254. (285) SENSITIVE ASSAYS FOR MONITORING NUCLEOLAR C23 IN THE CYTOSOL AND ENDOMEMBRANES OF MYCOBACTERIUM AVIUM-INFECTED MACROPHAGES AND TO STUDY ITS CLEAVAGE, KINASES AND INTERACTING PARTNERS

ZUPPAN KARIM; GARCÍA RODOLFO C.; ASENSIO CRISTIAN JORGE ALEJANDRO

virtual

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2021; 2021

SAIC, SAI, AAFE, NANOMED-AR

We had detected by cell-free in vitro proteome radiolabeling (RL) screening a decreased level of full-length nucleolar C23 but in the cytosol of M. avium-infected macrophages. C23 cytosolic roles were unknown in innate immunity, so, we searched alterations in proteins likely connected to C23 such as: a) CK2 and CDK kinases, b) p21waf1 (as partner of C23, CK2 and CDK), c) Pin1 as CDK target site phospho-dependent isomerase. We optimized assays to study CK2 and CDK as C23 kinases. Differentiated THP-1 cells ingested live or heat-killed M.avium. After time-courses, cells were harvested and cytosolic and membrane proteomes radiolabeled in reactions with or without inhibitors or substrates, then gel-resolved by 1D and 2D to analyze C23 RL level. Kinases activities were assayed by peptides containing C23 sites. Reproducible (in >95% replicates) significant time-dependent events were considered. The n for assays or for cell treatments was 5 to 9. We found that: 1) C23 downregulation quantitated sensitively by RL was likely caused by cleavage at 2 sites, not due to altered CK2 or CDKs and initially independent on bacterial viability. 2) cytosolic C23 was iv labeled by CK2 but not CDKs. 3) by WB, cytosolic p21 had cleavage. 4) C23 cleavage occurred also in endomembranes. 5) Pin1 was not controlling CDK sites in vitro. 6) C23 had different phospho-pattern in monocytes and macrophages. We conclude that the decreased C23 IV RL was due to cleavage but not kinases or Pin1. Since M. avium did not induce macrophage death, C23 cleavage might be a marker of sustained apoptosis signaling not resulting in death. Apoptotic bacteria should be compared. Our methods will allow finding protein complexes and innate receptors inducing p21 and C23 cleavage. C23 RNA-binding phosphoprotein might be a checkpoint hub integrating p21, CDK and CK2 with innate receptors and with pathways in nucleolus, membranes and cytosol. Infection cleaved C23, so, its fragments and other PTMs should be studied