validação dos dados de proteômica. enzimologia e

Prof. Jesús V. Jorrín Novo PESQUISADOR VISITANTE CNPq-CENARGEN-Brasilia

AGROFORESTRY AND PLANT BIOCHEMISTRY AND PROTEOMICS RESEARCH GROUP (AGR-164)

UNIVERSITY OF CORDOBA, CORDOBA, SPAIN

http://www.uco.es/investiga/grupos/probiveag/

[email protected]

Validação dos dados de proteômica. ENZIMOLOGIA e ENZIMOMICS

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18 Julio 2016




mailto:[email protected]

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A resposta está no nome do curso: VALIDAÇÃO?

Trends in Plant Sciences 2014, 19: 256-265.

Usaremos, como exemplo, com a pesquisa realizada por Cristina Romero (Universidad Nacional Asunción, Paraguai) para sua tese de doutorado em meu laboratório

A PERGUNTA É: POR QUE UMA "PALESTRA" SOBRE AS ENZIMAS E ATIVIDADES ENZIMÁTICAS EN UM CURSO SOBRE ABORDAGENS -ÓMICOS ?

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RubisCO L. We did not see the S subunit. In any case, RubisCO in seed (not green tissue)?

Some identified protein seems to correspond to degradation products (much lower Mr)

It appears in just a time point. Is it an artefact?

GEL E GEL-FREE ABORDAGENS DA RESULTADOS DIFERENTES OU CONTRADITÓRIAS. ARTEFACTS OU SIGNIFICADO BIOLÓGICO?

DO YOU AGREE? 1. Proteomics determine protein abundance.

2. Protein abundance does not mean increased gene expression.

3. Protein abundance does not mean protein activity.

4. The last two are common and big errors found in the current

literature.

5. The idenified (matched) protein corresponds to:

i) Protein species product of the same gene. ii) Allelic variants, including SNPs. iii) Protein species product of different genes (isoforms). iv) The case of oligomeric proteins coded corresponding to

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https://www.researchgate.net/publication/225096554_A_Nutrigenomics_View_of_Protein_Intake/figures?lo=1

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https://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj1ibqRzuHNAhUGDpAKHVcGA6EQjRwIBw&url=https://www.researchgate.net/figure/225096554_fig2_FIG-2-States-of-protein-turnover-Steady-state-protein-synthesis-equaling-degradation&bvm=bv.126130881,d.Y2I&psig=AFQjCNHmPRz3fyqgPoPpIUtk1ANfpP2kXg&ust=1467987791571573

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Os enzimas são proteínas (excepto ARN catalítica ou ribozimas) produzidos pelos organismos vivos que catalisam muito eficiente das reacções químicas que ocorrem na célula, e é altamente específico quanto ao substrato sobre o qual agem e produtos, e actuam in vivo de uma forma controlada.

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DADOS PROTEÓMICOS DEVEM SER VALIDADOS. O QUE É UMA ENZIMA?

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=jt6YIsRAJ8_0cM&tbnid=GJSJMTroOEiSBM:&ved=0CAUQjRw&url=http://en.wikipedia.org/wiki/Enzyme&ei=z2EnUoG4GbCa0QW9qYHABg&bvm=bv.51495398,d.d2k&psig=AFQjCNHt-Yel5D4V8wbsR9g-YGyD_zpshA&ust=1378399050797725

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=7vuOemayXB9bLM&tbnid=bissV2OFRvRmDM:&ved=0CAUQjRw&url=http://www.tokresource.org/tok_classes/biobiobio/biomenu/enzymes/&ei=-mEnUsmIMo_z0gXlr4DYDQ&bvm=bv.51495398,d.d2k&psig=AFQjCNHt-Yel5D4V8wbsR9g-YGyD_zpshA&ust=1378399050797725

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=IjKkuZEK1u7AbM&tbnid=625YIFrKZiSOoM:&ved=0CAUQjRw&url=http://www.jonbarron.org/article/proteolytic-enzyme-formula&ei=IWInUo6QIIja0QW20YGQCg&bvm=bv.51495398,d.d2k&psig=AFQjCNHt-Yel5D4V8wbsR9g-YGyD_zpshA&ust=1378399050797725

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=B_E1n2VHrV_7dM&tbnid=jubsk3Seg9KUOM:&ved=0CAUQjRw&url=http://products.mercola.com/digestive-enzymes/&ei=cGInUsaLO63w0gX1-4HgAw&bvm=bv.51495398,d.d2k&psig=AFQjCNHt-Yel5D4V8wbsR9g-YGyD_zpshA&ust=1378399050797725

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=zp7_SyvR_8fkwM&tbnid=fmWV40O1j9km6M:&ved=0CAUQjRw&url=http://depts.washington.edu/wmatkins/kinetics/michaelis-menten.html&ei=qGInUqCuM8Gm0AXOx4GgBg&bvm=bv.51495398,d.d2k&psig=AFQjCNFn74ZsMBVJidFIHHkPRa1_ZDo-EQ&ust=1378399266569912

http://www.google.es/url?sa=i&source=images&cd=&cad=rja&docid=IMyrf7RpQ799ZM&tbnid=ZBQHilSPzlb4RM:&ved=0CAgQjRwwAA&url=http://bernalybulla.blogspot.com/2013_06_01_archive.html&ei=xqVJUs3HHY-RhQfrh4GAAQ&psig=AFQjCNFsmfVKcXCYq_Y23PE-ZJuN5rj64A&ust=1380644678550795

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Pue0WdiylKHHrM&tbnid=YjmfVTh7b8ur3M:&ved=0CAUQjRw&url=http://vonneumannmachine.wordpress.com/tag/friedrich-nietzsche/&ei=G6ZJUrCtFYKk0QX99YCwBg&bvm=bv.53217764,d.ZG4&psig=AFQjCNE9HgXKr-bJKr-aZZYhaoiVtAHycA&ust=1380644749736729

LOS ENZIMAS DETERMINAR A COMPOSIÇÃO QUÍMICA DOS ORGANISMOS VIVOS E OS PROCESSOS DE REPLICAÇÃO, TRANSCRIÇÃO E TRANSDUÇÃO.

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http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjFut2B_azNAhXEuRQKHWd8B4YQjRwIBw&url=http://plantbiotechinfo.blogspot.com/2015/10/introduction-of-plant-secondary.html&bvm=bv.124272578,d.d24&psig=AFQjCNFJlBWExlBhWyWGMYGc1yi7SmdVng&ust=1466181270674917

BIOTECNOLOGÍA ROJA. Área de la salud (Biomedicina, antibióticos, vacunas, fármacos, terapias génicas).

BIOTECNOLOGÍA BLANCA

Área industrial (biocombustibles, plásticos, tejidos, papel, detergentes, madera, producción de compuestos de interés)

BIOTECNOLOGÍA AZUL. Biotecnología marina (acuicultura)

BIOTECNOLOGÍA VERDE. BIOTECNOLOGÍA VEGETAL. Área de la agricultura (cultivos mejorados). Área de alimentación (alimentos mejorados, conservación, nutraceúticos). Área medioambiental (biorremediación)

Biotecnología. Es definida por la “UN Convention on Biological Diversity” (1992) en su Art. 2, como: “The use of living systems and organisms to develop or make useful products, or "any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use".

TEM UM POTENCIAL BIOTECNOLÓGICO IMPORTANTE Pr

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ALGUNS EXEMPLOS Pr

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https://commons.wikimedia.org/wiki/File:RuBisCOL2S2.png

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjphK_b1eHNAhXIj5AKHYERAkUQjRwIBw&url=http://america.pink/rubisco_3826236.html&psig=AFQjCNH-x-QuV4Xw-RV6BC0bo_n21Ihh9Q&ust=1467991767615967

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiktYD-1eHNAhXEkJAKHZk3BnQQjRwIBw&url=http://slideplayer.es/slide/5304321/&bvm=bv.126130881,d.Y2I&psig=AFQjCNFT5iJDwkcDAE0yU6ESv1Jyt9YkWA&ust=1467991830835901

https://en.wikipedia.org/wiki/Taq_polymerase

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria.

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https://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjMn9jM1uHNAhXFQZAKHXBwB0UQjRwIBw&url=https://prezi.com/fw1d9bjmjmy5/bio-final-project-telomeres/&bvm=bv.126130881,d.Y2I&psig=AFQjCNFwn2LsSrjn-WCBdhDw6ZGNZZ2CmQ&ust=1467992023996732

https://en.wikipedia.org/wiki/RNA

https://en.wikipedia.org/wiki/DNA

https://en.wikipedia.org/wiki/Endonuclease

https://en.wikipedia.org/wiki/Enzyme

https://en.wikipedia.org/wiki/CRISPR

https://en.wikipedia.org/wiki/Streptococcus_pyogenes



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PROTEÔMICA E ENZYMOLOGY SÃO COMPLEMENTARES

What does enzyme specificity mean?

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QUEBRANDO PARADIGMAS¡¡¡¡¡¡¡

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An enzyme does what it can do: SUBSTRATE AVAILABILITY It is because plants can deal with xenobiotics they have never faced before.

Enzyme activity is modulated in vivo by multiple mechanisms. In vitro assays only reveal a minimum part of the catalytic potential of enzymes.

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjEsv-n79TNAhXIgpAKHUh8A98QjRwIBw&url=http://www.slideshare.net/drrajenarya/xenobiotics-and-cyt-p450-by-dr-rajender&bvm=bv.126130881,d.Y2I&psig=AFQjCNFwhEWLZ_abmCSXV2tu9n3d-Mlk6A&ust=1467551924174184

6. Enzyme promiscuity: multifunctional enzymes

Enzyme promiscuity is the ability of an enzyme to catalyse a side reaction in addition to its main biological reaction. Although enzymes are remarkably specific catalysts, they can often perform side reactions in addition to their main, native catalytic activity. These promiscuous activities are usually slow relative to the main activity and are under neutral selection.[1] Despite ordinarily being physiologically irrelevant, under new selective pressures these activities may confer a fitness benefit therefore prompting the evolution of the formerly promiscuous activity to become the new main activity

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https://en.wikipedia.org/wiki/Enzyme_promiscuity



LA BIOLOGÍA (BIOQUÍMICA Y BIOLOGÍA MOLECULAR, ENZIMOLOGÍA) ES UNA CIENCIA EXPERIMENTAL. EL MÉTODO CIENTÍFICO EL CIENTÍFICO Y EL TÉCNICO

ENZYMOLOGY. ABORDAGENS CLÁSSICAS EM PESQUISA COM ENZIMAS

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O FLUXO DE TRABALHO CLASSICO PARA ANÁLISE IN VITRO DO ENZIMAS

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1. PLANO EXPERIMENTAL.

2. PREPARAÇÃO DOS EXTRACTOS E TESTAR ATIVIDADE ENZIMÁTICA 3. PURIFICAÇÃO DA ENZIMA. 4. CARACTERIZAÇÃO: ESTRUTURAL, CATALÍTICO E CINÉTICA 5. BIOTECNOLOGIA ENZIMATICA 6. ENGENHARIA DO ENZIMAS: ENGENHARIA QUÍMICA, ENGENHARIA GENÉTICA

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCL73huyQgMgCFUK2FAod4ckBPg&url=http://armonicosdeconciencia.blogspot.com/2015/04/la-marihuana-y-el-girasol-absorben-la.html&psig=AFQjCNFQxEohB-IVv0bemzoxdSCmKnhoKg&ust=1442650157836741

Atividade enzimática e velocidade de reacção

v = -d[S]/dt = d[P]/dt

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http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCKuQ_9ywnsgCFcLVGgodptYIXg&url=http://www.100ciaquimica.net/temas/tema6/punto2a.htm&psig=AFQjCNEBjsV636IE3_VVzWp1UFAJ4vCMuA&ust=1443689506341843

TAMPÓN (pH, fuerza iónica, cte dieléctrica). EFECTORES ACTIVADORES INHIBIDORES

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A MISTURA DE ENSAIO

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Exatidão Precisão Especificidade Limite detecção Limite quantização Linearidade Robustez Facilidade Preço

CARACTERÍSTICAS E OS TIPOS DE TÉCNICAS ANALTICAS

http://www.google.es/url?sa=i&source=images&cd=&cad=rja&docid=AUTAopZptrDgPM&tbnid=oqio24aUJPqpHM:&ved=0CAgQjRwwAA&url=http://www.labotienda.com/es/catalogo/otros_polarimetros.aspx&ei=Cf9gUsDaDYmv7Ab96IDAAg&psig=AFQjCNHoUKS002LAysHZB59ZVn03TxMQ6w&ust=1382174857296239

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Western blot Pr

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https://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCOr1hpivocgCFUV-Ggod3KYAYA&url=https://www.emaze.com/@ALCWOTOZ/WESTERN-BLOT&psig=AFQjCNHOtZiOKNgK7BzxCcCWs1iuEq7zPg&ust=1443792187037051

https://commons.wikimedia.org/wiki/File:Deteci%C3%B3n_quimioluminiscente.jpg

Intracellular enzymes in erythrocytes can be made accessible for in situ kinetic studies by treating the cells with bifunctional reagents to crosslink proteins, thus creating a network that allows subsequent permeabilization by delipidation without escape of intracellular proteins. Dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate, and toluene- 2,4-diisocyanate have been used successfully as crosslinking reagents, and digitonin has been used for delipidation.

In a systematic study of the in situ behavior of the 11 glycolytic enzymes of rat erythrocytes, it was observed that Km and Vma, values for the majority of the enzymes are essentially the same in situ as in vitro. Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase,EC 1.1.1.27) is inhibited by excess of pyruvate as much in situ as in vitro. Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was allosterically inhibited by glucose 6-phosphate nearly as much in situ as in vitro but was not affected by 2,3-bisphosphoglycerate. The allosteric properties of 6-phosphofructo inase (ATP:D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase [D yceraldehydephosphate:NAD- oxidoreductase (phosphorylating), EC 1.2.1.121, and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) in situ were qualitatively similar to those observed in vitro, but some important quantitative differences were noticed. Particularly striking was the much greater activity of phosphofructokinase in situ compared to that in vitro at physiological concentrations of effector metabolites.

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Automatizacion

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HOLISTIC / HIGH THROUGHPUT ENZYMOLOGY: ENZYMOMICS Pr

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EU ESPERO QUE NOS ENCONTREMOS EM CÓRDOBA

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