ANÁLISIS DE PROTEÍNAS

Extracción de Proteínas

Purificación de Proteínas

Western Blot (detección de proteínas específicas con anticuerpos)

La detección de proteínas sirve para conocer:

1. Niveles de expresión (a nivel proteína)2. Isoformas3. Modificaciones postraduccionales4. Tiempo de vida media y degradación5. Localización subcelular

would display an increased cell size. To test this idea, stablecells were serum deprived for 24 h to induce G1 phase accu-mulation and then stimulated with serum containing medium(DMEM-FBS) for 24 h to promote cell cycle progression in theabsence or presence of rapamycin. The percentage of cells inG1 phase was then determined on a flow cytometer by mea-suring DNA content after propidium iodide staining. As ex-pected, WT-mTOR-expressing cells stimulated with serum inthe presence of rapamycin displayed increased G1-phase con-tent relative to cells stimulated in the absence of rapamycin,and expression of RR-mTOR completely rescued this G1-

phase delay (Fig. 6D). Expression of RR/AA-mTOR, however,led to impaired rescue, while expression of RR/DE-mTORconferred rescue (Fig. 6D). Thus, RR/AA-mTOR-expressingcells progress through G1 phase more slowly, thus having moretime to grow, while RR/DE-mTOR-expressing cells progressthrough G1 phase normally with an augmented rate of cellgrowth; in the end, both situations lead to increased cell size.

To confirm the idea that cells expressing RR/AA-mTORdisplay a larger cell size due to delayed cell cycle progression,we repeated our cell size analysis with cells experiencing a cellcycle block. First, we treated WT-mTOR-expressing cells in

FIG. 6. mTOR S2159/T2164 phosphorylation promotes mTORC1-mediated cell growth and G1-phase cell cycle progression. (A) Biochemicalanalysis of stable HEK293 Flp-In cell lines. mTOR S2159/T2164 phosphorylation promotes S6K1 and 4EBP1 phosphorylation in response toinsulin. Cells expressing various AU1-mTOR alleles were serum deprived (20 h), pretreated without or with rapamycin (20 ng/ml) for 30 min,incubated in the absence or presence of insulin (100 nM) for 30 min, and lysed. WCL was immunoblotted directly. (B) mTOR S2159/T2164phosphorylation promotes cell growth to increased cell size. Cells stably expressing various AU1-mTOR alleles were cultured for 96 h in theabsence or presence of rapamycin. The relative size of subconfluent cells was determined using a flow cytometer via the parameter mean FSC-H.The graph shows mean FSC-H (! standard deviation [SD]) of G1-phase cells from three experiments, two performed in quadruplicate and onein triplicate (n " 11). The size of cells expressing RR-mTOR and cultured in the presence of rapamycin was set to 100%. All other samples werenormalized to this value. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests.The letters (a to e) indicate significance at a P value of #0.05. (C) Expression of the various AU1-mTOR alleles and phosphorylation of rpS6 fromone representative cell size experiment. (D) mTOR S2159/T2164 phosphorylation promotes G1-phase cell cycle progression. Flp-In HEK293 cellsstably expressing various AU1-mTOR alleles were serum deprived for 24 h and then stimulated with serum-containing medium (DMEM-FBS) inthe absence or presence of rapamycin (20 ng/ml) for an additional 24 h. DNA content was determined on a flow cytometer after propidium iodidestaining. The graph shows the percentage of cells in G1 phase following serum stimulation in the absence or presence of rapamycin. Mean values(! SD) from one representative experiment performed in quadruplicate are shown (n " 4). Statistical significance was determined using one-wayanalysis of variance (ANOVA) followed by Tukey’s post hoc tests. The letters (a to c) indicate that the G1-phase percentage means are significantlydifferent at a P value of #0.05.

2796 EKIM ET AL. MOL. CELL. BIOL.

Anticuerpo-proteína

Anticuerpo-P-Ser (fosforilación)

Los antibióticos se dirigen a diferentes componentes/pasos de la Traducción:

- Subunidad grande- Subunidad

pequeña- Factores de

traducción- tRNAs

Inicio

Elongación

Terminación y recicladoEntrada del

aa-tRNA

Formación del enlace peptídico

Translocación

Elongación¿Por qué se hacen resistentes a los antibióticos las bacterias?

Aplicaciones en Estudios de Virus

Los coronavirus han sufrido mutaciones para adaptarse a nuevos hospederos

Ashour et al., 2020 Pathogens MDPI

Durante su ciclo los virus como SARS-Cov2 utilizan la maquinaria celular de traducción para su beneficio

ssRNA (+) Proteínas viralesssRNA (⎯)

vRdRP

vRdRP

vRdRP: RNA polimerasa dependiente de RNA viralssRNA (+): RNA de cadena sencilla positiva (codificante)ssRNA (⎯): RNA de cadena sencilla negativa

Ribosomas celulares

Virus de RNA de cadena sencilla positiva

Organización del genoma de SARS-Cov2

RNAs subgenómicosSe tienen que transcribir de la cadena ssRNA (-)

Se traducen diréctamente de la cadena (+) ORF1a y ORF1b

RNA (+) à RNA (-) à RNA (+)

Kim et al., 2020 Cell DOI: 10.1016/j.cell.2020.04.011 (pre-Proof)

Para traducir el ORF 2 hay un cambio de marco

• Frameshift: Cambio de marco

• (-1): El ribosoma se recorre 1 nt hacia el 5’

• A partir del cambio se leen nuevos codones

• Para el cambio se requiere una estructura secundaria definida en el RNA (Pseudonudo) y una proteína viral específica

Los ORFs genómicos codifican para poliproteínas que son cortadas por proteasas y generan proteínas

independientes

Esta polimerasa hace la cadena (-) de RNA

Tu et al., 10 Apr 2020IJMS. Algunos proyectos de tratamiento para COVID-19