered Ca(2+) homeostasis, transcriptional dysregulation, and impaired protein degradation in neurodegeneration conditions.

Humans↗
PubMed1985

Actin assembly and filament cross-linking in the presence of TW 260/240, the tissue-specific spectrin of the chicken intestinal brush border.

TW 260/240 is a tissue-specific spectrin found in the terminal web region of the chicken intestinal brush border. We have examined the effects of TW 260/240 on assembly rates and critical concentrations (Co's) for monomer addition at the barbed and pointed ends of the actin filament. For these studies, acrosomal processes (AP) from Limulus sperm were used as nuclei for actin assembly. Under conditions which favor the interaction of TW 260/240 for actin (20-75 mM KCl, 2 mM Mg++) no effect on either elongation rates or Co's at either end of the actin filament was observed in the presence of this spectrinlike protein. The Limulus AP nucleation assay also allowed visualization of the kinetics of filament binding and cross-linking by TW 260/240. Ultrastructural analysis of TW 260/240 binding to actin filaments at their growing ends indicates that TW 260/240 tetramers bind laterally to the filament. Finally, evidence is presented that indicates that filaments cross-linked by TW 260/240 are stabilized against shear-dependent breakage.

Acrosome↗
PubMed1991

Isolation and localization of a spectrin-like protein from echinoderm sperm.

Thyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 microns long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73-89 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal-like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS-PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated-calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca(++)-sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod-shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent-DNase-1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.

Acrosome↗
PubMed1983

The p36 substrate of tyrosine-specific protein kinases co-localizes with non-erythrocyte alpha-spectrin antigen, p230, in surface lamina of cultured fibroblasts.

Biochemical and immunofluorescence studies have demonstrated that p36, a major substrate for the tyrosine-specific protein kinases induced by several sarcoma viruses and epidermal growth factor, is associated with plasma membranes and detergent-resistant cytoskeletal structures of cultured cells. We have used here polyclonal antisera and monoclonal antibodies in indirect immunofluorescence microscopy to study the subcellular location of p36 and the p230, which is a subplasmalemmal polypeptide showing immunologic cross-reactivity with erythrocyte alpha-spectrin. Both p36 and p230 showed a diffuse distribution in fixed and permeabilized cells and were localized in a surface lamina-like network in Triton-extracted cells. In double-staining experiments, an extensive co-distribution between these proteins was seen in detergent-treated cultured fibroblasts. These results, together with our previous work, suggest that the p36 protein is an integral part of the detergent-resistant proteinaceous network at the cytoplasmic face of the plasma membrane.

Animals↗
PubMed1995

Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands.

Calcium is a universally employed cytosolic messenger in eukaryotic cells. Most of the proteins that bind signalling calcium are members of the calmodulin superfamily and share two or more helix-loop-helix motifs known as EF-hands. A model, based on structure comparison of different domains and supported by preliminary NMR data, has suggested that EF-hands involved in signal transduction undergo a major conformational change upon calcium binding from a 'closed' to an 'open' state allowing protein-protein interaction. We have determined the solution structures of the EF-hand pair from alpha-spectrin in the absence and in the presence of calcium. The structures are in the closed and open conformation respectively, providing a definite experimental proof for the closed-to-open model. Our results allow formulation of the rules which govern the movement induced by calcium. These rules may be generalized to other EF-hands since the key residues involved are conserved within the calmodulin family.

Amino Acid Sequence↗
PubMed1999

Polarized expression of immunoglobulin, spectrin, and protein kinase C beta II occurs in B cells from normal BALB/c, autoimmune lpr, and anti-ssDNA transgenic, tolerant mice.

The rapid redistribution of B cell surface immunoglobulin to a cap upon cross-linking treatment is a well-described phenomenon, the physiological significance of which is unknown. We describe the observation that splenic B cells from unimmunized normal, autoimmune, and tolerant mice express naturally occurring capped immunoglobulin in the absence of exogenous stimulation. The percentage of capped B cells increases to 20% of B cells by age 16 weeks in the progressive autoimmune lpr mouse. Transgenic, tolerant mice expressing lpr-derived genes for ssDNA-binding antibody also demonstrate a large percentage (35-75%) of immunoglobulin-capped splenic B cells. In these capped B cells, protein kinase C beta II, the cytoskeletal proteins spectrin and ankyrin, and the lipophilic probe diI are enriched beneath the site of the immunoglobulin cap. These data suggest that polarization of surface receptors, signaling molecules, anionic phospholipid domains, and cytoskeletal proteins may be an important part of the B cell immune response in vivo.

Animals↗
PubMed1978

Spectrin binding and the control of membrane protein mobility.

Transmembrane proteins of the human erythrocyte show restricted in-plane mobility. Many of the restrictions on mobility are attributable to the molecules of spectrin which are located on the protoplasmic surface of the erythrocyte membrane. These molecules are elongate, form end-to-end heterodimer associations, and bind selectively to protein (or proteins) accessible on inside-out, but not right-side out, membrane vesicles.

Actins↗
PubMed1978

Analysis of the major polypeptides of spectrin by tryptic digestion.

The two major polypeptides of erythrocyte membrane spectrin have been isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The tryptic peptide maps of the two polypeptides have been prepared by thin-layer chromatography and electrophoresis. Radioactive peptides have been prepared by 14C-carboxymethylation and chloramine T-catalysed 125I iodination. Maps of both sets of peptides demonstrate a marked similarity between the two parent polypeptides.

Animals↗
PubMed2000

Spectrin changes occur in erythrocytes from patients with Fanconi's anemia and their parents.

Fanconi's anemia (FA) is a clinically and genetically heterogeneous disease which has been hypothesized to be defective in the detoxification of reactive oxygen species. In this work we report the results obtained by morphometric analyses on the red blood cells (RBCs) from FA patients and their parents. We found that a high rate of erythrocytes from both homozygous and heterozygous subjects was significantly altered. RBCs underwent in fact cytoskeleton-dependent modifications, in particular of spectrin molecule, leading to cell shrinking and blebbing. We hypothesize that these changes may be the result of an oxidative imbalance that probably lead to alterations of RBC plasticity- and deformation-associated functions. Moreover, our results also suggest the possibility to identify FA carriers by the existence of RBC abnormalities.

Adolescent↗
PubMed1998

Dispensability of the actin-binding site and spectrin repeats for targeting sarcomeric alpha-actinin into maturing Z bands in vivo: implications for in vitro binding studies.

To explore the roles of specific domains of sarcomeric alpha-actinin (s-alpha-actinin) in the assembly and maintenance of striated myofibrils, myogenic cultures were transfected with four MYC-tagged s-alpha-actinin peptides. They were: (1) full-length sarcomeric alpha-actinin, (2) an N-terminal deletion that removed the actin-binding site only (MYC/A-), (3) a peptide that consisted of the actin-binding site only (MYC/A+), and (4) an N-terminal deletion that removed the EF-hands and titin-binding domains (MYC/EFT-). While cytotoxic in replicating myogenic cells, as they were in PtK2 cells, the four MYC peptides were not cytotoxic in postmitotic myotubes. In myotubes each of the four different MYC peptides were promptly and selectively incorporated into normal Z bands. The incorporation of MYC/A-, MYC/A+, and MYC/EFT- into Z bands suggests that (a) the actin-binding site, (b) the spectrin-repeats believed to be responsible for anti-parallel dimerization, and (c) the C-terminal EF-hands and titin-binding domains are each dispensable for targeting s-alpha-actinin/MYC peptides into Z bands. These findings could not have been predicted from the behavior of alpha-actinin (a) in binding assays in cell-free systems or (b) when expressed in transfected nonmuscle cells.

Actinin↗
PubMed1996

Conformational analysis of peptides corresponding to beta-hairpins and a beta-sheet that represent the entire sequence of the alpha-spectrin SH3 domain.

In an attempt to identify potential folding initiation sites for a small, all beta-protein domain, we have examined the conformational preferences in aqueous solution of peptides that span the entire length of the alpha-spectrin SH3 domain, using proton nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. Two of the peptides correspond to beta-hairpins (m6 and m8), one to the RT-loop (m4, which can be considered as a distorted beta-hairpin), one to a beta-hairpin created by joining the N and C-terminal strands via a small linker (m2) and the fifth one to a three-stranded antiparallel beta-sheet composed of beta-hairpins m6 and m8 (m68). To estimate the distorting effect of the aromatic side-chains of Trp41 and Trp42 on the CD and NMR spectra of peptides m6, m8 and m68, we have also analyzed a short, ten-residue random-coil peptide containing residues 39 to 44 (mC). The CD and NMR results indicate that none of the peptides populates to a large extent a particular secondary structure conformation. However, careful anlaysis of the NMR data reveals that peptides m6, m8 and m68 could adopt, to a small extent, native-like conformations, although in the case of peptide m68 there is also evidence of the presence of non-native helical conformations. Addition of 30% (v/v) 2,2,2-trifluoroethanol stabilizes the appearance of non-native helical populations in some small regions of peptides m2, m4, m8 and m68, while it induces a native-like conformation in peptide m6. Those fragments corresponding to the two real beta-hairpins in the protein are the ones which exhibit some tendency to populate native-like structures (m6 and m8), while the ones corresponding to the long RT-loop (m4) or the newly created one (m2) are mainly unstructured in water solution. Although there could be some local interactions that favor the acquisition of a native secondary structure in this domain, tertiary interactions should play a major role in defining its native secondary structure.

Amino Acid Sequence↗
PubMed1997

Non-native local interactions in protein folding and stability: introducing a helical tendency in the all beta-sheet alpha-spectrin SH3 domain.

The relative importance of secondary structure interactions versus tertiary interactions for stabilising and guiding the folding process is a matter for discussion. Phenomenological models of protein folding assign an important role to local contacts in protein folding and stability. On the other hand, simplistic lattice simulations find that secondary structure is mainly the product of protein compaction and that optimisation of folding speed seems to require small contributions of local contacts to the stability of the folded state. To examine the extent to which secondary structure propensities influence protein folding and stability, we have designed mutations that introduce a strong non-native helical propensity in the first 19 residues of the alpha-spectrin SH3 domain. The mutant proteins have the same three-dimensional structure as the wild-type, but they are less stable and have less co-operative folding transitions. There seems to be a relationship between the non-native helical propensity and the compaction of the denatured state. This suggests that in the denatured ensemble under native conditions there is a significant proportion of compact structures with non-native secondary structures. Our results demonstrate that non-local interactions can overcome strong non-native secondary structure propensities and, more important, that optimisation of folding speed and co-operativity requires the latter to be relatively small.

Amino Acid Sequence↗
PubMed1997

Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the pleckstrin homology domain from beta-spectrin.

We have used a novel, largely automated, calculation method to refine the NMR solution structure of the pleckstrin homology domain of beta-spectrin. The method is called ARIA for Ambiguous Restraints for Iterative Assignment. The starting point for ARIA is an almost complete assignment of the proton chemical shifts, and a list of partially assigned NOEs, mostly sequential and secondary structure NOEs. The restraint list is then augmented by automatically interpreting peak lists generated by automated peak-picking. The central task of ARIA is the assignment of ambiguous NOEs during the structure calculation using a combination of ambiguous distance restraints and an iterative assignment strategy. In addition, ARIA calibrates ambiguous NOEs to derive distance restraints, merges overlapping data sets to remove duplicate information, and uses empirical rules to identify erroneous peaks. While the distance restraints for the structure calculations were exclusively extracted from homonuclear 2D experiments, ARIA is especially suited for the analysis of multidimensional spectra. Applied to the pleckstrin homology domain, ARIA generated structures of good quality, and of sufficiently high accuracy to solve the X-ray crystal structure of the same domain by molecular replacement. The comparison of the free NMR solution structure to the X-ray structure, which is complexed to D-myo-inositol-1,4,5-triphosphate, shows that the ligand primarily induces a disorder-order transition in the binding loops, which are disordered in the NMR ensemble but well ordered in the crystal. The structural core of the protein is unaffected, as evidenced by a backbone root-mean-square difference between the average NMR coordinates and the X-ray crystal structure for the secondary structure elements of less than 0.6 A.

Binding Sites↗
PubMed1998

Heteronuclear relaxation study of the PH domain of beta-spectrin: restriction of loop motions upon binding inositol trisphosphate.

The structural dynamics of protein ligand binding sites is one factor determining the specificity towards related ligands. In this context, the spectrin PH domain, which binds to a number of phosphatidylinositol lipid head groups, was investigated with respect to the dynamics of the binding loops. The latter were found to be of intermediate flexibility on a picosecond to nanosecond time-scale in the free protein and become more rigid upon ligand binding. Significant 15N and proton chemical shift changes occur in the binding loops. The internal correlation time, determined from 15N heteronuclear relaxation data using the standard model-free approach, decreases upon ligand binding. For several residues a concomitant rise in the generalized order parameter is observed. This is interpreted as a dampening effect of the ligand on a slow loop motion, while a fast component is not affected. Molecular dynamics simulations were performed to further investigate this situation. In fact, two time-scales of loop motions in the free state are observed in a 9 ns molecular dynamics trajectory. Agreement with generalized order parameters obtained from the experiment improves when a subtrajectory is analyzed that excludes rare dihedral transitions.

Binding Sites↗