BRAFV600E causes an altered regulation of the MAPK pathway (mitogen-activated protein kinase or RAS / RAF / MEK / ERK pathway), involved in cell division and differentiation. This mutation consists of the substitution of a valine with glutamic acid at position 600 (V600E), resulting in a change in conformation, responsible for a constitutive activation of the protein, even in the presence of a low level of RAS, its activator. In this work we have demonstrated, for the first time, that hBRAFV600E kinase activity is preserved and can be studied in yeast. Indeed, hBRAFV600E complements the activity of MAPKKK kinases belonging to the osmotic stress (HOG) pathway and is toxic in yeast strains deleted for phosphatases of the same pathway. Moreover, we have demonstrated that a yeast genetic context allows the one-by-one analysis of the 3 BRAF protein isoforms that always coexist in human cells (BRAF-Ref, BRAF-X1 and BRAF-X2). In addition, we provide experimental evidence that yeast can be used to perform high throughput screenings and identify new BRAFV600E functional interactors. In fact, two screenings have been performed in this model system, one using a cDNA Library and another one taking advantage of the Yeast Deletion Pool collection. The screening performed with the cDNA library, deriving from HeLa cells, was performed in a yeast strain deleted for two phosphates PTC1 and PTP3. ~105 transformed cells have been screened. We obtained 16 complete CDS, 13 out of 16 have been validated using the spot assay. Among them, we found the Small Integral Membrane Protein 10 (SMIM10), a protein of unknown function that has been further characterized because of its different expression levels in human melanoma cell lines with mutated BRAF as compared to those with wild type (wt) BRAF. Interestingly, SMIM10 overexpression causes a dramatic decrease in the levels of BRAFV600E both in yeast and in human cells. These results suggest that SMIM10 is a negative functional interactors (FI) of BRAFV600E with a possible oncosuppressive role. The second screening was performed using the Yeast Deletion Pool, a pool of yeast S. cerevisiae clones, that has a deletion in non-essential genes (4,741 clones). Each clone is identifiable by means of two specific DNA sequences called "barcodes". 4 Through the use of the yeast deletion pool, it is possible to identify functional interactors of proteins related to diseases which do not have homologous counterparts in yeast, such as BRAF. As a result of this screening, we have identified 9 genes affecting the fitness of cells expressing BRAFV600E-X1/Ref, compared to cells transformed with the empty vector. Four out of nine affected the fitness with both isoforms. Interestingly, among deleted genes altering the fitness of BRAFV600E expressing cells when deleted, there are RAS, a SWI/SNF remodeling factor and Arf2 (GTPase). The addition of salt to the growth of the YDP highlighted differences between the two isoforms. In fact, while the comparison pYES2-BRAFV600E-Ref versus pYES2-BRAFV600E-Ref + NaCl showed differences in 4 clones, the comparison of pYES2-BRAFV600E-X1 versus pYES2-BRAFV600E-X1+ NaCl showed differences in 21 clones. Among them, an ABC transporter, a Rho GTPase, and a subunit of TORC2 membrane-associated complex have been found. Finally, this work has yielded a list of modulators of BRAFV600E to be further characterized experimentally and eventually translated into innovative therapeutic strategies that could be used in addition to the existing BRAF inhibitors.

Lubrano, S. (2018). Yeast S. cerevisiae as a tool to study BRAFV600E kinase isoforms.

Yeast S. cerevisiae as a tool to study BRAFV600E kinase isoforms

LUBRANO, SIMONE
2018

Abstract

BRAFV600E causes an altered regulation of the MAPK pathway (mitogen-activated protein kinase or RAS / RAF / MEK / ERK pathway), involved in cell division and differentiation. This mutation consists of the substitution of a valine with glutamic acid at position 600 (V600E), resulting in a change in conformation, responsible for a constitutive activation of the protein, even in the presence of a low level of RAS, its activator. In this work we have demonstrated, for the first time, that hBRAFV600E kinase activity is preserved and can be studied in yeast. Indeed, hBRAFV600E complements the activity of MAPKKK kinases belonging to the osmotic stress (HOG) pathway and is toxic in yeast strains deleted for phosphatases of the same pathway. Moreover, we have demonstrated that a yeast genetic context allows the one-by-one analysis of the 3 BRAF protein isoforms that always coexist in human cells (BRAF-Ref, BRAF-X1 and BRAF-X2). In addition, we provide experimental evidence that yeast can be used to perform high throughput screenings and identify new BRAFV600E functional interactors. In fact, two screenings have been performed in this model system, one using a cDNA Library and another one taking advantage of the Yeast Deletion Pool collection. The screening performed with the cDNA library, deriving from HeLa cells, was performed in a yeast strain deleted for two phosphates PTC1 and PTP3. ~105 transformed cells have been screened. We obtained 16 complete CDS, 13 out of 16 have been validated using the spot assay. Among them, we found the Small Integral Membrane Protein 10 (SMIM10), a protein of unknown function that has been further characterized because of its different expression levels in human melanoma cell lines with mutated BRAF as compared to those with wild type (wt) BRAF. Interestingly, SMIM10 overexpression causes a dramatic decrease in the levels of BRAFV600E both in yeast and in human cells. These results suggest that SMIM10 is a negative functional interactors (FI) of BRAFV600E with a possible oncosuppressive role. The second screening was performed using the Yeast Deletion Pool, a pool of yeast S. cerevisiae clones, that has a deletion in non-essential genes (4,741 clones). Each clone is identifiable by means of two specific DNA sequences called "barcodes". 4 Through the use of the yeast deletion pool, it is possible to identify functional interactors of proteins related to diseases which do not have homologous counterparts in yeast, such as BRAF. As a result of this screening, we have identified 9 genes affecting the fitness of cells expressing BRAFV600E-X1/Ref, compared to cells transformed with the empty vector. Four out of nine affected the fitness with both isoforms. Interestingly, among deleted genes altering the fitness of BRAFV600E expressing cells when deleted, there are RAS, a SWI/SNF remodeling factor and Arf2 (GTPase). The addition of salt to the growth of the YDP highlighted differences between the two isoforms. In fact, while the comparison pYES2-BRAFV600E-Ref versus pYES2-BRAFV600E-Ref + NaCl showed differences in 4 clones, the comparison of pYES2-BRAFV600E-X1 versus pYES2-BRAFV600E-X1+ NaCl showed differences in 21 clones. Among them, an ABC transporter, a Rho GTPase, and a subunit of TORC2 membrane-associated complex have been found. Finally, this work has yielded a list of modulators of BRAFV600E to be further characterized experimentally and eventually translated into innovative therapeutic strategies that could be used in addition to the existing BRAF inhibitors.
Lubrano, S. (2018). Yeast S. cerevisiae as a tool to study BRAFV600E kinase isoforms.
Lubrano, Simone
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11365/1040150
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