Luca Bini

Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering.

Signalling by immunoreceptors is orchestrated at specific plasma membrane microdomains, referred to as lipid rafts. Here we present a proteomics approach to the temporal analysis of protein association with lipid rafts following T-cell antigen receptor (TCR) triggering. We show that TCR engagement promotes the temporally regulated recruitment of proteins participating in the TCR signalling cascade to lipid rafts. Furthermore, TCR triggering results in profound modifications in the composition of lipid rafts involving a number of proteins associated either directly or indirectly with both plasma and intracellular membranes. Raft-associated proteins can be clustered according to their temporal profile of raft association. The data identify lipid rafts as highly dynamic structures and reveal a dramatic impact of surface TCR triggering not only on components of the TCR signalling machinery but also on proteins implicated in a number of diverse cellular processes.

Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis : a two-dimensional electrophoretic study

We used two‐dimensional (2‐D) electrophoresis to analyze the protein composition of fluid recovered by bronchoalveolar lavage (BALF) from patients with sarcoidosis and idiopathic pulmonary fibrosis, two forms of interstitial lung disease with different cellular composition and cytokine profile in BALF. They are also characterized by different pathogenesis and clinical evolution, idiopathic pulmonary fibrosis being less favorable than sarcoidosis due to rapidly progressive pulmonary fibrosis. Thirty‐eight proteins or protein fragments, never previously assigned in BALF samples, were identified by various methods including mass fingerprinting of tryptic digests. Comparison of the BALF protein maps of the two groups of patients showed 32 spots with statistically significant disease‐related variations in relative abundance. In sarcoidosis we found an increase in the amount of several plasma proteins, while in idiopathic pulmonary fibrosis we observed a statistically significant increase in low‐molecular‐weight proteins, many of which are involved in inflammatory processes (such as MIF and calgranulin) or antioxidant response (such as antioxidant peroxysomal enzyme and thioredoxin peroxidase 2). 2‐D electrophoresis allowed us to identify new BALF proteins and to characterize protein composition in patients with sarcoidosis and idiophatic pulmonary fibrosis. Comparison of the gels of the two diseases showed that they differ in BALF protein profiles as they do in type of immune response.

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two‐dimensional electrophoresis map with patient sera

Western blots of two‐dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot‐cluster due to the chlamydia‐specific antigen outer membrane protein‐2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL‐like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface‐exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK‐like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N‐terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha‐subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF‐Tu, seven (41%) a putative stress‐induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl‐like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.

The acid-stress response in Lactobacillus sanfranciscensis CB1

Lactobacillus sanfranciscensis CB1, an important sourdough lactic acid bacterium, can withstand low pH after initial exposure to sublethal acidic conditions. The sensitivity to low pH varied according to the type of acid used. Treatment of LB: sanfranciscensis CB1 with chloramphenicol during acid adaptation almost completely eliminated the protective effect, suggesting that induction of protein synthesis was required for the acid-tolerance response. Two constitutively acid-tolerant mutants, CB1-5R and CB1-7R, were isolated using natural selection techniques after sequential exposure to lactic acid (pH 3.2). Two-dimensional gel electrophoresis analysis of protein expression by non-adapted, acid-adapted and acid-tolerant mutant cells of LB: sanfranciscensis showed changes in the levels of 63 proteins. While some of the modifications were common to the acid-adapted and acid-tolerant mutant cells, several differences, especially regarding the induced proteins, were determined. The two mutants showed a very similar level of protein expression. Antibodies were used to identify heat-shock proteins DnaJ, DnaK, GroES and GrpE. Only GrpE showed an increased level of expression in the acid-adapted and acid-tolerant mutants as compared with non-adapted cells. The N-terminal sequence was determined for two proteins, one induced in both the acid-adapted and mutant cells and the other showing the highest induction factor of those proteins specifically induced in the acid-adapted cells. This second protein has 60% identity with the N-terminal portion of YhaH, a transmembrane protein of Bacillus subtilis, which has 54 and 47% homology with stress proteins identified in Listeria monocytogenes and Bacillus halodurans. The constitutively acid-tolerant mutants showed other different phenotypic features compared to the parental strain: (i) the aminopeptidase activity of CB1-5R decreased and that of CB1-7R markedly increased, especially in acid conditions; (ii) the growth in culture medium at 10 degrees C and in the presence of 5% NaCl was greater (the same was found for acid-adapted cells); and (iii) the acidification rate during sourdough fermentation in acid conditions was faster and greater.

Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation

AbstractWe previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, β-actin, tubulin-α-1-chain and enolase-α. Interestingly, all carbonylated proteins except calreticulin and enolase-α showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.

Two-Dimensional Electrophoresis: The State of the Art and Future Directions

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is the only method currently available which is capable of simultaneously separating thousands of proteins. Thus 2-D PAGE is the heart of proteome technology. The first dimension of 2-D PAGE is isoelectric focusing (IEF), during which proteins are separated in a pH gradient until they reach a stationary position where their net charge is zero. The pH at which a protein has zero net charge is called its isoelectric point (pI). In the second dimension the proteins separated by IEF are separated orthogonally by electrophoresis in the presence of sodium dodecyl sulphate (SDSPAGE). The surfactant SDS binds to proteins, overriding their intrinsic charge, such that they all have the same charge density and free solution electrophoretic mobility. When the SDS-coated proteins migrate in a sieving polyacrylamide gel they separate based on their molecular mass. The high resolution of 2-D PAGE results from the first and second dimension separations being based on independent protein parameters.

Identification of caseins in goat milk.

The importance of goat milk in infant diet is growing, because it is reported that goats milk in some cases is less allergenic than cows milk. This is due probably to the lower presence of caseins associated with a specific type of αs1‐casein. In caprine breeds, four types of αs1‐casein alleles are identified and associated with various amounts of this protein in milk. The contribution of strong alleles to the goat milk is approximately 3.6 g/L of αs1‐casein, while for middle alleles is only 1.6 g/L, weak alleles 0.6 g/L. The contribution of null allele is very low (or non‐existent). The quantity of total caseins in caprine milk is positively correlated with the amount of αs1‐casein. Milk from animals possessing strong alleles contain significantly more total caseins than milk from animals without those alleles. This is important because animals with mild alleles can be employed to produce milk for allergic subjects while the other animals can be used to produce milk for the dairy industry. This work shows casein profiles of two types of classified goat milk (B, strong αs1 allele, 0, null αs1 allele) with two‐dimensional electrophoresis coupled with matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry, and it confirms the different polymorphisms at locus αs1 casein.

Protein profile of capacitated versus ejaculated human sperm.

Freshly ejaculated sperm acquire the fertilizing potential by a continuing process that occurs during sperm transport through the female genital tract, and it is physiologically not complete until the spermatozoon reaches the oocyte. The process termed capacitation can be mimicked in vitro by using appropriate capacitation media. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. This work deals with a proteomic approach to the analysis of protein profile variations in human normospermic samples as a consequence of three hours in vitro capacitation. 2DE gels were produced per freshly ejaculated sperm and per capacitated sperm and several quantitative and qualitative significant variations were found. Among the MS obtained identifications, proteins with a significant decrease after capacitation were found to be involved in protein fate, metabolism, and flagellar organization; on the contrary, increasing proteins were found to be related to cellular stress. Interestingly, the detected flagellar organization proteins decreased during capacitation whereas their corresponding fragments increased. A swim-up selected and three-hour capacitated sperm subpopulation has also been resolved by 2DE, and its synthetic gel has been analyzed for the variations observed in the entire sperm population. An immunofluorescence analysis of this sperm typology was carried out with antiactin and antitubulin antibodies.

Characterization of membrane nongenomic receptors for progesterone in human spermatozoa.

Rapid, nongenomic actions of steroid hormones have been characterized only recently. They may be mediated by interaction with a poorly characterized membrane receptor, by classic receptor located to the plasma membrane, or by interaction of the classic receptor with other signaling effectors. Among these, rapid effects of progesterone on human spermatozoa have been shown to be mediated by interaction with one or more membrane receptors. Two proteins, respectively of 57 and 28 kDa, representing the possible surface progesterone receptors in human spermatozoa, have been identified by our group employing an antibody (c-262) directed against the progesterone binding domain of the genomic receptor. The two proteins have been immunoprecipitated using c-262, isolated by 2D gel electrophoresis and analyzed by Maldi-Tof. Preliminary results of the analysis in data bank of the obtained masses suggest that the two proteins represent previously unidentified ones since they do not match with any protein in the database. We have also performed RT-PCR analysis with RNA extracted from human spermatozoa, utilizing various oligoprimers in different regions of the human progesterone genomic receptor. Results indicate the presence of transcripts for the complete genomic receptor. However, several previously published studies in the literature indicate the absence of expression of the genomic receptor in human spermatozoa. In this light posttranscriptional/posttraductional modifications of the receptor can be hypothesized. Interestingly, with primers amplifying in the DNA-binding domain of the progesterone receptor gene, we detected a higher molecular weight transcript when compared to the placenta. Further studies are needed to determine whether the sequences of the transcripts obtained by RT-PCR analysis of human sperm RNA match exactly with the human genomic receptor gene and to define the sequence of the higher molecular weight transcript detected in the DNA-binding region.

Solubilization methods and reference 2-DE map of cow milk fat globules

Milk fat globules (MFGs) are secretory vesicles assembled and secreted by mammary epithelial cells during lactation. They consist of fat globules surrounded by a lipid bilayer membrane which is derived from the apical membrane of the lactating cells. MFGs contain, besides lipids, proteins from the apical plasma membrane and from the cytoplasmatic material. Their peculiar vesicle nature makes them a suitable and easily available source of biological material in monitoring the physiopathological state of the mammary gland. Unfortunately, the conspicuous lipidic component of MFGs consistently limits protein extraction and purification for MFG proteomic investigations. This work deals with the development of a suitable procedure for protein extraction from the cow MFGs in order to qualitatively and quantitatively improve 2-D electropherograms of the MFG. MFGs were purified from raw milk by centrifugation and then delipidated/precipitated. The resulting protein pellets were solubilised using four different 2-D SDS PAGE compatible lysis buffers. Applied methodological procedures for protein extraction and evaluation of the resulting 2-D protein-pattern are presented and discussed. Using these procedures a reference 2-D map of cow milk fat globules is also reported. The majority of the obtained identifications was represented by proteins involved in lipid synthesis or in fat globule secretion.