Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases.

BACKGROUND: Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. MATERIALS AND METHODS: Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. RESULTS: Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. CONCLUSIONS: This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide.

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism.

Differential proteomics of the plasma of individuals with sepsis caused by Acinetobacter baumannii.

This study examines alterations in the plasma proteome in ten adults affected by sepsis caused by Acinetobacter baumannii as compared to paired healthy controls. 2-DE profiles of plasma from patients and paired healthy donors, depleted of the six most abundant proteins, were analysed by the DIGE technique. Protein spot detection and quantification were performed with the Differential In-gel Analysis and Biological Variation Analysis modules of the DeCyder() software. Differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) after colloidal Coomassie blue staining. Almost 900 spots were detected on a unique 2-D gel by the DIGE technique. A total of 269 protein spots of differential abundance were shown to be statistically significant (2.5-fold) with p values of p< or =0.01 (135 spots) and p< or =0.05 (134 spots) as determined by the t test. Seventy-one spots were submitted to mass spectrometry and about 30% could be successfully identified. This multiplex approach significantly reduced experimental variability, allowing for the confident detection of small differences in protein levels. Results include differentially expressed lipoproteins as well as proteins belonging to inflammatory/coagulation pathways and the kallikrein-kinin system. These data improves the knowledge for future developments in sepsis diagnosis, staging and therapy.

Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A2 and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and Mr of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.

Proteome analysis of embryogenic cell suspensions of cowpea (Vigna unguiculata).

Using a combination of two-dimensional gel electrophoresis protein mapping and mass spectrometry analysis, we have established proteome reference maps of embryogenic cell suspensions of cowpea (Vigna unguiculata). The cell suspensions were generated from young primary leaves and contained basically pro-embryogenic masses, which enabled us to dissect their proteome composition while eliminating the complexity of too many cell types. Over 550 proteins could reproducibly be resolved over a pI range of 3-10. A total of 128 of the most abundant protein spots were excised, digested in-gel with trypsin and analyzed by tandem mass spectrometry. This enabled the identification of 67 protein spots. Two of the most abundant proteins were identified as a chitinase and as a ribonuclease belonging to the family of PR-4 and PR-10 proteins, respectively. The expression of the respective genes was confirmed by RT-PCR and the pattern of deposition of the PR-10 protein in cell suspensions as well as in developing cowpea seeds, roots, shoots and flowers were determined by Western blot experiments, using synthetic antibodies raised against a 14-amino acid synthetic peptide located close to the C-terminal region of the PR-10 protein.

BJ46a, a snake venom metalloproteinase inhibitor. Isolation, characterization, cloning and insights into its mechanism of action.

Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4-reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum.

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.

Inhibition of the hyperalgesic activity of Bothrops jararaca venom by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

The antibothropic fraction (ABF) already isolated from Didelphis marsupialis serum, inhibits the haemorrhagic, oedematogenic, myonecrotic and lethal activities of Bothrops jararaca venom (Bjv). The aim of this work was to verify the capability of ABF to inhibit the hyperalgesic activity of Bjv. Intraplantar injection of Bjv induced hyperalgesia in a time- and dose-dependent manner and ABF administered in situ concomitantly with Bjv or i.v. 30 min before venom injection reduced the induced hyperalgesia. This same effect was observed when ABF was intravenously injected at 5 and 15 min after Bjv. Our results show that ABF inhibits also the hyperalgesia induced by Bjv.

New methodology for the obtainment of antibothropic factors from the South American opossum (Didelphis marsupialis) and jararaca snake (Bothrops jararaca).

The antibothropic factor (ABF) from D. marsupialis was collected from perforated hollow plastic golf balls which were surgically implanted subcutaneously in anesthetized opossums, a technique originally described for the production of polyclonal antibodies. Two months after the implantation of the balls, approximately 15 ml of seromatous fluid from D. marsupialis (SFDm-50 mg total protein/ml) could be recovered monthly. Opossum serum as well as SFDm showed similar SDS-PAGE profiles and antihemorrhagic potencies against Bothrops jararaca snake venom (Bjv). The presence of ABF in SFDm was confirmed by immunoblotting, using rabbit polyclonal antibodies raised against ABF isolated from opossum serum. ABF isolated from SFDm or from serum by ion-exchange chromatography showed identical chromatographic and electrophoretic profiles. ABF fromboth sources displayed very similar antihemorrhagic and anticaseinolytic activities against Bjv. In the case of B. jararaca, polyethylene perforated tubes were inserted in the abdominal cavity and two months after implantation, approximately 4 ml of seromatous fluid from B. jararaca (SFBj-23 mg total protein/ml) were recovered. B.jararaca serum and SFBj showed the same native and SDS-PAGE band pattern. Both serum and SFBj inhibited Bjv hemorrhagic activity. We conclude that this new methodology is very suitable for continuously obtaining opossum ABF and SFBj, in large scale and in an easier way, avoiding animal suffering and eventual sacrifice.

Detection of an antibothropic fraction in opossum (Didelphis marsupialis) milk that neutralizes Bothrops jararaca venom.

An antibothropic fraction (ABF) from Didelphis marsupialis (opossum) serum, which is responsible for the neutralization of Bothrops jararaca venom was isolated by Perales et al. [Perales, J., Moussatché, H., Marangoni, S., Oliveira, B. and Domont, G. B. (1994). Isolation and partial characterization of an antibothropic complex from the serum of South American Didelphidae. Toxicon 32, 1237-1249]. The aim of this work was to verify the presence of this factor in opossum's milk, which could represent an additional protection for the neonatal opossum against bothropic venoms. An active milk fraction was isolated and showed similar physicochemical, structural, antigenic and biological properties when compared to ABF, indicating that they are probably the same protein.

Inhibitory properties of the antibothropic complex from the South American opossum (Didelphis marsupialis) serum.

The South American opossum Didelphis marsupialis is known to be highly resistant to snake envenomation. In this paper it is shown that the opossum serum inhibits haemorrhage induced by both Crotalinae and Viperinae venoms. Tested against Bothrops jararaca (jararaca) venom, the antibothropic complex (ABC) isolated from the opossum serum was at least six times more antihaemorrhagic than the commercial antivenom. ABC showed no proteolytic activity by itself and was not hydrolysed by the venom. It inhibited the hydrolysis of casein by B. jararaca venom, but did not inhibit its hydrolytic activities upon N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA). The inhibitor did not interfere with trypsin and bacterial collagenase activities on BAPNA and N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA), respectively. It reduced chymotrypsin hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) because ABC is also a substrate for this enzyme. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B. jararaca venom preferentially degraded fibrinogen A alpha-chain and fibrin alpha-chain. Tested on extracellular matrix proteins, the venom hydrolysed collagen IV, gelatins I and V, laminin and fibronectin, besides depolimerizing collagen I alpha-chain dimers. Fibrillar collagen V was not digested. These hydrolyses were inhibited by ABC and by EDTA. Our results show that the antibothropic complex is a venom metalloproteinase inhibitor, which could, at least partially, account for its antihaemorrhagic activity. Electrophoretic evidence indicated non-covalent complex formation between the antihaemorrhagic factor and component(s) of B. jararaca venom.

Isolation of protein factors from opossum (Didelphis albiventris) serum which protect against Bothrops jararaca venom.

The fractionation of Didelphis albiventris serum by DEAE-Sephadex A50 yields a fraction (DA2) which protects the opossum against Bothrops venom. One polypeptide (DA2-II) responsible for this protection was isolated from fraction DA2 by ion exchange chromatography and biochemically characterized. DA2-II is a 43,000 mol. wt glycoprotein with the following N-terminal sequence: LKAMDTTPPLKIKKEPVK. Pairwise comparison of the amino acid sequence with four anti-hemorrhagic factors isolated from other opossum species indicated that DA2-II possesses high similarity (60-80%) with these proteins.

Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durissus terrificus serum.

An antivenom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action. CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23-25-kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues. The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex. The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.

Isolation and partial characterization of an anti-bothropic complex from the serum of South American Didelphidae.

An anti-bothropic fraction (ABF) with anti-Bothrops jararaca venom activity tested in mice was isolated from the serum of some South American Didelphidae (Didelphis marsupialis, Philander opossum and Lutreolina crassicaudata) by DEAE-Sephacel chromatography. ABF from D. marsupialis was shown to be 12 times more active in protection assays on a weight basis than the serum proteins. A similar fraction obtained from Metachirus nudicaudatum serum was shown to be inactive. An anti-bothropic complex (ABC) was isolated from D. marsupialis ABF. HPLC gel permeation chromatography of ABC from D. marsupialis indicated the presence of a main peak with mol. wt of 84,000. SDS-PAGE of this ABC showed the presence of two subunits of 48,000 and 43,000. The active ABF isolated from P. opossum and L. crassicaudata also showed the presence of these subunits by SDS-PAGE. Isolation of the 48,000 mol. wt D. marsupialis subunit by HPLC-hydrophobic interaction chromatography demonstrated that the 43,000 subunit was essential for the protective action of the complex. Both subunits from D. marsupialis, P. opossum and L. crassicaudata were Western-blotted and N-terminal sequenced. No N-terminal amino acid was found for the 43,000 subunit, whereas for the 48,000 subunit a high degree of homology was found: D. marsupialis: H2N-L K A M D P T P P L W I K T E X P . ; L. crassicaudata: H2N-L K A M D P T P P L W I Q T E . . . ; P. opossum: H2N-L K A M D T T P E . . . No significant homology with known proteins was detected.

Biochemical characterization of a vascular smooth muscle contracting polypeptide purified from Phoneutria nigriventer (armed spider) venom.

Biochemical characterization of a vascular smooth muscle contracting polypeptide purified from Phoneutria nigriventer (armed spider) venom. Toxicon 31, 377-384, 1993. Crude Phoneutria nigriventer venom was fractionated by Sephadex, ion-exchange and reverse-phase high performance liquid chromatography. One protein (PNV1) with spasmogenic activity in rabbit vascular smooth muscle was isolated and biochemically characterized. PNV1 has 125 amino acid residues and a calculated mol. wt of 13,899. Special features of the amino acid composition of PNV1 are the presence of two disulfide bridges and the high percentage (27%) of Asx and Glx. The N-terminal amino acid sequence indicates that PNV1 is different from other polypeptides isolated from Phoneutria nigriventer venom.

Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

The pharmacological modulation of mice paw oedema produced by Bothrops jararaca venom (BJV) has been studied. Intraplantar injection of BJV (1-30 micrograms/paw) produced a dose- and time-related oedema, which was maximal 30 min after injection, reduced gradually thereafter and disappeared over 48 h. BJV heated at 100 degrees C for 5 or 15 min blocked local hemorrhage and caused partial inhibition of its oedematogenic activity. The BJV oedema was not inhibited by the anti-histamine meclizine, the inhibitor of histamine and serotonin, cyproheptadine, PAF-acether antagonist WEB 2170 or by the anti-leukotrienes C4/D4, LY 171883. Dexamethasone, aspirin, indomethacin, and the dual cyclooxygenase and lipoxygenase inhibitor BW 755C inhibited BJV-induced oedema indicating that arachidonic acid metabolism products via the cyclooxygenase pathway participate in its genesis and/or maintenance. The antibothropic fraction (ABF) (25-200 micrograms/paw) isolated from Didelphis marsupialis serum neutralized the oedema induced by the venom with and without heating, the hemorrhage induced by BJV and partially blocked the oedema induced by bradykinin and by cellulose sulphate. The oedema produced by histamine, serotonin, PAF-acether or leukotriene C4 was not inhibited.

Natural anti-snake venom proteins.

The resistance of several animals to snake venom has been reviewed. Some general concepts are introduced to allow the comparative evaluation of the resistance of different animals studied by different investigators. The purification and properties of several factors isolated from the serum of different animals by some researchers are described: Trimeresurus flavoviridis (Omori-Satoh et al., 1972); Vipera palaestinae (Ovadia et al., 1975, 1977); Sigmodon hispidus (Pichyangkul and Perez, 1981); Didelphis virginiana and Didelphis marsupialis (Menchaga and Perez, 1981; Moussatché et al., 1979, 1980, 1981; Perales et al., 1986, 1989a,b); Neotoma micropus (Garcia and Perez, 1984); Erinaceus europaeus (de Witt and Weströmm, 1987); Herpestes edwardsii (Tomihara et al., 1987); Dinodon semicarinatus (Tomihara et al., 1988); and Philander opossum (Domont et al., 1989). The protective antihemorrhagic and antineurotoxic factors have some common characteristics: they are acid proteins with isoelectric points ranging between 4.0 and 5.4; their molecular masses vary from 52 to 90 kDa, with one exception of 780 kDa; none has proteolytic activity; their pH and thermostabilities are high and they seem to be glycoproteins. No precipitation lines are formed between the neutralizing proteins and the venoms upon immunodiffusion, indicating that the serum protective factors are not immunoglobulins. The possible mode of action of the antineurotoxic factor isolated from Vipera palaestinae by Ovadia et al. (1977) is shortly discussed as well as the possibility that the antihemorrhagic factors may act by a similar mechanism.

Structural studies on allergen RC-13 from Ricinus communis L.: isolation and characterization of a major glycopeptide.

A glycoprotein, RC-13, isolated from Ricinus communis seeds was reduced, S-alkylated and cleaved by trypsin. The tryptic digest was fractionated by ion-exchange chromatography and a glycopeptide was isolated and purified by high-voltage paper electrophoresis. When submitted to amino acid and carbohydrate analyses this major glycopeptide showed the following chemical composition: Lys1, Asp1, Thr2, Ser4, Glu1, Pro2, Gly2, Ala2, Val2, GlcN6, Man6 and Gal8. Hydrazynolysis positioned Ser as the C-terminal residue. It is postulated that this glycopeptide belongs to the C-terminal region of the allergen.

Structural studies on allergen RC-13 from Ricinus communis L.: isolation and characterization of a major glycopeptide

A glycoprotein, RC-13, isolated from Ricinus communis seeds was reduced, S-alkylated and cleaved by trypsin. The tryptic digest was fractionated by ion-exchange chromatography and a glycopeptide was isolated and purified by high-voltage paper electrophoresis. When submitted to amino acid and carbohydrate analyses this major glycopeptide showed the following chemical composition: Lys1, Asp1, Thr2, Ser4, Glu1, Pro2, Gly2, Ala2, Val2, GlcN6, Man6 and Gal8. Hydrazynolysis positioned Ser as the C-terminal residue. It is postulated that this glycopeptide belongs to the C-terminal region of the allergen

Partial characterization of RC-13, a homogeneous fraction isolated from castor bean allergens (CB-1A).

Allergen RC-13's amino acid composition was determined-Lys3, His1, Arg7, Asp6, Thr2, Ser11, Glu29, Pro2, Gly7, Ala4, Cys6, Val5, Met1, Ile3, Leu3, Tyr1; 91 residues. Partial specific volume and minimum molecular weight evaluated from this composition were, respectively, 0.694 cm3/g and 10,194. SDS-polyacrylamide gel electrophoresis performed in the reduced and alkylated form of the allergen revealed a single band, suggesting the existence of only one polypeptide chain in the molecule. Results obtained for carbohydrate analysis by gas-liquid chromatography were: 22.2% galactose and 16.2% mannose--approximately 38% of neutral sugars. Employment of the amino acid methodology for analysis of amino sugars revealed two residues of glucosamine per molecule of the allergen. Hydrolyses with trypsin, alpha-chymotrypsin and type VII protease were tested for sequence studies. Trypsin was considered a suitable enzyme for such purpose.