Two-Dimensional Gel Electrophoresis as an Information Base for Human Proteome

The main intricacy in the human proteome is that it is tremendously complex and composed of diverse and heterogeneous gene products. These products are called protein species or proteoforms and are the smallest units of the proteome. In pursuit of the comprehensive profiling of the human proteome, significant advances should be performed. The approaches that allow disclosing and keeping the information about human proteome using two-dimensional gel electrophoresis (2DE) are described. Experimental identification methods such as mass spectrometry of high resolution and sensitivity (MALDI-TOF MS and ESI LC-MS/MS) or immunodetection in combination with bioinformatics and 2DE can be used for the development of a comprehensive knowledge base of the human proteome

Proliferating cell nuclear antigen in the cytoplasm interacts with components of glycolysis and cancer

AbstractProliferating cell nuclear antigen (PCNA) is involved in a wide range of functions in the nucleus. However, a substantial amount of PCNA is also present in the cytoplasm, although their function is unknown. Here we show, through Far-Western blotting and mass spectrometry, that PCNA is associated with several cytoplasmic oncoproteins, including elongation factor, malate dehydrogenase, and peptidyl-prolyl isomerase. Surprisingly, PCNA is also associated with six glycolytic enzymes that are involved in the regulation of steps 4–9 in the glycolysis pathway.Structured summaryMINT-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)

Acetylation of Dna2 Endonuclease/Helicase and Flap Endonuclease 1 by p300 Promotes DNA Stability by Creating Long Flap Intermediates

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α

A chromatin-bound kinase, ERK8, protects genomic integrity by inhibiting HDM2-mediated degradation of the DNA clamp PCNA

ERK8 prevents genome instability by blocking the association of the HDM2 E3 ubiquitin ligase with the genome-protecting protein PCNA

Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival

Cytosolic proliferating cell nuclear antigen (PCNA) binds to procaspases and protects human neutrophils from apoptosis

Biochemical properties and base excision repair complex formation of apurinic/apyrimidinic endonuclease from Pyrococcus furiosus

Apurinic/apyrimidinic (AP) sites are the most frequently found mutagenic lesions in DNA, and they arise mainly from spontaneous base loss or modified base removal by damage-specific DNA glycosylases. AP sites are cleaved by AP endonucleases, and the resultant gaps in the DNA are repaired by DNA polymerase/DNA ligase reactions. We identified the gene product that is responsible for the AP endonuclease activity in the hyperthermophilic euryarchaeon, Pyrococcus furiosus. Furthermore, we detected the physical interaction between P. furiosus AP endonuclease (PfuAPE) and proliferating cell nuclear antigen (PCNA; PfuPCNA) by a pull-down assay and a surface plasmon resonance analysis. Interestingly, the associated 3′–5′ exonuclease activity, but not the AP endonuclease activity, of PfuAPE was stimulated by PfuPCNA. Immunoprecipitation experiments using the P. furiosus cell extracts supported the interaction between PfuAPE and PfuPCNA in the cells. This is the first report describing the physical and functional interactions between an archaeal AP endonuclease and PCNA. We also detected the ternary complex of PfuPCNA, PfuAPE and Pfu uracil-DNA glycosylase. This complex probably functions to enhance the repair of uracil-containing DNA in P. furiosus cells

Protein Profile Changes during Porcine Oocyte Aging and Effects of Caffeine on Protein Expression Patterns

It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality

Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity

Functional Complexity of the Axonal Growth Cone: A Proteomic Analysis

The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions