Senescence-Associated Changes in Proteome andO-GlcNAcylation Pattern in Human Peritoneal Mesothelial Cells

Abstract

Introduction. Senescence of peritoneal mesothelial cells represents a biological program defined by arrested cell growth and altered cell secretory phenotype with potential impact in peritoneal dialysis. This study aims to characterize cellular senescence at the level of global protein expression profiles and modification of proteins with O-linked N-acetylglucosamine (O-GlcNAcylation). Methods. A comparative proteomics analysis between young and senescent human peritoneal mesothelial cells (HPMC) was performed using two-dimensional gel electrophoresis. O-GlcNAc status was assessed by Western blot under normal conditions and after modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease O-GlcNAcylation or O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAcylation. Results. Comparison of protein pattern of senescent and young HPMC revealed 29 differentially abundant protein spots, 11 of which were identified to be actin (cytoplasmic 1 and 2), cytokeratin-7, cofilin-2, transgelin-2, Hsp60, Hsc70, proteasome β-subunits (type-2 and type-3), nucleoside diphosphate kinase A, and cytosolic 5′(3′)-deoxyribonucleotidase. Although the global level of O-GlcNAcylation was comparable, senescent cells were not sensitive to modulation by PUGNAc. Discussion. This study identified changes of the proteome and altered dynamics of O-GlcNAc regulation in senescent mesothelial cells. Whereas changes in cytoskeleton-associated proteins likely reflect altered cell morphology, changes in chaperoning and housekeeping proteins may have functional impact on cellular stress response in peritoneal dialysis.

Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress

“…Of these proteins, 990 were identified and quantified in all three treatments (Table and Supplemental Table 1). Moreover, 318 proteins identified and quantified in this study have previously been annotated as O- GlcNAc-modified (Supplemental Table 1 ,,,,,− ). Finally, only 18 of the identified proteins are predicted to be membrane associated or secreted, suggesting that the majority of the proteins are likely modified by O- GlcNAc or associated with an O- GlcN...…”

Proteomic Research in Peritoneal Dialysis

“…Changes in the senescent phenotype of mesothelial cells were mainly found in cytoskeleton-associated proteins, which may contribute to the abnormal morphology of senescent cells. Changes were also found in housekeeping and chaperone proteins, with potential functional impact on cellular stress response in PD [28]. Modification of mesothelial cells with O-linked-N-acetylglucosamine (O-Glc-NAc), a posttranslational protein modification relevant in cell survival, did not differ between young and senescent cells, though in senescent cells altered dynamics of O-Glc-NAc regulation were identified.…”

“…In a more recent study, Herzog et al [28] performed a comparative proteomic analysis between young and senescent human mesothelial cells. Cellular senescence is a biological program initiated by various forms of stress, which is characterized by arrested cell growth and alterations in cell secretory phenotype, and that is of potential impact in PD.…”

Cellular functions of the ADF/cofilin family at a glance

“…Furthermore, lack of a readily available antibody against CFL2 has delayed deeper understanding of the role of this isoform. We now know that many tissues express all three isoforms, including oligodendrocytes, keratinocytes and cancerous tissue of non-muscle origin (Kanellos et al, 2015;Zuchero et al, 2015), and that CFL2 is a major constituent in human peritoneal mesothelial cells (Herzog et al, 2015). It is possible that tissues that express multiple isoforms might fine tune their expression levels individually in order to tightly control the rates of actin turnover, whereas the types of accessory protein that decorate actin filaments and the expression patterns of cofilin ancillary proteins (see Box 2) modify their efficiency.…”