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Proteomics of valve silica deposition vesicles from the diatom Thalassiosira pseudonana

ABSTRACT: Biomineral forming organisms produce inorganic materials with complex, genetically encoded morphologies that are inaccessible by current synthetic chemistry. It is poorly understood which genes are involved in biomineral morphogenesis and how the encoded proteins guide this process. We addressed these questions using diatoms, which are paradigms for the self-assembly of hierarchically meso- and macroporous silica under mild reaction conditions. By isolating the intracellular organelle for silica biosynthesis, we identified a suite of new biomineralization proteins. Three of these, dAnk1-3, are specific to diatoms and contain a common protein-protein interaction domain indicating a role in coordinating assembly of the silica biomineralization machinery. Knocking out individual dank genes led to characteristic structural aberrations in silica biogenesis that point to a liquid-liquid phase separation process as underlying mechanism for pore pattern morphogenesis. Our work provides an unprecedented path for the synthesis of tailored meso- and macroporous silicas using Synthetic Biology.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Thalassiosira Pseudonana Ccmp1335

TISSUE(S): Diploid Cell

SUBMITTER: Marc Gentzel

LAB HEAD: Prof. Dr. Nils Kröger

PROVIDER: PXD033943 | Pride | 2022-11-21

REPOSITORIES: pride

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			Action	DRS
	20200228_SVD01_0108_120min_01.raw	Raw
	20200302_SVD01_0108_120min_01.raw	Raw
	20200302_SVD02_0108_120min_01.raw	Raw
	20200302_SVD02_0108_120min_01_20200302225858.raw	Raw
	2020_SVD01_ALL_ProG_120min_01.mgf	Mgf

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Project description:Chitons are marine molluscs that posses a radula, a tongue like appendage containing many rows of microscopic teeth used for feeding. These teeth are hardened through the incorporation of biominerals. Since the original discovery of iron oxide and magnetite in these teeth, a wealth of chemical and structural knowledge has accumulated. The biomineralization process is known to be matrix mediated with precise control of the deposition of a number of different iron and calcium minerals at readily identifiable stages of tooth development. While much is known about the mechanisms of tooth mineralisation, there have been no studies undertaken to identify the genes that regulate this process. This investigation uses microarray technology to analyse the spatial expression of 493 expressed sequence tags (EST) derived from the radula sac of chiton, Acanthopleura hirtosa. A number of ESTs have been deemed significantly differentially expressed in relation to three designated areas of the radula sac. These spatial sections are defined by the variation in biomineral deposits of the radula, being immature radula which lacks any biomineral incorporation, iron deposition and calcium deposition. Additionally, foot muscle was used as a control to specify whether EST expression was specific to the radula and therefore likely involved biomineralization. A total of twelve A. hirtosa specimens were collected and sacrificed for microarray hybridizations. The radulae were removed from each animal immediately following sacrifice and dissected into three sections based on the biomineralisation development of the radula teeth. These sections are termed immature teeth, iron deposited teeth, and calcium deposited teeth. An additional sample of A. hirtosa foot muscle was taken from each specimen. RNA was extracted individually from these sections. Extracted RNA samples across subjects were pooled equally within sections in order to minimize the impact of biological variation and maximize the number of individual samples included in the study. To this effect, four single channel hybridizations were conducted for each radula and muscle section each representing a pooled biological replicate containing equal proportions of RNA from three A. hirtosa individuals. A total of sixteen microarray hybridizations were conducted in this manner.

Project description:Introduction: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with poor prognosis. Unlike many other cancers, MPM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several cancer types. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on MPM cells. Methods: Meso-CAFs were isolated from surgical specimens of MPM patients and analyzed by comparative genomic hybridization, transcriptomics and proteomics. Human MPM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, and response to pathway inhibitors and cisplatin was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis. Results: Meso-CAFs possess normal genomes without gene copy number aberrations typical for MPM cells. They express CAF markers and lack MPM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in the fraction of actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in increased proliferation and migration of MPM cells. A similar effect on cell growth was induced by conditioned medium. Met/PI3K or WNT signaling inhibition abolished the Meso-CAF-mediated growth stimulation. While Meso-CAFs reduced the efficacy of EGFR inhibition in co-cultures, they did not provide protection of tumor cells against cisplatin. Conclusion: Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on tumor cell growth and migration, two key aspects of MPM aggressiveness, indicating a substantial role of Meso-CAFs in driving MPM progression. Moreover, we show that Meso-CAFs significantly influence drug response and identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for improving therapeutic strategies in MPM.

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Proteomics of valve silica deposition vesicles from the diatom Thalassiosira pseudonana

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