74 research outputs found
The Sam domain of the lipid phosphatase Ship2 adopts a common model to interact with Arap3-Sam and EphA2-Sam
<p>Abstract</p> <p>Background</p> <p>Sterile alpha motif (Sam) domains are small protein modules that can be involved in homotypic or heterotypic associations and exhibit different functions. Previous studies have demonstrated that the Sam domain of the lipid phosphatase Ship2 can hetero-dimerize with the Sam domain of the PI3K effector protein Arap3.</p> <p>Results</p> <p>Here, we determine the NMR solution structure of Arap3-Sam and implement a multidisciplinary approach consisting of NMR spectroscopy, ITC (Isothermal Titration Calorimetry), mutagenesis and molecular modeling studies to analyze the interaction between Ship2-Sam and Arap3-Sam. This work reveals that Arap3-Sam may associate with Ship2-Sam by adopting a binding mode common to other Sam domains. This binding mode is identical to what we have very recently observed for the association between Ship2-Sam and the Sam domain from the Ephrin A2 receptor.</p> <p>Conclusion</p> <p>Our studies further clarify the structural features that are relevant for Sam-Sam interactions involving Ship2 and give additional hints that could be used for the identification of new molecules able to selectively inhibit Sam-Sam associations.</p
Conformational disorder in phosphopeptides: solution studies by CD and NMR techniques
In the last few years intrinsically disordered
proteins (IDPs) have received great attention from the
scientific community as they participate in several
important biological processes and diseases. The intrinsic
disorder and flexibility of IDPs grant them a number of
advantages with respect to ordered proteins, such as
conformational plasticity to bind several targets, a large
interaction surface, involvement in high specificity/low
affinity interactions, enhanced binding kinetics. It is
assumed that post-translational modifications such as
phosphorylation can stimulate structural rearrangement
in IDPs and facilitate their binding to partners. To
better understand at a structural level the multifaceted
mechanisms that govern molecular recognition processes
involving IDPs, we designed, synthesized by solid phase
methods, and structurally characterized unstructured
peptides. These molecules contain a putative disordered
module, flanked at either the N- or C-terminal ends by a
different phosphorylated amino acid (serine or threonine)
to mimick the effects of phosphorylation. The absence
of an ordered state in the designed peptides was proved
experimentally by CD and NMR conformational studies
that were carried out under different solution conditions
Rational Design and Real Time, In-Cell Detection of the Proapoptotic Activity of a Novel Compound Targeting Bcl-XL
AbstractAntiapoptotic Bcl-2-family proteins Bcl-2 and Bcl-XL have been recently validated as drug discovery targets for cancer. Here, by using a combination of molecular modeling, NMR-based structural analysis, fluorescence polarization assays, and cell-based assays, we have designed and characterized a novel proapoptotic compound targeting these proteins. Our compound, Apogossypol, is capable of binding and inhibiting Bcl-2 and Bcl-XL with high affinity and induces apoptosis of tumor cell lines. Mechanistic studies on the action of our compound were also performed via confocal microscopy that provided real-time detection of the interaction with Bcl-XL in intact cells. Finally, preliminary data on cells freshly isolated from patients affected by chronic lymphocytic leukemia strongly suggest potential applications of Bcl-2 antagonists as chemosensitizers in cancer therapy
Conformational Ensembles Explored Dynamically from Disordered Peptides Targeting Chemokine Receptor CXCR4
This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target
Intrinsically disordered amphiphilic peptides as potential targets in drug delivery vehicles
Intrinsically disordered proteins/peptides play a crucial role in many physiological and pathological
events and may assume a precise conformation upon binding to a specific target. Recently, we have
described the conformational and functional properties of two linear ester peptides provided with the
following sequences: Y-G-E-C-P-C-K-OAllyl (PepK) and Y-G-E-C-P-C-E-OAllyl (PepE). Both peptides
are characterized by the presence of the ‘‘CPC’’ motif together with a few amino acids able to promote
disorder. The CPC sequence is a binding motif for the CXCR4 receptor that represents a well-known
target for cancer therapies. In this paper, we report on synthetic amphiphilic peptides that consist of
lipophilic derivatives of PepE and PepK bearing two stearic alkyl chains and/or an ethoxylic spacer.
These peptide amphiphiles form stable supramolecular aggregates; they present conformational features
that are typical of intrinsically disordered molecules as shown by CD spectroscopy. Solution
fluorescence and DLS studies have been performed to evaluate Critical Micellar Concentrations and the
dimension of supramolecular aggregates. Moreover, preliminary in vitro cell-based assays have been
conducted to investigate the molecular recognition processes involving the CXCR4 receptor. In the
end, the results obtained have been compared with the previous data generated by the corresponding
non-amphiphilic peptides (PepE and PepK)
Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia
Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization
Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia
Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization
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