Down-regulation of BRCA2 expression by collagen type I promotes prostate cancer cell proliferation

BRCA2 is a tumor suppressor gene that when mutated confers an increased susceptibility to developing breast and prostate carcinoma. Besides its role in mediating DNA repair, new evidence suggests that BRCA2 may also play a role in suppressing cancer cell growth. Because altered interactions between neoplastic cells and the surrounding extracellular matrix (ECM) play a pivotal role in unchecked cancer cell proliferation and metastatic progression, we hypothesized that the ECM may have an effect in BRCA2 expression. By using normal and prostate carcinoma cell lines, we demonstrated that although normal cells transiently increase BRCA2 protein levels when adhering to the ECM protein collagen type I (COL1), carcinoma cells exhibit a significant reduction in BRCA2 protein. This aberrant effect is independent from de novo protein synthesis and results from COL1-beta(1) integrin signaling through phosphatidylinositol (PI) 3-kinase leading to BRCA2 ubiquitination and degradation in the proteasome. BRCA2 protein depletion after cancer cell adhesion to COL1 or in small RNA interference assays triggers new DNA synthesis, a trophic effect that is abrogated by recombinant BRCA2 expression. Blocking or inhibiting beta(1) integrin, PI 3-kinase, or proteasome activity all have a negative effect on COL1-mediated DNA synthesis in cancer cells. In normal cells, the transient increase in BRCA2 expression is independent from beta(1) integrin or PI 3-kinase and has no effect in cell proliferation. In summary, these results unravel a novel mechanism whereby prostate carcinoma cell proliferation is enhanced by the down-regulation of BRCA2 expression when interacting with COL1, a major component of the ECM at osseous metastatic sites.     

Overexpression, purification and characterization of the acidic ribosomal P-proteins from Candida albicans

In all eukaryotic cells, acidic ribosomal P-proteins form a lateral protuberance on the 60S ribosomal subunit-the so-called stalk-structure that plays an important role during protein synthesis. In this work, we report for the first time a full-length cloning of four genes encoding the P-proteins from Candida albicans, their expression in Escherichia coli, purification and characterization of the recombinant proteins. Considerable amino acid sequence similarity was found between the cloned proteins and other known fungal ribosomal P-proteins. On the basis of their phylogenetic relationship and amino acid similarity to their yeast counterparts, the C. albicans P-proteins were named P1A, P1B, P2A and P2B. Using three different approaches, namely: chemical cross-linking method, gel filtration and two-hybrid system, we analyzed mutual interactions among the C. albicans P-proteins. The obtained data showed all the four P-proteins able to form homo-oligomeric complexes. However, the ones found between P1B-P2A and P1A-P2B were dominant forms among the C. albicans P-proteins. Moreover, the strength of interactions between particular proteins was different in these two complexes; the strongest interactions were observed between P1B and P2A proteins, and a significantly weaker one between P1A and P2B proteins.     

Conformational properties of N-acetyl-L-alanine N',N'-dimethylamide

Ab initio/DFT analysis of the conformational properties of free Ac-Ala-NMe(2) (N-acetyl-L-alanine-N',N'-dimethylamide) in terms of the N-H.O, N-H.N, C-H.O hydrogen bonds and C(delta+) = O(delta-) dipole attractions was performed. The Ala residue combined with the C-terminal tertiary amide prefers an extended conformation and that characteristic of the (i + 1)th position of the betaVIb turn. These can be easily remodelled into a structure compatible with the (i + 1)th position of the betaII/betaVIa turn. The residue has also the potential to adopt the conformation accommodated at both central positions of the betaIII/betaIII' turn or the (i + 1)th position of the betaI/beta'I turn.     

Conformational properties of N-acetyl-N-methyl-alpha,beta-dehydroalanine N'-methylamide

The conformational properties of Ac-Delta(Me)Ala-NHMe (N-acetyl-N-methyl-alpha,beta-dehydroalanine N'-methylamide), as the simplest model of N-methyl-alpha,beta-dehydroamino acids, was examined with theoretical methods and in comparison with Ac-DeltaAla-NHMe and Ac-DeltaAla-NMe(2). The N-terminal amide of the Delta(Me)Ala residue easily adopts the configuration cis and the torsion angles phi, psi are highly flexible. The Delta(Me)Ala residue is a conformational flexibilizer as compared to the parent DeltaAla, which is a conformational stiffener. This seems to be the reason why Delta(Me)Ala is found in small natural cyclic peptides, where it ensures the conformational flexibility necessary for biological action.     

Implication of the disulfide bridge in trypsin inhibitor SFTI-1 in its interaction with serine proteinases

Fourteen monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds were synthesized by the solid-phase method. The purpose of this work was to establish the role of a disulfide bridge present in inhibitor’s side chains of Cys3 and Cys11 in association with serine proteinases. This cyclic fragment was replaced by the disulfide bridges formed by l-pencillamine (Pen), homo-l-cysteine (Hcy), N-sulfanylethylglycine (Nhcy) or combination of the three with Cys. As in the substrate specificity the P₁ position of the synthesized analogues Lys, Nlys [N-(4-aminobutyl)glycine], Phe or Nphe (N-benzylglycine) were present, and they were checked for trypsin and chymotrypsin inhibitory activity. The results clearly indicated that Pen and Nhcy were not acceptable at the position 3, yielding inactive analogues, whereas another residue (Cys11) could be substituted without any significant impact on the affinity towards proteinase. On the other hand, elongation of the Cys3 side chain by introduction of Hcy did not affect inhibitory activity, and an analogue with the Hcy–Hcy disulfide bridge was more than twice as effective as the reference compound ([Phe⁵] SFTI-1) in inhibition of bovine α-chymotrypsin.     

Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms: A NOVEL THERAPEUTIC STRATEGY FOR ANGIOGENESIS*

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF₁₆₅, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF₁₆₅b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF₁₆₅ and less VEGF₁₆₅b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.     

Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.