Olivares Sánchez, María Concepción

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Olivares Sánchez, María Concepción

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Olivares Sánchez, María Concepción

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Universidad de Murcia. Departamento de Bioquímica y Biología Molecular"B" e Inmunología

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Open Access
Mahogunin ring finger 1 is required for genomic stability and modulates the malignant phenotype of melanoma cells.
(MDPI, 2020-10-01) Martínez-Vicente, Idoya; Abrisqueta, Marta; Bennett, Dorothy C.; Sirés-Campos, Julia; Castejón Griñán, María; García-Borrón Martínez, José Carlos; Herraiz Serrano, Cecilia María; Jiménez-Cervantes Frigols, Celia; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología
The mouse mahoganoid mutation abrogating Mahogunin Ring Finger‐1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1‐knockout melanocytes with genetically matched controls and melan‐md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger γH2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea‐induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16‐F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1‐KO B16‐F10 cells had lower mitotic indices, fewer Ki67‐positive cells and showed a trend towards smaller size. In short‐term lung colonization assays Mgrn1‐KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.
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Functional characterization of a C-terminal splice variant of the human melanocortin 1 receptor.
(Wiley, 2020-05-31) Martínez-Vicente, Idoya; Abrisqueta, Marta; García-Borrón Martínez, José Carlos; Herraiz Serrano, Cecilia María; Jiménez-Cervantes Frigols, Celia; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología
The melanocortin 1 receptor (MC1R) is a major determinant of skin pigmentation and sensitivity to ultraviolet radiation. When stimulated by its natural agonists, it promotes the switch from synthesis of poorly photoprotective and lightly colored pheomelanins to production of photoprotective and darker eumelanins. In addition to an unusually high number of single nucleotide polymorphisms, the MC1R is expressed as 3 protein-coding splice variants. Two transcripts display different 5’ untranslated sequences but yield the same open reading frame corresponding to the canonical 317 aminoacids protein (termed MC1R). An alternative transcript named MC1R-203 encodes for a 382 amino acids protein of poorly characterized functional properties containing an additional 65 aminoacids C-terminal extension. Given the known roles of the MC1R C-terminal extension in forward trafficking, coupling to intracellular effectors and desensitization, the different structure of this domain in MC1R and MC1R-203 may lead to significant functional alteration(s). We have assessed the functional properties of MC1R-203, as compared with the canonical MC1R form. We show that unstimulated HBL human melanoma cells express the MC1R-203 spliceoform, although at much lower levels than canonical MC1R. When expressed in heterologous HEK293 cells, the presence of the 65 aminoacid-long cytosolic extension immediately after Cys316 in MC1R-203 did not impair the intracellular stability of the protein, but it interfered with functional coupling to the cAMP cascade and with the ubiquitylation of ARRB2 associated with MC1R desensitization. Conversely, MC1R-203 retained full capacity to activate ERK1/2 signaling. Accordingly, MC1R203 displays biased signaling when expressed in HEK293 cells.
Open Access
cAMP-independent non-pigmentary actions of variant melanocortin 1 receptor: AKT-mediated activation of protective responses to oxidative DNA damage
(Nature Publishing Group, 2018-07-05) Castejón Griñán, María; García-Borrón Martínez, José Carlos; Herraiz Serrano, Cecilia María; Jiménez-Cervantes Frigols, Celia; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología
Open Access
Determinantes estructurales del procesamiento y la actividad catalítica de tirosinasa
(Universidad de Murcia, 2010-06-23) Olivares Sánchez, María Concepción; Solano Muñoz, Francisco; García-Borrón Martínez, José Carlos; Departamentos y Servicios::Departamentos de la UMU::Bioquímica y Biología Molecular B e Inmunología
La melanogénesis es la ruta responsable de la biosíntesis de melaninas. Tirosinasa, la enzima limitante, presenta una alta homología con otras metaloenzimas implicadas, pero capacidades catalíticas diferentes (entre ellas y entre especies). Se han estudiado las relaciones estructura-función de estas proteínas. Las conclusiones más importantes han sido: -En humanos, tirosinasa es la enzima responsable de la actividad DHICA oxidasa de la ruta, en oposición a la enzima de ratón. -Se ha caracterizado el papel de dos importantes aminoácidos del centro activo de tirosinasa de ratón: His390 como tercer ligando del cobre en una región de unión a metal e His389 como responsable de la interacción enantioespecífica enzima-difenoles, pero no con monofenoles, introduciendo relevantes aportaciones al mecanismo del ciclo catalítico de tirosinasa. -La unión del cobre es un evento posterior en la maduración post-traduccional de la enzima, que además presenta dos sitios de glicosilación cuya ocupación es dependientes de conformación. ABSTRACT: Melanogenesis is the biochemical pathway responsible for melanin synthesis. Tyrosinase, the rate-limiting enzyme, shows high homology with other metalloenzymes involved. However, they differ in their enzymatic capabilities (also among species). In our attempt to characterize the structure-function relationships in mammalian melanogenic enzymes, we showed that: -In human, tyrosinase accounts for the DHICA oxidase activity prior to melanin formation, while the murine enzyme is unable to catalyze DHICA consumption. -Two important residues in the active site of the protein have been characterized: His390 as the third copper ligand in the metal-binding region and His389 as the amino acid responsible for the stereospecific interaction enzyme-diphenolic substrates, but not monophenols. This conclusion allowed for the modification of several aspects of the classical catalytic mechanism of the enzyme. -Neither glycosylation of all the potential sequons nor copper binding are sine qua non requirements for maturation of tyrosinase and processing beyond the endoplasmic reticulum.
Open Access
MGRN1 as a phenotypic determinant of human melanoma cells and a potential biomarker.
(MDPI, 2022-07-26) Abrisqueta, Marta; Sánchez-Beltrán, José; Martínez-Vicente, Idoya; Sevilla, Arrate; Alonso, Santos; Boyano, María Dolores; Cerdido Ochoa, Sonia; García-Borrón Martínez, José Carlos; Herraiz Serrano, Cecilia María; Jiménez-Cervantes Frigols, Celia; Lambertos Escudero, Ana; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología
Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.
Open Access
A Side-by-Side comparison of wildtype and variant melanocortin 1 receptor signaling with emphasis on protection against oxidative damage to DNA.
(MDPI, 2023-09-21) Sánchez-Beltrán, José; Abrisqueta, Marta; Padilla, Lidia; Herraiz Serrano, Cecilia María; Cerdido Ochoa, Sonia; García-Borrón Martínez, José Carlos; Herraiz Serrano, Cecilia María; Jiménez-Cervantes Frigols, Celia; Lambertos Escudero, Ana; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología
Common variants of the MC1R gene coding the α-melanocyte stimulating hormone receptor are associated with light skin, poor tanning, blond or red hair, and increased melanoma risk, due to pigment-dependent and -independent effects. This complex phenotype is usually attributed to impaired activation of cAMP signaling. However, several MC1R variants show significant residual coupling to cAMP and efficiently activate mitogenic extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Yet, residual signaling and the key actions of wildtype and variant MC1R have never been assessed under strictly comparable conditions in melanocytic cells of identical genetic background. We devised a strategy based on CRISPR-Cas9 knockout of endogenous MC1R in a human melanoma cell line wildtype for BRAF, NRAS and NF1, followed by reconstitution with epitope-labeled MC1R constructs, and functional analysis of clones expressing comparable levels of wildtype, R151C or D294H MC1R. The proliferation rate, shape, adhesion, motility and sensitivity to oxidative DNA damage were compared. The R151C and D294H RHC variants displayed impaired cAMP signaling, intracellular stability similar to the wildtype, triggered ERK1/2 activation as effectively as the wildtype, and afforded partial protection against oxidative DNA damage, although less efficiently than the wildtype. Therefore, common melanoma-associated MC1R variants display biased signaling and significant genoprotective activity.
Open Access
Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads.
(Springer Nature, 2019-11-29) Hamze Araujo, Julieta Gabriela; Canha-Gouveia, Analuce; Algarra, Blanca; Gómez-Torres, María José; Romar Andrés, Raquel; Jíménez Movilla, María; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología; Biotecnología (Universidad de Alicante)
The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte’s shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.
Open Access
Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells
(Springer Nature, 2021-12-18) Sirés-Campos, Julia; Delevoye, Cedric; Raposo, Graça; Bennett, Dorothy; Sviderskaya, Elena; Jiménez-Frigols, Celia; García-Borrón Martínez, José Carlos; Lambertos Escudero, Ana; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología
Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III-IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.
Open Access
MGRN1 depletion promotes intercellular adhesion in melanoma by upregulation of E-cadherin and inhibition of CDC42.
(Elsevier, 2023-11-25) Abrisqueta, Marta; Sánchez-Beltrán, José; Muñoz, Cristina; Castejón Griñán, María; Cerdido Ochoa, Sonia; García-Borrón Martínez, José Carlos; Herraiz Serrano, Cecilia María; Jiménez-Cervantes Frigols, Celia; Lambertos Escudero, Ana; Olivares Sánchez, María Concepción; Bioquímica y Biología Molecular B e Inmunología