Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function.

Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (although smaller values of nH are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role, safeguarding DNA from DNDGIC, display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs. Beside the well known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity.

The extreme hyper-reactivity of selected cysteines drives hierarchical disulfide bond formation in serum albumin.

After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pKa of the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.

Adult Human Biliary Tree Stem Cells Differentiate to ß-Pancreatic Islet Cells by Treatment with a Recombinant Human Pdx1 Peptide.

Generation of ß-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards ß-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the ß-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage ß-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional ß-pancreatic cells.

The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: a proposal for an evolutionary driving force.

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

The mitochondrial Italian Human Proteome Project initiative (mt-HPP).

Mitochondria carry maternally inherited genetic material, called the mitochondrial genome (mtDNA), which can be defined as the 25th human chromosome. The chromosome-centric Human Proteome Project (c-HPP) has initially focused its activities addressing the characterization and quantification of the nuclear encoded proteins. Following the last International HUPO Congress in Boston (September 2012) it was clear that however small the mitochondrial chromosome is, it plays an important role in many biological and physiopathological functions. Mutations in the mtDNA have been shown to be associated with dozens of unexplained disorders and the information contained in the mtDNA should be of major relevance to the understanding of many human diseases. Within this paper we describe the Italian initiative of the Human Proteome Project dedicated to mitochondria as part of both programs: chromosome-centric (c-HPP) and Biology/Disease (B/D-HPP). The mt-HPP has finally shifted the attention of the HUPO community outside the nuclear chromosomes with the general purpose to highlight the mitochondrial processes influencing the human health. Following this vision and considering the large interest and evidence collected on the non-Mendelian heredity of Homo sapiens associated with mt-chromosome and with the microbial commensal ecosystem constituting our organism we may speculate that this program will represent an initial step toward other HPP initiatives focusing on human phenotypic heredity.

Serological Proteome Analysis (SERPA) as a tool for the identification of new candidate autoantigens in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the presence of circulating autoantibodies directed against proteins of islet beta-cell. Autoantibody testing is used for diagnostic purposes; however, up to 2-5% of patients who are clinically diagnosed with T1D are found negative for known antibodies, suggesting that the T1D autoantigen panel is incomplete. With the aim of identifying new T1D autoantigen(s), we used sera from subjects clinically diagnosed with T1D, but who tested negative for the four T1D autoantibodies currently used in clinical practice and for genes responsible for sporadic cases of diabetes. Sera from these patients were challenged by Western blot against the proteome from human pancreatic beta-cells resolved by 2DE. Eleven proteins were identified by MS. A radiobinding assay (RBA) was developed to test the reactivity to Rab GDP dissociation inhibitor beta (GDIß) of T1D sera using an independent method. Depending on the construct used (open reading frame or COOH-terminus) 22% to 32% of fifty T1D sera showed increased binding to GDIß by RBA. In addition, 15% of patients with celiac disease had raised binding to the COOH-terminus GDIß. These results indicate that immunoproteomics is a feasible strategy for the identification of candidate T1D autoantigens. BIOLOGICAL SIGNIFICANCE: Several approaches have been previously used to look for new type 1 diabetes autoantigens. With the present work we show that carefully selected sera from rare patients with diabetes both negative for the 5 autoantibodies currently used in clinical practice and for genes responsible for sporadic cases of diabetes, may be exploited in experiments utilizing human pancreatic islets extracts as a target for SERPA to identify novel candidate T1D autoantigens.

NPY modulates miR-30a-5p and BDNF in opposite direction in an in vitro model of Alzheimer disease: a possible role in neuroprotection?

Using in vitro models of Alzheimer's disease (AD), we found that the toxic effects of amyloid beta 25-35 (Aß(25-35)) on the neurotrophin brain-derived neurotrophic factor (BDNF) were counteracted by pre-incubation with neuropeptide Y (NPY), a neuropeptide expressed within the central nervous system. Nonetheless, the mechanism of action of NPY on BDNF neuronal production in the presence of Aß is not known. BDNF expression might be directly regulated by microRNA (miRs), small non-coding DNA fragments that regulate the expression of target genes. Thus, there is the possibility that miRs alterations are present in AD-affected neurons and that NPY influences miR expression. To test this hypothesis, we exposed NPY-pretreated primary rat cortical neurons to Aß(25-35) and measured miR-30a-5p (a member of the miR-30a family involved in BDNF tuning expression) and BDNF mRNA and protein expression after 24 and 48 h. Our results demonstrated that pre-treatment with NPY decreased miR-30a-5p expression and increased BDNF mRNA and protein expression at 24 and 48 h of incubation with Aß. Therefore, this study demonstrates that NPY modulates BDNF and its regulating microRNA miR-30a-5p in opposite direction with a mechanism that possibly contributes to the neuroprotective effect of NPY in rat cortical neurons exposed to Aß.

Glutathione S-transferase P1-1 as a target for mesothelioma treatment.

Malignant pleural mesothelioma is a poorly responsive tumor known to overexpress the phase II detoxification enzyme glutathione-S-transferase, which catalyzes the conjugation between glutathione and platinum(II)-containing drugs. Therefore, we evaluated the effect of the strong glutathione S-transferase inhibitor NBDHEX on human mesothelioma cell lines (MSTO-211H, MPP89, MM-B1 and Mero 48a) featuring the most common mesothelioma phenotypes: epithelioid and biphasic. Even though a different response to NBDHEX was observed, the molecule was very effective on all cell lines tested, triggering a sustained activation of both JNK and p38, followed by caspase activation and apoptosis. NBDHEX also caused severe oxidative stress in the MPP89 cells and, to a lesser extent, in the MMB1 cells, while it did not cause a significant redox imbalance in the other cell lines. The efficacy of the drug was found to be comparable or even higher than that of cisplatin. Moreover, it showed synergistic or additive effects when used in combination with cisplatin. In conclusion, NBDHEX was effective on mesothelioma cell lines, with IC(50) values in the low micromolar range (IC(50) between 1 and 4 µM). These findings indicate that NBDHEX, alone or in combination with cisplatin, is a promising new strategy for treating this rare and aggressive malignancy.

Neuropeptide Y protects rat cortical neurons against ß-amyloid toxicity and re-establishes synthesis and release of nerve growth factor.

Neuropeptide Y (NPY) is a 36 amino acid peptide, widely distributed within central nervous system neurons. More recently, it has been shown that NPY is involved in Alzheimer's disease (AD), a disorder characterized by accumulation of amyloid ß-peptide (Aß) in neurons. In a previous study, we investigated the effect of NPY on neuronal damage by exposing SH-SY5Y cells (an established human derived neuroblastoma cell line) to Aß's pathogenic fragment 25-35 (Aß(25-35)). We found a NPY-neuroprotective action associated with changes in intracellular production of nerve growth factor (NGF), a member of the neurotrophin family. Since our results were encouraging, we decided to replicate our data using primary cortical neurons cultured in presence of Aß(25-35), and investigated whether NPY had similar neuroprotective action. Moreover, since cortical neurons are able to produce and release NGF, we investigated whether the synthesis and release of NGF were modified in such experimental conditions. Our results showed that a preincubation with NPY counteracted the toxic effect of Aß, as measured by increased cell viability. Moreover, NPY pretreatment had an effect on NGF since its intracellular synthesis was increased, release was normalized, and mRNA expression was downregulated. Notably, these effects on NGF were in the opposite direction of those produced by incubating the cells with Aß alone. This study in primary cortical neurons supports the hypothesis that NPY may be a neuroprotective agent against ß-amyloid neurotoxicity. These data also suggest that NPY may influence the synthesis and the release of NGF by cortical neurons.

Erythrocyte glutathione transferase: a novel biomarker to check environmental pollution hazardous for humans.

Glutathione transferase (GST) is an enzyme capable of protecting the body from a lot of toxic compounds. Previous studies demonstrated that the erythrocyte GST (e-GST) expression increases as the level of circulating toxins increases. Aim of the present study is to verify if e-GST may represent a biomarker able to signalize an environmental pollution hazardous for humans. The study involved about 500 healthy volunteers living in eight distinct areas at or near the Sacco river valley, a region of the Frosinone district (Lazio-Italy) well known for its environmental pollution. Subjects of six areas displayed increased levels of e-GST ranging from 18% to 44% compared to 400 volunteers living in the Rome hinterland. Higher levels of GSTs are present in the areas where the risk of pollution is higher (areas 7 and 8). Interestingly, women living in the Sacco valley display much higher expression of e-GST than men, possibly due to a greater time exposition to the environmental contamination. Possible oxidative alteration of GST activity has not been observed. In conclusion, e-GST may represent an early and sensitive bio-signal of dangerous pollution for humans.

Spectrophotometric assay for serum glutathione transferase: a re-examination.

OBJECTIVES: The aim of the present paper is a careful re-examination of an automated spectrophotometric procedure for glutathione transferase (GST) activity in human serum described previously and used in many laboratories. DESIGN AND METHODS: GST activity in human serum has been assayed spectrophotometrically under various experimental conditions. Recombinant human GSTs and specific inhibitors were also used to check the possible occurrence of artifacts. RESULTS: Basal level of the enzyme calculated using this method turns out to be much higher than that found using RIA and ELISA procedures. Relevant pH-dependent artifacts deeply affect this spectrophotometric assay. Notably, spectral changes previously interpreted as a measure of basal activity, are mainly due to an increase of the spontaneous reaction between the two substrates. CONCLUSION: GST activity in normal serum cannot be correctly determined with the spectrophotometric assay described previously because of the very low enzyme concentration and the pH-dependent artifacts.

Shotgun proteomics and network analysis of neuroblastoma cell lines treated with curcumin.

Curcumin is a natural compound with recognized anti-inflammatory properties, but its anticancer activity is still object of study. We provided an unsupervised molecular investigation of the main proteome rearrangements involved in the cellular response to curcumin in a human neuroblastoma cell line sensitive to cisplatin and its resistant counterpart by a comparative proteomic approach. Shotgun analysis demonstrated that 66 proteins were differentially expressed in response to 24 h treatment with 40 µM curcumin in sensitive cells, whereas 32 proteins were significantly modulated in treated resistant cells. Functional analysis revealed that proteins involved in cellular assembly and organization, biosynthesis and glycolysis were down-regulated by curcumin treatment. Proteome changes were associated to cell cycle arrest in the G2/M phase and accumulation of polyubiquitinated proteins, also confirmed by flow cytometry and immunoblotting analysis, but not to a significant increment of reactive oxygen species production. Since the polyubiquitination of proteins influences a wide range of cellular pathways, the inhibition of the ubiquitin-proteasome system may be the main way through which curcumin performs its multi-target activity.

Evaluation of DRB1 high resolution typing by a new SSO-based Luminex method.

HLA testing is an essential part of the process to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow, peripheral blood or cord blood and the DNA typing in high resolution is now recommended as the Scientific Societies also describe in their standards. Recently the new PCR-Luminex HLA typing method, based on the reverse sequence specific oligonucleotide probes coupled with a microsphere beads in an array platform, has been well established. We report the data from 146 samples previously typed to a four digits level and used to evaluate the accuracy, sensitivity and performance of the new high definition DRB1 by PCR-Luminex kit. One hundred and forty-six samples from unrelated healthy donors, haematological patients or external proficiency tests were used in this study. The Luminex high definition DRB1 typing represents a versatile method and may be easily introduced in the routine, particularly when the technical team has already acquired experience on the technique. Only few HLA allelic combinations need an additional typing by PCR-SSP or SBT to solve the ambiguous results thus reducing the time necessary to produce a final report.

Protein repertoire impact of Ubiquitin-Proteasome System impairment: insight into the protective role of beta-estradiol.

The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathways (ALP) are key mechanisms for cellular homeostasis sustenance and protein clearance. A wide number of Neurodegenerative Diseases (NDs) are tied with UPS impairment and have been also described as proteinopathies caused by aggregate-prone proteins, not efficiently removed by proteasome. Despite the large knowledge on proteasome biological role, molecular mechanisms associated with its impairment are still blur. We have pursued a comprehensive proteomic investigation to evaluate the phenotypic rearrangements in protein repertoires associated with a UPS blockage. Different functional proteomic approaches have been employed to tackle UPS impairment impact on human NeuroBlastoma (NB) cell lines responsive to proteasome inhibition by Epoxomicin. 2-Dimensional Electrophoresis (2-DE) separation combined with Mass Spectrometry and Shotgun Proteomics experiments have been employed to design a thorough picture of protein profile. Unsupervised meta-analysis of the collected proteomic data revealed that all the identified proteins relate each other in a functional network centered on beta-estradiol. Moreover we showed that treatment of cells with beta-estradiol resulted in aggregate removal and increased cell survival due to activation of the autophagic pathway. Our data may provide the molecular basis for the use of beta-estradiol in neurodegenerative disorders by induction of protein aggregate removal.

Erythrocyte glutathione transferase: a potential new biomarker in chronic kidney diseases which correlates with plasma homocysteine.

The erythrocyte glutathione S-transferase (e-GST) is a member of a superfamily of inducible enzymes involved in cell detoxification that shows an increased expression in chronic kidney disease (CKD) patients. We propose a new automated analysis procedure for e-GST activity that has been validated in 72 CKD patients and 62 maintenance hemodialysis patients (MHD). Regression analysis was carried out to assess association between e-GST activity data, main clinical variables, and plasma homocysteine (Hcy), a modified sulfur amino acid known as potential risk factor for cardiovascular disease that is increased above normal levels in more than 90% of the uremic patients. An increased e-GST activity was confirmed in MHD patients (N=62; 10.2±0.4 U/gHb) compared with healthy subjects (N=80; 5.8±0.4 U/gHb), and as an original finding, a significant increase of e-GST activity was observed in pre-dialysis CKD patients with a positive correlation with disease severity weighted according to the four stages of "Kidney Disease Outcomes Quality Initiative" classification (7.4±0.5, 8±1, 9.5±0.6, 12±1 U/gHb, respectively). No correlation was found between e-GST activity and hemoglobin, transferrin, blood iron and the markers of systemic inflammation and renal function such as alpha-1 acid glycoprotein and high-sensitive C-Reactive Protein, beta-2 microglobulin and the index of malnutrition-inflammation PINI, while a significant correlation was observed for the first time between plasma Hcy and e-GST activity (r2=0.64, P<0.0001) in MHD patients. Hcy, however, was not identified as an inhibitor of e-GST enzyme. The results in this study suggest the potential for automated e-GST analysis as a valuable tool to further explore phase II-related uremic toxicity in CKD and MHD patients.

Targeting GSTP1-1 induces JNK activation and leads to apoptosis in cisplatin-sensitive and -resistant human osteosarcoma cell lines.

The effect of the glutathione transferase P1-1 (GSTP1-1) targeting has been investigated in both sensitive (U-2OS) and cisplatin-resistant (U-2OS/CDDP4 µg) human osteosarcoma cell lines. Despite the different enzyme's content, inhibition of GSTP1-1 by 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) causes the activation of c-Jun N-terminal kinase (JNK) and apoptosis in both cell lines. However, different time courses of JNK activation and cell responses are observed. Whereas in the U-2OS/CDDP4 µg cell line drug treatment results in an early increase of caspase activity and secondary necrosis, in the U-2OS cells it mainly causes an early cell cycle arrest followed by apoptosis. In order to elucidate the action mechanism of NBDHEX we performed a proteomic investigation by label-free nLC-MS(E). The high-throughput analysis associated with a bioinformatic tool suggested the involvement of the TNF receptor associated factor (TRAF) family in the cellular response to the drug treatment. We report experimental evidence of the interaction between GSTP1-1 and TRAF2 and we demonstrate that NBDHEX is able to dissociate the GSTP1-1 : TRAF2 complex. This restores the TRAF2 : ASK1 signaling, thereby leading to the simultaneous and prolonged activation of JNK and p38. These mitogen-activated protein kinases (MAPKs) mediate different effects: JNK is crucial for apoptosis, whereas p38 causes an increase in the p21 level and a concomitant cell cycle arrest. Our study shows that GSTP1-1 plays an important regulatory role in TRAF signaling of osteosarcoma and discloses new features of the action mechanism of NBDHEX that suggest potentially practical consequences of these findings.

Platinum-(IV)-derivative satraplatin induced G2/M cell cycle perturbation via p53-p21(waf1/cip1)-independent pathway in human colorectal cancer cells.

AIM: Platinum-(IV)-derivative satraplatin represents a new generation of orally available anti-cancer drugs that are under development for the treatment of several cancers. Understanding the mechanisms of cell cycle modulation and apoptosis is necessary to define the mode of action of satraplatin. In this study, we investigate the ability of satraplatin to induce cell cycle perturbation, clonogenicity loss and apoptosis in colorectal cancer (CRC) cells. METHODS: CRC cells were treated with satraplatin, and the effects of satraplatin on apoptosis and the cell cycle were evaluated by flow cytometry. Western blot analysis was used to investigate the effects of satraplatin on cell cycle and apoptosis-related proteins. RT-qPCR was used to evaluate p53-related mRNA modulation. RESULTS: Satraplatin induced an accumulation of CRC cells predominantly in the G(2)/M phase. Increased p53 protein expression was observed in the p53 wild-type HCT116 and LoVo cells together with p21(waf1/cip1) protein up-regulation. However, p21(waf1/cip1) protein accumulation was not observed in the p53 mutant HCT15, HT29, and WiDr cells, even when p53 protein expression was compromised, suggesting that the cell cycle perturbation is p53-p21(waf1/cip1) independent. Following a candidate approach, we found an elevated expression of 14-3-3σ protein levels in CRC cells, which was independent of the status of p53, further supporting the role of satraplatin in the perturbation of the G(2)/M cell cycle phase. Moreover, satraplatin treatment induced apoptosis along with Bcl-2 protein down-regulation and abrogated the clonogenic formation of CRC cells in vitro. CONCLUSION: Collectively, our data suggest that satraplatin induces apoptosis in CRC cells, which is preceded by cell cycle arrest at G(2)/M due to the effect of 14-3-3σ and in a p53-p21(waf1/cip1)-independent manner. Taken together, these findings highlight the potential use of satraplatin for CRC treatment.

Differential expression of miR-144* as a novel fecal-based diagnostic marker for colorectal cancer.

BACKGROUND: MicroRNAs (miRNA) are tiny, noncoding, small, endogenous RNAs that play major roles in neoplastic transformation and could therefore offer a better quantitative and noninvasive method for the diagnosis and prognosis of colorectal cancer (CRC) using feces. In the present study, we screened feces for 648 miRNAs and analyzed the role of miR-144* as a potential CRC diagnostic marker. METHODS: Fecal miRNA expression was profiled with RT-pre-amplification-qPCR, and the stability was determined using both endogenous and exogenous miRNA by RT-qPCR. ROC analysis was performed to enhance the diagnosing power of the CRC patients' fecal specimens. RESULTS: We detected 39% of all the miRNAs screened in feces. Endogenous miRNAs are more stable over time and temperature, while exogenous miRNAs degraded rapidly. miR-144* was overexpressed in feces, suggesting that it could be a potent candidate diagnostic marker for CRC detection, with a sensitivity of 74% and a specificity of 87% (n = 75, p < 0.0001). Moreover, RT-qPCR analysis showed that miR-144* was also overexpressed in paired CRC tissues, thus suggesting its possible utilization as a diagnostic marker. CONCLUSIONS: We demonstrated that miRNAs are stable in the fecal microenvironment, and that, among them, miR-144* represents a novel fecal-based diagnostic marker for CRC screening. Nevertheless, our data need to be validated in a large cohort of subjects.

Treatment of doxorubicin-resistant MCF7/Dx cells with nitric oxide causes histone glutathionylation and reversal of drug resistance.

Acquired drug resistance was found to be suppressed in the doxorubicin-resistant breast cancer cell line MCF7/Dx after pre-treatment with GSNO (nitrosoglutathione). The effect was accompanied by enhanced protein glutathionylation and accumulation of doxorubicin in the nucleus. Among the glutathionylated proteins, we identified three members of the histone family; this is, to our knowledge, the first time that histone glutathionylation has been reported. Formation of the potential NO donor dinitrosyl-diglutathionyl-iron complex, bound to GSTP1-1 (glutathione transferase P1-1), was observed in both MCF7/Dx cells and drug-sensitive MCF7 cells to a similar extent. In contrast, histone glutathionylation was found to be markedly increased in the resistant MCF7/Dx cells, which also showed a 14-fold higher amount of GSTP1-1 and increased glutathione concentration compared with MCF7 cells. These results suggest that the increased cytotoxic effect of combined doxorubicin and GSNO treatment involves the glutathionylation of histones through a mechanism that requires high glutathione levels and increased expression of GSTP1-1. Owing to the critical role of histones in the regulation of gene expression, the implication of this finding may go beyond the phenomenon of doxorubicin resistance.

Protein-protein interaction heterogeneity of plasma apolipoprotein A1 in nephrotic syndrome.

Apolipoprotein A1 (apoA1) is a component of the high density lipoproteins (HDL) that regulates the transport of cholesterol between the liver and peripheral cells and modulates the removal of any excess of cholesterol from membranes. Any variation in apoA1 composition may modify the plasma lipid profile and be involved in atherogenesis. We investigated apoA1 composition in plasma of 6 children with nephrotic syndrome, a condition characterized by high levels of cholesterol in plasma and by a high risk to develop atherosclerosis. Non-denaturing two-dimensional electrophoresis (Nat/SDS-PAGE), mass spectrometry, western blot and pull down experiments were done to characterize proteins and define putative interactions. ApoA1 was resolved in 12 variants, 6 of which had a slightly lower molecular weight (18-19 KDa) and migrated on the same axes of the ß chain of haptoglobin (Hp). Low molecular weight apoA1 were observed in carriers of different Hp haplotypes (including one homozygous for the rare ßßα1α1) ruling out any contaminant effect of co-migration of apoA1 with Hp α2 chain. Overall, apoA1 isoforms were much more present in plasma of nephrotic patients compared to a normal profile. These findings show that apoA1 plasma in nephrotic syndrome is heterogeneous in terms of molecular weight. Low molecular weight fragments lack internal structural domains and likely form macro-aggregates with Hp. Fragmentation and transport of apoA1 may be involved in the general disorder of lipid metabolism that characterizes nephrotic syndrome.