Shell Matrix Protein N38 of Pinctada fucata, Inducing Vaterite Formation, Extends the DING Protein to the Mollusca World.

In the animal kingdom, DING proteins were only found in Chordata and Aschelminthes. At present study, a potential DING protein, matrix protein N38, was isolated and purified from the shell of Pinctada fucata. Tandem mass spectrometry analysis revealed that 14 peptide segments matched between N38 and human phosphate-binding protein (HPBP). HPBP belongs to the DING protein family and has a "DINGGG-" sequence, which is considered a "signature" of HPBP. In this study, the mass spectrometry analysis results showed that N38 had a "DIDGGG-" sequence; this structure is a mutation from the "DINGGG-" structure, which is a distinctive feature of the DING protein family. The role of N38 during calcium carbonate formation was explored through the in vitro crystallization experiment. The results of scanning electron microscopy and Raman spectrum analysis indicated that N38 induced vaterite formation. These findings revealed that N38 might regulate and participate in the precise control of the crystal growth of the shell, providing new clues for biomineralization mechanisms in P. fucata and DING protein family studies. In addition, this study helped extend the research of DING protein to the Mollusca world.

DING Proteins Extend to the Extremophilic World.

The DING proteins are ubiquitous in the three domains of life, from mesophiles to thermo- and hyperthermophiles. They belong to a family of more than sixty members and have a characteristic N-terminus, DINGGG, which is considered a "signature" of these proteins. Structurally, they share a highly conserved phosphate binding site, and a three dimensional organization resembling the "Venus Flytrap", both reminding the ones of PstS proteins. They have unusually high sequence conservation, even between distantly related species. Nevertheless despite that the genomes of most of these species have been sequenced, the DING gene has not been reported for all the relative characterized DING proteins. Identity of known DING proteins has been confirmed immunologically and, in some cases, by N-terminal sequence analysis. Only a few of the DING proteins have been purified and biochemically characterized. DING proteins are heterogeneous for their wide range of biological activities and some show different activities not always correlated with each other. Most of them have been originally identified for different biological properties, or rather for binding to phosphate and also to other ligands. Their involvement in pathologies is described. This review is an update of the most recent findings on old and new DING proteins.

In Sulfolobus solfataricus, the Poly(ADP-Ribose) Polymerase-Like Thermoprotein Is a Multifunctional Enzyme.

In Sulfolobus solfataricus, Sso, the ADP-ribosylating thermozyme is known to carry both auto- and heteromodification of target proteins via short chains of ADP-ribose. Here, we provide evidence that this thermoprotein is a multifunctional enzyme, also showing ATPase activity. Electrophoretic and kinetic analyses were performed using NAD+ and ATP as substrates. The results showed that ATP is acting as a negative effector on the NAD+-dependent reaction, and is also responsible for inducing the dimerization of the thermozyme. These findings enabled us to further investigate the kinetic of ADP-ribosylation activity in the presence of ATP, and to also assay its ability to work as a substrate. Moreover, since the heteroacceptor of ADP-ribose is the sulfolobal Sso7 protein, known as an ATPase, some reconstitution experiments were set up to study the reciprocal influence of the ADP-ribosylating thermozyme and the Sso7 protein on their activities, considering also the possibility of direct enzyme/Sso7 protein interactions. This study provides new insights into the ATP-ase activity of the ADP-ribosylating thermozyme, which is able to establish stable complexes with Sso7 protein.

Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts.

DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.

Human X-DING-CD4 mediates resistance to HIV-1 infection through novel paracrine-like signaling.

X-DING-CD4 is a novel phosphatase mediating antiviral responses to HIV-1 infection. This protein is constitutively expressed and secreted by HIV-1 resistant CD4(+) T cells and its mRNA transcription is up-regulated in peripheral blood mononuclear cells from HIV-1 elite controllers. The secreted/soluble X-DING-CD4 protein form is of particular importance because it blocks virus transcription when added to HIV-1 susceptible cells. The present study aimed to determine the contribution of this factor to the induction of the antiviral response in target cells. We found that soluble X-DING-CD4 enters cells by endocytosis and that influx of this protein induced transcription of interferon-α and endogenous X-DING-CD4 mRNA in transformed CD4(+) T cells and primary macrophages. Treatment of HIV-1 susceptible cells with exogenous X-DING-CD4 caused depletion of phosphorylated p50 and p65 nuclear factor kappa ß subunits and a significant reduction in p50/p65 nuclear factor kappa ß binding to the HIV-1 long terminal repeat. Taken together, these findings indicate a novel antiviral mechanism mediated by the influx of soluble X-DING-CD4, its signaling to promote self-amplification, and functional duality as an endogenous innate immunity effector and exogenous factor regulating gene expression in bystander cells.

The interplay between the X-DING-CD4, IFN-α and IL-8 gene activity in quiescent and mitogen- or HIV-1-exposed PBMCs from HIV-1 elite controllers, AIDS progressors and HIV-negative controls.

X-DING-CD4 blocks HIV-1 long terminal repeat (LTR) and pathogen induced pro-inflammatory response. Increased activity of the X-DING-CD4 gene is associated with cellular resistance to virus; therefore, HIV-1 elite controllers (ECs) should have higher X-DING-CD4 and reduced pro-inflammatory mRNA activity than viremic or uninfected individuals. Also, depending on the cell stimulating factor, expression of X-DING-CD4 mRNA in ECs might be autonomous or contingent on IFN signaling. We compared expression of X-DING-CD4, IFN-α and IL-8 mRNAs in naive, phytohemagglutinin- or HIV-1 exposed PBMCs from ECs, HIV progressors and negative controls; tested correlation between X-DING-CD4 and IFN-α expression; sensitivity of the X-DING-CD4 gene to IFN-α regulation; and evaluated interactions between innate and pro-inflammatory genes. We found that expression of X-DING-CD4 and IFN-α was up-regulated in ECs and correlated in cells stimulated with mitogen, but not HIV-1. The X-DING-CD4 gene was more sensitive to HIV-1 than rIFN-α stimulation. ECs had significantly less IL-8 mRNA when PBMCs were exposed to exogenous HIV-1. Two-way ANOVA showed that control of HIV-1 and virus-induced pro-inflammatory response by ECs stemmed from interactions between expression of innate immunity and pro-inflammatory genes, the state of cell stimulation and the status of virus control. Consequently, interaction of multiple host innate immune responses rather than a single mechanism regulates restriction of HIV-1 in ECs.