Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6–7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or β-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous β-glycosidase (EGIDFPOO_00532) with β-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.

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Cancer Immunomics: From Serological Proteome Analysis to Multiple Affinity Protein Profiling

Annals of the New York Academy of Sciences ◽

10.1196/annals.1381.024 ◽

2007 ◽

Vol 1107 (1) ◽

pp. 223-230 ◽

Cited By ~ 30

Author(s):

J. HARDOUIN ◽

J.-P. LASSERRE ◽

L. SYLVIUS ◽

R. JOUBERT-CARON ◽

M. CARON

Keyword(s):

Proteome Analysis ◽

Protein Profiling

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Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling

Analytical Chemistry ◽

10.1021/acs.analchem.8b03692 ◽

2018 ◽

Vol 90 (23) ◽

pp. 14003-14010 ◽

Cited By ~ 20

Author(s):

Xi Shao ◽

Xuantang Wang ◽

Sheng Guan ◽

Haizhu Lin ◽

Guoquan Yan ◽

...

Keyword(s):

Single Cell ◽

Proteome Analysis ◽

Single Cell Protein ◽

Protein Profiling ◽

Cell Protein

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Cell Shape and Cardiosphere Differentiation: A Revelation by Proteomic Profiling

Biochemistry Research International ◽

10.1155/2013/730874 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Nanako Kawaguchi ◽

Mitsuyo Machida ◽

Kota Hatta ◽

Toshio Nakanishi ◽

Yohtaroh Takagaki

Keyword(s):

Stem Cells ◽

Stress Proteins ◽

Embryonic Stem ◽

Proteome Analysis ◽

Protein Profiling ◽

Proteomic Profiling ◽

Low Oxygen ◽

Cellular Functions ◽

Proliferation And Differentiation ◽

Undifferentiated Cells

Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells) and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E) derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions.

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Kordiimonas gwangyangensis gen. nov., sp. nov., a marine bacterium isolated from marine sediments that forms a distinct phyletic lineage (Kordiimonadales ord. nov.) in the ‘Alphaproteobacteria’

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.63684-0 ◽

2005 ◽

Vol 55 (5) ◽

pp. 2033-2037 ◽

Cited By ~ 70

Author(s):

Kae Kyoung Kwon ◽

Hee-Soon Lee ◽

Sung Hyun Yang ◽

Sang-Jin Kim

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Marine Bacterium ◽

Enrichment Culture ◽

Sequence Similarity ◽

Bacterial Species ◽

Carbon Sources ◽

Optimal Growth ◽

Rrna Gene ◽

Respiratory Quinone

A marine bacterium, designated strain GW14-5T, capable of degrading high-molecular-mass polycyclic aromatic hydrocarbons was isolated from the sediments of Gwangyang Bay, Republic of Korea, after enrichment culture for 2 years with a mixture of benzo[a]pyrene and pyrene. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate forms a phyletic lineage that is distinct from the seven known orders within the ‘Alphaproteobacteria’. 16S rRNA gene sequence similarity of strain GW14-5T to all recognized bacterial species was not greater than 92 %. The dominant fatty acids of the isolate were i-17 : 1 (46·2 %), i-15 : 0 (15·1 %) and i-17 : 0 (12·6 %). The major respiratory quinone was MK-5, and the DNA G+C content was 39·3 mol%. Cells of strain GW14-5T were Gram-negative, motile, catalase-positive, oxidase-positive and weakly halophilic. Glucose, N-acetylglucosamine and maltose were utilized as sole carbon sources. The strain was positive for β-glucosidase activity. Optimal growth of strain GW14-5T was at pH 7·0 and 37–40 °C and required the presence of 2 % (w/v) NaCl. On the basis of this evidence, strain GW14-5T represents a novel genus and species in the ‘Alphaproteobacteria’ for which the name Kordiimonas gwangyangensis gen. nov., sp. nov. is proposed. The novel order Kordiimonadales is proposed for the distinct phyletic line represented by the genus Kordiimonas. The type strain is GW14-5T (=KCCM 42021T=JCM 12864T).

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Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47

Journal of Bacteriology ◽

10.1128/jb.00874-09 ◽

2009 ◽

Vol 192 (1) ◽

pp. 295-306 ◽

Cited By ~ 68

Author(s):

Draženka Selesi ◽

Nico Jehmlich ◽

Martin von Bergen ◽

Frank Schmidt ◽

Thomas Rattei ◽

...

Keyword(s):

Enrichment Culture ◽

Proteome Analysis ◽

Gene Clusters ◽

Comparative Sequence Analysis ◽

Open Reading Frames ◽

Ring Cleavage ◽

Sequence Motifs ◽

Oxidation Reactions ◽

Acetyl Coa ◽

Coa Reductase

ABSTRACT The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO2. The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of α-, β-, and γ-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe4S4] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers “Aromatoleum aromaticum” EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO2. Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation.

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In-depth human plasma proteome analysis captures tissue proteins and transfer of protein variants across the placenta

eLife ◽

10.7554/elife.41608 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Maria Pernemalm ◽

AnnSofi Sandberg ◽

Yafeng Zhu ◽

Jorrit Boekel ◽

Davide Tamburro ◽

...

Keyword(s):

Human Plasma ◽

Genomic Sequence ◽

Proteome Analysis ◽

Protein Profiling ◽

Single Amino Acid ◽

Sequence Information ◽

Plasma Proteome ◽

Multiple Protein ◽

Protein Variants ◽

Human Plasma Proteome

Here, we present a method for in-depth human plasma proteome analysis based on high-resolution isoelectric focusing HiRIEF LC-MS/MS, demonstrating high proteome coverage, reproducibility and the potential for liquid biopsy protein profiling. By integrating genomic sequence information to the MS-based plasma proteome analysis, we enable detection of single amino acid variants and for the first time demonstrate transfer of multiple protein variants between mother and fetus across the placenta. We further show that our method has the ability to detect both low abundance tissue-annotated proteins and phosphorylated proteins in plasma, as well as quantitate differences in plasma proteomes between the mother and the newborn as well as changes related to pregnancy.

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Comparative serum proteome analysis reveals potential early pregnancy-specific protein biomarkers in pigs

Reproduction Fertility and Development ◽

10.1071/rd18227 ◽

2019 ◽

Vol 31 (3) ◽

pp. 613 ◽

Cited By ~ 1

Author(s):

Ankan De ◽

Mohammad Ayub Ali ◽

Tukheswar Chutia ◽

Suneel Kumar Onteru ◽

Parthasarathi Behera ◽

...

Keyword(s):

Early Pregnancy ◽

Inflammatory Responses ◽

Proteome Analysis ◽

Specific Protein ◽

Protein Profiling ◽

Differentially Expressed ◽

Label Free ◽

Protein Biomarkers ◽

Serum Proteome ◽

Pregnancy Zone Protein

In this study, the comparative serum proteome profile of Day 5, 12 and 16 of gestation, representing three early embryonic events, namely formation, elongation and implantation of blastocysts, and non-pregnant control were explored by a label-free quantitation-based mass spectrometric approach to identify early pregnancy biomarkers in pigs. A total of 131 proteins were identified with respect to different groups, out of which 105 were found to be differentially expressed proteins (DEPs). Among the DEPs, 54 and 66 proteins were found to be up and downregulated respectively in early pregnancy groups (fold change >2) and the maximum number of upregulated proteins was observed in the Day 12 pregnancy stage. Functional classification and pathway analysis of the DEPs revealed involvement of most of the proteins in complement and coagulation cascades, metabolic processes and immune and inflammatory responses. Proteins such as glutathione peroxidise (GPX), pregnancy zone protein (PZP), thrombospondin-1 (THBS1), α-1-antitrypsin (AAT) and mannose-binding lectin C (MBLC) were differentially expressed during early pregnancy and actively involved in different pregnancy-related activities. To the best of our knowledge, this is the first report on comparative serum protein profiling of different early pregnancy stages in pigs and our results provide a set of proteins that can be used as potential biomarkers for early pregnancy diagnosis in pigs.

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Accelerated discovery of novel glycoside hydrolases using targeted functional profiling and selective pressure on the rumen microbiome

Microbiome ◽

10.1186/s40168-021-01147-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

André L. A. Neves ◽

Jiangkun Yu ◽

Yutaka Suzuki ◽

Marisol Baez-Magana ◽

Elena Arutyunova ◽

...

Keyword(s):

Selective Pressure ◽

De Novo ◽

Structural Diversity ◽

Glycoside Hydrolases ◽

Reference Database ◽

Xylanolytic Activity ◽

Rumen Microbiome ◽

Functional Profiling ◽

Microbial Environment ◽

Living Organisms

Abstract Background Carbohydrate-active enzymes (CAZymes) form the most widespread and structurally diverse set of enzymes involved in the breakdown, biosynthesis, or modification of lignocellulose that can be found in living organisms. However, the structural diversity of CAZymes has rendered the targeted discovery of novel enzymes extremely challenging, as these proteins catalyze many different chemical reactions and are sourced by a vast array of microbes. Consequently, many uncharacterized members of CAZyme families of interest have been overlooked by current methodologies (e.g., metagenomic screening) used to discover lignocellulolytic enzymes. Results In the present study, we combined phenotype-based selective pressure on the rumen microbiota with targeted functional profiling to guide the discovery of unknown CAZymes. In this study, we found 61 families of glycoside hydrolases (GH) (out of 182 CAZymes) from protein sequences deposited in the CAZy database—currently associated with more than 20,324 microbial genomes. Phenotype-based selective pressure on the rumen microbiome showed that lignocellulolytic bacteria (e.g., Fibrobacter succinogenes, Butyrivibrio proteoclasticus) and three GH families (e.g., GH11, GH13, GH45) exhibited an increased relative abundance in the rumen of feed efficient cattle when compared to their inefficient counterparts. These results paved the way for the application of targeted functional profiling to screen members of the GH11 and GH45 families against a de novo protein reference database comprised of 1184 uncharacterized enzymes, which led to the identification of 18 putative xylanases (GH11) and three putative endoglucanases (GH45). The biochemical proof of the xylanolytic activity of the newly discovered enzyme validated the computational simulations and demonstrated the stability of the most abundant xylanase. Conclusions These findings contribute to the discovery of novel enzymes for the breakdown, biosynthesis, or modification of lignocellulose and demonstrate that the rumen microbiome is a source of promising enzyme candidates for the biotechnology industry. The combined approaches conceptualized in this study can be adapted to any microbial environment, provided that the targeted microbiome is easy to manipulate and facilitates enrichment for the microbes of interest.

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Differential protein profiling of soil diazotroph Rhodococcus qingshengii S10107 towards low-temperature and nitrogen deficiency

Scientific Reports ◽

10.1038/s41598-019-56592-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Deep Chandra Suyal ◽

Divya Joshi ◽

Saurabh Kumar ◽

Ravindra Soni ◽

Reeta Goel

Keyword(s):

Low Temperature ◽

Nitrogen Deficiency ◽

Proteome Analysis ◽

Pentose Phosphate ◽

Protein Profiling ◽

Adaptive Responses ◽

Response Regulators ◽

Differential Proteomics ◽

First Time ◽

Atp Binding Protein

AbstractProtein-based biomarkers can be a promising approach for identification and real-time monitoring of the bio-inoculants employed under sustainable agricultural plans. In this perspective, differential proteomics of psychrophilic diazotroph Rhodococcus qingshengii S10107 (JX173283) was performed to unravel its adaptive responses towards low-temperature nitrogen deficiency and identification of a biomarker for respective physiological conditions. LC-MS/MS-based proteome analysis mapped more than 4830 proteins including 77 up-regulated and 47 down-regulated proteins (p ≤ 0.05). Differential expression of the structural genes of nif regulon viz. nifH, nifD, and nifK along with their response regulators i.e. nifA, nifL, and nifB indicated that the nitrogenase complex was activated successfully. Besides up-regulating the biosynthesis of certain amino acids viz. Leucine, Lysine, and Alanine; the expression of the peptidoglycan synthesis proteins were also increased; while, the enzymes involved in Lipid biosynthesis were found to decrease. Furthermore, two important enzymes of the pentose phosphate pathway viz. Transketolase and Transaldolase along with Ribose import ATP-binding protein RbsA were also found to induce significantly under low temperature a nitrogen deficient condition, which suggests the cellular need for ample ribose sugar instantly. Additionally, comparative protein profiling of S10107 strain with our previous studies revealed that CowN protein was significantly up-regulated in all the cases under low-temperature nitrogen deficient conditions and therefore, can be developed as a biomarker. Conclusively, present study for the first time provides an in-depth proteome profiling of R. qingshengii S10107 and proclaims CowN as a potential protein biomarker for monitoring BNF under cold niches.

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Yeosuana aromativorans gen. nov., sp. nov., a mesophilic marine bacterium belonging to the family Flavobacteriaceae, isolated from estuarine sediment of the South Sea, Korea

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.64073-0 ◽

2006 ◽

Vol 56 (4) ◽

pp. 727-732 ◽

Cited By ~ 43

Author(s):

Kae Kyoung Kwon ◽

Hee-Soon Lee ◽

Hong-Bae Jung ◽

Ji-Hyun Kang ◽

Sang-Jin Kim

Keyword(s):

Marine Bacterium ◽

Enrichment Culture ◽

Sequence Similarity ◽

Optimal Growth ◽

Estuarine Sediments ◽

Rrna Gene ◽

Strictly Aerobic ◽

The South ◽

The Family ◽

South Sea

A marine bacterium, GW1-1T, capable of degrading benzo[a]pyrene (BaP), was isolated from estuarine sediments of the South Sea (the Korea Strait), Korea, after an enrichment culture maintained for 2 years in a medium supplemented with a mixture of BaP and pyrene. The strain formed yellowish-brown colonies on marine agar 2216. Cells were strictly aerobic, non-motile, Gram-negative rods and produced non-diffusible carotenoid pigments. Optimal growth occurred in the presence of 1 % (w/v) NaCl and at pH 7 and 33–36 °C. No growth occurred without supplementation with either CaCl2 or MgCl2, even in the presence of NaCl. Phylogenetic analysis based on the nearly complete sequence of the 16S rRNA gene revealed that the isolate formed a phyletic lineage with the genera Gelidibacter (93·9–94·7 % gene sequence similarity), Subsaximicrobium (93·3 %) and Subsaxibacter (93·9 %). The isolate also showed high sequence similarities to Gaetbulibacter saemankumensis (94·5 %), Algibacter lectus (94·2 %), members of the genus Bizionia (93·6–94·3 %) and Formosa algae (93·2 %), even though it belonged to a different phyletic line. The major respiratory quinones of the isolate were menaquinones MK-5 and MK-6. The DNA G+C content was 51·4 mol%. Dominant fatty acids were i-15 : 0, a-15 : 0, i-15 : 1ω10c and 16 : 1. On the basis of this polyphasic taxonomic evidence, strain GW1-1T is classified as a member of a novel genus and species in the family Flavobacteriaceae, for which the name Yeosuana aromativorans gen. nov., sp. nov. is proposed. The type strain of the type species is GW1-1T (=KCCM 42019T=JCM 12862T).

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