Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The 1 JNC coupling constant for the (؊1) scissile peptide bond at the N-extein-intein junction was found to be Ϸ12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (؊1) scissile bond in its unusual conformation. Overall, these studies support the ''ground-state destabilization'' model as part of the mechanism of catalysis.

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

Conclusion:Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. General significance:The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipatic, since this feature seems to help the toroidal pores formation process. Highlights A new Temporin B analogue, TB_KKG6A, was designed.TB_KKG6A is active against Gram-positive and Gram-negative bacteria.The peptide strongly interacts with the LPS of E.coli.The peptide folds into a kinked helix upon interaction with LPS. The peptide shows very low hemolytic activity.

Peptide nucleic acids (PNAs) are DNA-mimicking molecules in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. We determined whether double-stranded molecules based on PNAs and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with specific transcription factors. We found that PNA-based hybrids do not inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging further experiments focused on the possible use of these molecules for the development of potential agents for a decoy approach in gene therapy. In this respect, the finding that PDPbased decoy molecules are more resistant than DNA/ DNA hybrids to enzymatic degradation appears to be of great interest. Furthermore, their resistance can even be improved after complexation with cationic liposomes to which PDP/PDP chimeras are able to bind by virtue of their internal DNA structure.In vitro transfection of cis elements that decoy against nuclear factors leads to alteration of gene expression and was recently proposed in molecular medicine as a novel tool for the possible therapy of several disorders (1-12). One of the most effective decoy approaches so far described involves nuclear proteins belonging to the NF-B 1 superfamily (7-9, 13-20).Decoy molecules against NF-B inhibit the expression of NF-B regulated genes (MHC complex genes, IL2 receptor ␣, Igk, IL6, ␦ opioid receptor, preprogalanin, adhesion molecule-1) (20). More recently, dumbell DNA decoy elements against NF-B were demonstrated to be active in inhibiting ex vivo transcription driven by the long terminal repeat (LTR) of human immunodeficiency type-1 virus (HIV-1) (19). In addition to proteins belonging to the NF-B superfamily, decoy molecules for other target transcription factors, such as HNF-1, RFX1, nuclear factor YB, E2F, cAMP-response element, and Sp1, were found to be effective (1-6, 10 -12, 21).As to the molecular targets for the decoy approach, proteins belonging to the Sp1 family are of great interest since these transcription factors are involved in the regulation of the expression of several genes relevant to human pathologies, including those encoding vascular endothelial growth factor, plasminogen-activator inhibitor type-1 (PAI-1), COL1A2, urokinase-type plasminogen activator (uPA), and uPA receptor (3,(21)(22)(23)(24)(25). Sp1 binding sites are also present in the HIV-1 LTR (26). Thus, the development of experimental approaches based on Sp1 decoys to modulate the transcription of Sp1-dependent genes appears to be of great interest (3).Rec...

The hypoxia-regulated alternative TrkAIII splice variant expressed by human neuroblastomas exhibits oncogenic potential, driven by in-frame exon 6 and 7 alternative splicing, leading to omission of the receptor extracellular immunoglobulin C 1 domain and several N-glycosylation sites. Here, we show that the TrkAIII oncogene promotes genetic instability by interacting with and exhibiting catalytic activity at the centrosome. This function depends upon intracellular TrkAIII accumulation and spontaneous interphase-restricted activation, in cytoplasmic tyrosine kinase (tk) domain orientation, predominantly within structures that closely associate with the fully assembled endoplasmic reticulum intermediate compartment and Golgi network. This facilitates TrkAIII tk-mediated binding of ␥-tubulin, which is regulated by endogenous protein tyrosine phosphatases and geldanamycin-sensitive interaction with Hsp90, paving the way for TrkAIII recruitment to the centrosome. At the centrosome, TrkAIII differentially phosphorylates several centrosome-associated components, increases centrosome interaction with polo kinase 4, and decreases centrosome interaction with separase, the net results of which are centrosome amplification and increased genetic instability. The data characterize TrkAIII as a novel internal membrane-associated centrosome kinase, unveiling an important alternative mechanism to "classical" cell surface oncogenic receptor tk signaling through which stressregulated alternative TrkAIII splicing influences the oncogenic process.Alternative splicing is fundamental for differential protein expression from the same gene and not only increases the proteomic complexity of higher organisms (29) but is also involved in cancer pathogenesis, activating several oncogenes and inactivating several oncosuppressors (17).The neurotrophin receptor tropomyosin-related kinase A (TrkA) is among the proto-oncogenes activated by alternative splicing, with a novel hypoxia-regulated oncogenic alternative TrkAIII splice variant recently identified in advanced-stage human neuroblastomas (NB) and primary glioblastomas (44,45). In contrast to wild-type TrkAI/TrkAII, the expression of which is associated with better prognosis for NB, induces NB cell differentiation, exhibits a tumor suppressor function in NB models in vivo (9,19,22,30,44,45), and may regulate both spontaneous and therapy-induced NB regression (30), TrkAIII is expressed by more-advanced-stage NB and exhibits oncogenic activity in NB models (44,45). This has challenged the hypothesis of an exclusively tumor-suppressing function for TrkA in NB by providing a way through which tumor-suppressing signals from TrkA can be converted to oncogenic signals from TrkAIII during tumor progression.The oncogenic potential of TrkAIII, characterized by NIH 3T3 cell transforming and NB xenograft primary and metastatic tumor-promoting activity (44,45), is driven by in-frame alternative splicing of exons 6 and 7. This results in the omission of the receptor extracellular immunoglobulin C 1 (Ig C...