Algorithmic computation of knot polynomials of secondary structure elements of proteins

The classification of protein structures is an important and still outstanding problem. The purpose of this paper is threefold. First, we utilize a relation between the Tutte and homfly polynomial to show that the Alexander-Conway polynomial can be algorithmically computed for a given planar graph. Second, as special cases of planar graphs, we use polymer graphs of protein structures. More precisely, we use three building blocks of the three-dimensional protein structure-alpha-helix, antiparallel beta-sheet, and parallel beta-sheet-and calculate, for their corresponding polymer graphs, the Tutte polynomials analytically by providing recurrence equations for all three secondary structure elements. Third, we present numerical results comparing the results from our analytical calculations with the numerical results of our algorithm-not only to test consistency, but also to demonstrate that all assigned polynomials are unique labels of the secondary structure elements. This paves the way for an automatic classification of protein structures.

Effect of antioxidants and ACE inhibition on chemical modification of proteins and progression of nephropathy in the streptozotocin diabetic rat

Aims/hypothesis. This study was designed to determine whether inhibition of formation of AGE and advanced lipoxidation end-products (ALE) is a mechanism of action common to a diverse group of therapeutic agents that limit the progress of diabetic nephropathy. We compared the effects of the ACE inhibitor enalapril, the antioxidant vitamin E, the thiol compound lipoic acid, and the AGE/ALE inhibitor pyridoxamine on the formation of AGE/ALE and protection against nephropathy in streptozotocin diabetic rats.

Molecular identity and cellular distribution of advanced glycation endproduct receptors: relationship of p60 to OST-48 and p90 to 80K-H membrane proteins.

Advanced glycation endproducts (AGEs) are derivatives of nonenzymatic reactions between sugars and protein or lipids, and together with AGE-specific receptors are involved in numerous pathogenic processes associated with aging and hyperglycemia. Two of the known AGE-binding proteins isolated from rat liver membranes, p60 and p90, have been partially sequenced. We now report that the N-terminal sequence of p60 exhibits 95% identity to OST-48, a 48-kDa member of the oligosaccharyltransferase complex found in microsomal membranes, while sequence analysis of p90 revealed 73% and 85% identity to the N-terminal and internal sequences, respectively, of human 80K-H, a 80- to 87-kDa protein substrate for protein kinase C. AGE-ligand and Western analyses of purified oligosaccharyltransferase complex, enriched rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membranes from rat liver or RAW 264.7 macrophages yielded a single protein of approximately 50 kDa recognized by both anti-p60 and anti-OST-48 antibodies, and also exhibited AGE-specific binding. Immunoprecipitated OST-48 from rat rough endoplasmic reticulum fractions exhibited both AGE binding and immunoreactivity to an anti-p60 antibody. Immune IgG raised to recombinant OST-48 and 80K-H inhibited binding of AGE-bovine serum albumin to cell membranes in a dose-dependent manner. Immunostaining and flow cytometry demonstrated the surface expression of OST-48 and 80K-H on numerous cell types and tissues, including mononuclear, endothelial, renal, and brain neuronal and glial cells. We conclude that the AGE receptor components p60 and p90 are identical to OST-48, and 80K-H, respectively, and that they together contribute to the processing of AGEs from extra- and intracellular compartments and in the cellular responses associated with these pathogenic substances.

The Delta Np63 Proteins Are Key Allies of BRCA1 in the Prevention of Basal-Like Breast Cancer

Little is known about the origin of basal-like breast cancers, an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues, and p63 expression may be a marker of basal-like breast cancers. Here we report that Delta Np63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless, we found that BRCA1 interacts with the specific p63 isoform Delta Np63 gamma along with transcription factor isoforms AP-2 alpha and AP-2 gamma. BRCA1 required Delta Np63 gamma and AP-2 gamma to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the Delta Np63 isoforms. In mammary stem/progenitor cells, siRNA- mediated knockdown of Delta Np63 expression resulted in genomic instability, increased cell proliferation, loss of DNA damage checkpoint control, and impaired growth control. Together, our findings establish that transcriptional upregulation of Delta Np63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-Delta Np63 signaling are key events in the pathogenesis of basal-like breast cancer. Cancer Res; 71( 5); 1933-44. (c) 2011 AACR.

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.

C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.

Use of synthetic peptides to locate novel integrin alpha(2)beta(1)-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triple-helical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

The GAL genetic switch: visualisation of the interacting proteins by split-EGFP bimolecular fluorescence complementation

A split-EGFP bimolecular fluorescence complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.