RESUMEN
Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Cynara/enzimología , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Concentración de Iones de Hidrógeno , Proteínas de Plantas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
The establishment of primary cell cultures is invaluable for studying cell and molecular biological questions. Although primary cell cultures more closely resemble and function like in the native environment, during the culture establishment the cells undergo several changes including the damage sustained during their removal from original tissue. The resultant cells have to rebalance the expression of their processing molecules to ascertain matrix signalling that ensure cell adaptation and consequent proliferation. Hence, we used cardosin, a novel plant enzyme for tissue disaggregation, for isolating and culturing neuronal cells from embryonic rats. The present investigation reports the molecular events, mainly related with matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) expression, which could substantiate the superior neurite outgrowth and dendritic extension previously described. It was observed that 24 h after primary culture establishment, MMP-2 and MMP-9 messenger RNA (mRNA) are significantly upregulated, while the expression of TIMP-1 and TIMP-2 is unaltered. Regarding the role of laminin in neuronal pathfinding, it was found that the use of anti-laminin antibody and arginine-glycine-aspartate (RGD) peptide exerted inhibitory effects on neurite outgrowth after mechanical lesion where the expression of MMP-9 and TIMP-1 is upregulated under non-permissive conditions in response to mechanical injury.
Asunto(s)
Ácido Aspártico Endopeptidasas/farmacología , Regeneración Nerviosa/efectos de los fármacos , Neuronas , Proteínas de Plantas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Células Cultivadas , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/fisiología , Flores/química , Expresión Génica/efectos de los fármacos , Laminina/fisiología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
The kinetics of the structural changes affecting cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculus L., in the presence of a mixture of acetonitrile (AN) in water (W) was studied. Incubation of cardosin A with 10% (v/v) AN resulted in a gradual increase in protein helicity, accompanied by changes in the tertiary structure, seen by changes in the intrinsic fluorescence of tryptophan. Differential scanning calorimetry (DSC) revealed that the temperature of denaturation of cardosin A decreased upon the addition of AN. With longer incubation times, the small chain of cardosin A denatured completely, consequent exposure of the single tryptophan residue accounting well for the observed spectral shift intrinsic fluorescence of the protein. Enzymatic activity assays demonstrated that the kinetically determined unfolding of the small chain of cardosin A does not result in loss of the activity of this enzyme.