RESUMO
Metastatic melanoma is among the most intractable cancers to treat; patients show resistance to therapy and limited survival time. A critical step in the development of metastatic melanoma is the acquisition of invasion and transition from thin to thick tumors on the skin, followed by invasion to lymph nodes. Prior studies have shown that metastatic melanoma is associated with dysregulation of RhoA and enhanced expression of a protein named "mediator of RhoA-dependent invasion (MRDI)". Importantly, MRDI is a "moonlighting" enzyme, with two distinct functions in melanoma cells. First, MRDI acts as a methylthioribose-1-phosphate (MTR-1-P) isomerase, catalyzing a critical step in methionine salvage. Second, MRDI promotes and is necessary for melanoma cell invasion, independent of its catalytic activity. This paper demonstrates that MtnA, a bacterial MTR-1-P isomerase, rescues the methionine salvage function of MRDI, but is unable to rescue its role in invasion. The crystal structure of MRDI was solved to a resolution of 2.5 Å to identify structural elements important for its invasion activity. This structure and its comparison with other MTR-1-P isomerases are presented, and mutations within a region separate from the MTR-1-P binding site, which interfere with invasion, are identified. Thus, structural elements in MRDI distal from the MTR-1-P catalytic site are responsible for the invasion phenotype.
Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Western Blotting , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Teste de Complementação Genética , Humanos , Isomerases/metabolismo , Melanoma/enzimologia , Melanoma/patologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Invasividade Neoplásica , Conformação Proteica , Ribulosefosfatos/metabolismo , Homologia de Sequência de Aminoácidos , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Relação Estrutura-AtividadeRESUMO
RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Melanoma , Metionina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Melanoma/enzimologia , Melanoma/patologia , Metionina/química , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Estrutura Molecular , Invasividade Neoplásica , Metástase Neoplásica , Proteômica/métodos , Interferência de RNA , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Transdução de Sinais/fisiologia , Transplante Heterólogo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial alpha-subunit of PheRS and the B8 domain of its beta-subunit. Together, the alpha-subunit and the 'RNP-domain' (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 55, b = 90, c = 96 A.
Assuntos
Mitocôndrias/enzimologia , Fenilalanina-tRNA Ligase/química , Trifosfato de Adenosina/metabolismo , Cristalização , Eletroforese em Gel de Poliacrilamida , Humanos , Fenilalanina/metabolismo , Fenilalanina-tRNA Ligase/isolamento & purificação , Fenilalanina-tRNA Ligase/metabolismo , Difração de Raios XRESUMO
Elongation factor G (EF-G) catalyzes the translocation step of protein biosynthesis. Genomic analysis suggests that two isoforms of this protein occur in mitochondria. The region of the cDNA coding for the mature sequence of isoform 1 of human mitochondrial EF-G (EF-G1(mt)) has been cloned and expressed in Escherichia coli. The recombinant protein has been purified to near homogeneity by chromatography on Ni-NTA resins and cation exchange high performance liquid chromatography. EF-G1(mt) is active on both bacterial and mitochondrial ribosomes. Human EF-G1(mt) is considerably more resistant to fusidic acid than many bacterial translocases. A molecular model for EF-G1(mt) has been created and analyzed in the context of its relationship to the translocases from other systems.