Structural Evaluation and Analyses of Tumor Differentiation Factor

Tumor differentiation factor (TDF) is a protein produced by the pituitary and secreted into the blood stream. The mechanism of its action has still not been elucidated, although the associated protein receptor was identified. Furthermore, the TDF protein does not have any homology with other known proteins, and the crystal structure of TDF also is not available at this time. To gain some insight into the structure of this rather underexplored protein, we have performed a molecular dynamics simulation of a model TDF structure. The structural stability of this protein is evaluated as a function of time. The time dependent structural changes of four cysteine residues present in this structure also are explored.     

Comparative two‐dimensional polyacrylamide gel electrophoresis of the salivary proteome of children with autism spectrum disorder

In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two‐dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P‐value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC‐MS/MS. Alpha‐amylase, CREB‐binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down‐regulated in ASD. Increased expression of proto‐oncogene Frequently rearranged in advanced T‐cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin‐inducible protein precursor, Mucin‐16, Ca binding protein migration inhibitory factor‐related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD.     

Purified mouse egg zona pellucida glycoproteins polymerize into homomeric fibrils under non-denaturing conditions

The mouse egg's zona pellucida (ZP) is composed of three glycoproteins, called ZP1, ZP2, and ZP3, that migrate as relatively broad, single bands on SDS-PAGE. The glycoproteins are organized within the ZP as a network of long interconnected fibrils that exhibit a structural periodicity. Here, ZP2 and ZP3 were purified by HPLC to homogeneity and analyzed by Blue Native- (BN-) PAGE and transmission electron microscopy (TEM), as well as by SDS-PAGE. As opposed to SDS-PAGE, BN-PAGE, and TEM permit analysis of ZP2 and ZP3 under non-denaturing conditions. ZP2 and ZP3 migrate on BN-PAGE, not as single bands, but as several discrete oligomers that give rise to larger structures which remain at the origin of the gel. Consistent with this, ZP2 and ZP3 are visualized by TEM as long interconnected fibrils that consist of contiguous beads. Therefore, under non-denaturing conditions both purified ZP2 and ZP3 polymerize into higher order structures. These findings are of interest since purified ZP3 inhibits binding of mouse sperm to eggs and induces sperm to undergo the acrosome reaction in vitro. Results presented here suggest that these biological effects of ZP3 are due to binding of homomeric fibrils of ZP3 to sperm.     

Selective ‘stencil’-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a ‘stencil’, a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil–wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.     

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.     

Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.     

Mass spectrometric evidence that proteolytic processing of rainbow trout egg vitelline envelope proteins takes place on the egg

The rainbow trout egg vitelline envelope (VE) is constructed of three proteins, called VEalpha,VEbeta, and VEgamma, that are synthesized and secreted by the liver and transported in the bloodstream to the ovary, the site of VE assembly around eggs. All three proteins possess an N-terminal signal peptide, a zona pellucida domain, a consensus furin-like cleavage site (CFLCS) close to the C terminus, and a short propeptide downstream of the CFLCS. Proteolytic processing at the CFLCS results in loss of the short C-terminal propeptide from precursor proteins and enables incorporation of mature proteins into the VE. Here mass spectrometry (matrix-assisted laser desorption ionization time-of-flight-mass spectrometry and liquid chromatography-mass spectrometry with a micromass-quadrupole TOF hybrid mass and a QSTAR Pulsar i mass spectrometer) was employed with VE proteins isolated from rainbow trout eggs in a peptidomics-based approach to determine the following: 1) the C-terminal amino acid of mature, proteolytically processed VE proteins; 2) the cellular site of proteolytic processing at the CFLCS of VE precursor proteins; and 3) the relationship between proteolytic processing and limited covalent cross-linking of VE proteins. Peptides derived from the C-terminal region were found for all three VE proteins isolated from eggs, indicating that processing at the CFLCS occurs after the arrival of VE precursor proteins at the egg. Consistent with this conclusion, peptides containing an intact CFLCS were also found for all three VE proteins isolated from eggs. Furthermore, peptides derived from the C-terminal propeptides of VE protein heterodimers VEalpha-VEgamma and VEbeta-VEgamma were found, suggesting that a small amount of VE protein can be covalently cross-linked on eggs prior to proteolytic processing at the CFLCS. Collectively, these results provide important evidence about the process of VE formation in rainbow trout and other non-cyprinoid fish and allow comparisons to be made with the process of zona pellucida formation in mammals.     

Cerebellar Oxidative DNA Damage and Altered DNA Methylation in the BTBR T+tf/J Mouse Model of Autism and Similarities with Human Post Mortem Cerebellum

The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (Ogg1) and increased expression of de novo DNA methyltransferases 3a and 3b (Dnmt3a and Dnmt3b). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression 1 (Tet1) and Tet2 genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of OGG1 alterations in pathogenesis of autism.     

Neophyte Invasion in Moldavia (Eastern Romania) in Different Habitat Types

The actual state of neophyte invasion in Moldavia (Eastern Romania) is described in this paper on the basis of 11,055 phytosociological relevés. We analyzed the i) proportion of relevés with neophytes, ii) mean proportion of neophytes per relevés, and iii) mean coverage of neophytes per relevé for 36 EUNIS habitat types to identify general plant invasion patterns. The level of invasion differed considerably between habitats. The invasion of neophytes especially affected habitats strongly determined or influenced by man, such as anthropogenic woodlands, ruderal habitats, arable lands or trampled areas. Most natural habitats are either slightly invaded or entirely free of neophytes. Only riverine willow stands and wet tall-herb stands are relatively highly invaded. However, the absence of neophytes in some natural habitats less represented in the phytosociological dataset could be of artifactual nature. No significant relationship between the number of neophytes and non-neophytes was found in the analysis across different habitats. When the analyses were made within-habitats, both negative and positive relationships were found, which confirm that the relationship between alien and native species richness depends on the habitats. A total number of 105 neophyte species were recorded in the phytosociological relevés used in this study. Among these, 13 species that are currently considered invasive in Moldavia occur in at least 10 types of habitats.