RESUMO
BACKGROUND: For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. METHODS: GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. RESULT: Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. CONCLUSION: PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.
Assuntos
Glucose-6-Fosfato Isomerase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Neoplasias/metabolismo , Piruvato Quinase/metabolismo , Animais , Carcinoma de Ehrlich/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/genética , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Espectrometria de Massas , Camundongos , Neoplasias/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Aldeído Pirúvico/farmacologia , Piruvato Quinase/química , Piruvato Quinase/genéticaRESUMO
Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion.
Assuntos
Miosina não Muscular Tipo IIA/metabolismo , Vírus Sendai/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Mutagênese , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Internalização do Vírus/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC20) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC20 phosphorylation may be associated with the fragmentation step of dedifferentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Metilcolantreno/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Miosina Tipo II/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Técnicas Imunoenzimáticas , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Miosina Tipo II/genética , Quinase de Cadeia Leve de Miosina/genética , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The functional role of the C2 insert of nonmuscle myosin II-C (NM II-C) is poorly understood. Here, we report for the first time that the expression of the C2 insert-containing isoform, NM II-C1C2, is inducible in Neuro-2a cells during differentiation both at mRNA and protein levels. Immunoblot and RT-PCR analysis reveal that expression of NM II-C1C2 peaks between days 3 and 6 of differentiation. Localization of NM II-C1C2 in Neuro-2a cells suggests that the C2 insert-containing isoform is localized in the cytosol and along the neurites, specifically at the adherence point to substratum. Inhibition of endogenous NM II-C1C2 using siRNA decreases the neurite length by 43% compared with control cells treated with nonspecific siRNA. Time lapse image analysis reveals that neurites of C2-siRNA-treated cells have a net negative change in neurite length per minute, leading to a reduction of overall neurite length. During neuritogenesis, NM II-C1C2 can interact and colocalize with ß1-integrin in neurites. Altogether, these studies indicate that NM II-C1C2 may be involved in stabilizing neurites by maintaining their structure at adhesion sites.
Assuntos
Cadeias Pesadas de Miosina/química , Miosina Tipo II/química , Processamento Alternativo , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Miosinas/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas , Pseudópodes/metabolismo , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
Herein we report that vesicular assembly from a simple non-ionic amphiphilic random copolymer initiates extremely efficient myotube formation from C2C12 myoblast cells in standard growth media lacking horse serum. Control experiments with structurally related polymers and a small molecule suggest the absolute necessity of the vesicular assembly as well as the particular hydrophilic functionality in mediating such high yielding muscle cell generation. The LDH assay indicates that the membrane integrity is retained during cell-cell fusion. Expression of various myogenic factors such as MyoD, myogenin and P-21 has been examined in the presence of the polymersome and control molecules to rationalize this serendipitous discovery.