Search

Protein science 32(2), e4563 (2023). doi:10.1002/pro.4563, Nerve growth factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR, and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+, known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis., Published by Protein Society, Bethesda, Md.

Nerve Growth Factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75

International journal of molecular sciences 23(12), 6596 (2022). doi:10.3390/ijms23126596, The human genetic variant BDNF (V66M) represents the first example of neurotrophin family member that has been linked to psychiatric disorders. In order to elucidate structural differences that account for the effects in cognitive function, this hproBDNF polymorph was expressed, refolded, purified, and compared directly to the WT variant for the first time for differences in their 3D structures by DSF, limited proteolysis, FT-IR, and SAXS measurements in solution. Our complementary studies revealed a deep impact of V66M polymorphism on hproBDNF conformations in solution. Although the mean conformation in solution appears to be more compact in the V66M variant, overall, we demonstrated a large increase in flexibility in solution upon V66M mutation. Thus, considering that plasticity in IDR is crucial for protein function, the observed alterations may be related to the functional alterations in hproBDNF binding to its receptors p75NTR, sortilin, HAP1, and SorCS2. These effects can provoke altered intracellular neuronal trafficking and/or affect proBDNF physiological functions, leading to many brain-associated diseases and conditions such as cognitive impairment and anxiety. The structural alterations highlighted in the present study may pave the way to the development of drug discovery strategies to provide greater therapeutic responses and of novel pharmacologic strategy in human populations with this common polymorphism, ultimately guiding personalized medicine for neuropsychiatric disorders., Published by Molecular Diversity Preservation International, Basel

LuxR solos are related to quorum sensing (QS) LuxR family regulators; however, they lack a cognate LuxI family protein. LuxR solos are widespread and almost exclusively found in proteobacteria. In this study, we investigated the distribution and conservation of LuxR solos in the fluorescent pseudomonads group. Our analysis of more than 600 genomes revealed that the majority of fluorescent Pseudomonas spp. carry one or more LuxR solos, occurring considerably more frequently than complete LuxI/LuxR archetypical QS systems. Based on the adjacent gene context and conservation of the primary structure, nine subgroups of LuxR solos have been identified that are likely to be involved in the establishment of communication networks. Modeling analysis revealed that the majority of subgroups shows some substitutions at the invariant amino acids of the ligand-binding pocket of QS LuxRs, raising the possibility of binding to non-acyl-homoserine lactone (AHL) ligands. Several mutants and gene expression studies on some LuxR solos belonging to different subgroups were performed in order to shed light on their response. The commonality of LuxR solos among fluorescent pseudomonads is an indication of their important role in cell-cell signaling. IMPORTANCE Cell-cell communication in bacteria is being extensively studied in simple settings and uses chemical signals and cognate regulators/receptors. Many Gram-negative proteobacteria use acyl-homoserine lactones (AHLs) synthesized by LuxI family proteins and cognate LuxR-type receptors to regulate their quorum sensing (QS) target loci. AHL-QS circuits are the best studied QS systems; however, many proteobacterial genomes also contain one or more LuxR solos, which are QS-related LuxR proteins which are unpaired to a cognate LuxI. A few LuxR solos have been implicated in intraspecies, interspecies, and interkingdom signaling. Here, we report that LuxR solo homologs occur considerably more frequently than complete LuxI/LuxR QS systems within the Pseudomonas fluorescens group of species and that they are characterized by different genomic organizations and primary structures and can be subdivided into several subgroups. The P. fluorescens group consists of more than 50 species, many of which are found in plant-associated environments. The role of LuxR solos in cell-cell signaling in fluorescent pseudomonads is discussed.

Graphical abstract, Highlights • Bioinformatics analysis unveiled the structural conservation domains of proNTs. • The dynamic behavior in solution of human proNTs has been exploited by SAXS and DSF. • The functional plasticity and evolutionary adaptation of proNTs have been assessed., The neurotrophins, i.e., Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT3) and Neurotrophin 4 (NT4), are known to play a range of crucial functions in the developing and adult peripheral and central nervous systems. Initially synthesized as precursors, i.e., proneurotrophins (proNTs), that are cleaved to release C-terminal mature forms, they act through two types of receptors, the specific Trk receptors (Tropomyosin-related kinases) and the pan-neurotrophin receptor p75NTR, to initiate survival and differentiative responses. Recently, all the proNTs but proNT4 have been demonstrated to be not just inactive precursors, but signaling ligands that mediate opposing actions in fundamental aspects of the nervous system with respect to the mature counterparts through dual-receptor complexes formation with a member of the VPS10 family and p75NTR. Despite the functional relevance, the molecular determinants underpinning the interactions between the pro-domains and their receptors are still elusive probably due to their intrinsically disordered nature. Here we present an evolutionary approach coupled to an experimental study aiming to uncover the structural and dynamical basis of the biological function displayed by proNGF, proBDNF and proNT3 but missing in proNT4. A bioinformatic analysis allowed to elucidate the functional adaptability of the proNTs family in vertebrates, identifying conserved key structural features. The combined biochemical and SAXS experiments shed lights on the structure and dynamic behavior of the human proNTs in solution, giving insights on the evolutionary conserved structural motifs, essential for the multifaceted roles of proNTs in physiological as well as in pathological contexts.

Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium-chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA-hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA-binding drugs allowed locating the high affinity binding site in sub-domain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications.

Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling., Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization. IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated alpha-DG and the transmembrane beta-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of alpha-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of alpha-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the alpha-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the beta-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of alpha-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 beta-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.

Dystroglycan (DG), composed of and subunits, belongs to the dystrophin-associated glycoprotein complex. alpha-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 alpha-DG, enabling its binding to laminin. We have already structurally analyzed the N-terminal region of murine alpha-DG (alpha-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 alpha-DG-Nt that has allowed us to assess the reliability of our murine crystallographic structure as alpha-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 alpha-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 alpha-DG-Nt in solution, as it can play an important role in the functional interactions with other binding partners.

The immobilization of biomolecules at screen printed electrodes for biosensing applications is still an open challenge. To enrich the toolbox of bioelectrochemists, graphite screen printed electrodes (G-SPE) were modified with an electropolymerized film of pyrrole-2-carboxilic acid (Py-2-COOH), a pyrrole derivative rich in carboxylic acid functional groups. These functionalities are suitable for the covalent immobilization of biomolecular recognition layers. The electropolymerization was first optimized to obtain stable and conductive polymeric films, comparing two different electrolytes: sodium dodecyl sulphate (SDS) and sodium perchlorate. The G-SPE modified with Py-2-COOH in 0.1 M SDS solution showed the required properties and were further tested. A proof-of-concept study for the development of an impedimetric sensor for perfluorooctanoic acid (PFOA) was carried out using the delipidated human serum albumin (hSA) as bioreceptor. The data interpretation was supported by size exclusion chromatography and small-angle X-ray scattering (SEC-SAXS) analysis of the bioreceptor-target complex and the preliminary results suggest the possibility to further develop this biosensing strategy for toxicological and analytical studies.

The dystroglycan (DG) adhesion complex has a crucial role for muscle stability and is involved in a number of autosomal recessive neuromuscular disorders ranging from severe congenital (Muscle-Eye-Brain and Walker-Warburg syndrome) to milder limb-girdle muscular dystrophies (LGMD2P). The complex undergoes a still poorly understood molecular maturation/targeting pathway, in which a precursor molecule undergoes an early proteolytic event in the endoplasmic reticulum and is cleaved into two subunits, alpha (extracellular) and beta (transmembrane); this is followed by a multistep glycosylation process which mainly takes place within the Golgi apparatus. Most of the currently identified dystroglycanopathies depend on an impaired sugar decoration of the alpha-dystroglycan subunit that, when hypoglycosylated, shows a reduced binding affinity towards laminin-2 (dubbed as secondary dystroglycanopathies). However, lately a novel subgroup of primary dystroglycanopathies, depending on missense mutations of the dystroglycan core protein, is emerging. We have established an array of diverse tools for the analysis of primary dystroglycanopathies. Namely, the production of novel mice lines, the analysis of transfected cellular lines and the recombinant expression of isolated domains for biochemical and crystallization analysis, as well as computational biochemistry. Our cellular, microscopic and biochemical analysis of the murine counterpart (C667F) of the recently identified missense mutation C669F (inducing a severe muscle-eye-brain phenotype) located within the ectodomain of human beta-DG has allowed us to show that i) the processing of the alpha-beta DG precursor is altered; ii) the variant protein is mainly stuck in the ER and iii) there is some degree of oligomerization and/or structural organization within these C667F aggregates. Following the award of an AFM grant to study primary dystroglycanopathies, we have strongly invested into the generation of a first mouse model. The company GenOway is currently taking care of preparing the knocked-in gametes which will be employed at Bonn University for generating the desired heterozygous and homozygous mouse colony that will be used for morphologic and histologic analysis in the brain and muscle.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix and it is composed of two subunits: the extracellular ?-DG and the transmembrane ?-DG. ?-DG is a highly glycosylated protein and its hypoglycosylation weakens its interaction with the extracellular matrix proteins, such as laminins, resulting in a functionally compromised protein. The DG complex undergoes a still poorly understood molecular maturation/targeting pathway, where a precursor is cleaved into ? and ? subunits. Furthermore, the ?-DG subunit is decorated with complex glycans in a multistep glycosylation process. Most of the dystroglycanopathies currently identified are due to an impaired functional state of the enzymes involved in ?-DG maturation (secondary dystroglycanopathies). More recently, missense mutations of the dystroglycan core protein has been identified as the cause of distroglycanopathies (primary dystroglycanopathies) of diverse severity. Indeed, a set of point mutations on the N-terminal region of ?-DG (a.a. 50-313 in mouse) determine the hypoglycosylation of the DG complex, due to the impairment of a key step in ?-DG glycosylation operated by the bifunctional glycosyltransferase LARGE1. Moreover, a point mutation affecting ?-DG, namely the mutation of cysteine 667 to phenylalanine results in a pathological retention of ?-DG at the endoplasmic reticulum level leading to a severe pathological state. With the aim of elucidating the structural implications of the pathological mutations leading to primary distroglycanopathies, we have undertaken a multi-technique study, involving biochemical and biophysical methods as well. Indeed, synchrotron radiation (SR) provides an invaluable tool for the elucidation of the molecular structure of biological macromolecules in both crystals and solution. We have used SR-based X-ray crystallography in order to determine the molecular structure, at the atomic resolution, of ?-DG point mutants. Moreover, SR-based Small Angle X-ray Scattering (SAXS) has been used as a low-resolution probe for the structural organization of ? and ? DG mutants at near-physiological conditions. The results of the SR-based experiments, combined with biochemical and microscopic analysis allowed to shed light on the molecular and structural basis of primary dystroglycanopathies.

A better understanding of the interactions between perfluroocatanoic acid (PFOA) and human serum albumin (HSA) can improve the design of protein-based electrochemical biosensors for fluorinated environmental contaminants, namely per- and poly-fluoroalkyl substances (PFAS). In the last decade, the affinity of PFOA for fatted and defatted serum albumin was confirmed by numerous toxicological studies 1-3 . The results showed that PFOA can bind strongly to serum albumins, such as HAS, mimicking fatty acids binding and affecting the functions of these transport proteins. To transpose these findings in the design of bioreceptors, a multi-analytical study was carried out comparing directly the performances of fatted and defatted HSA in terms of stability, number of binding sites and affinity towards PFOA and other PFAS . Isothermal titration calorimetry (ITC) measurements allowed to define the stoichiometry and the affinity constants, while native nano-electronspray ionization MS (nESI-MS) provided additional information about the stability of the proteins and the protein-target complex. To identify the binding site and discriminate between fatted and defatted HSA, vapor diffusion crystallization was performed. All analysis confirmed the higher affinity of PFOA for the defatted HSA even though the fatted HSA showed an higher stability. Both types of HSA were immobilized on the graphite-screen printed electrodes modified with graphene oxide (GO) or electropolymerized o-phenylene diamine (oPD). Impedance electrochemical spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the immobilization strategies performances. Also direct detection by EIS and indirect detection using a redox mediator were compared to evaluate the binding event between PFOA and HAS. The combination of toxicological data and electrochemistry allowed to devise a new detection strategy for environmental contaminants, mimicking a biological process happening in vivo for biosensing purposes.

Nerve Growth Factor (NGF) is the prototype of the neurotrophins family and induces cell growth and differentiation in neuronal cell types, both in the central and peripheral nervous system. Even though it was discovered almost 70 years ago, and its tertiary structure is known since 1991, many molecular and functional properties remain elusive. At the same time, its pharmaceutical interest is still high, since the protein is involved in many disease mechanisms, like chronic pain and neurodegenerative disorders. Small endogenous ligands that interact with NGF are of increasing interest, due to their likely capability of modulating its biological activity. Among these molecules, ATP was shown to mediate NGF neurotrophic activity through it receptors, TrkA and p75NTR. However, no structural information on the binding sites nor on the mechanism of the interaction is available so far. Aiming at gaining new information on this aspect, we undertook a biophysical study on NGF/ATP binding, by means of solution NMR. We have focused our studies on the recombinant human NGF (rhNGF), which is the molecule of medical interest, more that the better structural characterized mouse protein (mNGF). At first, we have obtained 15N- and 13C15N-labeled rhNGF, suitable for the NMR studies. We have optimized the protocols set up in our previous work on the mouse protein, to better adapt the expression conditions to the human protein. The 2D HSQC NMR collected spectra allowed us to identify structural features of the rhNGF in comparison to mNGF in solution. We know from our previously published data that the hNGF and mNGF do not overlap in their biochemical, biophysical and in vitro functional properties, reflected in their 3D structure in solution and highlighted by their available respective crystal structures. We therefore proceeded to the assignment of both the backbone and side chains of hNGF, to fully characterize the protein, by means of 3D NMR experiments (15N and 13C NOESYs). We investigated the binding effects of ATP and of a set of different divalent ions by means of Differential Scanning Fluorimetry and identified the suitable conditions for the NMR studies. We then moved to the investigation of the NGF/ATP binding, using a protein-based solution NMR approach. We recorded 2D HSQC spectra following a titration with increasing amounts of ATP or the more stable analogue, ATP-PCP. We could identify the binding site of ATP and the results will be described.

Plant cell walls are complex macromolecular composites and their degradation by microorganisms requires a large and diverse panel of hydrolytic enzymes. Moreover, the degradation and fermentation of complex polysaccharides by microbiota is essential for many ecosystem-processes including nutrient cycling and herbivores nutrition. Among the different structural polysaccharides composing the plant cell walls, xylan is the second most abundant one and its degradation in rumen involves the removal of O-acetyl esters decorating the O-2 and O-3 positions of the D-xylanopyranosyl moiety. A transcriptomic analysis of a gene cluster, present in the ruminal anaerobe Prevotella ruminicola 23, revealed a repertoire of xylanolytic enzymes with different functions and cellular localization [1]. Among them Axe7A, an acetylxylan esterase belonging to the CAZy CE7 carbohydrate esterase family, has been established [2]. Interestingly Axe7A amino acidic sequence includes, besides the characteristic catalytic domain, an additional 100 residues long N-terminal domain no previously found in the any other member of the CE7 family members, showing low similarity (around 10% identity) with any other structurally characterized protein annotated in the PDB. In order to gain insights into the functional role of its N-terminal domain, the crystal structure of Axe7XE7A has been solved by SAD phasing and refined at 2.6 Å resolution. Axe7A shares the 32 hexameric doughnut-shape commonly found in with all the CE7 deacetylases [3], while the unique protomer architecture has been disclosed in the CE7 family, for the first time. The N-terminal domain, characterized by a ?-sandwich fold, is actively engaged in the oligomer formation. We are further investigating, by SAXS, the behaviour of Axe7A in solution aiming at validate the crystallographic oligomeric assembly in nearly-physiological conditions.

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early ‘90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody aD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein which is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (aa 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the ?-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation states resulting in primary distroglycanopathies1. We have been investigating the structures of the three point mutants V72I (V72I-ma-DG), D109N (D109N-ma-DG) and T190M2 (T190M-ma-DG) of mouse alpha-DG (ma-DG), combining X-ray crystallography and in solution Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal ?-DG that are functionally relevant for its glycosylation pathway. The crystal structures of the three mutants display the same overall structure of WT-ma-DG. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations, which is mediated by the flexible peptide linking the Ig-like and S6 small-ribosomal domains, embodied in the alpha-DG N-terminal region. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein, whereas beta-DG is an integral membrane protein that also interacts with dystrophin in the cytoplasm. alpha-DG's glycosylation is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (a.a 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the alpha-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation state. We have been investigating the structures of N-terminal region of the WT human alpha-DG (WT-ha-DG) and of the two point mutants V72I (V72I-ma-DG) and D109N (D109N-ma-DG) of mouse alpha-DG by X-ray crystallography, and in solution by Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal alpha-DG that are functionally relevant for its glycosylation pathway. The crystal structure of the ha-DG does not significantly deviate from the already determined crystal structure of WT mouse alpha-DG (WT-ma-DG). The overall fold is conserved and differences are restricted to the most flexible part of the protein, i.e. the loop encompassing residues 159-179, which is only partially visible after crystal structure refinement. In addition, the crystal structures of the two mutants V72I-ma-DG and D109N-ma-DG display the same overall structure of WT-ma-DG, suggesting for negligible effects of the point mutations on the overall fold of alpha-DG. The two mutants show limited and local structural dissimilarities with respect to WT-ma-DG, probably influencing the interaction with potential binding partners. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation, with the most populated close to the crystal conformation and a second less populated one, which is more extended with respect to the principal conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.

A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.

The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and $p75^{NTR}$. Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary $TrkA/NGF/p75^{NTR}$ complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a $TrkA/NGF/p75^{NTR}$ ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of $p75^{NTR}$ with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and $p75^{NTR}/NGF$ complexes.

NMDA receptors (NMDARs) are multimeric proteins, the biological and functional characteristics of which depend on differential subunit assembly during postnatal development. In the present paper, we investigated whether the expression of NMDAR subunits NR1, NR2A, NR2B is influenced by neurotrophins in rat visual cortex. We used a soluble form of the TrkB receptor engineered as an immunoadhesin (TrkB-IgG) in order to block TrkB ligands. TrkB-IgG was released through a cannula implanted in the occipital pole and connected to a mini-osmotic pump. TrkB-IgG was continuously released from postnatal day 20-21 (P20-21) to P36-37. In a different group of animals used as controls, osmotic pumps were filled with saline. Different antibodies were used to stain neurons expressing NR1, NR2A and NR2B. We counted the number of neurons stained for NR2A and NR2B subunits and expressed this as percentage with respect to the total number of cresyl-violet stained neurons in each cortical layer. In the visual cortex of TrkB-IgG-treated rats, the percentage of neurons expressing NR2A was significantly increased in all cortical layers. Concerning the NR2B subunit, the percentage of stained neurons was not significantly different between TrkB-IgG-treated and control rats. The staining level for both NR2A and NR2B, but not NR1, was reduced in all cortical layers in TrkB-IgG-treated animals. In agreement with this result, the endogenous levels of NR2A and NR2B subunits were reduced in TrkB-IgG-treated animals as shown by Western blotting. Thus, TrkB signalling controls the cellular expression of NMDAR subunits in visual cortical neurons during postnatal development.

Objective: ADAMTS5 (aggrecanase-2) has been demonstrated to be crucial in the development of osteoarthritis (OA), by use of several mouse mutants carrying either truncated, catalytically inactive enzymes or aggrecanase-resistant mutant aggrecan. We have selected recombinant monoclonal antibodies directed against ADAMTS5, by using Intracellular Antibody Capture Technology (IACT). CRB0017 revealed very high affinity for the enzyme in Biacore analyses and very good specificity in a panel of binding assays. Therefore, we tested CRB0017 in a relevant spontaneous OA model, the STR/ort mouse. Design: STR/ort male mice were recruited at 5 months of age, and treated intra-articularly in each knee with CRB0017 1.2μg, CRB0017 12μg, or vehicle. After 6 weeks, the intra-articular administration of CRB0017 was repeated with the same doses. After 3 months from recruitment, the animals were sacrificed and the femorotibial joints processed for histology and scored in a blind fashion according to both Mankin's and the OARSI methods. Results and conclusions: All histological scores were significantly decreased in the CRB0017 12μg/knee group compared to vehicle, while administration of CRB0017 1.2μg was associated with a trend to a decrease in the same parameters. Therefore, CRB0017 administered twice in 3 months could modify the course of OA in the STR/ort mouse, by delaying cartilage breakdown as assessed histologically. The procedure of blind scoring of the histological samples clearly showed that knee intra-articular administration of CRB0017, an anti-ADAMTS5 antibody, dose-dependently improved disease progression in a relevant animal model of OA. © 2013 Osteoarthritis Research Society International.

Recent studies have identified a novel interkingdom signaling circuit, via plant signaling molecules, and a bacterial sub-family of LuxR proteins, bridging eukaryotes and prokaryotes. Indeed pivotal plant-bacteria interactions are regulated by the so called Plant Associated Bacteria (PAB) LuxR solo regulators that, although closely related to the quorum sensing (QS) LuxR family, do not bind or respond to canonical quorum sensing N-acyl homoserine lactones (AHLs), but only to specific host plant signal molecules. The large body of structural data available for several members of the QS LuxR family complexed with different classes of ligands (AHLs and other compounds), has been exploited to dissect the cartography of their regulatory domains through structure-based multiple sequence alignments, structural superimposition and a comparative analysis of the contact residues involved in ligand binding. In the absence of experimentally determined structures of members of the PAB LuxR solos subfamily, an homology model of its prototype OryR is presented, aiming to elucidate the architecture of its ligand-binding site. The obtained model, in combination with the cartography of the regulatory domains of the homologous QS LuxRs, provides novel insights into the 3D structure of its ligand-binding site and unveils the probable molecular determinants responsible for differences in selectivity towards specific host plant signal molecules, rather than to canonical QS compounds.

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability. Fil: Corbalan, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Runti, Giulia. Università degli Studi di Trieste; Italia Fil: Adler, Conrado. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Covaceuszach, Sonia. Consiglio Nazionale delle Ricerche; Italia Fil: Ford, Robert C.. University of Manchester; Reino Unido Fil: Lamba, Doriano. Consiglio Nazionale delle Ricerche; Italia Fil: Beis, Konstantinos. Imperial College London; Reino Unido Fil: Scocchi, Marco. Università degli Studi di Trieste; Italia Fil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8–10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.

Nerve Growth Factor (NGF) signalling is mediated by the TrkA and p75NTR receptors. Besides its neurotrophic and survival activities, NGF displays a potent pro-nociceptive activity. Recently, a missense point mutation was found in the NGFB gene (C661T, leading to the aminoacid substitution R100W) of individuals affected by a form of hereditary loss of pain perception (hereditary sensory and autonomic neuropathy type V, HSAN V). In order to gain insights into the functional consequences of the HSAN V NGF mutation, two sets of hNGFR100 mutants were expressed in Escherichia coli and purified, as mature NGF or proNGF, for in vitro receptor binding studies. Here, we show by Surface Plasmon Resonance analysis that the R100 mutation selectively disrupts binding of hNGF to p75NTR receptor, to an extent which depends on the substituting residue at position 100, while the affinity of hNGFR100 mutants for TrkA receptor is not affected. As for unprocessed hproNGF, the binding of the R100 variants to p75NTR receptor shows only a limited impairment, showing that the impact of the R100 mutation on p75NTR receptor binding is greater in the context of mature, processed hNGF. These results provide a basis for elucidating the mechanisms underlying the clinical manifestations of HSAN V patients, and provide a basis for the development of "painless" hNGF molecules with therapeutic potential.

Nerve growth factor (NGF) has a great potential for the treatment of Alzheimer's disease. However, the therapeutic administration of NGF represents a significant challenge, due to the difficulty to deliver relevant doses to the brain, in a safe and non-invasive way. We previously demonstrated the efficacy of a non-invasive delivery of NGF to the brain in animal models, by an intranasal route. Recently, topical eye application of NGF was proposed, as an option for the delivery of NGF to the brain. Here, we compare the efficacy of the two delivery routes of hNGF-61, a recombinant traceable form of human NGF, in the mouse neurodegeneration model AD11. The intranasal administration appeared to be significantly more effective than the ocular one, in rescuing the neurodegenerative phenotypic hallmarks in AD11 mice. The ocular administration of hNGF-61 showed a more limited efficacy, even at higher doses. Thus, NGF nasal drops represent a viable and effective option to successfully deliver therapeutic NGF to the brain in a non-invasive manner.

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm-proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron Small Angle X-ray Scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by Surface Plasmon Resonance (SPR) using, as structural probes, a panel of anti-NGF antibodies and the soluble forms of TrkA and p75NTR receptors. SAXS measurements revealed the rm-proNGF to be dimeric and anisometric, with the pro-peptide domain being intrinsically unstructured. Ab initio reconstructions assuming two-fold symmetry generated two types of structural models, a globular crab-like and an elongated shape, that resulted in equally good fits of the scattering data. A novel method accounting for possible co-existence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the crab-like to be a more likely proNGF conformation. In order to exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the pro-peptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75NTR receptors and a panel of neutralizing anti-NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners.

Nerve growth factor (NGF) deficits are linked to Alzheimer's Disease (AD), due to the role of NGF on basal forebrain cholinergic neurons (BFCN). We have further established that a disequilibrium in NGF signaling and/or processing from its precursor proNGF is also directly and causally related to the aberrant activation of an amyloidogenic route to neurodegeneration. The therapeutic potential of using human NGF to provide a long-lasting cholinergic trophic support, thereby preventing or slowing cognitive decline in AD patients, has therefore a strong rationale. However, a simple and practical means of delivering NGF to the brain in a safe and long-term manner, limiting the undesired adverse effects of NGF in activating nociceptive responses, has represented a significant challenge. For this reason, pilot clinical studies have been performed so far with invasive approaches requiring neurosurgery. We obtained a proof of principle, in neurodegeneration animal models, of an alternative, non-invasive delivery of NGF through an intranasal route, which facilitates access of NGF to the central nervous system (CNS), while minimizing the biodistribution of NGF to compartments where it activates undesired effects, such as pain. The ideal NGF product for a non invasive NGF-based therapy would be a recombinant NGF that, while exhibiting an identical biological activity to that of human NGF, can be traced, against the endogenous NGF, in order to optimize the therapeutical dose range and meet the required therapeutic window. We describe an engineered mutein of hNGF, hNGF-61, that is selectively recognized, against endogenous NGF, by a specific antibody. hNGF-61 mutein has an identical potency and bioactivity profile as hNGF, in vitro and in vivo. Moreover, hNGF-61 and hNGF are equally effective in rescuing the behavioral and neurodegenerative phenotype in adult and aged AD11 anti-NGF mice. Finally, we demonstrated that intranasally delivered hNGF-61 is significantly more effective than ocularly applied hNGF-61, to determine phenotypic rescue in AD11 mice. The development of hNGF-61 towards clinical applications in AD patients is under way.

The anti-nerve growth factor (NGF) monoclonal antibody alphaD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of alphaD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the alphaD11 Fab fragment was solved at 1.7 A resolution. A computational docking model of the alphaD11 Fab-NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X-ray scattering, provided the structural basis for identifying the residues involved in alphaD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor.

Nerve growth factor (NGF) is involved in pain transduction mechanisms and plays a key role in many persistent pain states, notably those associated with inflammation. On this basis, both the NGF ligand and its receptor TrkA (tyrosine kinase A) represent an eligible target for pain therapy. Although the direct involvement of NGF in pain modulation is well established, the effect of a direct functional block of the TrkA receptor is still unknown. In this study, we have demonstrated that MNAC13, the only anti-TrkA monoclonal antibody for which function neutralizing properties have been clearly shown both in vitro and in vivo , induces analgesia in both inflammatory and neuropathic pain models, with a surprisingly long-lasting effect in the latter. The formalin-evoked pain licking responses are significantly reduced by the MNAC13 antibody in CD1 mice. Remarkably, treatment with the anti-TrkA antibody also produces a significant antiallodynic effect on neuropathic pain: repeated i.p. injections of MNAC13 induce significant functional recovery in mice subjected to sciatic nerve ligation, with effects persisting after administration. Furthermore, a clear synergistic effect is observed when MNAC13 is administered in combination with opioids, at doses that are not efficacious per se . This study represents a direct demonstration that neutralizing antibodies directed against the TrkA receptor may display potent analgesic effects in inflammatory and chronic pain.

The rat monoclonal neuroantibody alphaD11 is a potent antagonist that prevents the binding of nerve growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems, most notably in two in vivo systems linked to crucial pathological states, such as Alzheimer's disease and HIV infection. To provide further insights into the mechanism of action of this potentially therapeutic monoclonal antibody, structural studies of the antigen-binding fragment (Fab) of alphaD11 were performed. alphaD11 IgG2a immunoglobulin was obtained from hybridomas by in vitro tissue culture. The alphaD11 Fab crystallizes in two crystal forms. Form I belongs to space group P1, with unit-cell parameters a = 42.7, b = 50.6, c = 102.7 A, alpha = 82.0, beta = 89.1, gamma = 86.0 degrees. With two molecules in the asymmetric unit, V(M) is 2.3 A(3) Da(-1) and the solvent content is 46%. A complete data set has been collected at 2.7 A resolution on beamline XRD-1 (ELETTRA, Trieste, Italy). Form II belongs to space group C2, with unit-cell parameters a = 114.8, b = 69.4, c = 64.10 A, beta = 117.0 degrees. With one molecule in the asymmetric unit, V(M) is 2.4 A(3) Da(-1) and the solvent content is 48%. A complete data set has been collected at 1.7 A resolution on beamline ID14-1 (ESRF, Grenoble, France). Phasing was successfully performed by Patterson search techniques and refinement of the structures is currently under way. Crystal forms I and II display a close-packing pattern.

Infection by HIV-1 causes persistent, long-term high virus production in macrophages. Major evidence, both in humans and in primate models, shows the crucial role of macrophages in sustaining virus production and in mediating a cytopathic effect on bystander CD4+T lymphocytes and neuronal cells. In the present study, we used severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID mice) to investigate thein vivoeffect of HIV-1-infected macrophages on virus spread and CD4+T lymphocyte depletion, and the ability of a mAb against nerve growth factor (NGF, a neurokine essential for the survival of HIV-1-infected macrophages) to suppress the pathogenetic events mediated by infected macrophages. Injection of mice with as few as 500 HIV-exposed macrophages causes (i) complete depletion of several millions of autologous CD4+T lymphocytes, (ii) sustained HIV viremia, and (iii) spreading of HIV-1 DNA in mouse lymphoid organs. In contrast,in vivotreatment with an anti-NGF Ab completely abrogates all effects mediated by HIV-infected macrophages. Taken together, the results demonstrate the remarkable power of macrophages in sustainingin vivoHIV-1 infection, and that such a phenomenon can be specifically abrogated by an anti-NGF Ab. This may open new perspectives of experimental approaches aimed at selectively eliminating persistently infected macrophages from the bodies of HIV-infected patients.