Biochemical and ultrastructural aspects of Mr 165,000 M-protein in cross-striated chicken muscle.

To better understand the relationship between the Mr 165,000 M-line protein (M-protein) and H-zone structure in skeletal and in cardiac muscle, as well as the possible interaction of M-protein with another skeletal muscle M-line component, the homodimeric creatine kinase isoenzyme composed of two M subunits (MM-CK), we performed biochemical, immunological, and ultrastructural studies on myofibrils extracted by different procedures. In contrast to MM-CK, M-protein could not be completely removed from myofibrils by low ionic strength extraction. Fab-fragments of antibodies against M-protein could not release M-protein quantitatively from either breast or heart myofibrils but remained bound to the myofibrillar structure, whereas monovalent antibodies against MM-CK cause the specific release of MM-CK and the concomitant disappearance of the M-line from chicken skeletal muscle myofibrils. When MM-CK was removed from skeletal myofibrils by low ionic strength extraction or, more specifically, by incubation with anti-MM-CK Fab, M-protein was still not released quantitatively upon treatment with anti-M-protein Fab as judged from immunofluorescence data. In the ultrastructural investigation of low ionic strength extracted muscle fibers, M protein could be localized in two stripes on both sides of the former M-line, suggesting a reduced attachment to the residual H-zone structure, whereas the specific removal of MM-CK resulted in the same dense staining pattern for M-protein within the M-line as observed in untreated fibers. However, the binding of M-protein to the residual M-line structure seemed to be reduced, as a considerable amount of this protein could be identified in the supernate of sequentially incubated myofibrils. The results indicate a strong binding of M-protein within the H-zone structure of skeletal as well as heart myofibrils.

Intracellular calcium-binding proteins: more sites than insights.

Calcium ions as biological regulators exert their effects in part via interaction with a wide variety of intracellular calcium-binding proteins. One class of these proteins shares a common calcium-binding motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of new members of this family of EF-hand proteins, which now has about 170 members. A few of these proteins are present in all cells, whereas the vast majority are expressed in a tissue-specific fashion. The physiological function of a few of these proteins is known to be achieved via a calcium-dependent interaction with other proteins, thereby regulating their activity. The elucidation of the interactions and functions of the majority of these proteins remains a challenging task for the coming years.

The S100 family of EF-hand calcium-binding proteins: functions and pathology.

Calcium lons as second messengers control many biological processes, at least in part, via interaction with a large number of Ca(2+)-binding proteins. One class of these proteins shares a common Ca(2+)-binding motif, the EF-hand, Here, we describe some functional aspects of EF-hand proteins, which have been found recently in different cellular compartments. Novel links between EF-hand proteins, particularly S100 proteins, and specific diseases are now emerging.

Pathologies involving the S100 proteins and RAGE.

The S100 proteins are exclusively expressed in vertebrates and are the largest subgroup within the superfamily of EF-hand Ca2(+)-binding proteins Generally, S100 proteins are organized as tight homodimers (some as heterodimers). Each subunit is composed of a C-terminal, 'canonical' EF-hand, common to all EF-hand proteins, and a N-terminal, 'pseudo' EF-hand, characteristic of S100 proteins. Upon Ca2(+)-binding, the C-terminal EF-hand undergoes a large conformational change resulting in the exposure of a hydrophobic surface responsible for target binding A unique feature of this protein family is that some members are secreted from cells upon stimulation, exerting cytokine- and chemokine-like extracellular activities via the Receptor for Advanced Glycation Endproducts, RAGE. Recently, larger assemblies of some S100 proteins (hexamers, tetramers, octamers) have been also observed and are suggested to be the active extracellular species required for receptor binding and activation through receptor multimerization Most S100 genes are located in a gene cluster on human chromosome 1q21, a region frequently rearranged in human cancer The functional diversification of S100 proteins is achieved by their specific cell- and tissue-expression patterns, structural variations, different metal ion binding properties (Ca2+, Zn2+ and Cu2+) as well as their ability to form homo-, hetero- and oligomeric assemblies Here, we review the most recent developments focussing on the biological functions of the S100 proteins and we discuss the presently available S100-specific mouse models and their possible use as human disease models In addition, the S100-RAGE interaction and the activation of various cellular pathways will be discussed. Finally, the close association of S100 proteins with cardiomyopathy, cancer, inflammation and brain diseases is summarized as well as their use in diagnosis and their potential as drug targets to improve therapies in the future.

S100A1 deficiency results in prolonged ventricular repolarization in response to sympathetic activation.

S100A1 is a Ca(2+)-binding protein and predominantly expressed in the heart. We have generated a mouse line of S100A1 deficiency by gene trap mutagenesis to investigate the impact of S100A1 ablation on heart function. Electrocardiogram recordings revealed that after beta-adrenergic stimulation S100A1-deficient mice had prolonged QT, QTc and ST intervals and intraventricular conduction disturbances reminiscent of 2 : 1 bundle branch block. In order to identify genes affected by the loss of S100A1, we profiled the mutant and wild type cardiac transcriptomes by gene array analysis. The expression of several genes functioning to the electrical activity of the heart were found to be significantly altered. Although the default prediction would be that mRNA and protein levels are highly correlated, comprehensive immunoblot analyses of salient up- or down-regulated candidate genes of any cellular network revealed no significant changes on protein level. Taken together, we found that S100A1 deficiency results in cardiac repolarization delay and alternating ventricular conduction defects in response to sympathetic activation accompanied by a significantly different transcriptional regulation.

Immunocytochemical localization of S100A1 in mitochondria on cryosections of the rat heart.

Immunocytochemical localization studies of S100A1 in muscle cells have so far yielded variable and conflicting results mainly due to different sample preparation techniques for immunoelectron microscopy. To minimize denaturation by fixation and embedding, cryofixation and cryosectioning followed by immunolabelling were used in the present study. Rat hearts were gently prefixed in a mixture of paraformaldehyde and glutaraldehyde. Samples from left and right ventricles and left and right atria were cryoprotected by sucrose and shock-frozen in liquid nitrogen. Ultrathin cryosections were labelled with rabbit polyclonal antiserum against S100A1. The sections were then incubated with secondary antibody conjugated to FITC (for fluorescence microscopy) or with protein A conjugated to 5 nm gold particles (for electron microscopy). The most prominent sites immunolabelled for S100A1 were mitochondria. In the fluorescence microscope the labelling of mitochondria was intense, suppressing the labelling in other compartments. In accordance with previous studies labelling of sarcoplasmic reticulum, Z-lines, actin and myosin filaments could also be detected in the electron microscope.

Changes in Ca(2+)-binding proteins in human neurodegenerative disorders.

The cellular distribution of Ca(2+)-binding proteins has been extensively studied during the past decade. These proteins have proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major roles are assumed to be Ca2+ buffering and transport, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca2+ homeostasis, a protective role for Ca(2+)-binding proteins in certain neuron populations has been postulated. As massive neuronal degeneration takes place in several brain diseases of humans, such as Alzheimer's disease, Parkinson's disease and epilepsy, changes in the expression of Ca(2+)-binding proteins have therefore been studied during the course of these diseases. Although the data from these studies are inconsistent, the detection and quantification of Ca(2+)-binding proteins and the neuron populations in which they occur may nevertheless be useful to estimate, for example, the location and extent of brain damage in the various neurological disorders. If future studies advance our knowledge about the physiological functions of these proteins, the neuronal systems in which they are expressed may become important therapeutical targets for preventing neuronal death in an array of neurodegenerative diseases.

Expression of the tumor-specific and calcium-binding protein oncomodulin during chemical transformation of rat fibroblasts.

An in vivo-in vitro approach to studying neoplastic development in carcinogen-exposed rat fibroblasts was evaluated. In the model described, oncomodulin (Mr 12,000; pI 3.9), a tumor-associated and Ca2+-binding protein, was used as a specific marker of malignant transformation. A rapidly proliferating granulation tissue was exposed in vivo or in vitro to potent carcinogens like N-methyl-N'-nitro-N-nitrosoguanidine and procarbazine. As an endpoint of transformation anchorage independent (AI) colony formation in the soft agar assay was chosen. Exposure to various doses of N-methyl-N'-nitro-N-nitrosoguanidine in vivo or in vitro, or to procarbazine in vivo, led to induction of AI, transformed cells. Exposure of the cells to various doses of procarbazine in vitro produced neither formation of AI cells in the agar nor expression of oncomodulin in extracts of the exposed cell population. Almost all of the chemically induced AI cell lines tested have been found to be tumorigenic in athymic mice. In contrast, a very low rate (zero to two colonies per 10(6) cells tested) of spontaneous AI populations derived from untreated cells. None of these control AI colonies yielded tumors. In our transformation assay the appearance of neoplastic phenotypes seems very rapid, probably due to the increased cell division at the time of carcinogen-exposure. Expression of oncomodulin was found in extracts of transformed cells harvested from agar colonies, derived from carcinogen-exposed granulation tissue, but not from normal, untreated fibroblasts, as shown by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatographic analysis, as well as 45Ca2+-transblot electrophoresis. The presence of oncomodulin in extracts of transformed cells correlates well with the chemically induced colony formation in the soft agar assay. Oncomodulin might be a suitable neoplastic marker to study chemical carcinogenesis.

Probing the structure of the human Ca2+- and Zn2+-binding protein S100A3: spectroscopic investigations of its transition metal ion complexes, and three-dimensional structural model.

A large-scale procedure was developed for the anaerobic purification of the human recombinant Ca2+- and Zn2+-binding protein S100A3 for spectroscopic studies. S100A3 eluted as a non-covalently bound dimer (20.8 kDa). It contained 7.5+/-0.1 free thiol groups/monomer, and bound Ca2+ with a Kd of approximately 4 mM, which corresponds to a tenfold increase in affinity compared to the aerobically purified protein. The transition metal ions Co2+, Zn2+ and Cd2+ were used as spectroscopic probes to investigate the role of the 10 cysteine residues per monomer S100A3 in metal binding. Spectrophotometric titrations suggest the formation of dinuclear thiolate-bridged clusters consisting of a Me2+(S(Cys))4 and a Me2+(S(Cys))3(N(His)) site as described for zinc finger proteins. A three-dimensional structural model of S100A3 was proposed on the basis of the NMR structure of the structurally related rabbit S100A6 protein, and taking into account the structural influence of cysteine residues.

Transcriptional regulation of S100A1 and expression during mouse heart development.

S100A1, a member of the large EF-hand family of Ca(2+)-binding proteins, is mainly expressed in the mammalian heart. To assess the underlying mechanisms for cell- and tissue-specific expression we isolated and characterized the mouse S100A1 gene. The gene displays a high degree of homology to the human and rat genes, especially in the exonic sequences. In its promoter region and the first intron, we identified regulatory elements characteristic for cardiac and slow skeletal muscle restricted genes. Transfection assays with luciferase constructs containing different parts of the S100A1 gene demonstrated the active expression in primary mouse cardiomyocytes and that its 5'-upstream region containing a putative cardiac enhancer showed a greatly increased activity. Furthermore, we investigated the expression of the S100A1 mRNA during embryonic mouse development, using in situ hybridization. S100A1 transcripts were first detected in the primitive heart at embryonic day (E) 8, with equal levels in the atrium and ventricle. During development up to E17.5 we detected a shift in the S100A1 expression pattern with lower levels in atrial and high levels in ventricular myocardium. The regulatory elements identified in the mouse S100A1 promoter correspond well with the observed expression pattern and suggest that S100A1 has an important function during heart muscle development.

Calcium-dependent translocation of S100A11 requires tubulin filaments.

Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.

Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide.

Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.

Expression of intracellular calcium-binding proteins in cultured skin fibroblasts from Alzheimer and normal aged donors.

Disturbed calcium homeostasis may play a role in the etiology in Alzheimer's and other neurodegenerative diseases. A protective role against cellular degeneration has been postulated for Ca(2+)-binding proteins in certain neuron populations. Recent data suggest that intracellular free calcium regulation is also altered in several non-neuronal cells, including skin fibroblasts, from patients with Alzheimer's disease. In this study we analyzed the expression of several EF-hand Ca(2+)-binding proteins in cultured skin fibroblasts from Alzheimer patients and age-matched normal donors. We detected a strong expression of some members of the S100 Ca(2+)-binding protein family and of calcineurin A. However, no significant differences were found between both types of donors by Northern blot and Western blot analysis. In addition, similar signals were detected on 45Ca(2+)-blots of fibroblasts extracts of Alzheimer patients and control donors. The present findings indicate that the altered level of some intracellular calcium-binding proteins in certain brain areas of Alzheimer patients is not found in skin fibroblasts of these patients.

S100A6, a calcium- and zinc-binding protein, is overexpressed in SOD1 mutant mice, a model for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by selective degeneration of motoneurones. Familial ALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1) is linked to the disease. An animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Recent electrophysiological data emphasised that the striking selective vulnerability of motoneurones might be due to their differential calcium buffering capacities. Therefore we have investigated, using immunohistochemistry, the expression of different calcium binding proteins in brainstem and spinal cord from normal and SOD1 mutated mice. Among the 13 calcium-binding proteins screened, only one, S100A6, a homodimeric calcium-binding protein able to bind four Zn(2+), appeared to be highly expressed in the SOD1 mutated mice. In brainstem, reactive astrocytes, but not motoneurones, from several regions, including nerve 12 root, were highly S100A6-positive. Hypoglossal nucleus was negative for S100A6. In dorsal root, reactive astrocytes from both white matter and anterior horn were highly reactive. If overexpression of S100A6 is specific for ALS, it will be a valuable diagnostic marker for this disease.

Human recombinant alpha-parvalbumin and nine mutants with individually inactivated calcium- and magnesium-binding sites: biochemical and immunological properties.

Human recombinant alpha-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca(2+)-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PVD51A,E62V,D90A,E101V), were expressed and purified. Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K'Ca, of 2.3 x 10(7) M-1 and that Mg2+ competes with a K'Mg.compet. of 4.9 x 10(3) M-1. The three mutants with an inactivated CD site bind 1 Ca2+ with K'Ca, of 2.0 to 2.3 x 10(7) M-1 and K'Mg.compet. of 3.4 to 4.6 x 10(3) M-1, i.e. very similar to those of PVwt. The mutants with an inactivated EF site bind 1 Ca2+ with K'Ca values of 7.9 x 10(6), 4.5 x 10(6) and 3.6 x 10(6) M-1 for PVD91A, PVE102V and PVE101V,D91A, respectively. The K'Mg.compet values of these mutants are about 4-times lower than in PVwt. The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+. After excitation at 259 nm, human PV, which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm. Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+. Specific antibodies against human alpha-parvalbumin were raised in rabbits. Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated.

Altered expression of the Ca(2+)-binding protein S100A1 in human cardiomyopathy.

The Ca(2+)-binding protein S100A1 displays a tissue-specific expression pattern with highest levels in myocardium and has been shown to interact with SR-proteins regulating the Ca(2+)-induced Ca(2+)-release. We, therefore, hypothesized that changes in S100A1 gene expression might correlate with the pathognomonic finding of altered SR Ca(2+)-transients in human end stage heart failure. To test this hypothesis, we established a specific and sensitive method to analyse S100A1 expression in cardiac tissues by employing hydrophobic interaction-chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) coupled with Electron-Ionisation-Mass-Spectrometry (ESI-MS). Porcine myocardium showed a differential expression of S100A1 with relative protein concentrations of 62 +/- 8% in the right ventricle (RV), 57 +/- 9% in the right atrium (RA), and 25 +/- 15% in the left atrium (LA) as compared to the left ventricle (LV) (100 +/- 10%; P < 0.001). Northern blot analyses confirmed a likewise distribution of porcine S100A1 mRNA implying a regulation on the transcriptional level. Analyses of left ventricular specimen of patients with end stage heart failure (CHF, n = 6; CHD, n = 6) revealed significantly reduced S100A1 protein levels, while integration of S100A1 peaks after RP-HPLC yielded two groups of patients with < 76% (69 +/- 7%, n = 6) and < 35% (23 +/- 12%, n = 6) respectively as compared to controls (100 +/- 8%, n = 3). These data demonstrate for the first time that S100A1 is differentially expressed in myocardium and that in human cardiomyopathy a reduced expression of S100A1 may contribute to a compromised contractility.

Clustered organization of S100 genes in human and mouse.

S100 Ca2+-binding proteins became of major interest because of their differential expression in tissues and their association with human diseases. Earlier studies showed that 13 S100 genes are located as a cluster on human chromosome 1q21. Since a number of mouse S 100 genes, such as S100A4 and S100A6, have been localized to a syntenic region on mouse chromosome 3, we investigated if the S100 gene cluster exists in mouse and is structurally conserved during evolution. First we identified the cDNA sequences of mouse S100A1, S100A3 and S100A5. Then we isolated a 490 kb mouse YAC clone which gives a specific signal by FISH most likely on chromosome 3. Hybridization studies with different mouse S100 cDNAs revealed that eight mouse S100 genes are arranged in a clustered organization similar to that in human. The linkage relationships between the genes S100A8-S100A9 and S100A3-S100A4-S100A5-S100A6 were conserved during divergence of human and mouse about 70 million years ago. However, the separation of the mouse S100 genes S100A1 and S100A13 in comparison to the human linkage group suggests rearrangement processes between human and mouse. Our data demonstrate that the S100 gene cluster is structurally conserved during evolution. Further studies on the genomic organization of the S100 genes including various species could generate new insights into gene regulatory processes and phylogenetic relationships.

Right ventricular upregulation of the Ca(2+) binding protein S100A1 in chronic pulmonary hypertension.

The Ca(2+) binding protein S100A1 increases the Ca(2+) release from the sarcoplasmatic reticulum by interacting with the ryanodine receptor. In order to understand whether this effect might be operative in the early course of hypertrophy, when myocardium is able to meet increased workload, we investigated the expression of S100A1 in a model of moderate right ventricular hypertrophy. The pulmonary arteries of nine pigs were embolised three times with Sephadex G-50. After 70 days, all pigs showed a moderate pulmonary hypertension. Right ventricular tissue of embolised animals showed a significant increase of connective tissue and enlargement of myocyte diameters. In controls, we found a differential expression of S100A1 with significantly lower S100A1 protein levels in right ventricular compared to left ventricular tissue. In pulmonary hypertension, S100A1 expression increased significantly in hypertrophied right ventricles while it was unchanged in left ventricular tissue. No change was observed in the expression of SERCA2a and phospholamban. Our data show, for the first time, that moderate pressure overload results in an upregulation of S100A1. This may reflect an adaptive response of myocardial Ca(2+) homeostasis to a higher workload.

The identification and differential expression of calcium-binding proteins associated with ocular melanoma.

Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding.

6-Pyruvoyl tetrahydropterin synthase (PTPS) is an enzyme involved in tetrahydrobiopterin biosynthesis, the cofactor for several aromatic amino acid monooxygenases and the nitric oxide synthases. The crystal structure of PTPS was recently solved and showed a homohexameric enzyme composed of a dimer of trimers. A transition metal binding site formed by the three histidine residues 23, 48 and 50 was found in each subunit. We showed by metal analysis and reconstitution of apo-PTPS that Zn(II) was the bound transition metal and responsible for the enzymatic activity. Site-directed mutagenesis of each of these three histidine residues resulted in a complete loss of metal binding and enzymatic activity. The three residues, Cys42, His89 and Glu133, located close to the metal binding site, were previously postulated to be involved in the catalytic reaction. We altered these residues and found a complete loss of enzymatic activity for the mutant C42A. The two mutants, H89N and E133Q, showed 4.3% and 1.3% enzymatic activity, respectively, but had similar KM values for the substrate as compared to wild-type PTPS. Based on these results we propose a model of the substrate fitted into the active site and we described a novel intersubunit catalytic triad motif composed of the amino acid residues Cys42, His89 and Asp88. Different from most other catalytic triads that catalyse the hydrolysis of an amide or ester bond, the catalytic triad in the active site of PTPS seems to be involved in the deprotonation of the substrate's side-chain carbons. Our model also proposes Zn(II) as the coordination site for the two substrate side-chain hydroxy groups as well as the involvement of Glu133 as putative stereospecific proton server.