Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

Structure-based design of small bicyclic peptide inhibitors of Cripto-1 activity.

Bicyclic peptides assembled around small organic scaffolds are gaining an increasing interest as new potent, stable and highly selective therapeutics because of their uncommon ability to specifically recognize protein targets, of their small size that favor tissue penetration and of the versatility and easiness of the synthesis. We have here rationally designed bicyclic peptides assembled around a common tri-bromo-methylbenzene moiety in order to mimic the structure of the CFC domain of the oncogene Cripto-1 and, more specifically, to orient in the most fruitful way the hot spot residues H120 and W123. Through the CFC domain, Cripto-1 binds the ALK4 receptor and other protein partners supporting uncontrolled cell growth and proliferation. Soluble variants of CFC have the potential to inhibit these interactions suppressing the protein activity. A CFC analog named B3 binds ALK4 in vitro with an affinity in the nanomolar range. Structural analyses in solution via NMR and CD show that B3 has rather flexible conformations, like the parent CFC domain. The functional effects of B3 on the Cripto-1-positive NTERA cancer cell line have been evaluated showing that both CFC and B3 are cytotoxic for the cells and block the Cripto-1 intracellular signaling. Altogether, the data suggest that the administration of the soluble CFC and of the structurally related analog has the potential to inhibit tumor growth.

Structural Perspective of Gliadin Peptides Active in Celiac Disease.

Gluten fragments released in gut of celiac individuals activate the innate or adaptive immune systems. The molecular mechanisms associated with the adaptive response involve a series of immunodominant gluten peptides which are mainly recognized by human leucocyte antigen (HLA)-DQ2.5 and HLA-DQ8. Other peptides, such as A-gliadin P31-43, are not recognized by HLA and trigger innate responses by several routes not yet well detailed. Among the gluten fragments known to be active in Celiac disease, here we focus on the properties of all gluten peptides with known tri-dimensional structure either those locked into HLA-DQ complexes whose crystals were X-ray analyzed or characterized in solution as free forms. The aim of this work was to find the structural reasons why some gluten peptides prompt the adaptive immune systems while others do not, by apparently involving just the innate immune routes. We propose that P31-43 is a non-adaptive prompter because it is not a good ligand for HLA-DQ. Even sharing a similar ability to adopt polyproline II structure with the adaptive ones, the way in which the proline residues are located along the sequence disfavors a productive P31-43-HLA-DQ binding.

Structural insights on P31-43, a gliadin peptide able to promote an innate but not an adaptive response in celiac disease.

Inflammation of intestinal tissue in patients affected by celiac disease (CD) originates from the adaptive and innate immune responses elicited by the undigested gliadin fragments through molecular mechanisms not yet completely described. Undigested A-gliadin peptide P31-43 is central to CD pathogenesis, entering enterocytes in vesicular compartments by endocytosis and inducing an innate immune response in CD intestinal mucosa. This study focused on the reasons why P31-43 does not behave as adaptive immunogenic agent. Once obtained by NMR analysis, the three-dimensional model of P31-43 was used to implement a series of in silico experiments aimed to explore the ability of the peptide to interact with HLA-DQ2 and the corresponding receptor onto T cells. Our results show that P31-43 is a poor ligand for DQ2 and/or T-cell receptor. This study was also aimed to investigate, from a structural point of view, the previous experimental findings by which P31-43 is able to enhance the phosphorylation level of the protein ERK2, while some P31-43 Ala-mutants decrease or totally inhibit that process. The molecular models of P31-43, P31-43 P36A, and F37A mutants were used for in silico docking experiments onto the ERK2 structure. The experiments support the hypothesis that P31-43 F37A works as an ERK2 phosphorylation inhibitor because it binds to the ERK2 phosphorylation site. This study reports on the structural properties of so far never NMR characterized gliadin peptides relevant in CD and explores details about their mechanisms of action.

PASTA sequence composition is a predictive tool for protein class identification.

PASTA domains are small modules expressed in bacteria and found in one or multiple copies at the C-terminal end of several penicillin binding proteins (PBPs) and Ser/Thr protein kinases (STPKs) and represent potential targets for a new class of antibiotics. PASTA domains are currently annotated as sensor domains, as they are thought to activate their cognate proteins in response to binding to opportune ligands. However, recent studies have shown that PASTA domains linked to proteins of different classes, STPKs or PBPs, do not share the same binding abilities. Despite this, there is currently no way to distinguish between PASTA domains from the two classes, since all of them share the same fold, independent of the class they belong to. To identify a predictive tool of class identification, we here analyse a pool of parameters, including amino acid compositions and total charges of PASTA domains either linked to PBPs or to STPKs. We screened sequences from Actinobacteria, Firmicutes and Bacteroidetes. The first two phyla include some of the most dangerous micro-organisms for human health such as Mycobacterium tuberculosis and Staphylococcus aureus. Based on this analysis, our study proposes a predictive method to assign PASTA domains with unknown origin to their corresponding enzyme class, based solely on sequence information.

Targeting VEGF receptors with non-neutralizing cyclopeptides for imaging applications.

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them 'mini-factors'. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.

Structural Basis for Mutations of Human Aquaporins Associated to Genetic Diseases.

Aquaporins (AQPs) are among the best structural-characterized membrane proteins, fulfilling the role of allowing water flux across cellular membranes. Thus far, 34 single amino acid polymorphisms have been reported in HUMSAVAR for human aquaporins as disease-related. They affect AQP2, AQP5 and AQP8, where they are associated with nephrogenic diabetes insipidus, keratoderma and colorectal cancer, respectively. For half of these mutations, although they are mostly experimentally characterized in their dysfunctional phenotypes, a structural characterization at a molecular level is still missing. In this work, we focus on such mutations and discuss what the structural defects are that they appear to cause. To achieve this aim, we built a 3D molecular model for each mutant and explored the effect of the mutation on all of their structural features. Based on these analyses, we could collect the structural defects of all the pathogenic mutations (here or previously analysed) under few main categories, that we found to nicely correlate with the experimental phenotypes reported for several of the analysed mutants. Some of the structural analyses we present here provide a rationale for previously experimentally observed phenotypes. Furthermore, our comprehensive overview can be used as a reference frame for the interpretation, on a structural basis, of defective phenotypes of other aquaporin pathogenic mutants.

A synthetic BMP-2 mimicking peptide induces glioblastoma stem cell differentiation.

BACKGROUND: Glioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. In this context, Bone Morphogenetic Proteins (BMPs) have been demonstrated to induce CSC differentiation and to sensitize GBM cells to treatments. METHODS: The BMP-2 mimicking peptide, named GBMP1a, was synthesized on solid-phase by Fmoc chemistry. Structural characterization and prediction of receptor binding were obtained by Circular Dicroism (CD) and NRM analyses. Activation of BMP signalling was evaluated by a luciferase reporter assay and western blot. Pro-differentiating effects of GBMP1a were verified by immunostaining and neurosphere assay in primary glioblastoma cultures. RESULTS: CD and NMR showed that GBMP1a correctly folds into expected tridimensional structures and predicted its binding to BMPR-IA to the same epitope as in the native complex. Reporter analysis disclosed that GBMP1a is able to activate BMP signalling in GBM cells. Moreover, BMP-signalling activation was specifically dependent on smad1/5/8 phosphorylation. Finally, we confirmed that GBMP1a treatment is sufficient to enhance osteogenic differentiation of Mesenchymal Stem Cells and to induce astroglial differentiation of glioma stem cells (GSCs) in vitro. CONCLUSIONS: GBMP1a was demonstrated to be a good inducer of GSC differentiation, thus being considered a potential anti-cancer tool to be further developed for GBM treatment. GENERAL SIGNIFICANCE: These data highlight the role of BMP-mimicking peptides as potential anti-cancer agents against GBM and stimulate the further development of GBMP1a-based structures in order to enhance its stability and activity.

Structural insights into the interaction of a monoclonal antibody and Nodal peptides by STD-NMR spectroscopy.

Nodal is a growth factor expressed during early embryonic development, but reactivated in several advanced-stage cancers. Targeting of Nodal signaling, which occurs via the binding to Cripto-1 co-receptor, results in inhibition of cell aggressiveness and reduced tumor growth. The Nodal binding region to Cripto-1 was identified and targeted with a high affinity monoclonal antibody (3D1). By STD-NMR technique, we investigated the interaction of Nodal fragments with 3D1 with the aim to elucidate at atomic level the interaction surface. Data indicate with high accuracy the antibody-antigen contact atoms and confirm the information previously obtained by immune-enzymatic methods. Main residues contacted by 3D1 are P46, V47, E49 and E50, which belong to the Nodal loop involved in the interaction with the co-receptor.

Analysis of the interface variability in NMR structure ensembles of protein-protein complexes.

NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html.

Essential dynamics analysis captures the concerted motion of the integrin-binding site in jerdostatin, an RTS disintegrin.

Disintegrins, small molecular weight proteins contained in the venom of vipers and rattlesnakes, are high-affinity and selectivity integrin antagonists. Disintegrins inhibitory epitope mainly consists in a tripeptide sequence localized in a mobile loop protruding from the protein core. RTS and/or KTS tripeptide characterizes the most recently discovered group of disintegrins that selectively block α1ß1 integrin receptor. A NMR study dedicated to structure and dynamics properties of jerdostatin, an RTS disintegrin, demonstrated that the substitution of the native RTS with KTS motif impaired flexibility and inhibitory activity of the molecule. Here we add atomic details to the experimental profiles of jerdostatin and its R24K mutant by analyzing the dynamics behavior of the molecules through computational methods. For jerdostatin wild type, molecular dynamics simulations and essential dynamics analyses showed that Y31 residue acts as hinge element in the concerted motions involving the active loop and the C-terminal tail. R24 side chain ability to engage both cation-π and H-bond interactions with Y31 residue was found crucial for that breathing mechanism. Less significant loop-tail concerted motions were observed for the R24K mutant. The description at atomic resolution of jerdostatin dynamics is useful for decoding the influence of specific residues on disintegrin functional properties.

Conformational features and binding affinities to Cripto, ALK7 and ALK4 of Nodal synthetic fragments.

Nodal, a member of the TGF-ß superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-ßs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44-67 of the Nodal protein, corresponding to the pre-helix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here, we show the structural characterization in solution by CD and NMR of the Nodal peptides and the measurement of binding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and binding measurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners.

Osteogenic properties of a short BMP-2 chimera peptide.

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide-receptor binding interface were highlighted by docking experiments. Peptide-receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73-92 and 30-34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2.

Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis.

PonA2 is one of the two class A penicillin binding proteins of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. It plays a complex role in mycobacterial physiology and is spotted as a promising target for inhibitors. PonA2 is involved in adaptation of M. tuberculosis to dormancy, an ability which has been attributed to the presence in its sequence of a C-terminal PASTA domain. Since PASTA modules are typically considered as ß-lactam antibiotic binding domains, we determined the solution structure of the PASTA domain from PonA2 and analyzed its binding properties versus a plethora of potential binders, including the ß-lactam antibiotics, two typical muropeptide mimics, and polymeric peptidoglycan. We show that, despite a high structural similarity with other PASTA domains, the PASTA domain of PonA2 displays different binding properties, as it is not able to bind muropeptides, or ß-lactams, or polymeric peptidoglycan. These results indicate that the role of PASTA domains cannot be generalized, as their specific binding properties strongly depend on surface residues, which are widely variable.

Dynamical properties of cold shock protein A from Mycobacterium tuberculosis.

Bacterial cold shock proteins (Csps) are over-expressed as response to cold stress. They have a role in transcriptional and translational events due to their ability to bind single stranded (ss) nucleic acids. Csps so far characterized show similar structures with a closed five stranded antiparallel ß-barrel. Here we report a structural and functional study of cold shock protein A from Mycobacterium tuberculosis, MTB-CspA. Structural investigations by CD and NMR reveal that MTB-CspA is less ordered than expected and is the least thermal stable cold shock protein so far characterized. However, electrophoretic mobility shift assays show that MTB-CspA is functionally active, as it is able to bind oligonucleotides. The dynamic behavior of MTB-CspA, compared to its homolog from Bacillus subtilis, was investigated by molecular dynamics simulations at room and high temperatures. Analysis of trajectories point to specific regions on ß1 and ß4 strands, likely responsible for the higher structural fragility of MTB-CspA. Also, they show that the nucleic-acid binding region of MTB-CspA is the least prone to unfolding, a finding which explains the ability of MTB-CspA to exert its function.

Structural and dynamic studies provide insights into specificity and allosteric regulation of ribonuclease as, a key enzyme in mycobacterial virulence.

Ribonuclease AS (RNase AS) is a crucial enzyme for virulence of Mycobacterium tuberculosis. We previously observed that RNase AS structurally resembles RNase T from Escherichia coli, an important enzyme for tRNA maturation and turnover. Here, we combine X-ray crystallography and molecular dynamics (MD) to investigate the specificity and dynamic properties of substrate binding. Both X-ray and MD data provide structural determinants that corroborate the strict substrate specificity of RNase AS to cleave only adenosine residues, due to the structural features of adenine base. Beside suggesting tRNA as most likely substrate of RNase AS, MD and modeling studies identify key enzyme-ligand interactions, both involving the catalytic site and the double helix region of tRNA, which is locked by interactions with a set of arginine residues. The MD data also evidence a ligand-induced conformational change of the enzyme which is transferred from one chain to the adjacent one. These data will explain the dimeric nature of both RNase AS and RNase T, with two catalytic grooves composed of both chains. Also, they account for the dichotomy of tRNA, which contains both the substrate poly(A) chain and an inhibiting double strand RNA. Indeed, they provide a possible mechanism of allosteric regulation, which unlocks one catalytic groove when the second groove is inhibited by the double strand region of tRNA. Finally, a full comprehension of the molecular details of tRNA maturation processes is essential to develop novel strategies to modulate RNA processing, for therapeutic purposes. AbbreviationsMDmolecular dynamicsPDBProtein Data BankRMSDroot mean square deviationRMSFroot mean square fluctuationRNAribonucleotidic acidRNase ASRibonuclease ASCommunicated by Ramasamy H. Sarma.

Insights into the Interaction Mechanism of DTP3 with MKK7 by Using STD-NMR and Computational Approaches.

GADD45ß/MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45ß/MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect; however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45ß/MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.

EAK Hydrogels Cross-Linked by Disulfide Bonds: Cys Number and Position Are Matched to Performances.

Hydrogels produced by self-assembling peptides are intrinsically biocompatible and thus appropriate for many biomedical purposes. Their application field may be even made wider by reducing the softness and improving the hydrogel mechanical properties through cross-linking treatments. To this aim, modifications of EAK16-II sequence by including Cys residues in its sequence were here investigated in order to obtain hydrogels cross-linkable through a disulfide bridge. Two sequences, namely, C-EAK and C-EAK-C, that contain Cys residues at the N-terminus or at both ends were characterized. Fiber-forming abilities and biological and dynamic mechanical properties were explored before and after the oxidative treatment. In particular, the oxidized version of C-EAK presents a good cell viability and sustains osteoblast proliferation. Furthermore, molecular dynamics (MD) simulations on monomeric and assembled forms of the peptides were performed. MD simulations explained how a specific Cys functionalization was better than the other one. In particular, the results suggested that EAK16-II functionalization with a single Cys residue, instead of two, together with biocompatible cross-linking may be considered an intriguing strategy to obtain a support with better dynamic mechanical properties and biological performances.

Identification and characterization of cytotoxic amyloid-like regions in human Pbx-regulating protein-1.

The ability of many proteins to fold into well-defined structures has been traditionally considered a prerequisite for fulfilling their functions. Protein folding is also regarded as a valuable loophole to escape uncontrolled and harmful aggregations. Here we show that the PBX-regulating protein-1 (PREP1), an important homeodomain transcription factor involved in cell growth and differentiation during embryogenesis, is endowed with an uncommon thermostability. Indeed, circular dichroism analyses indicate that it retains most of its secondary structure at very high temperatures. These findings have important implications for PREP1 functions since it is a stabilizing factor of its partner PBX1. Predictive analyses suggest that the observed PREP1 thermostability could be related to the presence of aggregation-prone regions. Interestingly, synthetic peptides corresponding to these regions exhibit a remarkable propensity to form toxic ß-rich amyloid-like aggregates in physiological conditions. On this basis, we suggest that PREP1 stability is an effective way to prevent or limit the formation of harmful aggregates. Notably, one of these PREP1 fragments (residues 117-132) is able to reversibly switch from α-helical to ß-rich states depending on the environmental conditions. The chameleon conformational behavior of this peptide makes it an ideal system to study this intriguing and widespread structural transition.

Generation and testing of engineered multimeric Fabs of trastuzumab.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.