Structural Change of Ribosomes during Apoptosis: Degradation and Externalization of Ribosomal Proteins in Doxorubicin-Treated Jurkat Cells

Abstract

Changes in the amount and localization of human ribosomal proteins during apoptosis were determined. When total lysates of Jurkat cells undergoing apoptosis induced by doxorubicin were analyzed by Western blotting, degradation of three ribosomal proteins, S18, L5, and L14, was detected at 48 h after the induction of apoptosis. Decreases in the amounts of these three ribosomal proteins were also observed in ribosome-enriched fractions. These changes were partly abolished by the addition of the pan-caspase inhibitor z-VAD-fmk. Moreover, formation of the 80S ribosome complex appeared to be inhibited at 48 h after apoptosis induction. On the other hand, the rate of protein synthesis, assessed by measuring the incorporation of [ 38 S]Met into bulk proteins, decreased as early as 12 h after the addition of doxorubicin. These results indicate that changes in the amount of ribosomal proteins and the overall structure of ribosomes in apoptosing cells occur after protein synthesis declines. Finally, analyses by flow cytometry, immunofluorescence, and Western blotting showed that six ribosomal proteins, S15, PO, L5, L6, L36a, and L41, were relocalized and expressed at the cell surface during apoptosis. The above results collectively indicate that ribosomes are structurally altered in apoptotic cells following inactivation of protein synthesis.Key words: apoptosis, protein degradation, protein synthesis, ribosomal protein, ribosome.Degradation of chromosomal DNA at the last stage in the though the precise function of individual ribosomal proteins process of apoptosis should result in the complete cessation and RNA has not yet been clarified, it is reasonable to of gene expression in cells fated to die. However, the rate of expect that the inhibition of protein synthesis in apoptotic protein synthesis decreases even before chromosomal DNA cells is the consequence of structural modification of these is degraded (1^1), suggesting the presence of an active molecules. In fact, the 28S ribosomal RNA is selectively mechanism that inhibits gene expression at the level of degraded in apoptotic cells (3,(5)(6)(7). This event occurs at relprotein synthesis in apoptotic cells. The molecular basis for atively early stages of apoptosis via both caspase-depenthis event, however, is not fully understood. dent and -independent mechanisms. It is, however, not A variety of proteins and nucleic acids are involved in clear whether degradation of the 28S RNA leads to strucprotein synthesis. The ribosome stands at the center of the tural changes of ribosomes or the inhibition of protein syntranslational machinery, and the eukaryotic 80S ribosome thesis. On the other hand, factors that are closely related to complex consists of two subunits, 40S and 60S, each of the translation reaction have been shown to undergo degrawhich contains a large number of proteins and RNA. Al-dation in apoptotic cells: eukaryotic translation initiation factors eIF2, eIF3, eIF4B, and eIF4G are degraded in a 1 This study was supported by Grante-in-Aid for Scientific Rese...

ZAK is required for doxorubicin, a novel ribotoxic stressor, to induce SAPK activation and apoptosis in HaCaT cells

“…Because doxorubicin also causes RNA damage 10 and inhibits DNA and RNA synthesis, 11,12 it is not unexpected that doxorubicin would also inhibit the synthesis of proteins. 13,14 In addition to inhibition of protein translation, doxorubicin induces the activation of SAPKs in a number of normal cell types, including hepatocytes, 6 primary mouse macrophages 7 and cardiomyocytes. 8,9 Our work presented here demonstrates that doxorubicin inhibits protein synthesis (Fig.…”

Rearrangement of nuclear ribonucleoprotein (RNP)‐containing structures during apoptosis and transcriptional arrest

“…Martelli et al (2000) reported that some nucleolar antigens (namely, PARP-1 and UBF) are cleaved during apoptosis, whereas others (fibrillarin, B23 and C23) are not; cleavage of topoisomerase I and B23 has been shown to occur in cis-DDP-induced apoptosis (Horky et al, 2001). Some autoantigens, such as ribosomal RNPs, previously described to resist proteolysis (Wu et al, 2001) have recently been shown to be cleaved and expressed at the cell surface: six proteins (S15, P0, L5, L6, L36a, L41) have been found to follow this pathway (Nishida et al, 2002). Nucleolin is one of the most studied protein in the nucleolus and it has recently been reported (Mi et al, 2003) to decrease in UV-induced apoptosis, in parallel with PARP-1 degradation.…”

Differential Expression of Ribosomal Proteins in Human Normal and Neoplastic Colorectum

“…However, the immunohistochemical (IHC) localization of ribosomal proteins in colorectal mucosa and cancer was largely unknown due to the limited availability of antibodies specific for human ribosomal proteins. In this study we have demonstrated the expression profile of 12 ribosomal proteins including Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a in human normal colorectal mucosa by IHC using a well-characterized antibody panel for the ribosomal proteins (Nadano et al 2000;Nishida et al 2002). We compared their expression patterns with those in colorectal adenocarcinoma cells.…”