Identification of new modulators of calcium-regulated processes using genomic and chemogenomic screens in yeast

• Executive summary

• Consortium structure

• State of the art

• Main goal

• Objectives

• Expected results

• Activity report – stage I

Title of the project: Identification of new modulators of calcium-regulated processes using genomic and chemogenomic screens in yeast

Funding source: „Unitatea Executivă pentru Finanţarea Învăţământului Superior, a Cercetării, Dezvoltării şi Inovării”

Acronym of the project: CalChemGen

Project cod: PN-II-PT-PCCA-2013-4-0291

Contract number: 203/ 01.07.2014

Project Director: Conf. Dr. Ileana Cornelia Fărcăşanu

Consortium structure

Coordinator – Bucharest University, The Research Center for Applied Organic Chemistry from the Faculty of Chemistry

Project Director: Ileana Cornelia Fărcăşanu, Asscociate Professor, Phd

The team members:

• Ioana Nicolau, PhD

• Lavinia Ruţă, PhD

• Claudia Valentina Popa, PhD

• Codruţa Paraschivescu, PhD

• Mihaela Matache, PhD

• Cristian Ene, PhD

Partener 1 - The Institute of Biochemistry of the Romanian Academy

Team Leader: Simona Ghenea, PhD

The team members:

• Gabriela Chiritoiu, student

• Octav Căldăraru, student

Partener 2 - The National Institute for Research Development in Microtechnology

Team Leader: Monica Simion, PhD

The team members:

• Mihaela Kusko, PhD

• Adina Brăgaru, postdoc

• Teodora Ignat, postdoc

• Iuliana Mihalache, student

• Răzvan Pascu, student

Partener 3 –SC APEL LASER SRL

Team Leader: Phys. Dan-Nicolae Becherescu Barbu

The team members:

• Virgil Mircea Udrea, PhD

• Bogdan Chiricuţă, postdoc

• Mioara Liliana Iacob

• Cristina Lungu

State of art

• Calcium ions are used by virtually all eukaryotic cells to signal information about the environment and the physiological state of the cell, or to regulate various cellular processes such as initiation of gene expression, alterations in cell shape, membrane fusion, or programmed cell death.

• Excessive or unregulated levels of calcium induce a variety of drastic defects, such as uncontrolled cell proliferation, aberrant cell morphology, or cell death, leading to disruption of normal metabolism and initiation of various diseases.

• The versatility of calcium-mediated regulation of key physiological processes requires extensive research to identify the interplay between calcium signaling, mechanisms of diseases and discovery of new drugs.

Main goals

• To unravel new aspects of the calcium-regulated cell mechanisms using Saccharomyces cerevisiae as model.

• To investigate the applicability of in house newly-synthesized chemicals selected through interactions with the calcium-dependent pathway components.

Objectives

• Systemic investigations such as genomic profiling paralleled by chemo-genomic screens designed to identify new interactions between small molecules and calcium-related biologic processes.

• Identification of in house synthesized organic compounds which interact with calcium-dependent biological processes.

The budget of the project (Ron)

Expected results

• Identification of the molecular components involved in regulating the phenotype caused by MID1 overexpression using the MODE screens.

• Elucidation of the mechanisms which underlie the aberrant morphology of wild type cells overexpressing MID1.

• Identification of the interactions between cell components and small molecules using AFM and AFM coupled with confocal microscopy.

• Synthesis of non-toxic lanthanide coordinative compounds designed to interact with calcium-related cellular processes.

• Identification of molecular targets of lanthanide ions and lanthanide complex compounds using chemo-genomic screens.

• Identification of other in house synthesized small molecules which augment or suppress the aberrant phenotype of MID1-overexpressing.

• Mapping of the yeast chemo-genetic network defining the interaction between the calcium-related metabolism and the chemicals used in the study.

• Strategies for implementing the applicative and market-oriented results.
  

Activity report –stage I - 01.07.2014 – 05.12.2014

• High-throughput transformation of non-essential S. cerevisiae deletome with a plasmid harboring the galactose-inducible MID1 gene.

• A collection of approximately 5000 individual non-essential gene knock-out mutants of S. cerevisiae BY4741 was transformed using high-throughput methods.

• A data base of the transgenic lines was created.

• Part of the MODE collection obtained was microscopically screened for the galactose induced phenotype caused by MID1 overexpression.

• In silico analysis of the identified genes.

• Technical support for high-troughput spectroscopic determinations.

• Market screening for devices supportive of single-cell analysis

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RESULTS

Ruta LL, Nicolau I, Popa CV, IC Farcasanu. Crosstalk between Calcium and Inositol dependent Pathways as seen by a chemogenomic screen. In preparation

Ene CD, Ruta LL, Nicolau I, Popa CV, Neagoe AD, IC Farcasanu. Interaction between Ln3+ and Calcium uptake by yeast cells. In preparation

Nicolau I, Ene CD, Paraschivescu C, Matache M, Ruta LL, Neagoe AD, IC Farcasanu, Interaction between Ln3+-based complexes and yeast cells with altered calcium homeostasis. In preparation

CV Popa, IC Farcasanu, Heat Shock, Visible Light or High Calcium Augment the Cytotoxic Effects of Ailanthus altissima (Swingle) Leaf Extracts against Saccharomyces cerevisiae Cells. Under revision