Publications by authors named "Luca Federici"

61 Publications

Phenotypic and Proteomic Analysis Identifies Hallmarks of Blood Circulating Extracellular Vesicles in NSCLC Responders to Immune Checkpoint Inhibitors.

Cancers (Basel) 2021 Feb 3;13(4). Epub 2021 Feb 3.

Department of Pharmacy, University "G. D'Annunzio" Chieti-Pescara, 66100 Chieti, Italy.

Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors ( = 31) or chemotherapy ( = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival ( = 0.0004) and higher disease control rate ( = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC.
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http://dx.doi.org/10.3390/cancers13040585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913165PMC
February 2021

Circulating extracellular vesicles as new inflammation marker in HIV infection.

AIDS 2021 03;35(4):595-604

Clinic of Infectious Diseases, Department of Medicine and Aging Sciences, University 'G. d'Annunzio' of Chieti-Pescara, Chieti.

Background: Extracellular vesicles, released by cell pullulation, are surrounded by a phospholipid bilayer and carry proteins as well and genetic material. It has been shown that extracellular vesicles mediate intercellular communication in several conditions, such as inflammation, immunodeficiency, tumor growth, and viral infections. Here, we analyzed circulating levels of extracellular vesicles in order to clarify their role in chronic inflammation mechanisms characterizing HIV patients.

Methods: We analyzed and subtyped circulating levels of extracellular vesicles, through a recently developed flow cytometry method. In detail, endothelial-derived extracellular vesicles (CD31+/CD41a-/CD45-, EMVs), extracellular vesicles stemming from leukocytes (CD45+, LMVs) and platelets (CD41a+/CD31+) were identified and enumerated. Moreover, we analyzed the extracellular vesicle protein cargo with proteomic analysis.

Results: Circulating levels of total extracellular vesicles, EMVs and LMVs were significantly lower in the HIV+ patients than in healthy subjects, whereas platelet-derived extracellular vesicles resulted higher in patients than in the healthy population. Proteomic analysis showed the upregulation of gammaIFN and IL1α, and down-regulation of OSM, NF-kB, LIF, and RXRA signaling resulted activated in this patients.

Conclusion: These data demonstrate, for the first time that HIV infection induces the production of extracellular vesicles containing mediators that possibly feed the chronic inflammation and the viral replication. These two effects are connected as the inflammation itself induces the viral replication. We, therefore, hypothesize that HIV infection inhibits the production of extracellular vesicles that carry anti-inflammatory molecules.
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http://dx.doi.org/10.1097/QAD.0000000000002794DOI Listing
March 2021

Proteostasis unbalance of nucleophosmin 1 in Acute Myeloid Leukemia: An aggregomic perspective.

Int J Biol Macromol 2020 Dec 2;164:3501-3507. Epub 2020 Sep 2.

Department of Pharmacy, University of Naples "Federico II", 80134, Italy. Electronic address:

The role exerted by the nucleus in the regulation of proteostasis in both health and disease is recognized of outmost importance, even though not fully understood. Many recent investigations are focused on its ability to modulate and coordinate protein quality control machineries in mammalian cells. Nucleophosmin 1 (NPM1) is one of the most abundant nucleolar proteins and its gene is mutated in ~30% of Acute Myeloid Leukemia (AML) patients. Mutations are localized in the C-terminal domain of the protein and cause cytoplasmatically delocalized and possibly aggregated forms of NPM1 (NPM1c+). Therapeutic interventions targeted on NPM1c+ are in demand and, to this end, deeper knowledge of NPM1c+ behavior in the blasts' cytosol is required. Here by means of complementary biophysical techniques we compared the conformational and aggregative behavior of the entire C-terminal domains of NPM1wt and type A NPM1c+ (bearing the most common mutation). Overall data show that only Cterm_mutA is able to form amyloid-like assemblies with fibrillar morphology and that the oligomers are toxic in human neuroblastoma SHSY cells. This study adds a novel piece of knowledge to the comprehension of the molecular roles exerted by cytoplasmatic NPM1c+ and suggests the exploitation of the amyloidogenic propensity of NPM1c+ as a new strategy for targeting AML with NPM1 mutations.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.08.248DOI Listing
December 2020

The Potential of Steroid Profiling by Mass Spectrometry in the Management of Adrenocortical Carcinoma.

Biomedicines 2020 Aug 28;8(9). Epub 2020 Aug 28.

Center for Advanced Studies and Technology (CAST), University "G. d'Annunzio" of Chieti-Pescara, 66100 Chieti, Italy.

Radiological and endocrinological work up of adrenal neoplasms is aimed at distinguishing between frequent non-functioning adenomas and rare but very aggressive adrenocortical carcinoma (ACC). Relevant research has addressed the identification of molecular, genetic and hormonal markers that could have clinical significance for malignancy, as well as a prognostic value. Regarding endocrine aspects, attention has been paid to the pattern of steroid secretion that can be affected by altered steroidogenic pathway in ACC. The advent of mass spectrometry techniques has overcome many limitations usually associated with immunoassays, allowing the determination of both common and rarely measured steroids in a single analysis with high specificity and sensitivity. Indeed, mass spectrometry strategies may be able to identify an individualized steroid profile of ACC, allowing a rapid diagnosis and a specific follow-up. In this review, insights, strengths and limitations of mass spectrometry-based approaches in steroid profiling, as well as of immunoassay in steroid measurements, will be specifically discussed. Moreover, the latest findings on steroid profiling by mass spectrometry-based techniques, the most promising analytical tool, will be summarized to evaluate if steroid profiling might be the clue for solving the clinical dilemma in differentiating ACC from non-functioning adrenocortical adenomas (ACA).
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http://dx.doi.org/10.3390/biomedicines8090314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555975PMC
August 2020

Nucleophosmin in Its Interaction with Ligands.

Int J Mol Sci 2020 Jul 10;21(14). Epub 2020 Jul 10.

Center for Advanced Studies and Technology (CAST), University of Chieti "G. d'Annunzio", Via Polacchi, 66100 Chieti, Italy.

Nucleophosmin (NPM1) is a mainly nucleolar protein that shuttles between nucleoli, nucleoplasm and cytoplasm to fulfill its many functions. It is a chaperone of both nucleic acids and proteins and plays a role in cell cycle control, centrosome duplication, ribosome maturation and export, as well as the cellular response to a variety of stress stimuli. NPM1 is a hub protein in nucleoli where it contributes to nucleolar organization through heterotypic and homotypic interactions. Furthermore, several alterations, including overexpression, chromosomal translocations and mutations are present in solid and hematological cancers. Recently, novel germline mutations that cause dyskeratosis congenita have also been described. This review focuses on NPM1 interactions and inhibition. Indeed, the list of NPM1 binding partners is ever-growing and, in recent years, many studies contributed to clarifying the structural basis for NPM1 recognition of both nucleic acids and several proteins. Intriguingly, a number of natural and synthetic ligands that interfere with NPM1 interactions have also been reported. The possible role of NPM1 inhibitors in the treatment of multiple cancers and other pathologies is emerging as a new therapeutic strategy.
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http://dx.doi.org/10.3390/ijms21144885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402337PMC
July 2020

Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites.

Sci Rep 2020 07 9;10(1):11276. Epub 2020 Jul 9.

Department of Science, Roma Tre University, Viale G. Marconi 446, 00146, Rome, Italy.

Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gram-negative bacteria. LPS is translocated to the OM by the LPS transport (Lpt) system. In the human pathogen Pseudomonas aeruginosa, the periplasmic Lpt component, LptH, is essential for LPS transport, planktonic and biofilm growth, OM stability and infectivity. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization. The relevance of these sites for LptH activity was verified through plasmid-mediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. Complementation and protein expression analyses provided evidence that all mutated sites are important for LptH activity in vivo. It was observed that the lptH conditional mutant overcomes the lethality of nonfunctional lptH variants through RecA-mediated homologous recombination between the wild-type lptH gene in the genome and mutated copies in the plasmid. Finally, biochemical assays on purified recombinant proteins showed that some LptH variants are indeed specifically impaired in oligomerization, while others appear to have defects in protein folding and/or stability.
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http://dx.doi.org/10.1038/s41598-020-68054-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347655PMC
July 2020

Extracellular Vesicles and Their Potential Use in Monitoring Cancer Progression and Therapy: The Contribution of Proteomics.

J Oncol 2019 9;2019:1639854. Epub 2019 Jun 9.

Department of Pharmacy, University "G. d'Annunzio" of Chieti-Pescara, Chieti, Italy.

Extracellular Vesicles (EVs) are small membrane-enclosed particles released by cells and able to vehiculate information between them. The term EVs categorizes many and different vesicles based on their biogenesis and release pathway, such as exosomes (Exo), ectosomes, or shedding microvesicles (SMVs), apoptotic blebs (ABs), and other EVs subsets, generating a heterogeneous group of components able to redistribute their cargo into the entire organism. Moreover EVs are becoming increasingly important in monitoring cancer progression and therapy, since they are able to carry specific disease biomarkers such as Glypican-1, colon cancer-associated transcript 2, CD63, CD24, and many others. The importance of their biological role together with their heterogeneity prompted researchers to adopt and standardize purification methods able to isolate EVs for characterizing their cargo. In this way, mass spectrometry (MS)-based proteomics approaches are emerging as promising tool for the identification and quantification of EVs protein cargoes, but this technique resulted to be deeply influenced by the low quality of the isolation techniques. This review presents the state-of-the-art of EVs isolation, purification, and characterization for omics studies, with a particular focus to their potential use in monitoring cancer progression and therapy.
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http://dx.doi.org/10.1155/2019/1639854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590542PMC
June 2019

Structural and functional investigation of the Small Ribosomal Subunit Biogenesis GTPase A (RsgA) from Pseudomonas aeruginosa.

FEBS J 2019 11 2;286(21):4245-4260. Epub 2019 Jul 2.

Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Roma, Italy.

The Small Ribosomal Subunit Biogenesis GTPase A (RsgA) is a bacterial assembly factor involved in the late stages of the 30S subunit maturation. It is a multidomain GTPase in which the central circularly permutated GTPase domain is flanked by an OB domain and a Zn-binding domain. All three domains participate in the interaction with the 30S particle thus ensuring an efficient coupling between catalytic activity and biological function. In vivo studies suggested the relevance of rsgA in bacterial growth and cellular viability, but other pleiotropic roles of RsgA are also emerging. Here, we report the 3D structure of RsgA from Pseudomonas aeruginosa (PaRsgA) in the GDP-bound form. We also report a biophysical and biochemical characterization of the protein in both the GDP-bound and its nucleotide-free form. In particular, we report a kinetic analysis of the RsgA binding to GTP and GDP. We found that PaRsgA is able to bind both nucleotides with submicromolar affinity. The higher affinity towards GDP (K  = 0.011 μm) with respect to GTP (K  = 0.16 μm) is mainly ascribed to a smaller GDP dissociation rate. Our results confirm that PaRsgA, like most other GTPases, has a weak intrinsic enzymatic activity (k  = 0.058 min ). Finally, the biological role of RsgA in P. aeruginosa was investigated, allowing us to conclude that rsgA is dispensable for P. aeruginosa growth but important for drug resistance and virulence in an animal infection model. DATABASES: Coordinates and structure factors for the protein structure described in this manuscript have been deposited in the Protein Data Bank (https://www.rcsb.org) with the accession code 6H4D.
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http://dx.doi.org/10.1111/febs.14959DOI Listing
November 2019

Integrated Lipidomics and Metabolomics Analysis of Tears in Multiple Sclerosis: An Insight into Diagnostic Potential of Lacrimal Fluid.

Int J Mol Sci 2019 Mar 13;20(6). Epub 2019 Mar 13.

Department of Pharmacy, University ''G. d'Annunzio'' of Chieti-Pescara, 66100 Chieti, Italy.

Metabolomics based on mass spectrometry represents an innovative approach to characterize multifactorial diseases, such as multiple sclerosis (MuS). To date, the most important biomarker source for MuS diagnosis is the cerebrospinal fluid. However, an important goal for research is to identify new molecules in more easily accessible biological fluids. A very interesting biofluid in MuS is represented by tears, considered as an intermediate fluid between the cerebrospinal fluid and serum. In this work, we developed a merged strategy for the analysis of lipids containing choline by Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS), as well as for the targeted analysis of free carnitine, acylcarnitines and aminoacids by direct infusion mass spectrometry. Samples for both metabolomics and lipidomics approaches were obtained in a single extraction procedure from tears of patients affected by MuS and healthy controls. Tear lipidomics showed 30 phospholipids significantly modulated and, notably, many sphingomyelins resulted lower in MuS. Moreover, the metabolomics approach carried out both on tears and serum highlighted the diagnostic potential of specific aminoacids and acylcarnitines. In conclusion, the metabolic profiling of tears appears to reflect the pathological conditions of the central nervous system, suggesting that the molecular repository of tears can be considered as a source of potential biomarkers for MuS.
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http://dx.doi.org/10.3390/ijms20061265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471885PMC
March 2019

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon .

Front Microbiol 2019 18;10. Epub 2019 Jan 18.

Department of Medical, Oral and Biotechnological Sciences, "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy.

Xi class glutathione transferases (GSTs) are a recently identified group, within this large superfamily of enzymes, specifically endowed with glutathione-dependent reductase activity on glutathionyl-hydroquinone. Enzymes belonging to this group are widely distributed in bacteria, fungi, and plants but not in higher eukaryotes. Xi class GSTs are also frequently found in archaea and here we focus on the enzyme produced by the extreme haloalkaliphilic archaeon (NmGHR). We investigated its function and stability and determined its 3D structure in the apo form by X-ray crystallography. NmGHR displays the same fold of its mesophilic counterparts, is enriched in negatively charged residues, which are evenly distributed along the surface of the protein, and is characterized by a peculiar distribution of hydrophobic residues. A distinctive feature of haloalkaliphilic archaea is their preference for γ-glutamyl-cysteine over glutathione as a reducing thiol. Indeed we found that the genome lacks a gene coding for glutathione synthase. Analysis of NmGHR structure suggests that the thiol binding site (G-site) of the enzyme is well suited for hosting γ-glutamyl-cysteine.
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http://dx.doi.org/10.3389/fmicb.2019.00009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345682PMC
January 2019

Metabolomic Signature in Sera of Multiple Sclerosis Patients during Pregnancy.

Int J Mol Sci 2018 Nov 14;19(11). Epub 2018 Nov 14.

Department of Medical, Oral and Biotechnological Sciences, "G. d'Annunzio" University of Chieti-Pescara, 66100 Chieti, Italy.

Multiple sclerosis (MuS) is an autoimmune disease of the central nervous system characterized by neuroinflammation, neurodegeneration, and degradation of the myelin sheath. Epidemiological studies have shown that the female gender is more susceptible than the male gender to MuS development, with a female-to-male ratio of 2:1. Despite this high onset, women have a better prognosis than men, and the frequency of the relapsing phase decreases during pregnancy, while it increases soon after birth. Therefore, it is interesting to investigate hormonal fluctuations during pregnancy and whether they correlate with metabolic signatures. To gain a deeper inside into the biochemical mechanism of such a multifactorial disease, we adopted targeted metabolomics approaches for the determination of many serum metabolites in 12 pregnant women affected by MuS by mass spectrometry analysis. Our data show a characteristic hormonal fluctuation for estrogens and progesterone, as expected. They also highlight other interesting hormonal alterations for cortisol, corticosterone, 11-deoxycortisol, 4-androstene-3,17-dione, testosterone, and 17α-hydroxyprogesterone. Furthermore, a negative correlation with progesterone levels was observed for amino acids and for acylcarnitines, while an imbalance of different sphingolipids pathways was found during pregnancy. In conclusion, these data are in agreement with the characteristic clinical signs of MuS patients during pregnancy and, if confirmed, they may add an important tessera in the complex mosaic of maternal neuroprotection.
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http://dx.doi.org/10.3390/ijms19113589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274842PMC
November 2018

Glutathione transferases: substrates, inihibitors and pro-drugs in cancer and neurodegenerative diseases.

Oncogenesis 2018 Jan 24;7(1). Epub 2018 Jan 24.

Department of Medical, Oral and Biotechnological Sciences, University "G. d'Annunzio", Chieti, Italy.

Glutathione transferase classical GSH conjugation activity plays a critical role in cellular detoxification against xenobiotics and noxious compounds as well as against oxidative stress. However, this feature is also exploited by cancer cells to acquire drug resistance and improve their survival. As a result, various members of the family were found overexpressed in a number of different cancers. Moreover several GST polymorphisms, ranging from null phenotypes to point mutations, were detected in members of the family and found to correlate with the onset of neuro-degenerative diseases. In the last decades, a great deal of research aimed at clarifying the role played by GSTs in drug resistance, at developing inhibitors to counteract this activity but also at exploiting GSTs for prodrugs specific activation in cancer cells. Here we summarize some of the most important achievements reached in this lively area of research.
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http://dx.doi.org/10.1038/s41389-017-0025-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833873PMC
January 2018

Nucleophosmin-1 regions associated with acute myeloid leukemia interact differently with lipid membranes.

Biochim Biophys Acta Gen Subj 2018 Apr 10;1862(4):967-978. Epub 2018 Jan 10.

Department of Pharmacy, CIRPEB: Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II", 80134, Naples, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.bbagen.2018.01.005DOI Listing
April 2018

Identification of a novel nucleophosmin-interaction motif in the tumor suppressor p14arf.

FEBS J 2018 03 15;285(5):832-847. Epub 2018 Jan 15.

Ce.S.I.-MeT Centro di Scienze dell'Invecchiamento e Medicina Traslazionale, Università "G. d'Annunzio" di Chieti, Italy.

The tumor suppressor p14arf interacts, in response to oncogenic signals, with the p53 E3-ubiquitin ligase HDM2, thereby resulting in p53 stabilization and activation. In addition, it also exerts tumor-suppressive functions in p53-independent contexts. The activities of p14arf are regulated by the nucleolar chaperone nucleophosmin (NPM1), which controls its levels and cellular localization. In acute myeloid leukemia with mutations in the NPM1 gene, mutated NPM1 aberrantly translocates in the cytosol carrying with itself p14arf that is subsequently degraded, thus impairing the p14arf-HDM2-p53 axis. In this work we investigated the complex between these two proteins by means of NMR and other techniques. We identified a novel NPM1-interacting motif in the C-terminal region of p14arf, which corresponds to its predicted nucleolar localization signal. This motif recognizes a specific region of the NPM1 N-terminal domain and, upon binding, the two proteins form soluble high molecular weight complexes. By NMR, we identified critical residues on both proteins involved in the interaction. Collectively, our data provide a structural framework to rationalize the overall assembly of the p14arf-NPM1 supramolecular complexes. A number of p14arf cancer-associated mutations cluster in this motif and their effect on the interaction with NPM1 was also analyzed.
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http://dx.doi.org/10.1111/febs.14373DOI Listing
March 2018

Targeted therapy of human glioblastoma via delivery of a toxin through a peptide directed to cell surface nucleolin.

J Cell Physiol 2018 05 18;233(5):4091-4105. Epub 2017 Dec 18.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

Targeted anticancer therapies demand discovery of new cellular targets to be exploited for the delivery of toxic molecules and drugs. In this perspective, in the last few years, nucleolin has been identified as an interesting surface marker to be used for the therapy of glioblastoma. In this study, we investigated whether a synthetic antagonist of cell-surface nucleolin known as N6L, previously reported to decrease both tumor growth and tumor angiogenesis in several cancer cell lines, including glioblastoma cells, as well as endothelial cells proliferation, could be exploited to deliver a protein toxin (saporin) to glioblastoma cells. The pseudopeptide N6L cross-linked to saporin-S6 induced internalization of the toxin inside glioblastoma cancer cells. Our results in vitro demonstrated the effectiveness of this conjugate in inducing cell death, with an ID four orders of magnitude lower than that observed for free N6L. Furthermore, the preliminary in vivo study demonstrated efficiency in reducing the tumor mass in an orthotopic mouse model of glioblastoma.
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http://dx.doi.org/10.1002/jcp.26205DOI Listing
May 2018

Therapeutic Efficacy of the Novel Stimuli-Sensitive Nano-Ferritins Containing Doxorubicin in a Head and Neck Cancer Model.

Int J Mol Sci 2017 Jul 18;18(7). Epub 2017 Jul 18.

Institute of Molecular Biology and Pathology, National Research Council of Italy (CNR), Rome 00185, Italy.

Doxorubicin is employed alone or in combination for the treatment of several hematological and solid malignancies; despite its efficacy, there are associated cardiotoxicity limits both in its application in patients with heart disease risk factors and also in its long-term use. HFt-MP-PAS40 is a genetically engineered human ferritin heavy chain (HFt)-based construct able to efficiently entrap and deliver doxorubicin to cancer cells. HF-MP-PAS contains a short motif sequence (defined as MP) responsive to proteolytic cleavage by tumor matrix metalloproteases (MMPs), located between each HFt subunit and a masking polypeptide sequence rich in proline (P), alanine (A), and serine (S) residues (PAS). This carrier displayed excellent therapeutic efficacy in a xenogenic pancreatic cancer model in vivo, leading to a significant increase in overall animal survival in treated mice. Herein, we describe the HFt-MP-PAS40-Dox efficacy against squamous cell carcinomas of the head and neck (HNSCC) with the goal of validating the application of our nano-drug for the treatment of different solid tumors. In addition, a tolerability study in healthy mice was also performed. The results indicate that HFt-MP-PAS40-Dox produced increased anti-tumor effects both in vitro and in vivo in comparison to the free drug in several HNSCC cell lines. In the acute toxicity studies, the maximum tolerated dose (MTD) of HFt-MP-PAS40-Dox was about 3.5 higher than the free drug: 25 mg/kg versus 7 mg/kg doxorubicin equivalents. Importantly, evaluation of heart tissues provided evidence that doxorubicin is less cardio-toxic when encapsulated inside the ferritin carrier. In conclusion, HFt-MP-PAS40-Dox may be administered safely at higher doses compared with the free drug, resulting in superior efficacy to control HNSCC malignancies.
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http://dx.doi.org/10.3390/ijms18071555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536043PMC
July 2017

The Glu331del mutation in the CYP17A1 gene causes atypical congenital adrenal hyperplasia in a 46,XX female.

Gynecol Endocrinol 2017 Dec 13;33(12):918-922. Epub 2017 Jun 13.

g Department of Life, Health and Environmental Sciences , University of L'Aquila , L'Aquila , Italy.

17α-Hydroxylase deficiency is an uncommon type of congenital adrenal hyperplasia (CAH) caused by mutations in the CYP17A1 gene encoding both 17α-hydroxylase and 17,20-lyase, essential for sex steroids production. Main clinical features include lack of pubertal development, hypertension, and hypokalemia. We report the first case of a 46,XX female homozygote for the p.Glu331del mutation in the CYP17A1 gene showing an atypical clinical presentation. She was evaluated the first time for primary amenorrhea and delayed puberty in the presence of low levels of androgens, 17β-estradiol, serum cortisol, and high levels of progesterone and gonadotropins. After puberty, the patient did not show hypocortisolism and/or hypertension. She started estrogen therapy for pubertal induction, followed by ethinylestradiol/gestodene with clinical and biochemical stability during the follow-up period. At the age of 40 years, she developed hypokalemia and clinical signs of hypocortisolism. Oral corticosteroid treatment was started showing a prompt clinical improvement. Modeling analysis predicted the main outcome of the E331 deletion to impair cytochrome b5 binding, according to a major effect on the enzyme's lyase activity. These data broaden the molecular and clinical spectrum of CAH caused by 17α-hydroxylase deficiency and adds to current genotype-phenotype correlations.
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http://dx.doi.org/10.1080/09513590.2017.1337097DOI Listing
December 2017

A New Homozygous Frameshift Mutation in the HSD3B2 Gene in an Apparently Nonconsanguineous Italian Family.

Horm Res Paediatr 2016 16;86(1):53-61. Epub 2016 Apr 16.

Background: 3β-Hydroxysteroid dehydrogenase (3β-HSD) deficiency is a rare cause of congenital adrenal hyperplasia (CAH) caused by inactivating mutations in the HSD3B2 gene.

Patient And Methods: We report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child born to apparently nonconsanguineous parents and presenting ambiguous genitalia and salt wasting. The steroid profile showed elevated concentrations of 17-hydroxyprogesterone, androstenedione, ACTH and plasma renin, but normal values of cortisol and dehydroepiandrosterone sulfate. Unexpectedly, plasma aldosterone was high. For structural and functional analyses, the three-dimensional structure of 3β-HSD2 was modeled using the crystal structure of the short-chain dehydrogenase Gox2253 from Gluconobacter oxydans as a template.

Results: The direct DNA sequence of the child revealed a new homozygous frameshift mutation in exon 4 of the HSD3B2 gene, a single nucleotide deletion at codon 319 [GTC(Val)x2192;GC], yielding premature stop codon in position 367. Molecular homology modeling and secondary structure predictions suggested that the variant sequence might both alter the substrate-binding cleft and compromise the overall stability of the enzyme.

Conclusion: We have described the first HSD3B2 gene mutation in the Italian population and analyzed its effect in the context of the 3β-HSD2 structure and function.
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http://dx.doi.org/10.1159/000444712DOI Listing
April 2017

Molecules that target nucleophosmin for cancer treatment: an update.

Oncotarget 2016 Jul;7(28):44821-44840

Dipartimento di Scienze Mediche, Orali e Biotecnologiche, Università di Chieti "G. d'Annunzio", Chieti, Italy.

Nucleophosmin is a highly and ubiquitously expressed protein, mainly localized in nucleoli but able to shuttle between nucleus and cytoplasm. Nucleophosmin plays crucial roles in ribosome maturation and export, centrosome duplication, cell cycle progression, histone assembly and response to a variety of stress stimuli. Much interest in this protein has arisen in the past ten years, since the discovery of heterozygous mutations in the terminal exon of the NPM1 gene, which are the most frequent genetic alteration in acute myeloid leukemia. Nucleophosmin is also frequently overexpressed in solid tumours and, in many cases, its overexpression correlates with mitotic index and metastatization. Therefore it is considered as a promising target for the treatment of both haematologic and solid malignancies. NPM1 targeting molecules may suppress different functions of the protein, interfere with its subcellular localization, with its oligomerization properties or drive its degradation. In the recent years, several such molecules have been described and here we review what is currently known about them, their interaction with nucleophosmin and the mechanistic basis of their toxicity. Collectively, these molecules exemplify a number of different strategies that can be adopted to target nucleophosmin and we summarize them at the end of the review.
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http://dx.doi.org/10.18632/oncotarget.8599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5190137PMC
July 2016

Complete loss of the DNAJB6 G/F domain and novel missense mutations cause distal-onset DNAJB6 myopathy.

Acta Neuropathol Commun 2015 Jul 25;3:44. Epub 2015 Jul 25.

The Centre for Applied Genomics and Program in Genetics and Genome Biology, The Hospital for Sick Children and University of Toronto, Toronto, Canada.

Introduction: Protein aggregation is a common cause of neuropathology. The protein aggregation myopathy Limb-Girdle Muscular Dystrophy 1D (LGMD1D) is caused by mutations of amino acids Phe89 or Phe93 of DNAJB6, a co-chaperone of the HSP70 anti-aggregation protein. Another DNAJB6 mutation, Pro96Arg, was found to cause a distal-onset myopathy in one family.

Results: We detail the mutational, neuropathological, neurophysiological, neurological and radiological features of five new DNAJB6-myopathy families. One has the known Phe93Leu mutation and classic late-onset slowly progressive LGMD1D. Two have different mutations of Phe91 causing a variant childhood-onset severe limb-girdle myopathy. One has a Phe100Val mutation and distal-onset myopathy, unique early bulbar involvement, and a gender-modified wide age-of-onset range. The last has childhood-onset severe distal-onset myopathy and the first non-missense DNAJB6 mutation, c.346 + 5G > A, causing a splicing defect that entirely eliminates DNAJB6's G/F domain (ΔG/F), the domain that harbours all other mutations. Clinical and imaging examinations reveal that muscles considered uninvolved in DNAJB6-myopathy, e.g. lateral gastrocnemii, are affected in our patients with new mutations. Mutational modelling based on the known structure of the bacterial DNAJ2 protein indicates that all past and present mutated residues cluster within 15 Å in the G/F domain and all disturb the interface of this domain with the protein's J domain that confers the interaction with HSP70.

Conclusions: Our patients expand the phenotypic spectrum of DNAJB6-myopathy and allow tentative genotype-phenotype specifications. Combining with previous studies, the clinical severity spectrum is as follows: ΔG/F and Phe91 mutations, most severe; Phe100, Pro96, Phe89 mutations, intermediate; and Phe93, least severe. As it stands presently, proximal G/F domain mutations (Phe89, Phe91, Phe93) cause proximal limb-girdle myopathy, while distal G/F mutations (Pro96, Phe100) cause distal-onset myopathy. While all mutations affect the G/F-J interaction, each likely does so in different unknown extents or ways. One mutation, ΔG/F, causes its associated severe distal-onset myopathy phenotype in a clear way, through generation of a G/F domain-lacking DNAJB6 protein.
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http://dx.doi.org/10.1186/s40478-015-0224-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513909PMC
July 2015

Nucleophosmin contains amyloidogenic regions that are able to form toxic aggregates under physiological conditions.

FASEB J 2015 Sep 14;29(9):3689-701. Epub 2015 May 14.

*Department of Pharmacy, Diagnostica e Farmaceutica Molecolari-Società Cooperativa a Responsabilità Limitata, Centro Interuniversitario di Ricerca sui Peptidi Bioattivi, University of Naples "Federico II," Naples, Italy; Section of Biochemistry, Department of Biomedical Experimental and Clinical Sciences "Mario Serio," University of Florence, Florence, Italy; Institute of Biostructures and Bioimaging, Consiglio Nazionale delle Ricerche, Naples, Italy; Department of Physics, University of Genoa, Genoa, Italy; Department of Medical, Oral, and Biotechnological Sciences, University of Chieti "G. d'Annunzio," Chieti, Italy; and Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome, Italy

Nucleophosmin (NPM)-1 is a multifunctional protein involved in a variety of biologic processes and has been implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments in NPM1 activities, we dissected the C-terminal domain (CTD) into its helical fragments. In this study, we observed the unexpected structural behavior of the peptide fragment corresponding to helix (H)2 (residues 264-277). This peptide has a strong tendency to form amyloidlike assemblies endowed with fibrillar morphology and β-sheet structure, under physiologic conditions, as shown by circular dichroism, thioflavin T, and Congo red binding assays; dynamic light scattering; and atomic force microscopy. The aggregates are also toxic to neuroblastoma cells, as determined using 3-(4;5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and Ca(2+) influx assays. We also found that the extension of the H2 sequence beyond its N terminus, comprising the connecting loop with H1, delayed aggregation and its associated cytotoxicity, suggesting that contiguous regions of H2 have a protective role in preventing aggregation. Our findings and those in the literature suggest that the helical structures present in the CTD are important in preventing harmful aggregation. These findings could elucidate the pathogenesis of acute myeloid leukemia (AML) caused by NPM1 mutants. Because the CTD is not properly folded in these mutants, we hypothesize that the aggregation propensity of this NPM1 region is involved in the pathogenesis of AML. Preliminary assays on NPM1-Cter-MutA, the most frequent AML-CTD mutation, revealed its significant propensity for aggregation. Thus, the aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology.
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http://dx.doi.org/10.1096/fj.14-269522DOI Listing
September 2015

Synergic role of nucleophosmin three-helix bundle and a flanking unstructured tail in the interaction with G-quadruplex DNA.

J Biol Chem 2014 Aug 21;289(31):21230-41. Epub 2014 Jun 21.

Ce.S.I. Centro Scienze dell'Invecchiamento, "Fondazione Università D'Annunzio," 66013 Chieti, Italy, Dipartimento di Scienze Sperimentali e Cliniche, Università di Chieti "G. D'Annunzio," 66013 Chieti, Italy

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.
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http://dx.doi.org/10.1074/jbc.M114.565010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118085PMC
August 2014

A single amino-acid substitution allows endo-polygalacturonase of Fusarium verticillioides to acquire recognition by PGIP2 from Phaseolus vulgaris.

PLoS One 2013 19;8(11):e80610. Epub 2013 Nov 19.

Dipartimento di Biologia e Biotecnologie "Charles Darwin", Sapienza Università di Roma, Roma, Italy.

Polygalacturonases (PGs) are secreted by phytopathogenic fungi to degrade the plant cell wall homogalacturonan during plant infection. To counteract Pgs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs) that slow down fungal infection and defend cell wall integrity. PGIPs favour the accumulation of oligogalacturonides, which are homogalacturonan fragments that act as endogenous elicitors of plant defence responses. We have previously shown that PGIP2 from Phaseolus vulgaris (PvPGIP2) forms a complex with PG from Fusarium phyllophilum (FpPG), hindering the enzyme active site cleft from substrate. Here we analyse by small angle X-ray scattering (SAXS) the interaction between PvPGIP2 and a PG from Colletotrichum lupini (CluPG1). We show a different shape of the PG-PGIP complex, which allows substrate entry and provides a structural explanation for the different inhibition kinetics exhibited by PvPGIP2 towards the two isoenzymes. The analysis of SAXS structures allowed us to investigate the basis of the inability of PG from Fusarium verticilloides (FvPG) to be inhibited by PvPGIP2 or by any other known PGIP. FvPG is 92.5% identical to FpPG, and we show here, by both loss- and gain-of-function mutations, that a single amino acid site acts as a switch for FvPG recognition by PvPGIP2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080610PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834070PMC
August 2014

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: protein stability and denatured state residual structure.

Biochem Biophys Res Commun 2013 May 22;435(1):64-8. Epub 2013 Apr 22.

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

Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.
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http://dx.doi.org/10.1016/j.bbrc.2013.04.038DOI Listing
May 2013

Nucleophosmin mutations in acute myeloid leukemia: a tale of protein unfolding and mislocalization.

Protein Sci 2013 May 18;22(5):545-56. Epub 2013 Mar 18.

Ce.S.I. Center of Excellence on Aging, University of Chieti "G. D'Annunzio", 66013 Chieti, Italy.

Nucleophosmin (NPM1) is an abundant, ubiquitously expressed protein mainly localized at nucleoli but continuously shuttling between nucleus and cytoplasm. NPM1 plays a role in several cellular functions, including ribosome biogenesis and export, centrosome duplication, chromatin remodeling, DNA repair, and response to stress stimuli. Much of the interest in this protein arises from its relevance in human malignancies. NPM1 is frequently overexpressed in solid tumors and is the target of several chromosomal translocations in hematologic neoplasms. Notably, NPM1 has been characterized as the most frequently mutated gene in acute myeloid leukemia (AML). Mutations alter the C-terminal DNA-binding domain of the protein and result in its aberrant nuclear export and stable cytosolic localization. In this review, we focus on the leukemia-associated NPM1 C-terminal domain and describe its structure, function, and the effect exerted by leukemic mutations. Finally, we discuss the possibility to target NPM1 for the treatment of cancer and, in particular, of AML patients with mutated NPM1 gene.
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http://dx.doi.org/10.1002/pro.2240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3649256PMC
May 2013

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA.

Nucleic Acids Res 2013 Mar 16;41(5):3228-39. Epub 2013 Jan 16.

Department of Biochemical Sciences, 'Sapienza' University of Rome, 00185 Rome, Italy.

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.
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http://dx.doi.org/10.1093/nar/gkt001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597674PMC
March 2013

New insights into the mechanism of JNK1 inhibition by glutathione transferase P1-1.

Biochemistry 2012 Sep 5;51(37):7304-12. Epub 2012 Sep 5.

Department of Chemical Sciences and Technologies, University of Tor Vergata, Rome, Italy.

The role played by glutathione transferase P1-1 (GSTP1-1) in modulating the c-Jun N-terminal kinase (JNK) pathway has been extensively investigated using JNK isoforms known to exert opposite effects in the cells. We have expressed isoform JNK1α2, which has been reported to transmit a pro-apoptotic signal, and we have analyzed both the phosphorylation level and the activity of this kinase in the presence of GSTP1-1. Contrary to what previous studies suggest, we found that GSTP1-1 is able to form a complex with the unphosphorylated and inactive JNK1α2 isoform, even in the absence of the substrate. We also analyzed the consequences of this interaction on the activity of both enzymes. The complex strongly reduced the extent of activation of JNK1α2 and preserved GSTP1-1 from inactivation. Unexpectedly, glutathione (GSH) exerted a negative effect on the affinity of GSTP1-1 for JNK1α2, suggesting that the intracellular levels of this thiol may allow a fine-tuning of the MAPK signaling pathway. Moreover, we found that the adduct formed by GSH and the strong GSTP1-1 inhibitor NBDHEX abolishes the interaction between GSTP1-1 and JNK1α2. These data confirm and extend at the molecular level previous evidence obtained in tumor cell lines.
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http://dx.doi.org/10.1021/bi300559mDOI Listing
September 2012

Structure of nucleophosmin DNA-binding domain and analysis of its complex with a G-quadruplex sequence from the c-MYC promoter.

J Biol Chem 2012 Aug 15;287(32):26539-48. Epub 2012 Jun 15.

Magnetic Resonance Center, Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Italy.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a key role in several cellular functions, including ribosome maturation and export, centrosome duplication, and response to stress stimuli. More than 50 mutations at the terminal exon of the NPM1 gene have been identified so far in acute myeloid leukemia; the mutated proteins are aberrantly and stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal domain and the appearance of a new nuclear export signal. We have shown previously that the 70-residue NPM1 C-terminal domain (NPM1-C70) is able to bind with high affinity a specific region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. Here we present the solution structure of the NPM1-C70 domain and NMR analysis of its interaction with a c-MYC-derived G-quadruplex. These data were used to calculate an experimentally restrained molecular docking model for the complex. The NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface between helices H1 and H2 through electrostatic interactions with the G-quadruplex phosphate backbone. Furthermore, we show that the 17-residue lysine-rich sequence at the N terminus of the three-helix bundle is disordered and, although necessary, does not participate directly in the contact surface in the complex.
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http://dx.doi.org/10.1074/jbc.M112.371013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410995PMC
August 2012

Distribution of glutathione transferases in Gram-positive bacteria and Archaea.

Biochimie 2012 Mar 19;94(3):588-96. Epub 2011 Sep 19.

Dipartimento di Scienze Biomediche, Università G. d'Annunzio, Via dei Vestini 31, I-66013 Chieti, Italy.

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.
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http://dx.doi.org/10.1016/j.biochi.2011.09.008DOI Listing
March 2012

Structural resolution of the complex between a fungal polygalacturonase and a plant polygalacturonase-inhibiting protein by small-angle X-ray scattering.

Plant Physiol 2011 Oct 22;157(2):599-607. Epub 2011 Aug 22.

Dipartimento di Biologia e Biotecnologie C. Darwin, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, 00185 Rome, Italy.

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.
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http://dx.doi.org/10.1104/pp.111.181057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192570PMC
October 2011