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J Kuźnicki

Publications and source records attributed to J Kuźnicki.

At least 19 recordsLinked to original sources
PubMed2001

Structural and biochemical characterization of neuronal calretinin domain I-II (residues 1-100). Comparison to homologous calbindin D28k domain I-II (residues 1-93).

This study characterizes the calcium-bound CR I-II domain (residues 1-100) of rat calretinin (CR). CR, with six EF-hand motifs, is believed to function as a neuronal intracellular calcium-buffer and/or calcium-sensor. The secondary structure of CR I-II, defined by standard NMR methods on 13C,15N-labeled protein, contains four helices and two short interacting segments of extended structure between the calcium-binding loops. The linker between the two helix-loop-helix, EF-hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I-II) confirms that the linker of CR I-II is solvent-exposed and that other potential sites are protected by regular secondary structure. 45Ca-overlay of glutathione S-transferase (GST)-CR(1-60) and GST-CR(61-100) fusion proteins confirm that both EF-hands of CR I-II have intrinsic calcium-binding properties. The primary sequence and NMR chemical shifts, including calcium-sensitive glycine residues, also suggest that both EF-hand loops of CR I-II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross-linking and NMR translation diffusion measurements indicate that CR I-II exists as a monomer. Calb I-II (the homologous domain of calbindin D28k) has the same EF-hand secondary structures as CR I-II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem. 262, 933-938]. In contrast, Calb I-II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I-II and Calb I-II probably have distinct tertiary structure features that suggest different cellular functions for the full-length proteins.

Amino Acid Sequence↗
PubMed2000

Upstream stimulatory factor is involved in the regulation of the human calcyclin (S100A6) gene.

Calcyclin (S100A6) is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity. The calcyclin gene promoter fragment -361/-167 activates transcription several fold when compared to the basal -167/+134 promoter fragment indicating the presence of enhancer element within -361/-167 bp region. By means of the electrophoretic mobility shift assay (EMSA) we found that this region contains a protein-binding site and mapped it to an E-box sequence at position -283/-278. Using antibodies against USF1 we identified the upstream stimulatory factor as the transcription factor bound to the E-box sequence in EMSA. This factor was also enriched in protein fractions obtained from Ehrlich ascites tumor cells nuclear extract by affinity chromatography using the E-box sequence as a ligand. Cotransfection of the USF1 expression vector with a plasmid carrying the luciferase gene under control of the -361/+134 calcyclin gene promoter fragment resulted in several fold activation of luciferase activity. On the other hand, mutations within the E-box led to a marked decrease in the efficiency of calcyclin gene promoter fragment. The results indicate that USF1 binds to an E-box sequence of the calcyclin gene promoter and enhances its transcription activity. This mechanism might be responsible for the upregulation of calcyclin gene expression in response to various stimuli and in tumors.

Base Sequence↗
PubMed2000

Regulation of cell specific expression of calcyclin (S100A6) in nerve cells and other tissues.

Many of the small, acidic, calcium binding S100 proteins present in the brain specifically map different anatomical regions and cell types and their overexpression is implicated in pathological changes. Similarly to other members of the S100 protein family, calcyclin (S100A6) is expressed in a cell specific manner and is found in subpopulations of neurons and astrocytes in the brain and in epithelial cells and fibroblasts. In this article we review data concerning the cell specific expression of S100 protein genes and present experimental results on the regulation of the calcyclin gene. We have performed promoter deletion studies to locate regions within the calcyclin gene promoter responsible for transcriptional regulation. The results demonstrate that the 3 kb long calcyclin gene promoter lacks a cell specific cis-acting element and drives the expression of the reporter gene also in cells that do not express endogenous calcyclin. The expression is modulated by positive and negative elements acting uniformly in the four different cell lines studied. The first intron of the calcyclin gene was found to have an inhibitory influence on expression regardless of cell type. It was also shown that calcyclin expression can be induced in calcyclin-negative cells by treatment with 5-azacytidine suggesting the involvement of gene methylation in its cell specific expression. The results are discussed in light of the data available on the regulation of other S100 genes.

Animals↗
PubMed1999

Measurements of [Ca2+] using fura-2 in glioma C6 cells expressing calretinin with GFP as a marker of transfection: no Ca2+-buffering provided by calretinin.

Glioma C6 cells were transfected with a plasmid containing the calretinin (CR) and green fluorescent protein (GFP) coding regions to analyze the effect of CR's presence on [Ca2+]i. Positive transfectants were identified by the detection of GFP and [Ca2+]i was measured using fura-2 as a probe. We found that neither the basic [Ca2+]i nor activated [Ca2+]i achieved by exposure to ionomycin, ADP or thapsigargin were affected by CR's presence in transfected cells, despite the ability of CR to bind Ca2+ as part of fusion protein. The level of expressed CR was estimated as at least 1 microM. The presented results suggest that CR's function is unlikely to be an intracellular Ca2+-buffer and support the hypothesis that CR might be involved in a specific Ca2+-dependent process. The results of this work also show that the S65T mutant of GFP is compatible with fura-2 measurements of intracellular [Ca2+]. We have demonstrated that the presence of GFP, as a transfection marker of glioma C6 cells, does not disturb fura-2 fluorescence, the basal or activated [Ca2+]i in these cells.

Animals↗
PubMed1999

[Regulation of protein S100 expression during transcription].

The S100 protein family is a group of homologous, small, calcium-binding proteins expressed in a cell specific manner. In man, the genes coding the majority of these proteins are localized in a cluster on chromosome 1. The cell specific expression of these proteins is mainly attained at the transcriptional level. It appears that the promoters of some S100 genes (rat S100A4, mouse and human S100B and rat CALB3) contain inhibitory sequences which reduce transcription level. Their effect may be counteracted by an interaction with cell specific transcription factor(s), which allows transcription in certain cell types. In other cases (human S100A2, mouse S100A4 and human S100A6), the main mechanism controlling cell specific expression seems to be methylation of the promoter or intronic sequences leading to gene silencing in some cells. The level of protein expression might be further modulated by different regulatory sequences that respond, via interactions with specific transcription factors, to various extracellular stimuli.

Animals↗
PubMed1998

A model for target protein binding to calcium-activated S100 dimers.

S100 proteins are a family of dimeric calcium-binding proteins implicated in several cancers and neurological diseases. Calbindin D9k is an unusual monomeric member of the S100 family. A calbindin D9k mutant containing a novel calcium-induced helix is characterized. Based on sequence comparison, this helix could be a component of other S100 proteins and a factor in target protein binding. The origin of structural differences between three reported apo S100 dimer structures is verified. We conclude that the differences are a result of modeling rather than a function of different target binding properties. A mechanism for target protein binding is suggested.

Animals↗
PubMed1998

Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcyclin.

A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4-kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of approximately 26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.

Amino Acid Sequence↗
PubMed1997

The mouse calretinin gene promoter region: structural and functional components.

The 5' flanking region of the mouse calretinin gene was cloned and a 1.8 kbp region adjacent to exon 1 was sequenced. Putative upstream promoter and enhancer elements were identified, including appropriately positioned TATA and CAAT boxes (positions -50 and -68, respectively). There was considerable sequence and structural homology between mouse and human upstream elements. Neuron-restrictive activity was demonstrated via transfection of calretinin promoter-reporter constructs into primary embryonic mouse brain cultures expressing calretinin. In promoterless reporter constructs, the proximal upstream 1.5 kbp of the mouse calretinin gene boosted luciferase activity (up to 100-fold) exclusively in the neuronal population. Deletion analysis revealed the minimal promoter to be within the 95-bp proximal to the transcription start site. Transfections with SV40 promoter constructs in these cultures resulted in reporter gene expression predominantly in non-neuronal cells. Inserting the proximal 1.5 kbp of mouse calretinin upstream in SV40 promoter-reporter constructs reduced luciferase activity. Thus, calretinin upstream sequences increased reporter expression in cultured neurons and decreased expression from the SV40 promoter in non-neuronal cultured brain cells. The calretinin promoter contained relevant regulatory element consensus motifs and demonstrated in vitro neuron-restrictive bioactivity.

3T3 Cells↗
PubMed1996

The expression of calretinin in transfected PC12 cells provides no protection against Ca(2+)-overload or trophic factor deprivation.

To address the question whether calretinin (CR) may protect cells against Ca2+ overload or trophic factor deprivation, PC12 cells were transfected with plasmids containing a CR coding region under control of a cytomegalovirus promoter. Nerve growth factor (NGF) treatment induced differentiation, increased transfection efficiency (at least 10-fold) and activated the CR gene (as found by RNase protection method and immunohistochemistry). Exogenous CR expression was identified either in living cells by fluorescence of green fluorescent protein (when the CR coding region was fused to this protein) or in fixed cells by CR immunoreactivity. Undifferentiated and NGF-differentiated populations of transfected cells were incubated in the presence of a Ca(2+)-ionophore or in media deprived of serum or NGF. Expression of exogenous CR in undifferentiated or NGF-treated cells (due to transfection) or endogenous CR (due to gene activation by NGF) did not render PC12 cells more resistant to insults such as Ca(2+)-overload and trophic factor deprivation.

Animals↗
PubMed1996

Antibody recognition of calcium-binding proteins depends on their calcium-binding status.

Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.

Animals↗
PubMed1995

Conformational changes and calcium binding by calretinin and its recombinant fragments containing different sets of EF hand motifs.

Four recombinant fragments, representing different sets of EF-hand motifs of rat calretinin (CR) (I-II, I-III, III-VI, IV-VI), were prepared, and their Ca2+ -induced conformational changes were compared with those of full-length recombinant CR. All fragments were able to bind calcium ions as shown by 45Ca2+ overlay method on nitrocellulose and fluorescence measurements. The intrinsic tryptophan fluorescence intensity (FI) of apo-CR reversibly increased about 3-fold upon addition of calcium, indicating a change of conformation. The FI of fragments I-II (Trp 25) and I-III (Trp 25 and 116) increased about 1.4-fold on calcium binding, but that of fragment III-VI (Trp 116) increased 3.5-fold. Calcium titration of CR monitored by Trp fluorescence intensity showed that recombinant CR and some fragments bound Ca2+ with high affinity (Kd below 0.4 microM) and with high cooperativity. An apparent Hill coefficient for Ca2+ -induced fluorescence changes in CR was about 3.7. CR bound to organomercurial-agarose Cys 101 and 266 did not form cystine. The fluorescence intensities of cysteine-linked fluorescent probes 5-iodoacetamidofluorescein and N-(1-pyreneiodoacetamide) were increased approximately 1.3-fold upon calcium binding by CR. These data indicate that CR binds Ca2+ with high affinity and cooperativity and that this binding induces a change of conformation that involves the interaction of different parts of the molecule. Taken together, our results suggest that CR works as an on/off switch within a narrow range of free Ca2+ by interacting with as yet unidentified targets.

Animals↗
PubMed1995

Localization of Ca(2+)-dependent conformational changes of calretinin by limited tryptic proteolysis.

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.

Animals↗
PubMed1995

Distribution of calretinin, calbindin D28k, and parvalbumin in subcellular fractions of rat cerebellum: effects of calcium.

The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane-associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.

Animals↗
PubMed1994

Calcyclin from mouse Ehrlich ascites tumor cells and rabbit lung form non-covalent dimers.

Crosslinking treatments of fresh cytosol from mouse Ehrlich ascites tumor (EAT) cells revealed the existence of calcyclin dimers which were sensitive to SDS, but not to reducing agents, which suggests the existence of non-covalent dimers. In stored EAT cell cytosol and preparations of purified calcyclin dimers were also formed by S-S bridging (covalent dimers). The S-S dimers did not bind to organomercurial Agarose and could be separated from reduced forms of calcyclin that bound to the resin. Calcyclin eluted from the resin with DTT was a mixture of monomers and non-covalent dimers as shown by crosslinking and subsequent immunoblotting. Calcyclin from rabbit lung, lacking a cysteine residue, could also be crosslinked as a dimer. It is suggested that the ability of calcyclin to form non-covalent dimers is of physiological significance.

Animals↗
PubMed1994

Expression and rapid purification of recombinant rat calretinin: similarity to native rat calretinin.

Rat calretinin coding region was subcloned into a prokaryotic expression vector (pGEX). The glutathione-S-transferase:calretinin fusion protein produced in Escherichia coli was purified on a glutathione-Sepharose affinity column. Recombinant rat calretinin was cleaved on the column by thrombin, eluted, and purified to homogeneity using DEAE-cellulose chromatography. Recombinant and native rat calretinin performed the same on DEAE columns, denaturing polyacrylamide gel electrophoresis (SDS-PAGE), Western blots, and 45Ca overlay on nitrocellulose blots. The recombinant calretinin migrated similarly to the more basic (pI 5.3) of two forms of native calretinin demonstrated by two-dimensional SDS-PAGE. Calcium binding equilibria revealed identical apparent binding affinity and capacity. Difference(s) between native and recombinant did not affect the binding of calcium to calretinin or antibody recognition. Thus recombinant calretinin may be useful in the elucidation of possible cellular targets of native calretinin.

Animals↗
PubMed1994

Ca(2+)-dependent and independent interactions of calretinin with hydrophobic resins.

The ability of rat calretinin to bind to hydrophobic resins in a Ca(2+)-dependent manner was examined. Both native calretinin present in cerebellum extract and purified recombinant calretinin bound similarly to hydrophobic resins such as phenyl-, hexyl-, octyl-, and W7-agarose. Hydrophobic interactions of calretinin were partially Ca(2+)-dependent since 1/3 of bound protein was released from the resins by EGTA under varied conditions. Some calretinin tryptic fragments bound to octyl-agarose in a manner similar to uncleaved calretinin, while others bound to the resin in a Ca(2+)-independent manner. These and other results suggest that calretinin has several hydrophobic regions of varied strength and sensitivity to Ca2+. It is proposed that the local changes in hydrophobicity induced by Ca2+ binding might be relevant for calretinin functions.

Animals↗
PubMed1993

Characterization of calcyclin fragments obtained by CNBr-cleavage.

1. Two calcyclin fragments were obtained by CNBr-cleavage. 2. One fragment represented N-terminal end of a molecule (residues 1-56), and another one a C-terminal end (residues 57-89). 3. Properties of intact calcyclin such as binding of calcium, binding to hydrophobic resins and interaction with calcyclin specific antibodies were not retained by these fragments. 4. However, both fragments were able to form dimers and higher forms of aggregates as seen for uncleaved calcyclin. 5. This indicates that both halves of the molecule contain the regions responsible for non-covalent interaction which might participate in dimer formation.

Amino Acid Sequence↗