Publications by authors named "Francesca Malagrinò"

24 Publications

  • Page 1 of 1

Folding and Misfolding of a PDZ Tandem Repeat.

J Mol Biol 2021 Apr 1;433(7):166862. Epub 2021 Feb 1.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy. Electronic address:

Although the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein Whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of Whirlin (PDZ1 and PDZ2 - namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics.
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http://dx.doi.org/10.1016/j.jmb.2021.166862DOI Listing
April 2021

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery.

Int J Mol Sci 2021 Jan 15;22(2). Epub 2021 Jan 15.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli' and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy.

Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted 'energetic coupling' describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein-ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.
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http://dx.doi.org/10.3390/ijms22020828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830974PMC
January 2021

Targeting the Interaction between the SH3 Domain of Grb2 and Gab2.

Cells 2020 11 7;9(11). Epub 2020 Nov 7.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy.

Gab2 is a scaffolding protein, overexpressed in many types of cancers, that plays a key role in the formation of signaling complexes involved in cellular proliferation, migration, and differentiation. The interaction between Gab2 and the C-terminal SH3 domain of the protein Grb2 is crucial for the activation of the proliferation-signaling pathway Ras/Erk, thus representing a potential pharmacological target. In this study, we identified, by virtual screening, seven potential inhibitor molecules that were experimentally tested through kinetic and equilibrium binding experiments. One compound showed a remarkable effect in lowering the affinity of the C-SH3 domain for Gab2. This inhibitory effect was subsequently validated in cellula by using lung cancer cell lines A549 and H1299. Our results are discussed under the light of previous works on the C-SH3:Gab2 interaction.
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http://dx.doi.org/10.3390/cells9112435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695167PMC
November 2020

Comparing the binding properties of peptides mimicking the Envelope protein of SARS-CoV and SARS-CoV-2 to the PDZ domain of the tight junction-associated PALS1 protein.

Protein Sci 2020 10 8;29(10):2038-2042. Epub 2020 Sep 8.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, Rome, Italy.

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS-CoV and SARS-CoV-2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ-binding motif at its C-terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS-CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS-CoV and SARS-CoV-2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS-CoV-2 compared to SARS-CoV may rely on the increased affinity of its Envelope protein for PALS1.
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http://dx.doi.org/10.1002/pro.3936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461438PMC
October 2020

Hidden kinetic traps in multidomain folding highlight the presence of a misfolded but functionally competent intermediate.

Proc Natl Acad Sci U S A 2020 08 3;117(33):19963-19969. Epub 2020 Aug 3.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy;

Although more than 75% of the proteome is composed of multidomain proteins, current knowledge of protein folding is based primarily on studies of isolated domains. In this work, we describe the folding mechanism of a multidomain tandem construct comprising two distinct covalently bound PDZ domains belonging to a protein called Whirlin, a scaffolding protein of the hearing apparatus. In particular, via a synergy between NMR and kinetic experiments, we demonstrate the presence of a misfolded intermediate that competes with productive folding. In agreement with the view that tandem domain swapping is a potential source of transient misfolding, we demonstrate that such a kinetic trap retains native-like functional activity, as shown by the preserved ability to bind its physiological ligand. Thus, despite the general knowledge that protein misfolding is intimately associated with dysfunction and diseases, we provide a direct example of a functionally competent misfolded state. Remarkably, a bioinformatics analysis of the amino acidic sequence of Whirlin from different species suggests that the tendency to perform tandem domain swapping between PDZ1 and PDZ2 is highly conserved, as demonstrated by their unexpectedly high sequence identity. On the basis of these observations, we discuss on a possible physiological role of such misfolded intermediate.
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http://dx.doi.org/10.1073/pnas.2004138117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443948PMC
August 2020

Understanding Binding-Induced Folding by Temperature Jump.

Methods Mol Biol 2020 ;2141:651-661

Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, Rome, Italy.

Temperature jump is a powerful technique for the characterization of fast kinetics and can be readily employed to understand both binding and folding reactions. Here we summarize briefly a temperature-jump prototypical experiment between an intrinsically disordered protein and its physiological partner. The model used is the N domain from Measles virus Nucleoprotein and its natural ligand, the globular P domain from Measles virus Phosphoprotein. We recapitulate how to set up the experiment and how to analyze data in order to extract the kinetic parameters of the reaction.
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http://dx.doi.org/10.1007/978-1-0716-0524-0_33DOI Listing
March 2021

Understanding the Mechanism of Recognition of Gab2 by the N-SH2 Domain of SHP2.

Life (Basel) 2020 Jun 11;10(6). Epub 2020 Jun 11.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy.

Gab2 is a scaffold protein with a crucial role in colocalizing signaling proteins and it is involved in the regulation of several important molecular pathways. SHP2 is a protein phosphatase that binds, through its two SH2 domains, specific consensus sequences presenting a phosphorylated tyrosine located on the disordered tail of Gab2. To shed light on the details of such a fundamental interaction for the physiology of the cell, we present a complete mutational analysis of the kinetics of binding between the N-SH2 domain of SHP2 and a peptide mimicking a specific region of Gab2. By analyzing kinetic data, we determined structural features of the transition state of the N-SH2 domain binding to Gab2, highlighting a remarkable cooperativity of the binding reaction. Furthermore, comparison of these data with ones previously obtained for another SH2 domain suggests the presence of underlying general features characterizing the binding process of SH2 domains. Data are discussed under the light of previous works on SH2 domains.
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http://dx.doi.org/10.3390/life10060085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345789PMC
June 2020

Demonstration of Binding Induced Structural Plasticity in a SH2 Domain.

Front Mol Biosci 2020 12;7:89. Epub 2020 May 12.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, Rome, Italy.

SH2 domains are common protein interaction domains able to recognize short aminoacidic sequences presenting a phosphorylated tyrosine (pY). In spite of their fundamental importance for cell physiology there is a lack of information about the mechanism by which these domains recognize and bind their natural ligands. The N-terminal SH2 (N-SH2) domain of PI3K mediates the interaction with different scaffolding proteins and is known to recognize a specific pY-X-X-M consensus sequence. These interactions are at the cross roads of different molecular pathways and play a key role for cell development and division. By combining mutagenesis, chemical kinetics and NMR, here we provide a complete characterization of the interaction between N-SH2 and a peptide mimicking the scaffolding protein Gab2. Our results highlight that N-SH2 is characterized by a remarkable structural plasticity, with the binding reaction being mediated by a diffused structural region and not solely by the residues located in the binding pocket. Furthermore, the analysis of kinetic data allow us to pinpoint an allosteric network involving residues far from the binding pocket involved in specificity. Results are discussed on the light of previous works on the binding properties of SH2 domains.
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http://dx.doi.org/10.3389/fmolb.2020.00089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247818PMC
May 2020

Understanding the Binding Induced Folding of Intrinsically Disordered Proteins by Protein Engineering: Caveats and Pitfalls.

Int J Mol Sci 2020 May 15;21(10). Epub 2020 May 15.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli' and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy.

Many proteins lack a well-defined three-dimensional structure in isolation. These proteins, typically denoted as intrinsically disordered proteins (IDPs), may display a characteristic disorder-to-order transition when binding their physiological partner(s). From an experimental perspective, it is of great importance to establish the general grounds to understand how such folding processes may be explored. Here we discuss the caveats and the pitfalls arising when applying to IDPs one of the key techniques to characterize the folding of globular proteins, the Φ value analysis. This method is based on measurements of the free energy changes of transition and native states upon conservative, non-disrupting, mutations. On the basis of available data, we reinforce the validity of Φ value analysis in the study of IDPs and suggest future experiments to further validate this powerful experimental method.
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http://dx.doi.org/10.3390/ijms21103484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279032PMC
May 2020

Templated folding of intrinsically disordered proteins.

J Biol Chem 2020 05 6;295(19):6586-6593. Epub 2020 Apr 6.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions to product, while ensuring a cooperative folding.
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http://dx.doi.org/10.1074/jbc.REV120.012413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212662PMC
May 2020

Unveiling the Molecular Basis of the Noonan Syndrome-Causing Mutation T42A of SHP2.

Int J Mol Sci 2020 Jan 10;21(2). Epub 2020 Jan 10.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy.

Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. Most NS-causing mutations influence the stability of the inactive form of SHP2. However, one NS-causing mutation, namely T42A, occurs in the binding pocket of the N-SH2 domain of the protein. Here, we present a quantitative characterization of the effect of the T42A mutation on the binding of the N-terminal SH2 domain of SHP2 with a peptide mimicking Gab2, a fundamental interaction that triggers the activation of the phosphatase in the cellular environment. Our results show that whilst the T42A mutation does not affect the association rate constant with the ligand, it causes a dramatic increase of the affinity for Gab2. This effect is due to a remarkable decrease of the microscopic dissociation rate constant of over two orders of magnitudes. In an effort to investigate the molecular basis of the T42A mutation in causing Noonan syndrome, we also compare the experimental results with a more conservative variant, T42S. Our findings are discussed in the context of the structural data available on SHP2.
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http://dx.doi.org/10.3390/ijms21020461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013464PMC
January 2020

The Effect of Proline - Isomerization on the Folding of the C-Terminal SH2 Domain from p85.

Int J Mol Sci 2019 Dec 23;21(1). Epub 2019 Dec 23.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", 00161 Roma, Italy.

SH2 domains are protein domains that modulate protein-protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.
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http://dx.doi.org/10.3390/ijms21010125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982175PMC
December 2019

-Acetylcysteine Serves as Substrate of 3-Mercaptopyruvate Sulfurtransferase and Stimulates Sulfide Metabolism in Colon Cancer Cells.

Cells 2019 08 4;8(8). Epub 2019 Aug 4.

CNR Institute of Molecular Biology and Pathology, Piazzale Aldo Moro 5, I-00185 Rome, Italy.

Hydrogen sulfide (HS) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of HS, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the HS biological effects. Reprogramming of HS metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and -acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the HS metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain HS synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates HS metabolism and that NAC can serve as a substrate for human MST.
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http://dx.doi.org/10.3390/cells8080828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721681PMC
August 2019

Binding induced folding: Lessons from the kinetics of interaction between N and XD.

Arch Biochem Biophys 2019 08 19;671:255-261. Epub 2019 Jul 19.

Istituto Pasteur, Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185, Rome, Italy. Electronic address:

Intrinsically Disordered Proteins (IDPs) are a class of protein that exert their function despite lacking a well-defined three-dimensional structure, which is sometimes achieved only upon binding to their natural ligands. This feature implies the folding of IDPs to be generally coupled with a binding event, representing an interesting challenge for kinetic studies. In this review, we recapitulate some of the most important findings of IDPs binding-induced folding mechanisms obtained by analyzing their binding kinetics. Furthermore, by focusing on the interaction between the Measles virus N protein, a prototypical IDP, and its physiological partner, the X domain, we recapitulate the major theoretical and experimental approaches that were used to describe binding induced folding.
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http://dx.doi.org/10.1016/j.abb.2019.07.011DOI Listing
August 2019

Structural characterization of an on-pathway intermediate and transition state in the folding of the N-terminal SH2 domain from SHP2.

FEBS J 2019 12 17;286(23):4769-4777. Epub 2019 Jul 17.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, Italy.

Src Homology 2 (SH2) domains are a class of protein domains that present a conserved three-dimensional structure and possess a crucial role in mediating protein-protein interactions. Despite their importance and abundance in the proteome, knowledge about the folding properties of SH2 domain is limited. Here we present an extensive mutational analysis (Φ value analysis) of the folding pathway of the N-SH2 domain of the Src homology region 2 domain-containing phosphatase-2 (SHP2) protein, a 104 residues domain that presents the classical SH2 domain fold (two α-helices flanking a central β-sheet composed of 3-5 antiparallel β-strands), with a fundamental role in mediating the interaction of SHP2 with its substrates and triggering key metabolic pathways in the cell. By analysing folding kinetic data we demonstrated that the folding pathway of N-SH2 presents an obligatory on-pathway intermediate that accumulates during the folding reaction. The production of 24 conservative site-directed variants allowed us to perform a Φ value analysis, by which we could fully characterize the intermediate and the transition state native-like interactions, providing a detailed quantitative analysis of the folding pathway of N-SH2. Results highlight the presence of a hydrophobic nucleus that stabilizes the intermediate, leading to a higher degree of native-like interactions in the transition state. Data are discussed and compared with previous works on SH2 domains.
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http://dx.doi.org/10.1111/febs.14990DOI Listing
December 2019

Mapping the allosteric network within a SH3 domain.

Sci Rep 2019 06 4;9(1):8279. Epub 2019 Jun 4.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185, Rome, Italy.

SH3 domains are very abundant protein-protein interactions modules, involved in the regulation of several cellular processes. Whilst they have been associated to allosteric communication pathways between contiguous domains in multi-domain proteins, there is lack of information regarding the intra-domain allosteric cross-talk within the SH3 moiety. Here we scrutinize the presence of an allosteric network in the C-terminal SH3 domain of Grb2 protein, upon binding the Grb2-associated binding 2 protein. To explore allostery, we performed double mutant cycle analysis, a powerful quantitative approach based on mutagenesis in conjunction with kinetic experiments. Data reveal the presence of an unexpected allosteric sparse network that modulates the affinity between the SH3 domain and its physiological partner.
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http://dx.doi.org/10.1038/s41598-019-44656-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547694PMC
June 2019

The kinetics of folding of the NSH2 domain from p85.

Sci Rep 2019 03 11;9(1):4058. Epub 2019 Mar 11.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185, Rome, Italy.

SH2 domains are protein domains that mediate protein-protein interaction through the recognition and binding of specific sequences containing phosphorylated tyrosines. The p85 protein is the regulatory subunit of the heterodimeric enzyme PI3K, an important enzyme involved in several molecular pathways. In this work we characterize the folding kinetics of the NSH2 domain of p85. Our data clearly reveal peculiar folding kinetics, characterized by an apparent mismatch between the observed folding and unfolding kinetics. Taking advantage of double mixing stopped flow experiments and site directed mutagenesis we demonstrate that such behavior is due to the cis/trans isomerization of the peptide bond between D73 and P74, being in a cis conformation in the native protein. Our data are discussed in comparison with previous works on the folding of other SH2 domains.
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http://dx.doi.org/10.1038/s41598-019-40480-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411737PMC
March 2019

Hydrogen Sulfide Oxidation: Adaptive Changes in Mitochondria of SW480 Colorectal Cancer Cells upon Exposure to Hypoxia.

Oxid Med Cell Longev 2019 29;2019:8102936. Epub 2019 Jan 29.

CNR Institute of Molecular Biology and Pathology, Rome, Italy.

Hydrogen sulfide (HS), a known inhibitor of cytochrome oxidase (CcOX), plays a key signaling role in human (patho)physiology. HS is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide:quinone oxidoreductase (SQR), whereby HS-derived electrons are injected into the respiratory chain stimulating O consumption and ATP synthesis. Under hypoxic conditions, HS has higher stability and is synthesized at higher levels with protective effects for the cell. Herein, working on SW480 colon cancer cells, we evaluated the effect of hypoxia on the ability of cells to metabolize HS. The sulfide-oxidizing activity was assessed by high-resolution respirometry, measuring the stimulatory effect of sulfide on rotenone-inhibited cell respiration in the absence or presence of antimycin A. Compared to cells grown under normoxic conditions (air O), cells exposed for 24 h to hypoxia (1% O) displayed a 1.3-fold reduction in maximal sulfide-oxidizing activity and 2.7-fold lower basal O respiration. Based on citrate synthase activity assays, mitochondria of hypoxia-treated cells were 1.8-fold less abundant and displayed 1.4-fold higher maximal sulfide-oxidizing activity and 2.6-fold enrichment in SQR as evaluated by immunoblotting. We speculate that under hypoxic conditions mitochondria undergo these adaptive changes to protect cell respiration from HS poisoning.
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http://dx.doi.org/10.1155/2019/8102936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374825PMC
March 2019

Investigating the Molecular Basis of the Aggregation Propensity of the Pathological D76N Mutant of Beta-2 Microglobulin: Role of the Denatured State.

Int J Mol Sci 2019 Jan 18;20(2). Epub 2019 Jan 18.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185 Rome, Italy.

Beta-2 microglobulin (β2m) is a protein responsible for a pathologic condition, known as dialysis-related amyloidosis (DRA), caused by its aggregation and subsequent amyloid formation. A naturally occurring mutation of β2m, D76N, presents a higher amyloidogenic propensity compared to the wild type counterpart. Since the three-dimensional structure of the protein is essentially unaffected by the mutation, the increased aggregation propensity of D76N has been generally ascribed to its lower thermodynamic stability and increased dynamics. In this study we compare the equilibrium unfolding and the aggregation propensity of wild type β2m and D76N variant at different experimental conditions. Our data revealed a surprising effect of the D76N mutation in the residual structure of the denatured state, which appears less compact than that of the wild type protein. A careful investigation of the structural malleability of the denatured state of wild type β2m and D76N pinpoint a clear role of the denatured state in triggering the amyloidogenic propensity of the protein. The experimental results are discussed in the light of the previous work on β2m and its role in disease.
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http://dx.doi.org/10.3390/ijms20020396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359115PMC
January 2019

A Carboxylate to Amide Substitution That Switches Protein Folds.

Angew Chem Int Ed Engl 2018 09 28;57(39):12795-12798. Epub 2018 Aug 28.

Department of Bioengineering, University of Washington, Seattle, WA, 98195-5013, USA.

Metamorphic proteins are biomolecules prone to adopting alternative conformations. Because of this feature, they represent ideal systems to investigate the general rules allowing primary structure to dictate protein topology. A comparative molecular dynamics study was performed on the denatured states of two proteins, sharing nearly identical amino-acid sequences (88 %) but different topologies, namely an all-α-helical bundle protein named G 88 and an α+β-protein named G 88. The analysis allowed successful design of and experimental validation of a site-directed mutant that promotes, at least in part, the switch in folding from G 88 to G 88. The mutated position, in which a glutamic acid was replaced by a glutamine, does not make any intramolecular interactions in the native state of G 88, such that its stabilization can be explained by considering the effects on the denatured state. The results represent a direct demonstration of the role of the denatured state in sculpting native structure.
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http://dx.doi.org/10.1002/anie.201807723DOI Listing
September 2018

The presence of glutamate residues on the PAS sequence of the stimuli-sensitive nano-ferritin improves in vivo biodistribution and mitoxantrone encapsulation homogeneity.

J Control Release 2018 04 20;275:177-185. Epub 2018 Feb 20.

Institute of Molecular Biology and Pathology, CNR - National Research Council of Italy, 00185 Rome, Italy. Electronic address:

A genetically engineered human ferritin heavy chain (HFt)-based construct has been recently shown by our group to efficiently entrap and deliver doxorubicin to cancer cells. This construct, named HFt-MP-PAS, contained a tumor-selective sequence (MP) responsive to proteolytic cleavage by tumor proteases (MMPs), located between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). HFt-MP-PAS displayed excellent therapeutic efficacy in xenogenic pancreatic and head and neck cancer models in vivo, leading to a significant increase in overall animal survivals. Here we report a new construct obtained by the genetic insertion of two glutamate residues in the PAS sequence of HFt-MP-PAS. Such new construct, named HFt-MP-PASE, is characterized by improved performances as drug biodistribution in a xenogenic pancreatic cancer model in vivo. Moreover, HFt-MP-PASE efficiently encapsulates the anti-cancer drug mitoxantrone (MIT), and the resulting MIT-loaded nanoparticles proved to be more soluble and monodispersed than the HFt-MP-PAS counterparts. Importantly, in vitro MIT-loaded HFt-MP-PASE kills several cancer cell lines of different origin (colon, breast, sarcoma and pancreas) at least as efficiently as the free drug. Finally, our MIT loaded protein nanocages allowed in vivo an impressive incrementing of the drug accumulation in the tumor with respect to the free drug.
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http://dx.doi.org/10.1016/j.jconrel.2018.02.025DOI Listing
April 2018

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine -Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

Oxid Med Cell Longev 2017 22;2017:8940321. Epub 2017 Mar 22.

CNR Institute of Molecular Biology and Pathology, Rome, Italy.

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5'-phosphate- (PLP-) dependent cystathionine -synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (HS). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired HS-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria.
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http://dx.doi.org/10.1155/2017/8940321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381205PMC
May 2017

Bioenergetic relevance of hydrogen sulfide and the interplay between gasotransmitters at human cystathionine β-synthase.

Biochim Biophys Acta 2016 Aug 31;1857(8):1127-1138. Epub 2016 Mar 31.

CNR Institute of Molecular Biology and Pathology, Piazzale Aldo Moro 5, I-00185 Rome, Italy. Electronic address:

Merely considered as a toxic gas in the past, hydrogen sulfide (H2S) is currently viewed as the third 'gasotransmitter' in addition to nitric oxide (NO) and carbon monoxide (CO), playing a key signalling role in human (patho)physiology. H2S can either act as a substrate or, similarly to CO and NO, an inhibitor of mitochondrial respiration, in the latter case by targeting cytochrome c oxidase (CcOX). The impact of H(2)S on mitochondrial energy metabolism crucially depends on the bioavailability of this gaseous molecule and its interplay with the other two gasotransmitters. The H(2)S-producing human enzyme cystathionine β-synthase (CBS), sustaining cellular bioenergetics in colorectal cancer cells, plays a role in the interplay between gasotransmitters. The enzyme was indeed recently shown to be negatively modulated by physiological concentrations of CO and NO, particularly in the presence of its allosteric activator S-adenosyl-l-methionine (AdoMet). These newly discovered regulatory mechanisms are herein reviewed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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http://dx.doi.org/10.1016/j.bbabio.2016.03.030DOI Listing
August 2016

N-Methylated α-Amino Acids And Peptides: Synthesis And Biological Activity.

Mini Rev Med Chem 2016 ;16(9):683-90

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende (CS) Italy.

The simplest and minimal modification of a single amino acid or peptide bonds is represented by N-methylation. This can improve the pharmacokinetic properties of biologically active peptides as well as resulting in analogues that show specific biological activity such as enzyme inhibitors, receptor antagonists and agonists, building blocks in combinatorial chemistry for the screening of new potential drugs. Further, structural and conformational studies performed with N-methylated analogues of natural amino acids and peptides enabled to (i) produce stable foldamers with different topology with respect to the helix of natural and endogenous peptides, (ii) confer to modified peptides high stability against proteases and (iii) enhance lipophilicity and bioavailability for pharmacological purposes. Consequentially, it is crucial to provide optically pure N-methyl-amino acids and N-methylated peptides with a large supply. The present report will focus on the results obtained in the last decade in the field of chemical synthetic methodologies for the N-methylation of amino acids.
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http://dx.doi.org/10.2174/1389557516666160322152457DOI Listing
December 2016