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1

Interaction of an anticancer ruthenium complex HInd[RuInd2Cl4] with cytochrome c.

100%

Trynda-Lemiesz L.

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nr 1

199-205

EN

Cytochrome c is an important electron transfer protein in the respiratory chain, shuttling electrons from cytochrome c reductase to cytochrome c oxidase. Extensive chemical modification studies indicate significant electrostatic interactions between these proteins and show that all structural and conformational changes of cytochrome c can influence the electron transport. In the present work we examine the effect of an anticancer ruthenium complex, trans-Indazolium (bisindazole) tetrachlororuthenate(III) (HInd[RuInd2Cl4]), on the conformation of cytochrome c, the state of the heme moiety, formation of the protein dimer and on the folding state of apocytochrome c. For this purpose, gel-filtration chromatography, absorption second derivative spectroscopy, circular dichroism (CD) and inductively coupled plasma atomic emission spectroscopy (ICP(AES)) were used. The present data have revealed that binding of the potential anticancer drug HInd[RuInd2Cl4] complex to cytochrome c induces a conformation of the protein with less organized secondary and tertiary structure.

2

Overexpression of BimSs3, the novel isoform of Bim, can trigger cell apoptosis by inducing cytochrome c release from mitochondria

100%

Liu L. , Chen J. , Zhang J. , Ji C. , Zhang X. , Gu S. , Xie Y. , Mao Y.

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tom 54

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nr 3

603-610

EN

Bim is defined as the pro-apoptotic BH3-only protein of the Bcl-2 family, which is a critical sensor and mediator in the mitochondrial-dependent apoptosis. In a previous work, we have cloned a novel transcript of Bim (GenBank accession number: AY305716) from the fetal brain cDNA, which is widely expressed in some carcinoma tissues and normal human tissues. According to the sequence analysis and the newly-defined nomenclature system of Bim isoforms (Adachi et al., 2005, Cell Death Differ 2: 192), we term it BimSs3 according to its characteristic structure. The subcellular location analysis indicated that the fused protein GFP-BimSs3 is distributed in the whole cell, mainly to the nucleus. Overexpression of BimSs3 in HEK293 cells causes apoptosis (28.16 ± 1.55%) compared to the negative control (5.44 ± 2.63%). It also causes cytochrome c release from the mitochondrial fraction to the cytosolic fraction during apoptosis. Western blotting assay indicates the molecular mass of GFP-BimSs3 is approximately 31.0 kDa (GFP: 27 kDa). Hence the open reading frame of BimSs3 may initiate at the second ATG and encodes a 36 amino-acid peptide with BH3 domain.

3

Probing protein structure by limited proteolysis.

51%

Fontana A. , de Laureto P. P. , Spolaore B. , Frare E. , Picotti P. , Zambonin M.

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nr 2

299-321

EN

Limited proteolysis experiments can be successfully used to probe conformational features of proteins. In a number of studies it has been demonstrated that the sites of limited proteolysis along the polypeptide chain of a protein are characterized by enhanced backbone flexibility, implying that proteolytic probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis was used to analyze the partly folded (molten globule) states of several proteins, such as apomyoglobin, α-lactalbumin, calcium-binding lysozymes, cytochrome c and human growth hormone. These proteins were induced to acquire the molten globule state under specific solvent conditions, such as low pH. In general, the protein conformational features deduced from limited proteolysis experiments nicely correlate with those deriving from other biophysical and spectroscopic techniques. Limited proteolysis is also most useful for isolating protein fragments that can fold autonomously and thus behave as protein domains. Moreover, the technique can be used to identify and prepare protein fragments that are able to associate into a native-like and often functional protein complex. Overall, our results underscore the utility of the limited proteolysis approach for unravelling molecular features of proteins and appear to prompt its systematic use as a simple first step in the elucidation of structure-dynamics-function relationships of a novel and rare protein, especially if available in minute amounts.

4

Pharmacological inhibition of GSK3 attenuates DNA damage-induced apoptosis via reduction of p53 mitochondrial translocation and Bax oligomerization in neuroblastoma SH-SY5Y cells

44%

Ngok-Ngam P. , Watcharasit P. , Thiantanawat A. , Satayavivad J.

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tom 18

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nr 1

EN

Glycogen synthase kinase-3 (GSK3) and p53 play crucial roles in the mitochondrial apoptotic pathway and are known to interact in the nucleus. However, it is not known if GSK3 has a regulatory role in the mitochondrial translocation of p53 that participates in apoptotic signaling following DNA damage. In this study, we demonstrated that lithium and SB216763, which are pharmacological inhibitors of GSK3, attenuated p53 accumulation and caspase-3 activation, as shown by PARP cleavage induced by the DNA-damaging agents doxorubicin, etoposide and camptothecin. Furthermore, each of these agents induced translocation of p53 to the mitochondria and activated the mitochondrial pathway of apoptosis, as evidenced by the release of cytochrome C from the mitochondria. Both mitochondrial translocation of p53 and mitochondrial release of cytochrome C were attenuated by inhibition of GSK3, indicating that GSK3 promotes the DNA damage-induced mitochondrial translocation of p53 and the mitochondrial apoptosis pathway. Interestingly, the regulation of p53 mitochondrial translocation by GSK3 was only evident with wild-type p53, not with mutated p53. GSK3 inhibition also reduced the phosphorylation of wild-type p53 at serine 33, which is induced by doxorubicin, etoposide and camptothecin in the mitochondria. Moreover, inhibition of GSK3 reduced etoposide-induced association of p53 with Bcl2 and Bax oligomerization. These findings show that GSK3 promotes the mitochondrial translocation of p53, enabling its interaction with Bcl2 to allow Bax oligomerization and the subsequent release of cytochrome C. This leads to caspase activation in the mitochondrial pathway of intrinsic apoptotic signaling.