Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis

Aburaya, Shunsuke; Esaka, Kohei; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

doi:10.1186/s13568-015-0115-6

Cited by 25 publications

(19 citation statements)

References 33 publications

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“…3). The proteome analysis covered approximately 50% of all gene products of C. cellulovorans ; this proteome coverage is so far the highest reported in C. cellulovorans research [16–18]. The identified proteins and their quantitative values corresponding to carbon sources are summarized in Additional file 1.…”

Section: Resultsmentioning

confidence: 99%

“…A previous study on C. cellulovorans used a defined time point for proteomic analyses of the secreted and cellular proteins to understand mechanisms underlying polysaccharide degradation and metabolism [16, 17]. Another proteome analysis, also performed at a defined time point, analyzed signal transition and metabolism-related proteins [18]. However, these analyses could not reveal temporal dynamics of secretory proteins.…”

Section: Introductionmentioning

confidence: 99%

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Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides

et al. 2019

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Background Clostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation. Results We cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k -means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k -means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k -means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8–19 cellulosomal proteins and 9–16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant. Conclusion In this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans . This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides. Electronic supple...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides

et al. 2019

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“…colleagues (21). Proteome analyses were performed using an LC-MS system (LC, UltiMate 3000 RSLCnano System, and MS, LTQ Velos Orbitrap mass spectrometer; Thermo Fisher Scientific) equipped with a long monolithic silica capillary column (490 cm in length, 0.075 mm ID; Kyoto Monotech).…”

Section: Phosphoproteome Analysismentioning

confidence: 99%

Alectinib Resistance in ALK-Rearranged Lung Cancer by Dual Salvage Signaling in a Clinically Paired Resistance Model

Tsuji

Ozasa

Aoki

et al. 2019

Molecular Cancer Research

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The mechanisms responsible for the development of resistance to alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, are still unclear, and few cell lines are currently available for investigating ALK-rearranged lung cancer. To identify the mechanisms underlying acquired resistance to alectinib, two patient-derived cell lines were established from an alectinib-naïve ALK-rearranged lung cancer and then after development of alectinib resistance. The properties acquired during treatments were detected by comparisons of the two cell lines, and then functional analyses were performed. Coactivation of c-Src and MET was identified after the development of alectinib resistance. Combinatorial therapy against Src and MET significantly restored alectinib sensitivity (17.2-fold). Increased apoptosis, reduction of tumor volume, and inhibition of MAPK and PI3K/AKT signaling molecules for proliferation and survival were observed when the three kinases (Src, MET, and ALK) were inhibited. A patient-derived xenograft from the alectinib-resistant cells indicated that combination therapy with a saracatinib and crizotinib significantly decreased tumor size To confirm the generality, a conventional alectinib-resistant cell line model (H2228-AR1S) was established from NCI-H2228 cells (EML4-ALK variant 3a/b). In H2228-AR1S, combination inhibition of Src and MET also restored alectinib sensitivity. These data reveal that dual salvage signaling from MET and Src is a potential therapeutic target in alectinib-resistant patients. This study demonstrates the feasibility to elucidate personalized drug-resistance mechanisms from individual patient samples.

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“…A meter-scale monolithic silica capillary column has a higher separation capacity than a particle-packed column. [11][12][13] A gradient was achieved by changing the ratio of the following two eluents: eluent A, 0.1% (v/v) formic acid (Wako); eluent B, 80% acetonitrile (Wako) containing 0.1% (v/v) formic acid. The separated peptides were directly electrosprayed into the mass spectrometer using a Fortis tip (AMR).…”

Section: Monolithic Capillary Lc-esi-ms/ms Analysismentioning

confidence: 99%

“…We selected standard parameter from previous proteome analysis using monolithic capillary LC-ESI-MS/MS. 11 Definitive screening design. The definitive screening design was adopted using JMP 11(SAS Institute Inc., Cary, NC.).…”

Section: Monolithic Capillary Lc-esi-ms/ms Analysismentioning

confidence: 99%

Definitive screening design enables optimization of LC–ESI–MS/MS parameters in proteomics

Aburaya

Aoki

Minakuchi

2017

Bioscience, Biotechnology, and Biochemistry

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In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC-ESI-MS/MS to comprehensively identify these peptides. However, there are many parameters for LC-ESI-MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC-ESI-MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC-ESI-MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC-ESI-MS/MS systems.

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Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis

Cited by 25 publications

References 33 publications

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides

Alectinib Resistance in ALK-Rearranged Lung Cancer by Dual Salvage Signaling in a Clinically Paired Resistance Model

Definitive screening design enables optimization of LC–ESI–MS/MS parameters in proteomics

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