Biomedical subjects

M Michalak

Publications and source records attributed to M Michalak.

At least 37 records · Page 2Linked to original sources

PubMed1999

alpha-actinin-2 is a new component of the dystrophin-glycoprotein complex.

The human skeletal muscle yeast two-hybrid cDNA library was screened with the carboxyl-terminal region (the last 200 amino acids) of dystrophin. Two interacting clones were identified corresponding to alpha-actinin-2 and actin. Interactions between alpha-actinin, actin, and dystrophin were confirmed by the ligand-blotting technique, by colocalization of dystrophin and alpha-actinin-2 to the isolated skeletal muscle sarcolemmal vesicles and to the plasma membranes isolated from C2C12 myoblasts, and by indirect immunolocalization of dystrophin and alpha-actinin-2 in skeletal muscle cells. This is the first identification of a direct interaction between alpha-actinin, actin, and the carboxyl-terminal region of dystrophin. We propose that dystrophin forms lateral, multicontact association with actin and that binding of alpha-actinin-2 to the carboxyl-terminus of dystrophin is the communication link between the integrins and the dystrophin/dystrophin-glycoprotein complex.

Actinin↗

PubMed1999

Calreticulin is essential for cardiac development.

Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt-/- cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.

Animals↗

PubMed1999

Ca2+ regulation of interactions between endoplasmic reticulum chaperones.

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.

Animals↗

PubMed1999

Identification of common epitopes on gliadin, enterocytes, and calreticulin recognised by antigliadin antibodies of patients with coeliac disease.

BACKGROUND: Sera of patients with coeliac disease, containing IgA and IgG antigliadin antibodies (AGA) and various IgA autoantibodies, react with isolated enterocytes. AGA cross react with enterocyte antigens, one of which has been identified as calreticulin. AIMS: To characterise the antigenic structures of gliadin, enterocytes, and calreticulin recognised by AGA from patients with active coeliac disease. METHODS: AGA were isolated from sera of nine patients by affinity chromatography and tested by competitive ELISA using 40 alpha-gliadin synthetic dodecapeptides (A1-F6). RESULTS: Reactivity of gliadin with all purified AGA tested was inhibited by peptide A4 at the N-terminal region; by C2, C3, and D4 at the central region; and by F3 and F4 at the C-terminal region of the gliadin molecule. AGA cross reactivity with enterocytes was inhibited by peptides A4, D1-D4, and F6 and with calreticulin by peptides A4, D3, and D4. As dominant epitopes AGA of coeliac patients recognise similar structures corresponding to peptides A4, D3, D4, and F6 present on gliadin, enterocytes, and calreticulin. Substitution of glutamine in the A4 peptide by glutamic acid caused loss of inhibitory capacity. Shortening of peptide A4 on the N-terminal by three amino acids increased its inhibitory effect. CONCLUSIONS: AGA of patients with coeliac disease react with similar structures on gliadin and potential autoantigens on enterocytes.

Adult↗

PubMed1998

Interaction between a Ca2+-binding protein calreticulin and perforin, a component of the cytotoxic T-cell granules.

Calreticulin is a component of cytotoxic T-lymphocyte and NK lymphocyte granules. We report here that granule-associated calreticulin terminates with the KDEL endoplasmic reticulum retrieval amino acid sequence and somehow escapes the KDEL retrieval system. In perforin knock-out mice calreticulin is still targeted into the granules. Thus, calreticulin will traffic without perforin to cytotoxic granules. In the granules, calreticulin and perforin are associated as documented by (i) copurification of calreticulin with perforin but not with granzymes and (ii) immunoprecipitation of a calreticulin-perforin complex using specific antibodies. By using calreticulin affinity chromatography and protein ligand blotting we show that perforin binds to calreticulin in the absence of Ca2+ and the two proteins dissociate upon exposure to 0.1 mM or higher Ca2+ concentration. Perforin interacts strongly with the P-domain of calreticulin (the domain which has high Ca2+-binding affinity and chaperone function) as revealed by direct protein-protein interaction, ligand blotting, and the yeast two-hybrid techniques. Our results suggest that calreticulin may act as Ca2+-regulated chaperone for perforin. This action will serve to protect the CTL during biogenesis of granules and may also serve to regulate perforin lytic action after release.

Amino Acid Sequence↗

PubMed1998

Enhanced catalysis of ribonuclease B folding by the interaction of calnexin or calreticulin with ERp57.

The endoplasmic reticulum is the site of folding, disulfide bond formation, and N-glycosylation of secretory proteins. Correctly folded proteins are exported from the endoplasmic reticulum, whereas incorrectly folded proteins are retained by a quality control system. The type I membrane-protein calnexin and its soluble homologue calreticulin are constituents of this system that recognize monoglucosylated N-linked glycans that are present on unfolded glycoproteins. Although several components of the quality control apparatus are well characterized, it is not known whether and how they interact with enzymes that catalyze protein folding. The endoplasmic reticulum protein ERp57 is homologous to protein-disulfide isomerase and can be cross-linked to the same monoglucosylated glycoproteins that bind to calnexin and calreticulin. The present study demonstrates that the disulfide isomerase activity of ERp57 on the refolding of monoglucosylated ribonuclease B is much greater when this glycoprotein is associated with calnexin or calreticulin. This result is in contrast to protein-disulfide isomerase, whose activity on monoglucosylated ribonuclease B is decreased in the presence of these lectins. No direct binding of monoglucosylated ribonuclease B or monoglucosylated glycans to ERp57 could be detected, but we show that ERp57 interacts directly with calnexin.

Calcium-Binding Proteins↗

PubMed1998

Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin.

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.

Adenosine Triphosphate↗

PubMed1998

Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum.

Calreticulin is a ubiquitous endoplasmic reticulum Ca2+ binding chaperone. The protein has been implicated in a variety of diverse functions. Calreticulin is a lectin-like chaperone and, together with calnexin, it plays an important role in quality control during protein synthesis, folding, and posttranslational modification. Calreticulin binds Ca2+ and affects cellular Ca2+ homeostasis. The protein increases the Ca2+ storage capacity of the endoplasmic reticulum and modulates the function of endoplasmic reticulum Ca2+-ATPase. Calreticulin also plays a role in the control of cell adhesion and steroid-sensitive gene expression. Recently, the protein has been identified and characterized in higher plants but its precise role in plant cells awaits further investigation.

Animals↗

PubMed1998

Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.

Calcium-Binding Proteins↗

PubMed1998

Calreticulin inhibits vitamin D's action on the PTH gene in vitro and may prevent vitamin D's effect in vivo in hypocalcemic rats.

1,25-dihydroxyvitaminD3 [1,25-(OH)2D3] and PTH both act to increase serum calcium. In addition, 1,25-(OH)2D3 decreases PTH gene transcription, which is relevant both to the physiology of calcium homeostasis and to the management of the secondary hyperparathyroidism of patients with chronic renal failure. In chronic hypocalcemia there is secondary hyperparathyroidism with increased levels of PTH mRNA and serum PTH despite markedly increased levels of 1,25-(OH)2D3. We have studied the role of calreticulin in this resistance to 1,25-(OH)2D3. Weanling rats fed a low-calcium diet were hypocalcemic and had increased PTH mRNA levels despite high serum 1,25-(OH)2D3 levels. 1,25-(OH)2D3 given by continuous minipump infusion to normal rats led to the expected decrease in PTH mRNA. The hypocalcemic rats had an increased concentration of calreticulin in the nuclear fraction of their parathyroids, but not in other tissues. Gel shift assays showed that a purified vitamin D receptor and retinoid X receptor-beta bound to the PTH promoter's chicken and rat vitamin D response element (VDRE), and this binding was inhibited by added pure calreticulin. Transfection studies with a PTH VDRE-chloramphenicol acetyltransferase (CAT) construct showed that 1,25-(OH)2D3 decreased CAT transcription. Cotransfection of PTH VDRE-CAT with a calreticulin expression vector in the sense orientation prevented the transcriptional effect of 1,25-(OH)2D3, but a calreticulin vector in the antisense orientation had no effect. These results show that calreticulin prevents the binding of vitamin D receptor-retinoid X receptor-beta to the PTH VDRE in gel retardation assays and prevents the transcriptional effect of 1,25-(OH)2D3 on the PTH gene. This is the first report of calreticulin inhibiting a down-regulatory function of a sterol hormone and may help explain the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to 1,25-(OH)2D3.

Animals↗

PubMed1997

Interactions of a high mobility group-like protein with human mitochondrial DNA.

A number of nuclear-encoded proteins are known to bind to the mitochondrial genome and may be involved in mitochondrial replication and in mitochondrial diseases. Mitochondrial diseases such as Kearns-Sayre syndrome (KSS) are flanked by common direct repeats. To study protein binding to these mitochondria DNA regions we used gel mobility shift binding assays. Proteins present in nuclear or in mitochondrial extracts from normal or KSS-derived fibroblasts bound to a 280-bp mitochondrial DNA fragment encompassing a deletion breakpoint in the mitochondrial genome. In addition, nuclear and mitochondrial protein bound to a nearby surrounding fragment and to a synthetic oligonucleotide with a related consensus DNA sequence. Southwestern blot analysis showed that the DNA binding ability resided in a 35-kDa protein. The amino terminal sequence of the 35-kDa protein was very similar to human high mobility group proteins. These results suggest that this protein may be involved in mitochondrial DNA deletions.

Amino Acid Sequence↗

PubMed1997

Regulation of calreticulin gene expression by calcium.

We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter region. The mouse calreticulin gene consists of nine exons and eight introns, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/ 3T3 cells. Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin promoter was also increased by fourfold in NIH/3T3 cells treated with bradykinin, a hormone that induces Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (-115 to -260 and -685 to -1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. Northern blot analysis of cells treated with A23187, or with thapsigargin, revealed a fivefold increase in calreticulin mRNA levels. Thapsigargin also induced a fourfold increase in calreticulun protein levels. Importantly, we show by nuclear run-on transcription analysis that calreticulin gene transcription is increased in NIH/3T3 cells treated with A23187 and thapsigargin in vivo. This increase in gene expression required over 4 h of continuous incubation with the drugs and was also sensitive to treatment with cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular and cytoplasmic Ca2+ did not affect the increased expression of the calreticulin gene. These studies suggest that stress response to the depletion of intracellular Ca2+ stores induces expression of the calreticulin gene in vitro and in vivo.

3T3 Cells↗

PubMed1997

Phosphorylation and regulation of the Na+/H+ exchanger through mitogen-activated protein kinase.

We examined mitogen-activated protein kinase-mediated phosphorylation and activation of the Na+/H+ exchanger isoform type 1. A rabbit skeletal muscle extract was fractionated by FPLC chromatography. Four main fractions had the ability to phosphorylate the carboxyl-terminal region of NHE1. Western blot analysis and immunoprecipitation showed that three of these were associated with MAP kinase-dependent phosphorylation. Phosphorylation studies using purified MAP kinase showed that the region involved was the carboxyl-terminal 178 amino acids of the protein and that the stoichiometry was 1 phosphate/mol of protein. In-gel kinase assays showed that cytosolic extracts from smooth muscle cells also phosphorylate the carboxyl-terminal of NHE1 and that the MAP kinase-dependent phosphorylation could be activated by PDGF and AngII. Mutant cell lines with an inducible dominant negative MAP kinase showed decreased serum activation of Na+/H+ exchange but normal hypertonic activation of the protein. The results show that MAP kinase is intimately involved in regulation of the Na+/H+ exchanger, possibly through phosphorylation of one amino acid of the carboxyl-terminal cytosolic domain.

Animals↗

PubMed1997

Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells.

The loss of tyrosinase, the key enzyme in melanin synthesis, has been implicated in the dedifferentiation of malignant melanocytes. The presence of tyrosinase transcripts and antigenic peptides in melanoma tumors prompted us to investigate whether the basis for the loss of the enzyme was proteolytic degradation. Toward this aim, we followed the kinetics of synthesis, degradation, processing, chaperone binding, inhibitor sensitivity, and subcellular localization of tyrosinase in normal and malignant melanocytes. We found that, in amelanotic melanoma cell lines, tyrosinase failed to reach the melanosome, the organelle for melanin synthesis, because it was retained in the endoplasmic reticulum (ER) and then degraded. Tyrosinase appeared mostly as a 70-kDa core-glycosylated, endoglycosidase H-sensitive, immature form bound to the ER chaperone calnexin and had a life-span of only 25% of normal. Maturation and transit from the ER to the Golgi compartment was facilitated by lowering the temperature of incubation to 31 degrees C. Several proteasome inhibitors caused the accumulation of an approximately 60-kDa tyrosinase doublet that was more prominent in malignant than in normal melanocytes and promoted, to various degrees, the maturation of tyrosinase in melanoma cells and the translocation of the enzyme to melanosomes. The appearance of ubiquitinated tyrosinase after treatment of normal melanocytes with N-acetyl-L-leucinyl-L-leucinal-L-norleucinal reinforced our notion that some tyrosinase is normally degraded by proteasomes. Proteolysis of tyrosinase by proteasomes is consistent with the production of antigenic tyrosinase peptides that are presented to the immune system by major histocompatibility complex class I molecules.

Adult↗

PubMed1997

Calreticulin.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1997

Anti-gliadin antibodies in patients with celiac disease cross-react with enterocytes and human calreticulin.

One of the characteristic features of celiac disease is an increase in anti-gliadin antibodies (Abs). Recently we found that some of the monoclonal Abs to gliadin cross-react with molecules on rat enterocytes. One of these cross-reacting molecules was identified as rat calreticulin. This study shows that the levels of serum IgA Abs to gliadin, rat, and human enterocytes; purified enterocyte antigens; and calreticulin in sera from patients with active disease were significantly higher than in patients on a gluten-free diet and healthy controls (P < 0.001). Anti-gliadin Abs were isolated by affinity chromatography from the sera of six active celiac patients. The reactivity of these anti-gliadin Abs was demonstrated to be significantly higher (P < 0.05) with human enterocytes and human calreticulin than with other antigens tested. Furthermore, using isolated patients' anti-gliadin Abs bound to Sepharose 2B, two main proteins of molecular mass 62 and 66 kDa were purified from a lysate of human enterocytes. The 62-kDa enterocyte antigen was identified as human calreticulin. These findings suggest that anti-gliadin Abs may play a pathogenic role in celiac disease by cross-reacting with enterocytes. Calreticulin in enterocytes may be one of the putative targets for autoimmune reactions.

Adolescent↗

PubMed1997

Calreticulin inhibits glucocorticoid- but not cAMP-sensitive expression of tyrosine aminotransferase gene in cultured McA-RH7777 hepatocytes.

Calreticulin is a ubiquitously expressed Ca2+ binding protein of the endoplasmic reticulum which inhibits DNA binding and transcriptional activation by steroid hormone receptors. In this study the effects of calreticulin on tyrosine aminotransferase (TAT) gene expression in cultured McA-RH7777 hepatocytes was investigated. McA-RH7777 cells were stably transfected with calreticulin expression vector to generate cells overexpressing the protein. The transcriptional activity of the TAT gene, which is glucocorticoid-sensitive and cAMP-dependent, was investigated in the mock transfected McA-RH7777 and in cells overexpressing calreticulin (designated McA-11 and McA-17). In the presence of dexamethasone or the cAMP analog (CTP-cAMP) expression of the TAT gene was induced in mock transfected McA-RH7777 cells by approximately 4.5 and 5 fold, respectively. In McA-11 and McA-17 cells, overexpressing calreticulin, glucocorticoid-sensitive expression of the TAT gene was significantly inhibited, however, the CTP-cAMP-dependent expression of the TAT gene was not affected. The ability of calreticulin to inhibit glucocorticoid-sensitive TAT gene expression but not the cAMP-dependent expression of the gene suggests that the protein affects specifically the action of transcription pathways involving steroid receptors or transcription factors containing KxFF(K/R)R-like motifs. Calreticulin may play an important role in the regulation of glucocorticoid-sensitive pathway of expression of the hepatocytes specific genes during development.

Animals↗

PubMed1997

Heat shock-regulated expression of calreticulin in retinal pigment epithelium.

Calreticulin is a major Ca2+ binding protein in the endoplasmic reticulum of non-muscle cells. In this report we show that calreticulin protein is strongly induced by heat shock. Activation and attenuation of the heat shock transcriptional response is caused by heat shock factor that binds to 5'-flanking sequences of heat shock responsive genes, the heat shock element. The smallest stretch of DNA that shows detectable binding of heat shock factor in vitro contains a two-sequence unit nGAAnnTTCn which exists in the 5'-flanking region of calreticulin DNA (5'-gGAAccCAGcgTTC-3'). The present data provide direct evidence that calreticulin expression can be modulated by heat shock. Thus, our results strengthen the hypothesis that calreticulin, in addition to its function as a cellular Ca2+ store, is a multifunctional protein which performs at least some of its functions from the lumen of the ER.

Animals↗