DING Proteins from Phylogenetically Different Species Share High Degrees of Sequence and Structure Homology and Block Transcription of HIV-1 LTR Promoter

Independent research groups reported that DING protein homologues isolated from bacterial, plant and human cells demonstrate the anti-HIV-1 activity. This might indicate that diverse organisms utilize a DING-mediated broad-range protective innate immunity response to pathogen invasion, and that this mechanism is effective also against HIV-1. We performed structural analyses and evaluated the anti-HIV-1 activity for four DING protein homologues isolated from different species. Our data show that bacterial PfluDING, plant p38SJ (pDING), human phosphate binding protein (HPBP) and human extracellular DING from CD4 T cells (X-DING-CD4) share high degrees of structure and sequence homology. According to earlier reports on the anti-HIV-1 activity of pDING and X-DING-CD4, other members of this protein family from bacteria and humans were able to block transcription of HIV-1 and replication of virus in cell based assays. The efficacy studies for DING-mediated HIV-1 LTR and HIV-1 replication blocking activity showed that the LTR transcription inhibitory concentration 50 (IC50) values ranged from 0.052–0.449 ng/ml; and the HIV-1 replication IC50 values ranged from 0.075–0.311 ng/ml. Treatment of cells with DING protein alters the interaction between p65-NF-κB and HIV-1 LTR. Our data suggest that DING proteins may be part of an innate immunity defense against pathogen invasion; the conserved structure and activity makes them appealing candidates for development of a novel therapeutics targeting HIV-1 transcription

The Level of DING Proteins Is Increased in HIV-Infected Patients: In Vitro and In Vivo Studies

DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins

Structural and functional study of phosphate-binding proteins : Illustration with ding and ding-like proteins and their involvment in hiv infection/inhibition

L’acquisition du phosphate du milieu extracellulaire est un processus biologique primordial pour toute les cellules vivantes. Durant ce travail nous nous sommes intéressé à deux famille de phosphate-binding proteins, les protéines DING et DING-like. Les protéines DING sont des protéines ubiquitaires nommées ainsi en raison de leur extrémité N-terminal très conservée DINGGG-. Bien que largement représentées chez les bactéries, ces protéines ont été identifiées initialement chez l’homme en vertu de leurs propriétés biologiques ou leur implication dans les maladies, y compris dans l’inhibition du VIH. La présence chez l’homme de telles protéines soulève plusieurs questions quant à leur rôle lors de l’infection par ce virus. Nous avons entrepris une étude visant à quantifier ces protéines chez une population d’individus infectés par le VIH. Nos résultats montrent une augmentation surprenante de leur concentration chez ces derniers par rapport aux sujet sains. D’autre part, nos travaux sur les protéines DING nous ont amené à nous intéresser à la famille des DING-like, très proche de la famille des protéines DING. Inversement, les protéines DING-like sont exclusivement bactériennes. En plus de leur capacité à fixer le phosphate, certains membres de cette famille semblent jouir d’une activité phosphatase. Durant ce travail nous avons caractérisé structuralement et enzymatiquement deux représentants de ces protéines isolés à partir de Pseudomonas aeruginosa. L’analyse structurale de ces protéines ainsi de la caractérisation de leurs activités pourraient être d’un grand intérêt afin de comprendre leur rôle dans le schéma d’acquisition du phosphate chez cette bactérie.Phosphate acquisition from the extracellular environment is a key process for all living cells. In bacteria, its importation is ensured, in part, by high affinity phosphate binding proteins (PBP). During this work, we are interested to two families of PBPs named DING and DING like proteins. DING proteins are ubiquitous proteins, so named due to their highly conserved N-terminal sequence DINGGG-. Although widely represented in bacteria, these proteins were initially identified in human by virtue of their biological properties or their involvement in diseases, including in HIV-inhibition. Currently, the anti-HIV activity has been shown for two human members of the DING family, HPBP and X-DING CD4. The presence in human of such proteins raises numerous questions about their role during HIV-infection. Thus, we undertook a study to evaluate their serum concentration in HIV-infected patients. Our results shows a surprising increase of the concentration of DING proteins in this population, compared to controls. On the other hand, our work on the DING proteins family led us to be interested on the family of DING-like proteins, closely related to the DING family. Unlike DING proteins, DING-like members were identified exclusively in bacteria. In addition to their ability to bind phosphate, some DING-like members appear to have a phosphatase activity. In this work we have characterize structurally and enzymatically two members of this family isolated from Pseudomonas aeruginosa. The structural analysis of these proteins combined with activities characterization could be of a great interest in order to understand their role in phosphate acquisition in this bacterium

Structural and functional study of phosphate-binding proteins : Illustration with ding and ding-like proteins and their involvment in hiv infection/inhibition

L’acquisition du phosphate du milieu extracellulaire est un processus biologique primordial pour toute les cellules vivantes. Durant ce travail nous nous sommes intéressé à deux famille de phosphate-binding proteins, les protéines DING et DING-like. Les protéines DING sont des protéines ubiquitaires nommées ainsi en raison de leur extrémité N-terminal très conservée DINGGG-. Bien que largement représentées chez les bactéries, ces protéines ont été identifiées initialement chez l’homme en vertu de leurs propriétés biologiques ou leur implication dans les maladies, y compris dans l’inhibition du VIH. La présence chez l’homme de telles protéines soulève plusieurs questions quant à leur rôle lors de l’infection par ce virus. Nous avons entrepris une étude visant à quantifier ces protéines chez une population d’individus infectés par le VIH. Nos résultats montrent une augmentation surprenante de leur concentration chez ces derniers par rapport aux sujet sains. D’autre part, nos travaux sur les protéines DING nous ont amené à nous intéresser à la famille des DING-like, très proche de la famille des protéines DING. Inversement, les protéines DING-like sont exclusivement bactériennes. En plus de leur capacité à fixer le phosphate, certains membres de cette famille semblent jouir d’une activité phosphatase. Durant ce travail nous avons caractérisé structuralement et enzymatiquement deux représentants de ces protéines isolés à partir de Pseudomonas aeruginosa. L’analyse structurale de ces protéines ainsi de la caractérisation de leurs activités pourraient être d’un grand intérêt afin de comprendre leur rôle dans le schéma d’acquisition du phosphate chez cette bactérie.Phosphate acquisition from the extracellular environment is a key process for all living cells. In bacteria, its importation is ensured, in part, by high affinity phosphate binding proteins (PBP). During this work, we are interested to two families of PBPs named DING and DING like proteins. DING proteins are ubiquitous proteins, so named due to their highly conserved N-terminal sequence DINGGG-. Although widely represented in bacteria, these proteins were initially identified in human by virtue of their biological properties or their involvement in diseases, including in HIV-inhibition. Currently, the anti-HIV activity has been shown for two human members of the DING family, HPBP and X-DING CD4. The presence in human of such proteins raises numerous questions about their role during HIV-infection. Thus, we undertook a study to evaluate their serum concentration in HIV-infected patients. Our results shows a surprising increase of the concentration of DING proteins in this population, compared to controls. On the other hand, our work on the DING proteins family led us to be interested on the family of DING-like proteins, closely related to the DING family. Unlike DING proteins, DING-like members were identified exclusively in bacteria. In addition to their ability to bind phosphate, some DING-like members appear to have a phosphatase activity. In this work we have characterize structurally and enzymatically two members of this family isolated from Pseudomonas aeruginosa. The structural analysis of these proteins combined with activities characterization could be of a great interest in order to understand their role in phosphate acquisition in this bacterium

Insights into aldehyde dehydrogenase enzymes: A structural perspective

Aldehyde dehydrogenases engage in many cellular functions, however their dysfunction resulting in accumulation of their substrates can be cytotoxic. ALDHs are responsible for the NAD(P)-dependent oxidation of aldehydes to carboxylic acids, participating in detoxification, biosynthesis, antioxidant and regulatory functions. Severe diseases, including alcohol intolerance, cancer, cardiovascular and neurological diseases, were linked to dysfunctional ALDH enzymes, relating back to key enzyme structure. An in-depth understanding of the ALDH structure-function relationship and mechanism of action is key to the understanding of associated diseases. Principal structural features 1) cofactor binding domain, 2) active site and 3) oligomerization mechanism proved critical in maintaining ALDH normal activity. Emerging research based on the combination of structural, functional and biophysical studies of bacterial and eukaryotic ALDHs contributed to the appreciation of diversity within the superfamily. Herewith, we discuss these studies and provide our interpretation for a global understanding of ALDH structure and its purpose–including correct function and role in disease. Our analysis provides a synopsis of a common structure-function relationship to bridge the gap between the highly studied human ALDHs and lesser so prokaryotic models

The quaternary structure of Thermus thermophilus aldehyde dehydrogenase is stabilized by an evolutionary distinct C-terminal arm extension

Aldehyde dehydrogenases (ALDH) form a superfamily of dimeric or tetrameric enzymes that catalyze the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the concomitant reduction of the cofactor NAD(P) into NAD(P)H. Despite their varied polypeptide chain length and oligomerisation states, ALDHs possess a conserved architecture of three domains: the catalytic domain, NAD(P)+ binding domain, and the oligomerization domain. Here, we describe the structure and function of the ALDH from Thermus thermophilus (ALDHTt) which exhibits non-canonical features of both dimeric and tetrameric ALDH and a previously uncharacterized C-terminal arm extension forming novel interactions with the N-terminus in the quaternary structure. This unusual tail also interacts closely with the substrate entry tunnel in each monomer providing further mechanistic detail for the recent discovery of tail-mediated activity regulation in ALDH. However, due to the novel distal extension of the tail of ALDHTt and stabilizing termini-interactions, the current model of tailmediated substrate access is not apparent in ALDHTt. The discovery of such a long tail in a deeply and early branching phylum such as Deinococcus-Thermus indicates that ALDHTt may be an ancestral or primordial metabolic model of study. This structure provides invaluable evidence of how metabolic regulation has evolved and provides a link to early enzyme regulatory adaptations

Modulating the release of pharmaceuticals from lipid cubic phases using a lipase inhibitor

Lipid cubic phase formulations have gained recognition as potential controlled delivery systems for a range of active pharmaceutical and biological agents on account of their desirable physiochemical properties and ability to encapsulate both hydrophobic and hydrophilic molecules. The most widely studied lipid cubic systems are those of the monoacylglycerol lipid family. These formulations are susceptible to lipolysis by a variety of enzymes, including lipases and esterases, which attack the ester bond present on the lipid chain bridging the oleic acid component to the glycerol backbone. The release of poorly soluble molecules residing in the lipid membrane portions of the phase is limited by the breakdown of the matrix; thus, presenting a potential means for further controlling and sustaining the release of therapeutic agents by targeting the matrix stability and its rate of degradation. The aims of the present study were twofold: to evaluate an approach to regulate the rate of degradation of lipid cubic phase drug delivery systems by targeting the enzyme interactions responsible for their demise; and to study the subsequent drug release profiles from bulk lipid cubic gels using model drugs of contrasting hydrophobicity. Here, hybrid materials consisting of cubic phases with monoacylglycerol lipids of different chain lengths formulated with a potent lipase inhibitor tetrahydrolipstatin were designed. Modulation of the release ofa hydrophobic model pharmaceutical, a clofazimine salt, was obtained by exploiting the matrices’ enzyme-driven digestion. A stable cubic phase is described, displaying controlled degradation with at least a 4-fold improvement compared to the blank systems shown in inhibitor-containing cubic systems. Sustained release of the model hydrophobic pharmaceutical was studied over 30 days to highlight the advantage of incorporating an inhibitor into the cubic network to achieve tunable lipid release systems. This is done without negatively affecting the structure of the matrix itself, as shown by comprehensive small-angle x-ray scattering experiments

Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase†

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase.This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductase