Jacek Kuźnicki

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

Abstract: 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 ∼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.

Similarity in Ca2+-induced changes between troponin-C and protein activator of 3′:5′-cyclic nucleotide phosphodiesterase and their tryptic fragments

Abstract 1. 1.|The Ca2+-dependent protein activator of 3′:5′-cyclic nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) has been isolated from bovine brain and its properties have been compared with those of troponin-C. It has been shown that bovine brain does not contain troponin-C, but only the protein activator of phosphodiesterase. Its molecular weight is 16 500 as compared with 18 000 for troponin-C. 2. 2.|Similarly to troponin-C, calcium has a pronounced effect on the rate of cleavage of protein activator by trypsin and on the obtained peptide pattern. In the presence of 0.1 mM CaCl2 two large peptides are formed, essentially resistant to further splitting, whereas upon removal of calcium by chelators a fast cleavage of protein activator into small peptides takes place. 3. 3.|Similarly to troponin-C, tyrosine fluorescence intensity of protein activator is markedly enhanced in the presence of Ca2+. The apparent binding constant for Ca2+ calculated from the transition midpoint of fluorescence changes is about 1 · 107 M−1. During digestion of protein activator with trypsin in the presence of CaCl2 essentially no change of fluorescence intensity takes place and the subsequent decrease upon removal of calcium is reversible. 4. 4.|Two large peptides that accumulate during digestion by trypsin in the presence of Ca2+ of both troponin-C and protein activator have been isolated and their properties have been compared with those of corresponding parent molecules. One of the peptides obtained from both proteins retains the ability of the intact molecular to change the mobility on the polyacrylamide gel electrophoresis in the presence of urea depending on concentration of Ca2+ and to interacted with the inhibitory component of troponin in the presence of Ca2+. This peptide also shows Ca2+-dependent fluorescence changes, characteristic for parent molecule. 5. 5.|On the basis of similarity between the two proteins and their tryptic fragments it is suggested that in the presence of Ca2+ protein activator is cleaved in the same area as troponin-C. The peptide obtained from protein activator, which shows Ca2+-dependent changes characteristics for intact molecules, corresponds to the troponin-C peptide containing calcium binding sites 3 and 4. All these results furnish new pieces of evidence for a pronounced structural similarity between troponin-C and protein activator of cyclic nucleotide phosphodiesterase.

Mutations in presenilin 1, presenilin 2 and amyloid precursor protein genes in patients with early-onset Alzheimer's disease in Poland

Mutations in three causative genes have been identified in patients with an autosomal-dominant form of early-onset Alzheimers disease (EOAD). To determine the spectrum of mutations in a group consisting of 40 Polish patients with clinically diagnosed familial EOAD and 1 patient with mild cognitive impairment (MCI) and family history of AD, we performed a screening for mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2) and amyloid precursor protein (APP) genes. Four previously recognized pathogenic mutations in PSEN1 gene (H163R, M139V) and APP gene (T714A, V715A), and three novel putative mutations in PSEN1 gene (P117R and I213F) and PSEN2 gene (Q228L) were identified. The 34 patients with no mutations detected were older than the patients with mutations. A frequency of APOE4 allele was higher in this group. Frequency of mutations is relatively low (17%), possibly due to used operational definition of a patient with familial EOAD (a patient having at least one relative with early-onset dementia). It could be concluded that screening for mutations in the three genes could be included in a diagnostic program directed at patients with a positive family history or age of onset before 55 years.

Two novel presenilin 1 gene mutations connected with frontotemporal dementia-like clinical phenotype : Genetic and bioinformatic assessment

Mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes are associated with early-onset familial Alzheimers disease (EOAD). There are several reports describing mutations in PSEN1 in cases with frontotemporal dementia (FTD). We identified two novel mutations in the PSEN1 gene: L226F and L424H. The first mutation was detected in a patient with a clinical diagnosis of FTD and a post-mortem diagnosis of AD. The second mutation is connected with a clinical phenotype of variant AD with strong FTD signs. In silico modeling revealed that the mutations, as well as mutations used for comparison (F177L and L424R), change the local structure, stability and/or properties of the transmembrane regions of the presenilin 1 protein (PS1). In contrast, a silent non-synonymous substitution F175S is eclipsed by external residues and has no influence on PS1 interfacial surface. We suggest that in silico analysis of PS1 substitutions can be used to characterize novel PSEN1 mutations, to discriminate between silent polymorphisms and a potential disease-causing mutation. We also propose that PSEN1 mutations should be considered in FTD patients with no MAPT mutations.

Calcyclin (S100A6) Binding Protein (CacyBP) Is Highly Expressed in Brain Neurons

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0.34 ng/μg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein.

Calcyclin--Ca(2+)-binding protein homologous to glial S-100 beta is present in neurones.

The aim of this work was to confirm the presence of calcylin in brain and to identify calcyclin positive cells. Calcyclin was identified in brain soluble proteins that were bound to phenyl-sepharose in a calcium dependent fashion. Specific antibodies against calcylin were found to stain subsets of brain neurones such as neurones in Ammons horn of hippocampus, granule cells in cerebellum, and neurones in brain stem. Glial cells which contain large amounts of S-100 beta protein were calcyclin negative. These results indicate that calcyclin is present in neurones, but not in glial cells.

Tissue specific distribution of calcyclin — 10.5 kDa Ca2+ -binding protein

Expression of calcyclin in different cell lines and mouse tissues was determined with polyclonal antibodies raised against calcyclin from Ehrlich ascites tumour (EAT) cells. The protein was detected in mouse skeletal and cardiac muscle, in lung, kidney and spleen, and was especially enriched in mouse smooth muscle as well as in rat fibroblasts. No positive immunological reaction was detected in mouse brain, liver and intestine and some tumourigenic cell lines. The level of calcyclin mRNA found in different cells and tissues corresponded well to the calcyclin level estimated by immunoblotting. The calcyclin‐like protein was purified from mouse stomach and appeared to be very similar to the EAT protein.

Sgt1 has co-chaperone properties and is up-regulated by heat shock.

The Sgt1 protein is a binding partner of heat shock proteins such as Hsp90, Hsp70 or Hsc70. In this work we show that the level of Sgt1 is increased in HEp-2 cells exposed to heat shock or radicicol. The citrate synthase aggregation assay shows that Sgt1 attenuates aggregation of the enzyme induced by increased temperature as efficiently as p23, a known co-chaperone of Hsp90. We have cloned two fragments of the human Sgt1 gene promoter (-708/+98 and -351/+98) into pGL3-luciferase vector and found that both fragments generated a 2-fold increase in luciferase activity upon heat shock. Furthermore, electrophoretic mobility shift assay revealed binding of the HSF-1 transcription factor to the heat shock element in the proximal (-42/-2) Sgt1 gene promoter fragment. These results indicate that Sgt1 is a co-chaperone protein with an expression pattern matching that of the well known heat shock proteins.

Calretinin and calbindin D28k have different domain organizations.

The domain organization of calretinin (CR) was predicted to involve all six EF‐hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed 1H,15N HSQC NMR spectra of a 15N‐labeled calretinin fragment (CR III–VI, residues 100–271) in the presence of the unlabeled complimentary fragment (CR I–II, residues 1–100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of 15N‐labeled CR is similar to the overlaid spectra of individual 15N‐labeled CR fragments (CR I–II and CR III–VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1–60) with CR II–VI (61–271), and CR I–III (1–142) with CR IV–VI (145–271). We conclude that CR is formed from at least two independent domains consisting of CR I–II and CR III–VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase‐3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF‐hand proteins, and perhaps of other protein families.

Strong association between Saitohin gene polymorphism and tau haplotype in the Polish population

The saitohin (STH) gene is located in intron 9 of the tau protein gene. It has been postulated that the R allele of Q7R polymorphism at the Saitohin gene is over-represented in the homozygous state in sporadic Alzheimers disease (AD). Tau protein was implicated in AD pathophysiology and the tau gene haplotype is probably connected with sporadic late-onset Parkinsons disease (PD). We analyzed the STH polymorphism and tau gene haplotype in 100 clinically diagnosed AD cases, 100 PD cases and 100 age-matched healthy controls. We found that the R allele of the STH gene is associated with the H2 haplotype of tau in all cases. Additionally we observed no correlation between R allele frequency and AD or PD.