471. (528) CELL-FREE, IN VITRO RADIOLABELING ASSAYS SENSITIVELY UNCOVERING INFECTION-DEPENDENT ALTERATIONS IN CYTOSOLIC FORMS OF NUCLEOLAR PROTEINS IN HUMAN MACROPHAGES.

ASENSIO CRISTIAN JORGE ALEJANDRO; RODOLFO C GARCÍA

virtual

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2020; 2020

SAIC , SAI, SAFIS

AIMS: To developed and test cell free, in vitro (iv) radiolabeling (RL) assays detecting infection time-dependent proteome alterations in macrophage cytosols uncovering novel altered proteins and pathways. METHODS: Macrophages were mycobacterial infected comparing live vs killed bacteria and time points. The cytosolic fraction was studied for being the purest. Cell-free, iv RL phosphorylating available sites in proteins and others nucleotide-based assays were used. The iv reactions were resolved in 1D and 2D gels comparing staining vs RL patterns. Reproducibly (>95 % cases) altered proteins were identified by MS and WB. The n for iv assays and treatments was 3 to 9. RESULTS: The assays were sensitive, reproducible and quick. Many proteins were labelled but 2 NUCLEOLAR proteins, C23 and B23, were detected in cytosols with a highly reproducible time-dependency of level alteration. Bacterial viability was not required for triggering that, but >4 days post-infection only live bacteria sustained it. MW alterations in both, but mainly C23, were compatible with partial cleavage to shorter fragments. The same kinase iv phosphorylating both was unaffected in activity by infection. RL assays other than phosphorylation confirmed the altered levels. Coupled iv phosphatase assays and anti-P WB revealed the C23 in vivo phosphorylation status. For detecting minor C23 changes and fragments, RL was more sensitive or linear than gel staining or WB. C23 has reported roles in mRNA translation/degradation, in ribosomes, in hubs for kinases and NEMO, etc. B23 in ribosome, PIDDosome and casp2 regulation. CONCLUSIONS: the assays tracked alterations in low-abundant cytosolic C23 and B23 forms and might be useful for: a) infection studies assessing their multiple roles, b) for cancer (C23 as aptamer target and B23 in leukemia), c) for C23 kinase assays. The RL approach would serve in tracking their intracellular trafficking and roles in nucleolar, cytosolic and membrane surveillance pathways