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Exploring roles of the chitinase ChiC in modulating Pseudomonas aeruginosa virulence phenotypes

ABSTRACT: Proteins were precipitated using chloroform and methanol, by the protocol described by Wessel and Flügge (1984). Following precipitation, the proteins were reduced and alkylated before being digested, cleaned and desalted using STrap tips (Zougman A, Selby PJ, Banks RE. 2014). The peptide samples were analyzed by a nano UPLC (nanoElute, Bruker) coupled to a trapped ion mobility spectrometry and a quadrupole time of flight mass spectrometer (timsTOF Pro, Bruker).

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Pseudomonas Aeruginosa (strain Ucbpp-pa14) Bacteria

SUBMITTER: Per Kristian Edvardsen

LAB HEAD: Gustav Vaaje-Kolstad

PROVIDER: PXD048597 | Pride | 2024-04-30

REPOSITORIES: Pride

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Exploring roles of the chitinase ChiC in modulating Pseudomonas aeruginosa virulence phenotypes

Project description:Chitinases are ubiquitous enzymes involved in biomass degradation and chitin turnover in nature. Pseudomonas aeruginosa (PA), an opportunistic human pathogen, expresses ChiC, a secreted glycoside hydrolase 18 (GH18) family chitinase. Despite speculation about ChiC's role in PA disease pathogenesis, there is scant evidence supporting this hypothesis. Since PA cannot catabolize chitin, we investigated the potential function(s) of ChiC in PA pathophysiology

2024-04-30 | PXD048320 | Pride

Proteomic insight into arabinogalactan utilization by particle-associated Maribacter sp. MAR_2009_72

Project description:Arabinose and galactose are major components of both particulate and dissolved organic matter. They often constitute polysaccharides such as arabinogalactan. Field studies have demonstrated a turnover of arabinogalactan, but enzymes and organisms involved have not been explored. In this study, the degradation of arabinogalactan by the marine flavobacterium Maribacter sp. MAR_2009_72 was investigated with growth experiments and proteomic analyses. Growth of Maribacter sp. MAR_2009_72 on arabinogalactan displayed its ability to utilize arabinogalactan. Proteomic analysis of cells grown on arabinogalactan, arabinose, galactose or glucose revealed expression of specific proteins in presence of arabinogalactan, mainly glycoside hydrolases. Candidates for extracellular glycan hydrolysis are five alpha-L-arabinofuranosidases affiliating with glycosyl hydrolase (GH) families 43 and 51, four unsaturated rhamnogalacturonyl hydrolases (GH105) and a protein with a glycosyl hydrolase family like domain. We detected expression of three induced TonB-dependent SusCD transporter systems, one SusC and nine glycosyl hydrolases with a predicted periplasmatic location. These are affiliated with the families GH3, GH10, GH29, GH31, GH67, GH78 and GH115. The genes are located within three canonical polysaccharide utilization loci (PUL), but also outside of the defined loci. PULs can be classified as specific for arabinogalactan, galactose-containing glycans and for arabinose-containing glycans. The breadth of enzymatic functions expressed in Maribacter sp. MAR_2009_72 as response to arabinogalactan from terrestrial plant larch suggests that Flavobacteriia are main catalysts of the rapid turnover of arabinogalactans in the marine environment.

2024-05-02 | PXD049074 | Pride

Exploring roles of the chitinase ChiC in modulating Pseudomonas aeruginosa virulence phenotypes

2024-04-30 | PXD048596 | Pride

Serine-Threonine Kinase Receptor-Associated Protein (STRAP) Knockout Decreases the Malignant Phenotype in Neuroblastoma

Project description:Purpose: Serine-threonine kinase receptor-associated protein (STRAP) plays an important role in neural development but also in tumor growth. Neuroblastoma, a tumor of neural crest origin, is the most common extracranial malignancy of childhood, and it continues to carry a poor prognosis. The recent discovery of the role of STRAP in another pediatric solid tumor, osteosarcoma, and its known function in neural development, led us to investigate the role of STRAP in neuroblastoma tumorigenesis. Methods: STRAP protein expression was abrogated in two human neuroblastoma cell lines, SK-N-AS (AS) and SK-N-BE(2). Methods included transient knockdown with siRNA, stable knockdown with shRNA lentiviral transfection, and CRISPR-Cas9 genetic knockout of STRAP. STRAP knockdown and knockout cells were examined for phenotypic alterations in vitro and tumor growth in vivo. Results: Cell proliferation, viability, motility, and growth were significantly decreased in STRAP knockout compared to wild-type cells. Indicators of stemness, including mRNA abundance of common stem cell markers Oct4, Nanog, and Nestin, the percentage of cells expressing CD133 on their surface, and the ability to form tumorspheres, were significantly decreased in the STRAP KO cells. In vivo, AS STRAP knockout cells formed tumors less readily than wild-type tumor cells. Conclusion: These novel findings demonstrated that STRAP plays a role in tumorigenesis and maintenance of neuroblastoma stemness.

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Transcriptome-wide maps of STRAP-RNA interactions in mouse early embryo and lineage-committed cells.

Project description:We report the application of enhanced cross-linking immunoprecipitation (eCLIP) sequencing technology for high-throughput profiling of STRAP-RNA interactions in mouse embryos (E9.5) or embryoid body (EB) cells. Based on the eCLIP-seq data, we computationally identified a transcriptome-wide set of high-confidence peaks (over ten thousands) and most of STRAP-peaks distributed in the murine genome by defining certain exons and introns as well as their intersection. STRAP eCLIP signals in embryos were dramatically enriched at exon/intron and intron/exon borders. Numerous STRAP peaks are significantly enriched on the genes with critical roles in early organ development. Finally, we show two examples of STRAP-RNA binding that link to its regulated genes at the alternative splicing level. Together, we uncover the position- and context- dependent effects of STRAP on splicing regulation during mouse embryonic stem cells lineage commitment. Our studies provide a very useful platform to decipher STRAP’s role in the many steps in RNA biogenesis for future studies.

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STRAP is involved in mutated Apc-induced stemness and tumorigenesis in colon cancer

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Oncogenic Serine-Threonine Kinase Receptor Associated Protein Supports Hepatocellular Carcinoma Cell Growth by Enhancing Wnt/β-catenin Signaling

Project description:Serine-threonine kinase receptor-associated protein (STRAP) is upregulated in breast, colorectal and lung cancers, promoting their growth. We identify the upregulation of STRAP in hepatocellular carcinomas. Elevated STRAP endows tumor cells with growth advantage by reprograming a variety of metabolic processes and signaling pathways critical for hepatocellular carcinoma progression. Especially, enhanced Wnt/β-catenin signaling is likely to be a major effector of its tumor-promoting role.

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Next generation sequencing facilitates quantitative analysis of STRAP-dependent alternative splicing events

Project description:We report the application of next generation sequencing for evaluating alternative splicing (AS) events on lineage-committed WT and Strap-KO embryoid body (EB) cells. We also analyzed AS during mouse embryo development using RNA-seq data. Of all STRAP-regulated AS, exon-skipping events were the most frequent ones with significant changes in 49% (240 out of 489) cases. Retained introns were also overrepresented (20%, 97 out of 489) as compared to other types of AS STRAP appears to function in promoting both exon inclusion and skipping at different exons. GO function revealed STRAP-regulated AS events involve in protein catabolism, molecular binding and cell motility. More specifically, they included neuronal migration and development. Together, these analyses uncovered the previous unknown role of STRAP involved in the substantial transcriptional diversity at the exon level in certain development stage. Our study also represents the first detailed analysis of AS events in the mouse early organogenesis with biologic replicates, generated by RNA-seq technology.

2020-09-16 | GSE131471 | GEO

Single-shot DDA analysis of HeLa using a 1-m particle-packed capillary column

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