Giulia Greco

Low density lipoprotein misfolding and amyloidogenesis

In early atherogenesis, subendothelial retention of lipidic droplets is associated with an inflammatory response‐to‐injury, culminating in the formation of foam cells and plaque. Low density lipoprotein (LDL) is the main constituent of subendothelial lipidic droplets. The process is believed to occur following LDL modification. Searching for a modified LDL in plasma, electronegative LDL [LDL(—)] was identified and found to be associated with major risk biomarkers. The apoprotein in LDL(—) is misfolded, and we show here that this modification primes the aggregation of native LDL, conforming to the typical pattern of protein amyloidogenesis. After a lag phase, whose length depends on LDL(—) concentration, light scattering and atomic force microscopy reveal early exponential growth of intermediate globules, which evolve into fibrils. These globules are remarkably similar to subendothelial droplets in atheromatous lesions and different from those produced by oxidation or biochemical manipulation. During aggregation, ellipticity and tryptophan fluorescence measurements reveal a domino‐style spread of apoprotein misfolding from LDL(—) to all of the LDL. Computational analysis of the apoprotein primary sequence predicts an unstable, aggregation‐prone domain in the regulatory α2 region. Apoprotein misfolding well represents an LDL modification able to transform this cholesterol carrier into a trigger for a response‐to‐injury in the artery wall.—Parasassi, T., De Spirito, M., Mei, G., Brunelli, R., Greco, G., Lenzi, L., Maulucci, G., Nicolai, E., Papi, M., Arcovito, G., Tosatto, S. C. E., Ursini, F. Low density lipoprotein misfolding and amyloidogenesis. FASEB J. 22, 2350–2356 (2008)

Differentiation of normal and cancer cells induced by sulfhydryl reduction : biochemical and molecular mechanisms

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1–10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell–cell and cell–substratum junctions; (iv) a relocation of the oncogenic β-catenin at the cell–cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro.

BackgroundCancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process.MethodsIn order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point.ResultsMicroarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action.The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform.ConclusionNAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action.

Generation in human plasma of misfolded, aggregation-prone electronegative low density lipoprotein

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(-), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(-) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(-) during incubation of unprocessed human plasma at 37 degrees C. In addition to a higher fraction of amyloidogenic LDL(-), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoproteins structure, which misfolds and becomes aggregation-prone.

Gene expression analysis of human epidermal keratinocytes after N-acetyl L-cysteine treatment demonstrates cell cycle arrest and increased differentiation.

Objectives: Several cancer prevention programmes have previously been executed using treatment of antioxidant compounds. The antioxidant N-acetyl L-cysteine (NAC), a membrane-permeable aminothiol, is a sulfhydryl reductant reducing oxidised glutathione, as well as being a precursor of intracellular cysteine and glutathione. A previous report based on the cellular response to NAC treatment showed that NAC induced a 10-fold more rapid differentiation in normal primary keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation. In order to investigate molecular events underlying thechanges in proliferation and differentiation induced by NAC treatment, we performed global gene expression analysis of normal human epidermal keratinocytes in a time series. Methods: Treated samples were compared to untreated samples through a reference design using a spotted cDNA array comprising approximately 30,000 features. B statistics was used to identify differentially expressed genes, and RT-PCR of a selected set of genes was performed to verify differential expression. Results: The number of differentially expressed genes increased over time, starting with 0 at 30 min, 73 at 3 h and increasing to 952 genes at 48 h. Results of the expression analysis showed arrest of the cell cycle and an upregulation of cytoskeletal reorganisation, implicating increased differentiation. A comparison to gene ontology groups indicated downregulation of a large number of genes involved in cell proliferation and regulation of the cell cycle. Conclusions: A significant fraction of the differentially expressed genes could be classified according to their role in the differentiation process, demonstrating that NAC regulates the conversion from proliferation to differentiation at a transcriptional level.

Estradiol Binding Prevents ApoB-100 Misfolding in Electronegative LDL(−)

Seeking for a modified lipoprotein present in plasma that could account for the atherogenic effect of high cholesterol, several years ago electronegative LDL(-) was identified. The peculiar feature of LDL(-) is an apoprotein misfolding that triggers the formation of aggregates, perfectly fitting in size the subendothelial droplets observed in early phases of atherogenesis. Apoprotein misfolding was therefore proposed as a possible atherogenic modification. LDL(-) can be spontaneously produced in vitro by plasma incubation through phospholipid hydrolysis catalyzed by the activity of endogenous phospholipases. As a consequence, apoprotein is misfolded. 17beta-Estradiol (E2), a specific ligand to apoB-100, was used to unravel the relationship between negative charge of the lipoprotein and apoprotein structural/conformational shift. Although E2 addition to plasma does not prevent LDL(-) generation nor phospholipase activity, it deeply stabilizes apoB-100 structure, thus preventing its structural and conformational shift. Apoprotein stabilization extends to lipids. Indeed, while a loosening of lipid packing is observed together with apoprotein misfolding, conversely, when E2 stabilizes apoprotein, lipid structure is preserved. Finally, even in the presence of LDL(-), the E2-stabilized LDL is resistant to aggregation, unambiguously demonstrating that misfolding, but not negative charge, primes aggregation. In conclusion, electronegative charge and misfolding are independent and distinct features of LDL(-), and apoprotein misfolding rather than the increase in the negative charge emerges both as a valid biomarker and as an appealing pharmacological and nutritional target.

One site on the apoB-100 specifically binds 17-β-estradiol and regulates the overall structure of LDL

The major protein component (apoB‐100) of low‐density lipoprotein (LDL) is known as a multipotential molecule the several functional regions of which can all be affected by key structural modifications driven by specific domains. Based on our previous report on structural and conformational modifications of apoB‐100 in the presence of 17‐β‐estradiol (E2), we characterized the interaction between E2 and the apoB‐100 and further explored the induced alterations in terms of the structural arrangement of the whole LDL particle. We report evidence for the existence on apoB‐100 of a single specific and saturable binding site for E2, the occupancy of which modifies the overall structure of the protein, inducing an increase in the α‐helix fraction. As a consequence, the structure of the LDL particle is deeply perturbed, with a change in the arrangement of both the outer shell and lipid core and an overall volume shrinkage. The evidence of a regulation of apoB‐100 structure by a physiological ligand opens new perspectives in the study of the biological addressing of the LDL particle and suggests a novel rationale in the search for mechanisms underlying the beneficial role of E2 in decreasing the risk of early lesions in atherosclerosis.

Impact of electronegative low-density lipoprotein on angiographic coronary atherosclerotic burden

OBJECTIVE Low density lipoproteins (LDL) with an electronegative charge [LDL(-)] may cause endothelial injury. We assessed the association between serum LDL(-) levels and coronary artery disease (CAD) severity. METHODS We prospectively enrolled patients with CAD angiographic evidence [stable angina (SA) or non-ST-elevation-acute coronary syndrome (NSTE-ACS)], or with normal coronary arteries (NCA). Baseline LDL(-) serum levels were measured in all patients. Angiographic CAD extent was assessed by using the Bogaty extent index, while CAD severity by evaluating the presence of multi-vessel disease. RESULTS Forty-seven patients (age 61 ± 9 years, male sex 60%) were enrolled (17 SA, 15 NSTE-ACS and 15 NCA patients). LDL(-) levels were significantly higher in SA [21% (18-34) p = 0.0001] and NSTE-ACS [22% (18-28), p = 0.0001] as compared to NCA [6% (5-8)], without significant differences between SA and NSTE-ACS (p = 0.92). Multi-vessel disease patients had higher LDL(-) levels as compared to single-vessel disease patients (p = 0.002) but similar total LDL levels (p = 0.66). LDL(-) significantly correlated with extent index (r = 0.38, p = 0.03), while total LDL did not (p = 0.24). CONCLUSION LDL(-) serum levels are associated with CAD angiographic severity and extent. This exploratory analysis should prime further larger studies in order to assess LDL(-) proatherogenic role.