Annamaria Sandomenico - Academia.edu (original) (raw)

Papers by Annamaria Sandomenico

Research paper thumbnail of Peptides as modulators of protein-protein interactions

Research paper thumbnail of Probing the interaction interface of the GADD45β/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry

International Journal of Biological Macromolecules, Jul 1, 2018

GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expressio... more GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Δ(101-136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.

Research paper thumbnail of Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

Cell Death and Disease, Nov 7, 2013

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and a... more Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.

Research paper thumbnail of High sensitive reflection type long period fiber grating biosensor for real time detection of thyroglobulin, a differentiated thyroid cancer biomarker: the Smart Health project

Proceedings of SPIE, Sep 28, 2015

We report the development of a reflection-type long period fiber grating (LPG) biosensor able to ... more We report the development of a reflection-type long period fiber grating (LPG) biosensor able to perform the real time detection of thyroid cancer markers in the needle washout of fine-needle aspiration biopsy. A standard LPG is first transformed in a practical probe working in reflection mode, then it is coated by an atactic-polystyrene overlay in order to increase its surrounding refractive index sensitivity and to provide, at the same time, the desired interfacial properties for a stable bioreceptor immobilization. The results provide a clear demonstration of the effectiveness and sensitivity of the developed biosensing platform, allowing the in vitro detection of human Thyroglobulin at sub-nanomolar concentrations.

Research paper thumbnail of Branched Peptides for the Modulation of Protein-Protein Interactions: More Arms are Better than One?

Current Medicinal Chemistry, Jun 1, 2011

Research paper thumbnail of Intrinsic structural versatility of the highly conserved 412–423 epitope of the Hepatitis C Virus E2 protein

International Journal of Biological Macromolecules, Sep 1, 2018

HCV infection is a major threaten for human health as it affects hundreds of million people world... more HCV infection is a major threaten for human health as it affects hundreds of million people worldwide. Here we investigated the conformational properties of the 412-423 fragment of the envelope E2 protein, one of the most immunogenic regions of the virus proteome whose characterization may provide interesting insights for anti-HCV vaccine development. The spectroscopic characterization of the polypeptide unravels its unexpected tendency to form amyloid-like aggregates. When kept in monomeric state, it shows a limited tendency to adopt regular secondary structure. Enhanced molecular dynamics simulations, starting from four distinct conformational states, highlight its structural versatility. Interestingly, all multiform conformational states of the polypeptide detected in crystallographic complexes with antibodies are present in the structural ensemble of all simulations. This observation corroborates the idea that known antibodies recognize this region through a conformational selection mechanism. Accordingly, the design of effective anti-HCV vaccines should consider the intrinsic flexibility of this region. The structural versatility of the 412-423 region is particularly puzzling if its remarkable sequence conservation is considered. It is likely that flexibility and sequence conservation are important features that endow this epitope with the ability to accomplish distinct functions such as immunity escape and interaction with host receptors.

Research paper thumbnail of Long period fiber grating nano-optrode for cancer biomarker detection

Biosensors and Bioelectronics, Jun 1, 2016

We report an innovative fiber optic nano-optrode based on Long Period Gratings (LPGs) working in ... more We report an innovative fiber optic nano-optrode based on Long Period Gratings (LPGs) working in reflection mode for the detection of human Thyroglobulin (TG), a protein marker of differentiated thyroid cancer. The reflection-type LPG (RT-LPG) biosensor, coated with a single layer of atactic polystyrene (aPS) onto which a specific, high affinity anti-Tg antibody was adsorbed, allowed the label-free detection of Tg in the needle washouts of fine-needle aspiration biopsies, at concentrations useful for pre-and postoperative assessment of the biomarker levels. Analyte recognition and capture were confirmed with a parallel on fiber ELISA-like assay using, in pilot tests, the biotinylated protein and HRP-labeled streptavidin for its detection. Dose-dependent experiments showed that the detection is linearly dependent on concentration within the range between 0 and 4 ng/mL, while antibody saturation occurs for higher protein levels. The system is characterized by a very high sensitivity and specificity allowing the ex-vivo detection of sub ng/ml concentrations of human Tg from needle washouts of fine-needle aspiration biopsies of thyroid nodule from different patients.

Research paper thumbnail of The role of Nodal and Cripto‐1 in human oral squamous cell carcinoma

Oral Diseases, 2020

Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal a... more Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal and Cripto‐1 (CR‐1) are important developmental morphogens expressed in several adult cancers and are associated with disease progression. Whether Nodal and CR‐1 are simultaneously expressed in the same tumor and how this affects cancer biology are unclear. We investigate the expression and potential role of both Nodal and CR‐1 in human OSCC. Immunohistochemistry results show that Nodal and CR‐1 are both expressed in the same human OSCC sample and that intensity of Nodal staining is correlated with advanced‐stage disease. However, this was not observed with CR‐1 staining. Western blot analysis of lysates from two human OSCC line experiments shows expression of CR‐1 and Nodal, and their respective signaling molecules, Src and ERK1/2. Treatment of SCC25 and SCC15 cells with both Nodal and CR‐1 inhibitors simultaneously resulted in reduced cell viability and reduced levels of P‐Src and P‐ERK...

Research paper thumbnail of Protein-Protein Interactions: A Simple Strategy to Identify Binding Sites and Peptide Antagonists

Chemical Biology & Drug Design, May 1, 2009

Abbreviations: CARD, caspase recruitment domain; CD, circular dichroism; DED, death effector doma... more Abbreviations: CARD, caspase recruitment domain; CD, circular dichroism; DED, death effector domain; Gadd45b, growth arrest and DNA damage-inducible factor 45b; IC 50 , concentration giving 50% inhibition; LC-MS, liquid chromatography-mass spectrometry; PED ⁄ PEA-15, phosphoprotein enriched in diabetes ⁄ phosphoprotein enriched in astrocytes; PLD1, phospholipase 1; TFE, trifluoroethanol.

Research paper thumbnail of Expression and purification of the D4 region of PLD1 and characterization of its interaction with PED-PEA15

Protein Expression and Purification, Jun 1, 2008

PLD&a... more PLD's (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx(4)Dx(6)GSxN motifs. PLD1 interacts with the small phosphoprotein PED-PEA15 by an unknown mechanism that, by enhancing PLD1 stability, apparently increases its enzymatic activity; the minimum interacting region of PLD1 was previously identified as spanning residues 712-1074 (D4 region). Since the D4/PED-PEA15 interaction has been claimed to be one of the multiple molecular events that can trigger type 2 diabetes, we purified the two recombinant proteins to study in vitro this binding by both ELISA and SPR techniques. Whilst PED-PEA15 was easily expressed and purified, expression of recombinant D4 was more problematic and only the fusion protein with Thioredoxin A and a six Histidine Tag (Trx-His(6)-D4) demonstrated sufficient stability for further characterization. We have found that Trx-His(6)-D4 is present as two different oligomeric forms, though only the monomeric variant is able to interact with PED-PEA15. All these findings may have important implications for both the mechanisms of phospholipase activity and PED-PEA15 regulative functions.

Research paper thumbnail of High sensitivity lab-on-fiber biosensing platform assisted by oriented antibody immobilization strategy

Research paper thumbnail of An IgE receptor mimetic peptide (PepE) protects mice from IgE mediated anaphylaxis

Molecular BioSystems, 2013

Crosslinking of receptor-bound Immunoglobulin E (IgE) triggers immediate hypersensitivity reactio... more Crosslinking of receptor-bound Immunoglobulin E (IgE) triggers immediate hypersensitivity reactions including anaphylaxis. Blocking the interaction of IgE with its high-affinity receptor, FcεRI, on mast cells and basophils is an attractive strategy for the treatment of allergies. This approach has seen clinical success using the anti-IgE monoclonal antibody, omalizumab. We recently designed and characterized a novel FcεRI-mimetic peptide (PepE) which contains the two key FcεRI α-chain receptor loops known to interact with the ε-heavy chain of IgE, C′-E and B-C, with an optimized linker for joining them. PepE has high specificity and affinity for IgE, blocks IgE binding to FcεRI and prevents IgE induced mediator release from RBL2H3 cells. We have now investigated the biological effects of this peptide in vivo using a line of mice (BALB/c Il4raF709) very sensitive to IgE-mediated systemic anaphylaxis. IgE-deficient (IgE −/−) Il4raF709 mice were passively sensitized with the anti-DNP IgE monoclonal antibody (SPE-7) and subsequently challenged i.v. with DNP-BSA. Mice receiving a single dose of PepE prior to sensitization with SPE-7 IgE, were fully protected from anaphylaxis while vehicle control-treated mice displayed strong reactions with significant core body temperature drops and elevated levels of mouse mast cell protease-1 (mMCP-1) in the serum. However, PepE had no effect on IgEmediated anaphylaxis if given after IgE administration in IgE −/− mice, suggesting that PepE can block binding of free IgE to FcεRI but cannot compete with the receptor for already bound IgE in vivo. A single dose of PepE treatment did not protect IgE sufficient mice from IgE mediated anaphylaxis. However, a 3-week long course of PepE treatment protected IgE sufficient Il4raF709 mice from body temperature drops and elevation of serum mMCP-1. Our findings establish the potential of this type of structure for blocking IgE binding to mast cells in vivo and suggest that related peptides might have the potential to attenuate clinical allergic reactions.

Research paper thumbnail of Unveiling CD59-Antibody Interactions to Design Paratope-Mimicking Peptides for Complement Modulation

International Journal of Molecular Sciences, May 10, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Targeting Nodal and Cripto-1: Perspectives Inside Dual Potential Theranostic Cancer Biomarkers

Current Medicinal Chemistry, Jun 28, 2019

Research paper thumbnail of Automatic procedures for the synthesis of difficult peptides using oxyma as activating reagent: A comparative study on the use of bases and on different deprotection and agitation conditions

Peptides, Apr 1, 2018

Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthe... more Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as "difficult" poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well-known "difficult peptides" prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N-methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of "difficult peptides".

Research paper thumbnail of An Engineered RNA Platform to Neutralize DNMT1 Function and Control DNA Methylation for Myelodysplastic Syndrome

Blood, Nov 5, 2020

DNA methylation is a major signature involved in the regulation of gene expression. Numerous stud... more DNA methylation is a major signature involved in the regulation of gene expression. Numerous studies have established a link between aberrant DNA methylation and cancer (Herman and Baylin 2003, Baylin and Jones 2011, Feinberg 2018). Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic malignancies, characterized by ineffective hematopoiesis, cytopenia and risk of progression to acute myeloid leukemia (AML) in approximately 30% of the cases (Khan, Vale et al. 2013, Arber, Orazi et al. 2016). Abnormal DNA methylation is considered the molecular lesion leading to tumor suppressor gene silencing and clonal variation in MDS and evolution to AML (Figueroa, Skrabanek et al. 2009, Jiang, Dunbar et al. 2009, Feinberg 2018). In the past decades two nucleoside-based compounds, 5-azacytidine and 5-aza 2'-deoxycytidine have been extensively tested to reduce global DNA methylation levels and received approval by the U.S. Food and Drug Administration (FDA) for the treatment of MDS. Both drugs have been used as the frontline therapy for the management of higher-risk-to-transform MDS, that are ineligible for more aggressive treatment such as: allogenic transplants and standard chemotherapy. Unfortunately, cytotoxic and global non-specific demethylation effects, have limited their clinical application, revealing the need for smarter and safer epigenetic drugs. Nucleic-acid aptamers are a new class of specific targeting molecules and represent high affinity ligands and potential antagonists of disease-associated proteins. Herein, we present an innovative RNA aptamer-based approach to target DNMT1 function by translating the inherent neutralizing properties of RNA (Di Ruscio, Ebralidze et al. Nature, 2013, 503(7476):371-6) into an aptamer platform. Through a combinatorial chemistry strategy named SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Mercier, Dontenwill et al. 2017), we shortlisted the most specific DNMT1-neutralizing RNA aptamers. These molecules display high specificity and serum stability and reveal strong structural affinity in targeting DNMT1, by molecular modelling and dynamic simulations. Foremost, we found that the selected aptamers specifically inhibit DNMT1 enzymatic activity both in vitro and in cells, thus impairing cell viability and leading to global demethylation. Collectively, this study provides a proof-of-concept for the generation of the first RNA-based epigenetic therapy as well as an attractive and scalable tool of precision medicine. Such a tool will overcome the toxicity of in-use demethylating protocols and greatly improve care and treatment of patients with MDS and other disease triggered by aberrant DNA methylation. Figure No relevant conflicts of interest to declare.

Research paper thumbnail of Peptides binding the type E immunoglobulins

Advances in Experimental Medicine and Biology, 2009

Research paper thumbnail of The Screening of Combinatorial Peptide Libraries for Targeting Key Molecules or Protein–Protein Interactions in the NF-κB Pathway

Springer eBooks, 2021

Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of can... more Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45β, in multiple myeloma (MM). We showed that the GADD45β/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45β/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.

Research paper thumbnail of Members of the GADD45 Protein Family Show Distinct Propensities to form Toxic Amyloid-Like Aggregates in Physiological Conditions

International Journal of Molecular Sciences, Oct 2, 2021

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Structural Versatility of Hepatitis C Virus Proteins: Implications for the Design of Novel Anti-HCV Intervention Strategies

Current Medicinal Chemistry, Nov 24, 2017

Research paper thumbnail of Peptides as modulators of protein-protein interactions

Research paper thumbnail of Probing the interaction interface of the GADD45β/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry

International Journal of Biological Macromolecules, Jul 1, 2018

GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expressio... more GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Δ(101-136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.

Research paper thumbnail of Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

Cell Death and Disease, Nov 7, 2013

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and a... more Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.

Research paper thumbnail of High sensitive reflection type long period fiber grating biosensor for real time detection of thyroglobulin, a differentiated thyroid cancer biomarker: the Smart Health project

Proceedings of SPIE, Sep 28, 2015

We report the development of a reflection-type long period fiber grating (LPG) biosensor able to ... more We report the development of a reflection-type long period fiber grating (LPG) biosensor able to perform the real time detection of thyroid cancer markers in the needle washout of fine-needle aspiration biopsy. A standard LPG is first transformed in a practical probe working in reflection mode, then it is coated by an atactic-polystyrene overlay in order to increase its surrounding refractive index sensitivity and to provide, at the same time, the desired interfacial properties for a stable bioreceptor immobilization. The results provide a clear demonstration of the effectiveness and sensitivity of the developed biosensing platform, allowing the in vitro detection of human Thyroglobulin at sub-nanomolar concentrations.

Research paper thumbnail of Branched Peptides for the Modulation of Protein-Protein Interactions: More Arms are Better than One?

Current Medicinal Chemistry, Jun 1, 2011

Research paper thumbnail of Intrinsic structural versatility of the highly conserved 412–423 epitope of the Hepatitis C Virus E2 protein

International Journal of Biological Macromolecules, Sep 1, 2018

HCV infection is a major threaten for human health as it affects hundreds of million people world... more HCV infection is a major threaten for human health as it affects hundreds of million people worldwide. Here we investigated the conformational properties of the 412-423 fragment of the envelope E2 protein, one of the most immunogenic regions of the virus proteome whose characterization may provide interesting insights for anti-HCV vaccine development. The spectroscopic characterization of the polypeptide unravels its unexpected tendency to form amyloid-like aggregates. When kept in monomeric state, it shows a limited tendency to adopt regular secondary structure. Enhanced molecular dynamics simulations, starting from four distinct conformational states, highlight its structural versatility. Interestingly, all multiform conformational states of the polypeptide detected in crystallographic complexes with antibodies are present in the structural ensemble of all simulations. This observation corroborates the idea that known antibodies recognize this region through a conformational selection mechanism. Accordingly, the design of effective anti-HCV vaccines should consider the intrinsic flexibility of this region. The structural versatility of the 412-423 region is particularly puzzling if its remarkable sequence conservation is considered. It is likely that flexibility and sequence conservation are important features that endow this epitope with the ability to accomplish distinct functions such as immunity escape and interaction with host receptors.

Research paper thumbnail of Long period fiber grating nano-optrode for cancer biomarker detection

Biosensors and Bioelectronics, Jun 1, 2016

We report an innovative fiber optic nano-optrode based on Long Period Gratings (LPGs) working in ... more We report an innovative fiber optic nano-optrode based on Long Period Gratings (LPGs) working in reflection mode for the detection of human Thyroglobulin (TG), a protein marker of differentiated thyroid cancer. The reflection-type LPG (RT-LPG) biosensor, coated with a single layer of atactic polystyrene (aPS) onto which a specific, high affinity anti-Tg antibody was adsorbed, allowed the label-free detection of Tg in the needle washouts of fine-needle aspiration biopsies, at concentrations useful for pre-and postoperative assessment of the biomarker levels. Analyte recognition and capture were confirmed with a parallel on fiber ELISA-like assay using, in pilot tests, the biotinylated protein and HRP-labeled streptavidin for its detection. Dose-dependent experiments showed that the detection is linearly dependent on concentration within the range between 0 and 4 ng/mL, while antibody saturation occurs for higher protein levels. The system is characterized by a very high sensitivity and specificity allowing the ex-vivo detection of sub ng/ml concentrations of human Tg from needle washouts of fine-needle aspiration biopsies of thyroid nodule from different patients.

Research paper thumbnail of The role of Nodal and Cripto‐1 in human oral squamous cell carcinoma

Oral Diseases, 2020

Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal a... more Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal and Cripto‐1 (CR‐1) are important developmental morphogens expressed in several adult cancers and are associated with disease progression. Whether Nodal and CR‐1 are simultaneously expressed in the same tumor and how this affects cancer biology are unclear. We investigate the expression and potential role of both Nodal and CR‐1 in human OSCC. Immunohistochemistry results show that Nodal and CR‐1 are both expressed in the same human OSCC sample and that intensity of Nodal staining is correlated with advanced‐stage disease. However, this was not observed with CR‐1 staining. Western blot analysis of lysates from two human OSCC line experiments shows expression of CR‐1 and Nodal, and their respective signaling molecules, Src and ERK1/2. Treatment of SCC25 and SCC15 cells with both Nodal and CR‐1 inhibitors simultaneously resulted in reduced cell viability and reduced levels of P‐Src and P‐ERK...

Research paper thumbnail of Protein-Protein Interactions: A Simple Strategy to Identify Binding Sites and Peptide Antagonists

Chemical Biology & Drug Design, May 1, 2009

Abbreviations: CARD, caspase recruitment domain; CD, circular dichroism; DED, death effector doma... more Abbreviations: CARD, caspase recruitment domain; CD, circular dichroism; DED, death effector domain; Gadd45b, growth arrest and DNA damage-inducible factor 45b; IC 50 , concentration giving 50% inhibition; LC-MS, liquid chromatography-mass spectrometry; PED ⁄ PEA-15, phosphoprotein enriched in diabetes ⁄ phosphoprotein enriched in astrocytes; PLD1, phospholipase 1; TFE, trifluoroethanol.

Research paper thumbnail of Expression and purification of the D4 region of PLD1 and characterization of its interaction with PED-PEA15

Protein Expression and Purification, Jun 1, 2008

PLD&a... more PLD's (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx(4)Dx(6)GSxN motifs. PLD1 interacts with the small phosphoprotein PED-PEA15 by an unknown mechanism that, by enhancing PLD1 stability, apparently increases its enzymatic activity; the minimum interacting region of PLD1 was previously identified as spanning residues 712-1074 (D4 region). Since the D4/PED-PEA15 interaction has been claimed to be one of the multiple molecular events that can trigger type 2 diabetes, we purified the two recombinant proteins to study in vitro this binding by both ELISA and SPR techniques. Whilst PED-PEA15 was easily expressed and purified, expression of recombinant D4 was more problematic and only the fusion protein with Thioredoxin A and a six Histidine Tag (Trx-His(6)-D4) demonstrated sufficient stability for further characterization. We have found that Trx-His(6)-D4 is present as two different oligomeric forms, though only the monomeric variant is able to interact with PED-PEA15. All these findings may have important implications for both the mechanisms of phospholipase activity and PED-PEA15 regulative functions.

Research paper thumbnail of High sensitivity lab-on-fiber biosensing platform assisted by oriented antibody immobilization strategy

Research paper thumbnail of An IgE receptor mimetic peptide (PepE) protects mice from IgE mediated anaphylaxis

Molecular BioSystems, 2013

Crosslinking of receptor-bound Immunoglobulin E (IgE) triggers immediate hypersensitivity reactio... more Crosslinking of receptor-bound Immunoglobulin E (IgE) triggers immediate hypersensitivity reactions including anaphylaxis. Blocking the interaction of IgE with its high-affinity receptor, FcεRI, on mast cells and basophils is an attractive strategy for the treatment of allergies. This approach has seen clinical success using the anti-IgE monoclonal antibody, omalizumab. We recently designed and characterized a novel FcεRI-mimetic peptide (PepE) which contains the two key FcεRI α-chain receptor loops known to interact with the ε-heavy chain of IgE, C′-E and B-C, with an optimized linker for joining them. PepE has high specificity and affinity for IgE, blocks IgE binding to FcεRI and prevents IgE induced mediator release from RBL2H3 cells. We have now investigated the biological effects of this peptide in vivo using a line of mice (BALB/c Il4raF709) very sensitive to IgE-mediated systemic anaphylaxis. IgE-deficient (IgE −/−) Il4raF709 mice were passively sensitized with the anti-DNP IgE monoclonal antibody (SPE-7) and subsequently challenged i.v. with DNP-BSA. Mice receiving a single dose of PepE prior to sensitization with SPE-7 IgE, were fully protected from anaphylaxis while vehicle control-treated mice displayed strong reactions with significant core body temperature drops and elevated levels of mouse mast cell protease-1 (mMCP-1) in the serum. However, PepE had no effect on IgEmediated anaphylaxis if given after IgE administration in IgE −/− mice, suggesting that PepE can block binding of free IgE to FcεRI but cannot compete with the receptor for already bound IgE in vivo. A single dose of PepE treatment did not protect IgE sufficient mice from IgE mediated anaphylaxis. However, a 3-week long course of PepE treatment protected IgE sufficient Il4raF709 mice from body temperature drops and elevation of serum mMCP-1. Our findings establish the potential of this type of structure for blocking IgE binding to mast cells in vivo and suggest that related peptides might have the potential to attenuate clinical allergic reactions.

Research paper thumbnail of Unveiling CD59-Antibody Interactions to Design Paratope-Mimicking Peptides for Complement Modulation

International Journal of Molecular Sciences, May 10, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Targeting Nodal and Cripto-1: Perspectives Inside Dual Potential Theranostic Cancer Biomarkers

Current Medicinal Chemistry, Jun 28, 2019

Research paper thumbnail of Automatic procedures for the synthesis of difficult peptides using oxyma as activating reagent: A comparative study on the use of bases and on different deprotection and agitation conditions

Peptides, Apr 1, 2018

Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthe... more Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as "difficult" poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well-known "difficult peptides" prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N-methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of "difficult peptides".

Research paper thumbnail of An Engineered RNA Platform to Neutralize DNMT1 Function and Control DNA Methylation for Myelodysplastic Syndrome

Blood, Nov 5, 2020

DNA methylation is a major signature involved in the regulation of gene expression. Numerous stud... more DNA methylation is a major signature involved in the regulation of gene expression. Numerous studies have established a link between aberrant DNA methylation and cancer (Herman and Baylin 2003, Baylin and Jones 2011, Feinberg 2018). Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic malignancies, characterized by ineffective hematopoiesis, cytopenia and risk of progression to acute myeloid leukemia (AML) in approximately 30% of the cases (Khan, Vale et al. 2013, Arber, Orazi et al. 2016). Abnormal DNA methylation is considered the molecular lesion leading to tumor suppressor gene silencing and clonal variation in MDS and evolution to AML (Figueroa, Skrabanek et al. 2009, Jiang, Dunbar et al. 2009, Feinberg 2018). In the past decades two nucleoside-based compounds, 5-azacytidine and 5-aza 2'-deoxycytidine have been extensively tested to reduce global DNA methylation levels and received approval by the U.S. Food and Drug Administration (FDA) for the treatment of MDS. Both drugs have been used as the frontline therapy for the management of higher-risk-to-transform MDS, that are ineligible for more aggressive treatment such as: allogenic transplants and standard chemotherapy. Unfortunately, cytotoxic and global non-specific demethylation effects, have limited their clinical application, revealing the need for smarter and safer epigenetic drugs. Nucleic-acid aptamers are a new class of specific targeting molecules and represent high affinity ligands and potential antagonists of disease-associated proteins. Herein, we present an innovative RNA aptamer-based approach to target DNMT1 function by translating the inherent neutralizing properties of RNA (Di Ruscio, Ebralidze et al. Nature, 2013, 503(7476):371-6) into an aptamer platform. Through a combinatorial chemistry strategy named SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Mercier, Dontenwill et al. 2017), we shortlisted the most specific DNMT1-neutralizing RNA aptamers. These molecules display high specificity and serum stability and reveal strong structural affinity in targeting DNMT1, by molecular modelling and dynamic simulations. Foremost, we found that the selected aptamers specifically inhibit DNMT1 enzymatic activity both in vitro and in cells, thus impairing cell viability and leading to global demethylation. Collectively, this study provides a proof-of-concept for the generation of the first RNA-based epigenetic therapy as well as an attractive and scalable tool of precision medicine. Such a tool will overcome the toxicity of in-use demethylating protocols and greatly improve care and treatment of patients with MDS and other disease triggered by aberrant DNA methylation. Figure No relevant conflicts of interest to declare.

Research paper thumbnail of Peptides binding the type E immunoglobulins

Advances in Experimental Medicine and Biology, 2009

Research paper thumbnail of The Screening of Combinatorial Peptide Libraries for Targeting Key Molecules or Protein–Protein Interactions in the NF-κB Pathway

Springer eBooks, 2021

Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of can... more Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45β, in multiple myeloma (MM). We showed that the GADD45β/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45β/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.

Research paper thumbnail of Members of the GADD45 Protein Family Show Distinct Propensities to form Toxic Amyloid-Like Aggregates in Physiological Conditions

International Journal of Molecular Sciences, Oct 2, 2021

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Structural Versatility of Hepatitis C Virus Proteins: Implications for the Design of Novel Anti-HCV Intervention Strategies

Current Medicinal Chemistry, Nov 24, 2017