Lunasin is a 43-amino acid peptide from seeds and grains with bioavailability in humans and potent chemotherapeutic action against several cancer cell lines. Here, we investigate new information ...about the physicochemical and structural properties of lunasin using circular dichroism (CD), fluorescence spectroscopy, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), size exclusion chromatography (SEC), molecular dynamics (MD), and bioinformatics. CD analysis and disorder prediction obtained by PONDR indicate that lunasin has a mostly unordered structure. Double wavelength θ222nm x θ200nm plot data suggests that lunasin is an intrinsically disordered peptide (IDP) in a pre-molten globule-like (PMG-like) state, while CD spectrum deconvolution and MD simulation indicate small β-strand content. The presence of residual structure was supported by loss of CD signal at 222 nm after treatment with urea and by increasing fluorescence emission upon bis-ANS binding. Lunasin also demonstrated stability to heating up to the temperature of 100 °C, as verified by CD. MD and CD analyses in the presence of TFE and MoRFpred prediction indicated the helix propensity of lunasin. ESI-IMS-MS data revealed that lunasin shows a propensity to form disulfide bonds at the conditions used. MD data also indicated that disulfide bond formation affects the adopted structure, showing a possible role of aspartyl-end in structure stabilization and compaction. In conclusion, our data support a characterization of lunasin as a peptide with an intrinsic disorder in a PMG-like state and reveal new aspects about its structural stability and plasticity, as well as the effects of disulfide bond formation and electrostatic attractions.
•Lunasin exhibits an intrinsic disorder profile in a pre-molten globule-like state.•Lunasin has structural plasticity and propensity to α-helix gain.•Lunasin is prone to disulfide bond formation in Cys10-Cys22.•Lunasin compaction can be related to disulfide bond and electrostatic attractions.•Structural plasticity can play an important role in multiple properties of lunasin.
Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and ...aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance.
Microcystis aeruginosa causes severe problems in freshwater environments worldwide due to its capacity to form blooms and produce toxins. Natural populations contain a mixture of strains that vary in ...morphology, genotype and microcystin production. Beyond its toxic effect in animals, the possible role of microcystin in the metabolism of the producing cell is also a topic of interest. Recent studies pointed to a protective role against cellular oxidative stress. Since natural populations include microcystin producing and non-producing strains, the latter must rely on other ecophysiological traits to thrive. Here, we compared a toxic and a non-toxic strain of M. aeruginosa displaying different morphotypes and growth behaviors, aiming to explore the role of microcystin in an integrated way to the primary metabolism. The toxic strain formed large floating colonies (150–1850 μm) with densely aggregated cells, numerous gas vesicles and lipid inclusions and a typical irregular thylakoid arrangement. The non-toxic strain remained submerged, formed small colonies (< 100 μm) with a dense mucilage covering few sparse cells with rare gas vesicles and various types of thylakoids. It showed a higher abundance of carboxysomes, cyanophycin and polyphosphate granules and also greater amounts of pigments than the toxic strain. The antioxidant potential was higher for the toxic strain and it presented a higher expression of proteins related to photosynthesis, protein folding and cellular redox homeostasis. These traits of the toxic strain were compatible with growth under higher irradiance and microcystin can be a key factor to this adaptation. Growing submerged, the non-toxic strain showed preference for a lower light intensity and invested in intracellular reserves. This study illustrates different adaptive strategies of M. aeruginosa morphotypes that can be valuable in nature, enabling this species to widely exploit freshwater ecosystems.
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•Toxic/non-toxic strains grow similarly but prefer different strata of the water column.•Toxic strain exploited the surface minimizing harmful effects of photooxidation.•Microcystin can be part of cellular adaptation to higher irradiance.•Non-toxic strain allocated resources to intracellular reserves.•Differences disclose intraspecific variability, crucial for the success of this species.
Lunasin is a soybean peptide with promising therapeutic applications in cancer and other diseases. It is described as an intrinsically disordered peptide, monomeric, and that can form an ...intramolecular disulfide bond between Cys
10
and Cys
22
. In this study, we tested a new approach to obtain recombinant lunasin by
Escherichia coli
expression and performed its structural characterization. We expressed a lunasin sequence with an N-terminal 6× His-tag, B1 domain of
Streptococcal
protein G (GB1), and
Tobacco etch virus
(TEV) cleavage site (His
6
-GB1-lunasin), using pET-25b(+) vector. His
6
-GB1-lunasin purification using immobilized metal affinity chromatography achieved high recovery. We obtained a final yield of 12.0 (± 0.39) mg/L of recombinant lunasin with high purity. The molecular mass and conformation were as expected, as verified by liquid chromatography–mass spectrometry (LC–MS), electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI–IMS–MS), electrospray ionization–mass spectrometry (ESI–MS), size-exclusion chromatography, circular dichroism, fluorescence spectroscopy, and one-dimensional
1
H nuclear magnetic resonance. Additionally, the identity of lunasin and its complete amino acid sequence was confirmed by LC–MS/MS. Recombinant lunasin also showed antioxidant activity (2.92 ± 0.16 µmol of trolox equivalents/µmol lunasin) by oxygen radical absorbance capacity and inhibited cell migration of MDA-MB-231 cell line. On the other hand, for the first time, LC–MS, ESI–IMS–MS and ESI–MS analyses revealed, in addition to the reduced and oxidized monomeric forms, the presence of small populations of disulfide cross-linked dimeric species of lunasin. Overall, our data demonstrate the effectiveness of the GB1-tagging system to obtain lunasin, that lunasin can form disulfide cross-linked dimers, and that LC–MS, ESI–IMS–MS and ESI–MS are efficient analytical chemistry techniques to assess reduced and oxidized (monomer and dimer) species.
Surface-associated proteins from
BCG Moreau RDJ are important components of the live Brazilian vaccine against tuberculosis. They are important targets during initial BCG vaccine stimulation and ...modulation of the host's immune response, especially in the bacterial-host interaction. These proteins might also be involved in cellular communication, chemical response to the environment, pathogenesis processes through mobility, colonization, and adherence to the host cell, therefore performing multiple functions. In this study, the proteomic profile of the surface-associated proteins from
BCG Moreau was compared to the BCG Pasteur reference strain. The methodology used was 2DE gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF), leading to the identification of 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits. Moonlighting proteins are multifunctional proteins in which two or more biological functions are fulfilled by a single polypeptide chain. Therefore, the identification of such proteins with moonlighting predicted functions can contribute to a better understanding of the molecular mechanisms unleashed by live BCG Moreau RDJ vaccine components.
Unveiling the Trypanosoma cruzi Nuclear Proteome dos Santos Júnior, Agenor de Castro Moreira; Kalume, Dário Eluan; Camargo, Ricardo ...
PloS one,
09/2015, Letnik:
10, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Replication of Trypanosoma cruzi, the etiological agent of Chagas disease, displays peculiar features, such as absence of chromosome condensation and closed mitosis. Although previous proteome and ...subproteome analyses of T. cruzi have been carried out, the nuclear subproteome of this protozoan has not been described. Here, we report, for the first time to the best of our knowledge, the isolation and proteome analysis of T. cruzi nuclear fraction. For that, T. cruzi epimastigote cells were lysed and subjected to cell fractionation using two steps of sucrose density gradient centrifugation. The purity of the nuclear fraction was confirmed by phase contrast and fluorescence microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 864 proteins. Among those, 272 proteins were annotated as putative uncharacterized, and 275 had not been previously reported on global T. cruzi proteome analysis. Additionally, to support our enrichment method, bioinformatics analysis in DAVID was carried out. It grouped the nuclear proteins in 65 gene clusters, wherein the clusters with the highest enrichment scores harbor members with chromatin organization and DNA binding functions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Objective The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass ...spectrometry–based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. Methods and materials Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry–based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MSE (1D LC-MSE ), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. Results All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MSE quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. Conclusions The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MSE and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.
The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by ...database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.
The taeniasis/cysticercosis complex is a zoonosis caused by the presence of the parasite
Taenia solium
in humans. It is considered a neglected disease that causes serious public health and economic ...problems in developing countries. In humans, the most common locations for the larval form are the skeletal muscles, ocular system, and the central nervous system, which is the most clinically important. Several glycoproteins of
T. solium
and
Taenia crassiceps
cysticerci have been characterized and studied for their use in the immunodiagnosis of neurocysticercosis and/or the development of synthetic or recombinant vaccines against cysticercosis. The aim of this study was to perform a gel-free shotgun proteomic analysis to identify saline vesicular extract (SVE) proteins of
T. solium
and
T. crassiceps
cysticerci. After solubilization of the SVE with and without surfactant reagent and in-solution digestion, the proteins were analyzed by LC–MS/MS. Use of a surfactant resulted in a significantly higher number of proteins that were able to be identified by LC–MS/MS. Novel proteins were identified in
T. solium
and
T. crassiceps
SVE. The qualitative analysis revealed a total of 79 proteins in the
Taenia
species: 29 in
T. solium
alone, 11 in
T. crassiceps
alone, and 39 in both. These results are an important contribution to support future investigations and for establishing a
Taenia
proteomic profile to study candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.
Pseudallescheria boydii is a filamentous fungus that causes a wide array of infections that can affect practically all the organs of the human body. The treatment of pseudallescheriosis is difficult ...since P. boydii exhibits intrinsic resistance to the majority of antifungal drugs used in the clinic and the virulence attributes expressed by this fungus are unknown. The study of the secretion of molecules is an important approach for understanding the pathogenicity of fungi. With this task in mind, we have shown that mycelial cells of P. boydii were able to actively secrete proteins into the extracellular environment; some of them were recognized by antibodies present in the serum of a patient with pseudallescheriosis. Additionally, molecules secreted by P. boydii induced in vitro irreversible damage in pulmonary epithelial cells. Subsequently, two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of secreted proteins from P. boydii mycelial cells. The two-dimensional map showed that most of the proteins (around 100 spots) were focused at pH ranging from 4 to 7 with molecular masses ranging from 14 to >117 kDa. Fifty spots were randomly selected, of which 30 (60%) were consistently identified, while 20 (40%) spots generated peptides that showed no resemblance to any known protein from other fungi and/or MS with low quality. Notably, we identified proteins involved in metabolic pathways (energy/carbohydrate, nucleotide, and fatty acid), cell wall remodeling, RNA processing, signaling, protein degradation/nutrition, translation machinery, drug elimination and/or detoxification, protection against environmental stress, cytoskeleton/movement proteins, and immunogenic molecules. Since the genome of this fungus is not sequenced, we performed enzymatic and immunodetection assays in order to corroborate the presence of some released proteins. The identification of proteins actively secreted by P. boydii provides important new information for understanding immune modulation and provides important new perspectives on the biology of this intriguing fungus.
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