Guarino, Andrea Maria (2018) CHARACTERIZATION OF YB-1 AS A NOVEL STRESS BIOMARKER AND PARACRINE SIGNALING POLYPEPTIDE. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Resource language: English
Title: CHARACTERIZATION OF YB-1 AS A NOVEL STRESS BIOMARKER AND PARACRINE SIGNALING POLYPEPTIDE
Creators:
CreatorsEmail
Guarino, Andrea Mariaandreamaria.guarino@unina.it
Date: 13 December 2018
Number of Pages: 214
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Biotecnologie
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannisannia@unina.it
Tutor:
nomeemail
Calabrò, ViolaUNSPECIFIED
Date: 13 December 2018
Number of Pages: 214
Keywords: YB-1; Secretion; Stress Granules; Zebrafish; HEK293T.
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 05 - Scienze biologiche > BIO/13 - Biologia applicata
Area 05 - Scienze biologiche > BIO/18 - Genetica
Date Deposited: 16 Jan 2019 10:07
Last Modified: 16 Jun 2020 09:27
URI: http://www.fedoa.unina.it/id/eprint/12699

Collection description

Cell signalling is the complex network of connections between cells and inside the single cell. Protein trafficking is deeply involved in cell signalling. Different proteins are ordered in clusters of receptors of extracellular signals, transducers, sensors and biological response effectors. Together, they are arranged in molecular pathways that are relevant for maintenance of cell homeostasis. Protein secretion is a relevant component of eukaryotic cell signalling. The prototype cold-shock Y-box binding protein 1 (YB-1) is a multifunctional protein which regulates a variety of fundamental biological processes. Recently YB-1 has been found in human extracellular fluids and shown to be secreted by different cell types. This PhD project is focused on YB-1 protein secretion to investigate on its role as a potential paracrine signal to elicit changes or responses in nearby cells, altering their behaviour. During the first part of the project I focused on stimuli and conditions triggering YB-1 secretion. Low amounts of extracellular YB-1 are released by HEK293T cells in physiological condition. Interestingly, I found that YB-1 secretion was enhanced following oxidative insults. Secreted YB-1 protein was purified and analysed by mass spectrometry to confirm its identity. In parallel, I verified the assembly of YB-1 in stress granules (SGs), cytoplasmic foci where untranslated mRNAs are sorted or processed for reinitiation, degradation, or packaging into mRNPs. To understand the mechanism beneath YB-1 secretion and uptake by receiving cells, I tried several experimental approaches; among them the production and characterization of a stable HEK293T cell line expressing a YB-1-GFP fusion protein. The properties of this cell line as a stress biosensor were evaluated. The second part of the project was devoted to the production of purified recombinant human YB-1 (rYB-1) in bacterial host. In parallel I produced enriched fractions of YB-1 from HEK293T cell culture medium (CCM-YB-1). I found that both CCM-YB-1 and rYB-1 have anti-proliferative activity on receiving cells. Both forms of YB-1 protein were effective on different recipient cell lines, including HaCaT cells. In particular, inhibition of human keratinocytes proliferation by extracellular YB-1 was associated to a G2/M cell cycle arrest, induction of p21WAF and reduction of Np63 protein level. The obtained results suggest that sustained release of full length YB-1 protein or YB-1 derived peptides, by stress stimuli acts as paracrine/autocrine signal stimulating cell cycle arrest. Finally, I spent six months of my PhD at the Institute of Toxicology and Genetics (ITG-KIT), in Karlsruhe, Germany, where I had the opportunity to study the behaviour of the evolutionarily conserved YB-1 in zebrafish (Danio rerio), one of the most versatile genetic models for environmental studies. Unexpectedly, I found that zebrafish cells assemble YB-1 positive aggregates only in response to heat, but not oxidative stress or copper treatment. YB-1-positive aggregates were confirmed as SGs as they contained the G3BP1 protein, a well assessed SG marker in mammalian. I found that zfYB-1 gene silencing compromised cell viability under heat shock, but not in normal conditions highlighting the essential role played by YB-1 in cell survival following heat shock. My findings point to a fundamental role of YB-1 as a valuable biomarker for thermal stress.

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