Proliferating cell nuclear antigen (PCNA), a cell cycle marker protein, is well known as a DNA sliding clamp for DNA polymerase delta and as an essential component for eukaryotic chromosomal DNA replication and repair. Due to its mobility inside nuclei, PCNA is dynamically presented in a soluble or chromatin-associated form. The heterogeneity and specific modifications of PCNA may reflect its multiple functions and the presence of many binding partners in the cell. The recent proteomics approaches applied to characterizing PCNA interactions revealed multiple PCNA partners with a wide spectrum of activity and unveiled the possible existence of new PCNA functions. Since more than 100 PCNA-interacting proteins and several PCNA modifications have already been reported, a proteomics point of view seems exactly suitable to better understand the role of PCNA in cellular functions.

Persistent infection with high-risk human papillomaviruses (HPVs), especially type 16 has been undeniably linked to cervical cancer. The Asian-American (AA) variant of HPV16 is more common in the Americas than the prototype in cervical cancer. The different prevalence is based on three amino acid changes within the E6 protein denoted Q14H/H78Y/L83V. To investigate the mechanism(s) behind this observation, both E6 proteins, in the presence of E7, were evaluated for their ability to extend the life span of and transform primary human foreskin keratinocytes (PHFKs). Longterm cell culture studies resulted in death at passage 9 of vector-transduced PHFKs (negative control), but survival of both E6 PHFKs to passage 65 (and beyond). Compared with E6/E7 PHFKs, AA/E7 PHFKs were significantly faster dividing, developed larger cells in monolayer cultures, showed double the epithelial thickness and expressed cytokeratin 10 when grown as organotypic raft cultures. Telomerase activation and p53 inactivation, two hallmarks of immortalization, were not significantly different between the two populations. Both were resistant to anoikis at later passages, but only AA/E7 PHFKs acquired the capacity for in vitro transformation. Proteomic analysis revealed markedly different protein patterns between E6/E7 and AA/E7, particularly with respect to key cellular metabolic enzymes. Our results provide new insights into the reasons underlying the greater prevalence of the AA variant in cervical cancer as evidenced by characteristics associated with higher oncogenic potential.

The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini ("front") sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase ␦. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex.

The final goal of the Russian part of the Chromosome-centric Human Proteome Project (C-HPP) was established as the analysis of the chromosome 18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells with the sensitivity of 10(-18) M. Using SRM, we have recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2 cells. On the basis of the results of the survey, the SRM assays were drafted for 250 proteins: 41 proteins were found only in the liver tissue, 82 proteins were specifically detected in depleted plasma, and 127 proteins were mapped in both samples. The targeted analysis of HepG2 cells was carried out for 49 proteins; 41 of them were successfully registered using ordinary SRM and 5 additional proteins were registered using a combination of irreversible binding of proteins on CN-Br Sepharose 4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq and RT-PCR has shown a significant correlation (r = 0.78) for 42 gene transcripts. A pilot affinity-based interactome analysis was performed for cytochrome b5 using analytical and preparative optical biosensor fishing followed by MS analysis of the fished proteins. All of the data on the proteome complement of the Chr 18 have been integrated into our gene-centric knowledgebase ( www.kb18.ru ).

MINT‐7995351: G3P (uniprotkb:P04406) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT‐7995334: ENOA (uniprotkb:P06733) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT‐7995368: ALDOA (uniprotkb:P04075) and PCNA (uniprotkb:P12004) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT‐7995141: G3P (uniprotkb:P04406) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995182: ENOA (uniprotkb:P06733) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995132: G3P (uniprotkb:P04406) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995228: PRDX6 (uniprotkb:P30041) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995220: CAH2 (uniprotkb:P00918) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995114: Triosephosphate isomerase (uniprotkb:P60174) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995244: K2C7 (uniprotkb:P08729) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995252: ANXA2 (uniprotkb:P07355) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995122: Triosephosphate isomerase (uniprotkb:P60174) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995093: ALDOA (uniprotkb:P04075) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995148: PGK1 (uniprotkb:P00558) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995158: PGAM1 (uniprotkb:P18669) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995166: PGAM1 (uniprotkb:P18669) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995105: ALDOA (uniprotkb:P04075) binds (MI:0407) to PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995260: PPIA (uniprotkb:P62937) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995173: ENOA (uniprotkb:P06733) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995268: EF1A (uniprotkb:P68104) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995236: MDHM (uniprotkb:P40926) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995189: RSSA (uniprotkb:P08865) physically interacts (MI:0915) with PCNA (uniprotkb:P12004) by far western blotting (MI:0047)MINT‐7995282: PCNA (uniprotkb:P12004) physically interacts (MI:0915) with ALDOA (uniprotkb:P00883) and G3P (uniprotkb:P46406) by anti bait coimmunoprecipitation (MI:0006).

The diverse function of proliferating cell nuclear antigen (PCNA) is thought to be due, in large part, to posttranslational modifications. Here we show by high resolution two-dimensional PAGE analysis that there are three distinct PCNA isoforms that differ in their acetylation status. The moderately acetylated main (M) form was found in all of the subcellular compartments of cycling cells, whereas the highly acetylated acidic form was primarily found in the nucleoplasm, nuclear matrix, and chromatin. Interestingly, the deacetylated basic form was most pronounced in the nucleoplasm of cycling cells. The cells in G 0 and the cytoplasm of cycling cells contained primarily the M form only. Because p300 and histone deacetylase (HDAC1) were co-immunoprecipitated with PCNA, they are likely responsible for the acetylation and deacetylation of PCNA, respectively. We also found that deacetylation reduced the ability of PCNA to bind to DNA polymerases ␤ and ␦. Taken together, our data support a model where the acidic and M forms participate in DNA replication, whereas the basic form is associated with the termination of DNA replication. PCNA1 participates in a broad spectrum of biological activities, including DNA replication, repair, cell cycle control, apoptosis, and chromatin remodeling (1-3). Several reports (4 -7) previously suggested the existence of different PCNA species, which is thought to be relevant to the diverse functions of PCNA. Furthermore, we have shown recently by high resolution two-dimensional PAGE and immunoblot analyses that both hamster and human cells contain three different PCNA isoforms, the acidic (A), the main (M), and the basic (B) forms (8). The different PCNA isoforms could be the result of posttranslational modifications, especially phosphorylation status (9 -12). Consistent with this hypothesis, Prosperi et al. (13) presented data that support the phosphorylation of PCNA. In contrast, however, Bravo and Celis (14) did not find any evidence of PCNA phosphorylation. We wanted to address this issue by carrying out several different experimental approaches, including in vivo labeling, immunoprecipitation, and immunoblot analysis in combination with high resolution twodimensional PAGE. Here we show that mammalian PCNA is regulated not by phosphorylation but by acetylation. We have also shown that three distinct PCNA isoforms differ in their acetylation status. Our data further show that acetylation is associated with subcellular localization of PCNA. Moreover, deacetylated PCNA by HDAC1 has lower affinity to DNA polymerases ␤ and ␦ than that of PCNA treated with trichostatin A (TSA, an HDAC inhibitor). Most important, the PCNA-DNA polymerase complex treated with HDAC1 showed lower polymerization activity than a TSA-treated control. Taken together, our data are consistent with the idea that acetylated PCNA is involved in the DNA replication process, whereas deacetylation of PCNA is associated with the termination of DNA replication. MATERIALS AND METHODSReagents-All the reagents used are from ...

In order to clarify the status of PCNA in normal and transformed cells, we performed analysis of this protein by 2D-PAGE, Western blot and mass spectrometry. All the cell lines examined contained the major PCNA form (pI 4.57/30 kDa), that is not post-translationally modified. In addition to the major form, two minor isoforms (pI 4.52/30 kDa and pI 4.62/30 kDa) were also detected in all the cell lines examined. However, the level of PCNA in cancer cells is 5-6 folds higher than those in primary and most of the immortalized cells. Taken together, the significant difference in PCNA status between cancer and normal cells is not at the post-translational modifications but in the overall levels of PCNA. Crown

Virtual and experimental 2DE coupled with ESI LC-MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI-TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC-MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw (http://web.expasy.org/compute_pi/pi_tool-doc.html). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.