Publications by authors named "Manuela Bozzi"

31 Publications

Discovery of 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids as novel PPARα agonists with anti-hyperlipidemic and antioxidant activities: Design, synthesis and in vitro/in vivo biological evaluation.

Bioorg Chem 2021 Jul 16;115:105170. Epub 2021 Jul 16.

Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12622, Egypt. Electronic address:

In the current work, a series of novel 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids (10a-o) and (11a-e) were synthesized and evaluated as new PPARα agonists in order to find new agents with higher activity and fewer side effects. The 2-propanoic acid derivative 10a and the 2-butanoic acid congener 10i showed the best overall PPARα agonistic activity showing E% values of 50.80 and 90.55%, respectively, and EC values of 8.9 and 25.0 μM, respectively, compared to fenofibric acid with E = 100% and EC = 23.22 μM, respectively. These two compounds also stimulated carnitine palmitoyltransferase 1A gene transcription in HepG2 cells and PPARα protein expression. Molecular docking simulations were performed for the newly synthesized compounds to study their predicted binding pattern and energies in PPARα active site to rationalize their promising activity. In vivo, compounds 10a and 10i elicited a significant hypolipidemic activity improving the lipid profile in triton WR-1339-induced hyperlipidemic rats, including serum triglycerides, total cholesterol, LDL, HDL and VLDL levels. Compound 10i possessed better anti-hyperlipidemic activity than 10a. At a dose of 200 mg/kg, it demonstrated significantly lower TC, TG, LDL and VLDL levels than that of fenofibrate at the same dose with similar HDL levels. Compounds 10i and 10a possessed atherogenic indices (CRR, AC, AI, CRI-II) like that of fenofibrate. Additionally, a promising antioxidant activity indicated by the increased tissue reduced glutathione and plasma total antioxidant capacity with decreased plasma malondialdehyde levels was demonstrated by compounds 10a and 10i. No histopathological alterations were recorded in the hepatic tissue of compound 10i (200 mg/kg).
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http://dx.doi.org/10.1016/j.bioorg.2021.105170DOI Listing
July 2021

Molecular Mechanisms Underlying Muscle Wasting in Huntington's Disease.

Int J Mol Sci 2020 Nov 5;21(21). Epub 2020 Nov 5.

Istituto di Scienze e Tecnologie Chimiche "Giulio Natta"- SCITEC Sede di Roma, Largo F. Vito 1, 00168 Roma, Italy.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by pathogenic expansions of the triplet cytosine-adenosine-guanosine (CAG) within the Huntingtin gene. These expansions lead to a prolongation of the poly-glutamine stretch at the N-terminus of Huntingtin causing protein misfolding and aggregation. Huntingtin and its pathological variants are widely expressed, but the central nervous system is mainly affected, as proved by the wide spectrum of neurological symptoms, including behavioral anomalies, cognitive decline and motor disorders. Other hallmarks of HD are loss of body weight and muscle atrophy. This review highlights some key elements that likely provide a major contribution to muscle atrophy, namely, alteration of the transcriptional processes, mitochondrial dysfunction, which is strictly correlated to loss of energy homeostasis, inflammation, apoptosis and defects in the processes responsible for the protein quality control. The improvement of muscular symptoms has proven to slow the disease progression and extend the life span of animal models of HD, underlining the importance of a deep comprehension of the molecular mechanisms driving deterioration of muscular tissue.
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http://dx.doi.org/10.3390/ijms21218314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664236PMC
November 2020

3D Graphene Scaffolds for Skeletal Muscle Regeneration: Future Perspectives.

Front Bioeng Biotechnol 2020 5;8:383. Epub 2020 May 5.

Dipartimento di Neuroscienze, Università Cattolica del Sacro Cuore, Rome, Italy.

Although skeletal muscle can regenerate after injury, in chronic damages or in traumatic injuries its endogenous self-regeneration is impaired. Consequently, tissue engineering approaches are promising tools for improving skeletal muscle cells proliferation and engraftment. In the last decade, graphene and its derivates are being explored as novel biomaterials for scaffolds production for skeletal muscle repair. This review describes 3D graphene-based materials that are currently used to generate complex structures able not only to guide cell alignment and fusion but also to stimulate muscle contraction thanks to their electrical conductivity. Graphene is an allotrope of carbon that has indeed unique mechanical, electrical and surface properties and has been functionalized to interact with a wide range of synthetic and natural polymers resembling native musculoskeletal tissue. More importantly, graphene can stimulate stem cell differentiation and has been studied for cardiac, neuronal, bone, skin, adipose, and cartilage tissue regeneration. Here we recapitulate recent findings on 3D scaffolds for skeletal muscle repairing and give some hints for future research in multifunctional graphene implants.
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http://dx.doi.org/10.3389/fbioe.2020.00383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214535PMC
May 2020

Identification and Modeling of a GT-A Fold in the α-Dystroglycan Glycosylating Enzyme LARGE1.

J Chem Inf Model 2020 06 14;60(6):3145-3156. Epub 2020 May 14.

Institute of Chemical Sciences and Technologies "Giulio Natta" (SCITEC)-CNR, L.go F. Vito 1, 00168 Rome, Italy.

The acetylglucosaminyltransferase-like protein LARGE1 is an enzyme that is responsible for the final steps of the post-translational modifications of dystroglycan (DG), a membrane receptor that links the cytoskeleton with the extracellular matrix in the skeletal muscle and in a variety of other tissues. LARGE1 acts by adding the repeating disaccharide unit [-3Xyl-α1,3GlcAβ1-] to the extracellular portion of the DG complex (α-DG); defects in the gene result in an aberrant glycosylation of α-DG and consequent impairment of its binding to laminin, eventually affecting the connection between the cell and the extracellular environment. In the skeletal muscle, this leads to degeneration of the muscular tissue and muscular dystrophy. So far, a few missense mutations have been identified within the LARGE1 protein and linked to congenital muscular dystrophy, and because no structural information is available on this enzyme, our understanding of the molecular mechanisms underlying these pathologies is still very limited. Here, we generated a 3D model structure of the two catalytic domains of LARGE1, combining different molecular modeling approaches. Furthermore, by using molecular dynamics simulations, we analyzed the effect on the structure and stability of the first catalytic domain of the pathological missense mutation S331F that gives rise to a severe form of muscle-eye-brain disease.
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http://dx.doi.org/10.1021/acs.jcim.0c00281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340341PMC
June 2020

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site.

PLoS One 2018 15;13(2):e0192651. Epub 2018 Feb 15.

CNR Institute for Molecular Recognition, Roma Italy.

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813964PMC
April 2018

Evaluation of the effect of a floxed Neo cassette within the dystroglycan (Dag1) gene.

BMC Res Notes 2017 Nov 21;10(1):601. Epub 2017 Nov 21.

Istituto di Chimica del Riconoscimento Molecolare (CNR), c/o Università Cattolica del Sacro Cuore, Rome, Italy.

Objective: Dystroglycan (DG) is an adhesion complex formed by two subunits, α-DG and β-DG. In skeletal muscle, DG is part of the dystrophin-glycoprotein complex that is crucial for sarcolemma stability and it is involved in a plethora of muscular dystrophy phenotypes. Due to the important role played by DG in skeletal muscle stability as well as in a wide variety of other tissues including brain and the peripheral nervous system, it is essential to investigate its genetic assembly and transcriptional regulation.

Results: Herein, we analyze the effect of the insertion of a floxed neomycin (Neo) cassette within the 3' portion of the universally conserved IG1-intron of the DG gene (Dag1). We analyzed the transcription level of Dag1 and the expression of the DG protein in skeletal muscle of targeted mice compared to wild-type and we did not find any alterations that might be attributed to the gene targeting. However, we found an increase of the cross-sectional areas of tibialis anterior that might have some physiological significance that needs to be assessed in the future. Moreover, in targeted mice the skeletal muscle morphology and its regeneration capacity after injury did not show any evident alterations. We confirmed that the targeting of Dag1 with a floxed Neo-cassette did not produce any gross undesired effects.
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http://dx.doi.org/10.1186/s13104-017-2926-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696793PMC
November 2017

A dystroglycan mutation (p.Cys667Phe) associated to muscle-eye-brain disease with multicystic leucodystrophy results in ER-retention of the mutant protein.

Hum Mutat 2018 02 7;39(2):266-280. Epub 2017 Dec 7.

Istituto di Chimica del Riconoscimento Molecolare - CNR c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Roma, Italy.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated α-DG and the transmembrane β-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of α-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of α-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the α-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the β-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of α-DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high-resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small-angle X-ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the β-DG ectodomain overall folding, resulting in disulfide-associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum-retention, underlying dystroglycanopathy.
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http://dx.doi.org/10.1002/humu.23370DOI Listing
February 2018

The effect of the pathological V72I, D109N and T190M missense mutations on the molecular structure of α-dystroglycan.

PLoS One 2017 16;12(10):e0186110. Epub 2017 Oct 16.

Istituto di Cristallografia-CNR, Trieste Outstation, Trieste, Italy.

Dystroglycan (DG) is a highly glycosylated protein complex that links the cytoskeleton with the extracellular matrix, mediating fundamental physiological functions such as mechanical stability of tissues, matrix organization and cell polarity. A crucial role in the glycosylation of the DG α subunit is played by its own N-terminal region that is required by the glycosyltransferase LARGE. Alteration in this O-glycosylation deeply impairs the high affinity binding to other extracellular matrix proteins such as laminins. Recently, three missense mutations in the gene encoding DG, mapped in the α-DG N-terminal region, were found to be responsible for hypoglycosylated states, causing congenital diseases of different severity referred as primary dystroglycanopaties.To gain insight on the molecular basis of these disorders, we investigated the crystallographic and solution structures of these pathological point mutants, namely V72I, D109N and T190M. Small Angle X-ray Scattering analysis reveals that these mutations affect the structures in solution, altering the distribution between compact and more elongated conformations. These results, supported by biochemical and biophysical assays, point to an altered structural flexibility of the mutant α-DG N-terminal region that may have repercussions on its interaction with LARGE and/or other DG-modifying enzymes, eventually reducing their catalytic efficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186110PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643065PMC
October 2017

Structural flexibility of human α-dystroglycan.

FEBS Open Bio 2017 08 17;7(8):1064-1077. Epub 2017 Jul 17.

Istituto di Cristallografia CNR, Trieste Outstation Italy.

Dystroglycan (DG), composed of α and β subunits, belongs to the dystrophin-associated glycoprotein complex. α-DG is an extracellular matrix protein that undergoes a complex post-translational glycosylation process. The bifunctional glycosyltransferase like-acetylglucosaminyltransferase (LARGE) plays a crucial role in the maturation of α-DG, enabling its binding to laminin. We have already structurally analyzed the N-terminal region of murine α-DG (α-DG-Nt) and of a pathological single point mutant that may affect recognition of LARGE, although the structural features of the potential interaction between LARGE and DG remain elusive. We now report on the crystal structure of the wild-type human α-DG-Nt that has allowed us to assess the reliability of our murine crystallographic structure as a α-DG-Nt general model. Moreover, we address for the first time both structures in solution. Interestingly, small-angle X-ray scattering (SAXS) reveals the existence of two main protein conformations ensembles. The predominant species is reminiscent of the crystal structure, while the less populated one assumes a more extended fold. A comparative analysis of the human and murine α-DG-Nt solution structures reveals that the two proteins share a common interdomain flexibility and population distribution of the two conformers. This is confirmed by the very similar stability displayed by the two orthologs as assessed by biochemical and biophysical experiments. These results highlight the need to take into account the molecular plasticity of α-DG-Nt in solution, as it can play an important role in the functional interactions with other binding partners.
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http://dx.doi.org/10.1002/2211-5463.12259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537065PMC
August 2017

Genetic Engineering of Dystroglycan in Animal Models of Muscular Dystrophy.

Biomed Res Int 2015 24;2015:635792. Epub 2015 Aug 24.

Istituto di Chimica del Riconoscimento Molecolare, CNR c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, 00168 Roma, Italy ; School of Biochemistry, Bristol University, Bristol B58 1TD, UK.

In skeletal muscle, dystroglycan (DG) is the central component of the dystrophin-glycoprotein complex (DGC), a multimeric protein complex that ensures a strong mechanical link between the extracellular matrix and the cytoskeleton. Several muscular dystrophies arise from mutations hitting most of the components of the DGC. Mutations within the DG gene (DAG1) have been recently associated with two forms of muscular dystrophy, one displaying a milder and one a more severe phenotype. This review focuses specifically on the animal (murine and others) model systems that have been developed with the aim of directly engineering DAG1 in order to study the DG function in skeletal muscle as well as in other tissues. In the last years, conditional animal models overcoming the embryonic lethality of the DG knock-out in mouse have been generated and helped clarifying the crucial role of DG in skeletal muscle, while an increasing number of studies on knock-in mice are aimed at understanding the contribution of single amino acids to the stability of DG and to the possible development of muscular dystrophy.
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http://dx.doi.org/10.1155/2015/635792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561298PMC
June 2016

Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

PLoS One 2015 17;10(7):e0132455. Epub 2015 Jul 17.

Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Via Montpellier 1, I-00133, Rome, Italy; Center for Space Biomedicine, University of Roma Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy.

Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132455PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506093PMC
May 2016

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy.

PLoS One 2015 1;10(5):e0124277. Epub 2015 May 1.

School of Biochemistry, University of Bristol, Bristol, BS8 1TD, United Kingdom; Istituto di Chimica del Riconoscimento Molecolare-CNR, c/o Università Cattolica del Sacro Cuore, Roma, 00168, Italy.

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159-180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1-B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124277PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416926PMC
January 2016

Role of gelatinases in pathological and physiological processes involving the dystrophin-glycoprotein complex.

Matrix Biol 2015 May-Jul;44-46:130-7. Epub 2015 Feb 17.

Istituto di Chimica del Riconoscimento Molecolare (CNR) c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy.

Dystrophin is a cytosolic protein belonging to a membrane-spanning glycoprotein complex, called dystrophin-glycoprotein complex (DGC) that is expressed in many tissues, especially in skeletal muscle and in the nervous system. The DGC connects the cytoskeleton to the extracellular matrix and, although none of the proteins of the DGC displays kinase or phosphatase activity, it is involved in many signal transduction pathways. Mutations in some components of the DGC are linked to many forms of inherited muscular dystrophies. In particular, a mutation in the dystrophin gene, leading to a complete loss of the protein, provokes one of the most prominent muscular dystrophies, the Duchenne muscular dystrophy, which affects 1 out of 3500 newborn males. What is observed in these circumstances, is a dramatic alteration of the expression levels of a multitude of metalloproteinases (MMPs), a family of extracellular Zn(2+)-dependent endopeptidases, in particular of MMP-2 and MMP-9, also called gelatinases. Indeed, the enzymatic activity of MMP-2 and MMP-9 on dystroglycan, an important member of the DGC, plays a significant role also in physiological processes taking place in the central and peripheral nervous system. This mini-review discusses the role of MMP-2 and MMP-9, in physiological as well as pathological processes involving members of the DGC.
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http://dx.doi.org/10.1016/j.matbio.2015.02.005DOI Listing
March 2016

α-dystroglycan is a potential target of matrix metalloproteinase MMP-2.

Matrix Biol 2015 Jan 4;41:2-7. Epub 2014 Dec 4.

Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy. Electronic address:

Dystroglycan (DG) is a member of the glycoprotein complex associated to dystrophin and composed by two subunits, the β-DG, a transmembrane protein, and the α-DG, an extensively glycosylated extracellular protein. The β-DG ectodomain degradation by the matrix metallo-proteinases (i.e., MMP-2 and MMP-9) in both, pathological and physiological conditions, has been characterized in detail in previous publications. Since the amounts of α-DG and β-DG at the cell surface decrease when gelatinases are up-regulated, we investigated the degradation of α-DG subunit by MMP-2. Present data show, for the first time, that the proteolysis of α-DG indeed occurs on a native glycosylated molecule enriched from rabbit skeletal muscle. In order to characterize the α-DG portion, which is more prone to cleavage by MMP-2, we performed different degradations on tailored recombinant domains of α-DG spanning the whole subunit. The overall bulk of results casts light on a relevant susceptibility of the α-DG to MMP-2 degradation with particular reference to its C-terminal domain, thus opening a new scenario on the role of gelatinases (in particular of MMP-2) in the degradation of this glycoprotein complex, taking place in the course of pathological processes.
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http://dx.doi.org/10.1016/j.matbio.2014.11.007DOI Listing
January 2015

Insights from molecular dynamics simulations: structural basis for the V567D mutation-induced instability of zebrafish alpha-dystroglycan and comparison with the murine model.

PLoS One 2014 31;9(7):e103866. Epub 2014 Jul 31.

Istituto di Chimica del Riconoscimento Molecolare (ICRM) - CNR c/o Università Cattolica del Sacro Cuore, Rome, Italy.

A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103866PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117597PMC
November 2015

The multiple affinities of α-dystroglycan.

Curr Protein Pept Sci 2013 Nov;14(7):626-34

CNR-Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, I-00168 Rome, Italy.

The dystroglycan (DG) adhesion complex is formed by the peripheral α-DG and the transmembrane β-DG, both originating from the same precursor. α-DG plays a crucial role for tissue stability since it binds with high affinity a variety of proteins and proteoglycans in many different cell types. One common molecular feature of most of the α-DG ligands is the presence of laminin globular (LG) domains that are likely to interact with some of the carbohydrates protruding from the mucin-like region of α-DG. Every tissue is supposed to produce a specific α-DG harboring a particular sugar moiety that will enable it to bind a specific ligand, but often several α-DG ligands are co-expressed within the same tissue. It is therefore very important to assess all these different interactions, ultimately measuring the affinity constants (KDs) underlying them. Herein, we present an updated list of α-DG interactors, including non LG-domains containing ligands, offering both a historic perspective on the original contributions made by several laboratories and an update on the different techniques used and the KD values obtained so far. For the cure of some muscular dystrophies, the reinstatement of a prominent affinity between α-DG and one of its vicarious ligands is becoming an increasingly popular choice for strengthening the basement membrane-tissue connection. An update on the current available information about α- DG's multiple, and often "concomitant" affinities, may be of interest for those wishing to better direct their molecular therapy approaches. A final paragraph is dedicated to comment on the evidence that an increase in affinity is not always advantageous.
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http://dx.doi.org/10.2174/1389203711209070644DOI Listing
November 2013

Probing the stability of the "naked" mucin-like domain of human α-dystroglycan.

BMC Biochem 2013 Jul 1;14:15. Epub 2013 Jul 1.

Background: α-Dystroglycan (α-DG) is heavily glycosylated within its central mucin-like domain. The glycosylation shell of α-dystroglycan is known to largely influence its functional properties toward extracellular ligands. The structural features of this α-dystroglycan domain have been poorly studied so far. For the first time, we have attempted a recombinant expression approach in E. coli cells, in order to analyze by biochemical and biophysical techniques this important domain of the α-dystroglycan core protein.

Results: We expressed the recombinant mucin-like domain of human α-dystroglycan in E. coli cells, and purified it as a soluble peptide of 174 aa. A cleavage event, that progressively emerges under repeated cycles of freeze/thaw, occurs at the carboxy side of Arg461, liberating a 151 aa fragment as revealed by mass spectrometry analysis. The mucin-like peptide lacks any particular fold, as confirmed by its hydrodynamic properties and its fluorescence behavior under guanidine hydrochloride denaturation. Dynamic light scattering has been used to demonstrate that this mucin-like peptide is arranged in a conformation that is prone to aggregation at room temperature, with a melting temperature of ~40°C, which indicates a pronounced instability. Such a conclusion has been corroborated by trypsin limited proteolysis, upon which the protein has been fully degraded in less than 60 min.

Conclusions: Our analysis indirectly confirms the idea that the mucin-like domain of α-dystroglycan needs to be extensively glycosylated in order to reach a stable conformation. The absence/reduction of glycosylation by itself may greatly reduce the stability of the dystroglycan complex. Although an altered pattern of α-dystroglycan O-mannosylation, that is not significantly changing its overall glycosylation fraction, represents the primary molecular clue behind currently known dystroglycanopathies, it cannot be ruled out that still unidentified forms of αDG-related dystrophy might originate by a more substantial reduction of α-dystroglycan glycosylation and by its consequent destabilization.
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http://dx.doi.org/10.1186/1471-2091-14-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3704865PMC
July 2013

Enzymatic processing by MMP-2 and MMP-9 of wild-type and mutated mouse β-dystroglycan.

IUBMB Life 2012 Dec 5;64(12):988-94. Epub 2012 Nov 5.

Dipartimento di Scienze Cliniche e Medicina Traslazionale, Università di Roma Tor Vergata, Roma, Italy.

Dystroglycan (DG) is a membrane-associated protein complex formed by two noncovalently linked subunits, α-DG, a highly glycosylated extracellular protein, and β-DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C-terminal domain of α-DG and the N-terminal extracellular domain of β-DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP-9 and/or MMP-2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine β-DG ectodomain by gelatinases, identifying a main cleavage site on the β-DG ectodomain produced by MMP-9. In this article, we have deepened the pattern of the β-DG ectodomain digestion by MMP-2 by using a combined approach based on SDS-PAGE, Orbitrap, and HPLC-ESI-IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some β-DG ectodomain mutants by gelatinases.
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http://dx.doi.org/10.1002/iub.1095DOI Listing
December 2012

Insertion of a myc-tag within α-dystroglycan domains improves its biochemical and microscopic detection.

BMC Biochem 2012 Jul 26;13:14. Epub 2012 Jul 26.

Istituto di Chimica del Riconoscimento Molecolare (CNR) c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo F, Vito 1, Rome, Italy.

Background: Epitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology. The knowledge collected on the subdomain organization of the two subunits of the adhesion complex dystroglycan (DG) enabled us to insert the 10 amino acids myc-tag at different locations along the α-subunit, in order to better visualize and investigate the DG complex in eukaryotic cells.

Results: We have generated two forms of DG polypeptides via the insertion of the myc-tag 1) within a flexible loop (between a.a. 170 and 171) that separates two autonomous subdomains, and 2) within the C-terminal domain in position 500. Their analysis showed that double-tagging (the β-subunit is linked to GFP) does not significantly interfere with the correct processing of the DG precursor (pre-DG) and confirmed that the α-DG N-terminal domain is processed in the cell before α-DG reaches its plasma membrane localization. In addition, myc insertion in position 500, right before the second Ig-like domain of α-DG, proved to be an efficient tool for the detection and pulling-down of glycosylated α-DG molecules targeted at the membrane.

Conclusions: Further characterization of these and other myc-permissive site(s) will represent a valid support for the study of the maturation process of pre-DG and could result in the creation of a new class of intrinsic doubly-fluorescent DG molecules that would allow the monitoring of the two DG subunits, or of pre-DG, in cells without the need of antibodies.
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http://dx.doi.org/10.1186/1471-2091-13-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432625PMC
July 2012

Dystroglycan is associated to the disulfide isomerase ERp57.

Exp Cell Res 2012 Nov 16;318(19):2460-9. Epub 2012 Jul 16.

Istituto di Chimica del Riconoscimento Molecolare (CNR), c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, 00168 Roma, Italy.

Dystroglycan (DG) is an extracellular receptor composed of two subunits, α-DG and β-DG, connected through the α-DG C-terminal domain and the β-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the β-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with β-DG in the ER and at the plasma membrane of 293-Ebna cells. The β-DG-ERp57 complex also included α-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. β-DG and ERp57 were also co-immunoprecipitated in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both α-DG and β-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated.
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http://dx.doi.org/10.1016/j.yexcr.2012.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459099PMC
November 2012

An immunological analysis of dystroglycan subunits: lessons learned from a small cohort of non-congenital dystrophic patients.

Open Neurol J 2011 20;5:68-74. Epub 2011 Oct 20.

CNR - Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Catholic University, Rome, Italy.

The dystroglycan (DG) expression pattern can be altered in severe muscular dystrophies. In fact, some congenital muscular dystrophies (CMDs) and limb-girdle muscular dystrophies (LGMDs) are caused by point mutations identified in six glycosyltransferase genes which are likely to target different steps along the posttranslational "O-glycosylation route" leading to a fully decorated and functional α-DG subunit. Indeed, hypoglycosylation of α-DG is thought to represent a major pathological event, in that it could reduce the DG's ability to bind the basement membrane components, thus leading to sarcolemmal instability and necrosis. In order to set up an efficient standard immunological protocol, taking advantage of a wide panel of antibodies, we have analyzed the two DG subunits in a small cohort of adult dystrophic patients, whom an extensive medical examination had already clinically classified as affected by LGMD (5), Miyoshi (1) or distal (1) myopathy. Immunofluorescence analysis of skeletal muscle tissue sections revealed a proper sarcolemmal localization of the DG subunits in all the patients analyzed. However, Western blot analysis of lectin enriched skeletal muscle samples revealed an abnormal glycosylation of α-DG in two patients. Our work reinforces the notion that a careful immunological and biochemical analysis of the two DG subunits should be always considered as a prerequisite for the identification of new putative cases of dystroglycanopathy.
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http://dx.doi.org/10.2174/1874205X01105010068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204415PMC
November 2011

A second Ig-like domain identified in dystroglycan by molecular modelling and dynamics.

J Mol Graph Model 2011 Aug 5;29(8):1015-24. Epub 2011 May 5.

CNR - Istituto di Chimica del Riconoscimento Molecolare, c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, I-00168 Rome, Italy.

Dystroglycan (DG) is a cell surface receptor which is composed of two subunits that interact noncovalently, namely α- and β-DG. In skeletal muscle, DG is the central component of the dystrophin-glycoprotein complex (DGC) that anchors the actin cytoskeleton to the extracellular matrix. To date only the three-dimensional structure of the N-terminal region of α-DG has been solved by X-ray crystallography. To expand such a structural analysis, a theoretical molecular model of the murine α-DG C-terminal region was built based on folding recognition/threading techniques. Although there is no a significant (<30%) sequence homology with the N-terminal region of α-DG, protein fold recognition methods found a significant resemblance to the α-DG N-terminal crystallographic structure. Our in silico structural prediction identified two subdomains in this region. Amino acid residues ∼ 500-600 of α-DG were predicted to adopt an immunoglobulin-like (Ig-like) β-sandwich fold. Such modeled domain includes the β-DG binding epitope of α-DG and, confirming our previous experimental results, suggests that the linear epitope (residues 550-565) assumes a β-strand conformation. The remaining segment of the α-DG C-terminal region (residues 601-653) is organized in a coil-helix-coil motif. A 20-ns molecular dynamics simulation in explicit water solvent provided support to the predicted Ig-like model structure. The identification of a second Ig-like domain in DG represents another important step towards a full structural and functional description of the α/β DG interface. Preliminary characterization of a novel recombinant peptide (505-600) encompassing this second Ig-like domain demonstrates that it is soluble and stable, further corroborating our in silico analysis.
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http://dx.doi.org/10.1016/j.jmgm.2011.04.008DOI Listing
August 2011

Enzymatic processing of beta-dystroglycan recombinant ectodomain by MMP-9: identification of the main cleavage site.

IUBMB Life 2009 Dec;61(12):1143-52

Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, Rome, Italy.

Dystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, alpha-DG, a highly glycosylated extracellular matrix protein, and beta-DG, a transmembrane protein. The two DG subunits interact through the C-terminal domain of alpha-DG and the N-terminal extracellular domain of beta-DG in a noncovalent way. Such interaction is crucial to maintain the integrity of the plasma membrane. In some pathological conditions, the interaction between the two DG subunits may be disrupted by the proteolytic activity of gelatinases (i.e. MMP-9 and/or MMP-2) that removes a portion or the whole beta-DG ectodomain producing a 30 kDa truncated form of beta-DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of beta-DG, beta-DG(654-750) with human MMP-9, characterizing the catalytic parameters of its cleavage. Furthermore, using a combined approach based on SDS-PAGE, MALDI-TOF and HPLC-ESI-IT mass spectrometry, we were able to identify one main MMP-9 cleavage site that is localized between the amino acids His-715 and Leu-716 of beta-DG, and we analysed the proteolytic fragments of beta-DG(654-750) produced by MMP-9 enzymatic activity.
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http://dx.doi.org/10.1002/iub.273DOI Listing
December 2009

Mutagenesis at the alpha-beta interface impairs the cleavage of the dystroglycan precursor.

FEBS J 2009 Sep 4;276(17):4933-45. Epub 2009 Aug 4.

Istituto di Chimica del Riconoscimento Molecolare (CNR), c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy.

The interaction between a-dystroglycan (alpha-DG) and beta-dystroglycan (beta-DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial in maintaining the integrity of the dystrophin-glycoprotein complex. The importance of the alpha-beta interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the alpha-beta interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis. The reciprocal binding epitopes of the two subunits reside within the C-terminus of alpha-DG and the ectodomain of beta-DG. As no ultimate structural data are yet available on the alpha-beta interface, site-directed mutagenesis was used to identify which specific amino acids are involved in the interaction. A previous alanine-scanning analysis of the recombinant beta-DG ectodomain allowed the identification of two phenylalanines important for alpha-DG binding, namely F692 and F718. In this article, similar experiments performed on the alpha-DG C-terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two beta-DG phenylalanines. In 293-Ebna cells, the introduction of alanine residues instead of F692, F718, G563 and P565 prevented the cleavage of the DG precursor that liberates alpha- and beta-DG, generating a pre-DG of about 160 kDa. This uncleaved pre-DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pull-down assays. These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07196.xDOI Listing
September 2009

Functional diversity of dystroglycan.

Matrix Biol 2009 May 19;28(4):179-87. Epub 2009 Mar 19.

Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo F. Vito 1, Rome, Italy.

During the last 15 years, following its identification and first detailed molecular characterization, the dystroglycan (DG) complex has taken centre stage in biology and biomedicine. Functions in different cells and tissues have been identified for this complex, ranging from its typical role in skeletal muscle as a sarcolemmal stabilizer, highlighted by the recently identified "secondary dystroglycanopathies", to a variety of very diverse functions including embryogenesis, cancer progression, virus particle entry and cell signalling. Such functional promiscuity can be in part explained when considering the multiple domain organization of the two DG subunits, the extracellular alpha-DG and the transmembrane beta-DG, that has been largely scrutinized, but only in part unraveled, exploiting a variety of recombinant and transgenic approaches. Herein, while rapidly recapitulating some of the functions that nowadays can be assigned safely to each DG domain, we also try to envisage a sort of worry list featuring and dwelling on some of the most compelling "mysteries" that should be solved to finally understand DG's functional diversity.
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http://dx.doi.org/10.1016/j.matbio.2009.03.003DOI Listing
May 2009

First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle.

Matrix Biol 2008 May 23;27(4):360-70. Epub 2007 Dec 23.

Istituto di Chimica del Riconoscimento Molecolare (CNR) c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore L.go F. Vito 1, 00168 Rome, Italy.

Keratoepithelin (KE) is an extracellular matrix protein that binds collagens, fibronectin, decorin, biglycan and integrins, interconnecting extracellular matrix components with resident cells in several tissues. KE has a molecular mass of 68 kDa and harbours four FAS1 domains named after those identified in the insect cell adhesion molecule fasciclin I. In humans, KE is preferentially expressed by the corneal epithelial layer and liberated towards the corneal stroma but it was also detected in the lung and in the bladder smooth muscle. No detailed information is available on the distribution of this protein in other human tissues. In this work, we have raised a polyclonal antibody against the recombinantly expressed human fourth FAS1 domain which is able to specifically detect KE in human skeletal muscle tissue extracts. Immunofluorescence experiments indicate that KE is localized around the perimysium and endomysium of each skeletal muscle fiber. The same kind of analysis shows that in muscle sections from patients affected by different forms of muscular dystrophy KE is upregulated and widely distributed in fibrotic tissues. The muscle specific expression of KE was also demonstrated by RT-PCR. In human skeletal muscle, KE may help to build up a bridge between collagen VI and yet unidentified muscle receptor(s), adding to the complexity of the adhesive molecular network established between muscle fibers and the surrounding basement membrane.
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http://dx.doi.org/10.1016/j.matbio.2007.12.003DOI Listing
May 2008

Concerted mutation of Phe residues belonging to the beta-dystroglycan ectodomain strongly inhibits the interaction with alpha-dystroglycan in vitro.

FEBS J 2006 Nov 3;273(21):4929-43. Epub 2006 Oct 3.

Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy.

The dystroglycan adhesion complex consists of two noncovalently interacting proteins: alpha-dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane beta-dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F-actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post-translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of beta-dystroglycan (691-719), and approximately 15 amino acids of alpha-dystroglycan (550-565). Exploiting alanine scanning, we have produced a panel of site-directed mutants of our two consolidated recombinant peptides beta-dystroglycan (654-750), corresponding to the ectodomain of beta-dystroglycan, and alpha-dystroglycan (485-630), spanning the C-terminal domain of alpha-dystroglycan. By solid-phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild-type alpha-dystroglycan and beta-dystroglycan, and shown the crucial role of two beta-dystroglycan phenylalanines, namely Phe692 and Phe718, for the alpha-beta interaction. Substitution of the alpha-dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro. As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan-transfected 293-Ebna cells.
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http://dx.doi.org/10.1111/j.1742-4658.2006.05492.xDOI Listing
November 2006

The effect of an ionic detergent on the natively unfolded beta-dystroglycan ectodomain and on its interaction with alpha-dystroglycan.

Protein Sci 2004 Sep 4;13(9):2437-45. Epub 2004 Aug 4.

Consiglio Nazionale delle Richerche (CNR), Istituto di Chimica del Riconoscimento Molecolare, c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.

Dystroglycan (DG) is an adhesion complex, expressed in a wide variety of tissues, formed by an extracellular and a transmembrane subunit, alpha-DG and beta-DG, respectively, interacting noncovalently. Recently, we have shown that the recombinant ectodomain of beta-DG, beta-DG(654-750), behaves as a natively unfolded protein, as it is able to bind the C-terminal domain of alpha-DG, while not displaying a defined structural organization. We monitored the effect of a commonly used denaturing agent, the anionic detergent sodium dodecylsulphate (SDS), on beta-DG(654-750) using a number of biophysical techniques. Very low concentrations of SDS (< or =2 mM) affect both tryptophan fluorescence and circular dichroism of beta-DG, and significantly perturb the interaction with the alpha-DG subunit as shown by solid-phase binding assays and fluorescence titrations in solution. This result confirms, as recently proposed for natively unfolded proteins, that beta-DG(654-750) exists in a native state, which is crucial to fulfill its biological function. Two-dimensional NMR analysis shows that SDS does not induce any evident conformational rearrangement within the ectodomain of beta-DG. Its first 70 amino acids, which show a lower degree of mobility, interact with the detergent, but this does not change the amount of secondary structure, whereas the highly flexible and mobile C-terminal region of beta-DG(654-750) remains largely unaffected, even at a very high SDS concentration (up to 50 mM). Our data indicate that SDS can be used as a useful tool for investigating natively unfolded proteins, and confirm that the beta-DG ectodomain is an interesting model system.
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http://dx.doi.org/10.1110/ps.04762504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2280000PMC
September 2004

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.

Biochemistry 2003 Nov;42(46):13717-24

Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Consiglio Nazionale della Ricerche, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy.

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.
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http://dx.doi.org/10.1021/bi034867wDOI Listing
November 2003

Dystroglycan and muscular dystrophies related to the dystrophin-glycoprotein complex.

Ann Ist Super Sanita 2003 ;39(2):173-81

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy.

Dystroglycan (DG) is an adhesion molecule composed of two subunits, alpha and beta, that are produced by the post-translational cleavage of a single precursor molecule. DG is a pivotal component of the dystrophin-glycoprotein complex (DGC), which connects the extracellular matrix to the cytoskeleton in skeletal muscle and many other tissues. Some muscular dystrophies are caused by mutations of DGC components, such as dystrophin, sarcoglycan or laminin-2, or also of DGC-associated molecules, such as caveolin-3. DG-null mice died during early embriogenesis and no neuromuscular diseases directly associated to genetic abnormalities of DG were identified so far. However, DG plays a crucial role for muscle integrity since its targeting at the sarcolemma is often perturbed in DGC-related neuromuscular disorders.
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December 2003
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