Identification of proteins involved in starch and polygalacturonic acid degradation using LC/MS

Petkauskaite, Raimonda; Lukosius, Dangiras; Dębski, Janusz; Jasilionis, Andrius; Dadlez, Michał; Kieraite, Ieva; Timonina, Ana; Kuisienė, Nomeda

doi:10.2478/s11535-014-0303-4

Cited by 4 publications

(3 citation statements)

References 15 publications

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“…LC-MS analysis of gel slices was conducted as described in [ 36 ]. The raw data were pre-processed with Mascot Distiller software (v. 2.3, Matrix Science) and obtained peak lists were searched against the database of human protein sequences from SwissProt combined with its randomized version (40464 sequences) using Mascot search engine (version 2.4, 8-processors onsite license) (Matrix Science) with the following search parameters: enzyme specificity – semi-trypsin, missed cleavages – 1, variable modifications – oxidation (M), carbamidomethylation (CK), SK053(C), peptide mass tolerance – ± 20 ppm, fragment mass tolerance – ± 0.6 Da.…”

Section: Methodsmentioning

confidence: 99%

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

et al. 2015

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Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.

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Section: Methodsmentioning

confidence: 99%

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

et al. 2015

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“…Prior to peptide analysis, a single subunit gel band obtained was subjected to in-gel digestion procedures during which gel band was cut and reduced with 5 mM TCEP (tris (2-carboxyethyl) phosphine). Before trypsin (1:50, Trypsin/lysate ratio) digestion for 16 h at 37 °C, the gel band was further alkylated with 50 mM iodoacetamide (Petkauskaite et al, 2014). Digested materials were cleaned with a C18 silica cartridge to remove the salt.…”

Section: Sample Preparationmentioning

confidence: 99%

Biochemical Characterization of Cellulase From Bacillus subtilis Strain and its Effect on Digestibility and Structural Modifications of Lignocellulose Rich Biomass

Malik

Javed

2021

Front. Bioeng. Biotechnol.

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Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. The present study reports the partial purification and characterization of cellulase from Bacillus subtilis CD001 and its application in biomass saccharification. Out of four different substrates, carboxymethyl cellulose, when amended as fermentation substrate, induced the highest cellulase production from B. subtilis CD001. The optimum activity of CMCase, FPase, and amylase was 2.4 U/ml, 1.5 U/ml, and 1.45 U/ml, respectively. The enzyme was partially purified by (NH4)2SO4 precipitation and sequenced through LC-MS/MS. The cellulase was found to be approximately 55 kDa by SDS-PAGE and capable of hydrolyzing cellulose, as confirmed by zymogram analysis. The enzyme was assigned an accession number AOR98335.1 and displayed 46% sequence homology with 14 peptide-spectrum matches having 12 unique peptide sequences. Characterization of the enzyme revealed it to be an acidothermophilic cellulase, having an optimum activity at pH 5 and a temperature of 60°C. Kinetic analysis of partially purified enzyme showed the Km and Vmax values of 0.996 mM and 1.647 U/ml, respectively. The enzyme activity was accelerated by ZnSO4, MnSO4, and MgSO4, whereas inhibited significantly by EDTA and moderately by β-mercaptoethanol and urea. Further, characterization of the enzyme saccharified sugarcane bagasse, wheat straw, and filter paper by SEM, ATR-FTIR, and XRD revealed efficient hydrolysis and structural modifications of cellulosic materials, indicating the potential industrial application of the B. subtilis CD001 cellulase. The findings demonstrated the potential suitability of cellulase from B. subtilis CD001 for use in current mainstream biomass conversion into fuels and other industrial processes.

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“…The strain was isolated via enrichment culture with polygalacturonic acid and subsequently apple pomace as the main source of carbon and energy. The potential of a recombinant thermostable pectate lyase (locus_tag GEPA3_0510) from G. thermodenitrificans DSM 101594 has already been demonstrated for the enzymatic production of long-chain pectic oligosaccharides, which serves as a valuable prebiotics obtained from the agro-industrial wastes 2, 3…”

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confidence: 99%

Draft genome sequence of pectic polysaccharide-degrading moderate thermophilic bacterium Geobacillus thermodenitrificans DSM 101594

Petkauskaite

Blom

Goesmann

et al. 2017

Brazilian Journal of Microbiology

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Identification of proteins involved in starch and polygalacturonic acid degradation using LC/MS

Abstract: Abbreviations:ACN -acetonitrile; GH -glycoside hydrolase; LC/MS -liquid chromatography/mass spectrometry; MS -mass spectrometry; PGA -polygalacturonic acid; PL -polysaccharide lyase.

Cited by 4 publications

References 15 publications

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

Biochemical Characterization of Cellulase From Bacillus subtilis Strain and its Effect on Digestibility and Structural Modifications of Lignocellulose Rich Biomass

Draft genome sequence of pectic polysaccharide-degrading moderate thermophilic bacterium Geobacillus thermodenitrificans DSM 101594

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