Structure-function relationships in a bacterial DING protein

A recombinant DING protein from Pseudomonas fluorescens has been previously shown to have a phosphate-binding site, and to be mitogenic for human cells. Here we report the three-dimensional structure of the protein, confirming a close similarity to the "Venus flytrap" structure seen in other human and bacterial phosphate-binding proteins. Site-directed mutagenesis confirms the role of a key residue involved in phosphate binding, and that the mitogenic activity is not dependent on this property. Deletion of one of the two hinged domains that constitute the Venus flytrap also eliminates phosphate binding whilst enhancing mitogenic activity.     

Eukaryotic DING Proteins Are Endogenous: An Immunohistological Study in Mouse Tissues

Background

DING proteins encompass an intriguing protein family first characterized by their conserved N-terminal sequences. Some of these proteins seem to have key roles in various human diseases, e.g., rheumatoid arthritis, atherosclerosis, HIV suppression. Although this protein family seems to be ubiquitous in eukaryotes, their genes are consistently lacking from genomic databases. Such a lack has considerably hampered functional studies and has fostered therefore the hypothesis that DING proteins isolated from eukaryotes were in fact prokaryotic contaminants.

Principal Findings

In the framework of our study, we have performed a comprehensive immunological detection of DING proteins in mice. We demonstrate that DING proteins are present in all tissues tested as isoforms of various molecular weights (MWs). Their intracellular localization is tissue-dependant, being exclusively nuclear in neurons, but cytoplasmic and nuclear in other tissues. We also provide evidence that germ-free mouse plasma contains as much DING protein as wild-type.

Significance

Hence, data herein provide a valuable basis for future investigations aimed at eukaryotic DING proteins, revealing that these proteins seem ubiquitous in mouse tissue. Our results strongly suggest that mouse DING proteins are endogenous. Moreover, the determination in this study of the precise cellular localization of DING proteins constitute a precious evidence to understand their molecular involvements in their related human diseases.     

DING proteins are from Pseudomonas

DING proteins have been described as animal and plant proteins with potential biomineralisation, receptor or signalling roles that have been characterised by an N-terminal DINGGG-sequence. However, these sequences have only ever been identified as either N-terminal peptides or partial cDNA sequences, and have yet to be detected in any of the many genomic animal and plant genomes now available. Microbial relatives of the DING proteins have been described, which appear to be periplasmic phosphate-binding proteins. Recently, full-length Pseudomonas aeruginosa UCBPP-PA14 and Hypericum perforatum genes have been sequenced that show high homology to the published DING protein N-terminal sequences, and small peptides previously identified in conjunction with the peptide sequencing of DING proteins can also be mapped to regions across these full-length sequences. Searching with these sequences identifies other plant and animal cDNA fragments in the public nucleotide databases, and, additionally, an unordered rat genomic contig that contains a DING-like sequence on a small fragment. Analysing the codon usage of these DNA sequences identifies all of these sequences as of Pseudomonas origin, suggesting that DING proteins do not exist in eukaryotes, but instead are potentially due to microbial contamination or infection.     

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.

     

DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom

PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.     

Genetic characterization of <Emphasis Type="Italic">psp</Emphasis> encoding the DING protein in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> SBW25

Background  

DING proteins constitute a conserved and broadly distributed set of proteins found in bacteria, fungi, plants and animals (including humans). Characterization of DING proteins from animal and plant tissues indicated ligand-binding ability suggesting a role for DING proteins in cell signaling and biomineralization. Surprisingly, the genes encoding DING proteins in eukaryotes have not been identified in the eukaryotic genome or EST databases. Recent discovery of a DING homologue (named Psp here) in the genome of Pseudomonas fluorescens SBW25 provided a unique opportunity to investigate the physiological roles of DING proteins. P. fluorescens SBW25 is a model bacterium that can efficiently colonize plant surfaces and enhance plant health. In this report we genetically characterize Psp with a focus on conditions under which psp is expressed and the protein exported.     

Human cells: new platform for recombinant therapeutic protein production

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production.     

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.     

Serendipitous discovery and X-ray structure of a human phosphate binding apolipoprotein

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is copurified with the enzyme paraoxonase. Its X-ray structure is similar to the prokaryotic phosphate solute binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The systematic absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation between genes belonging to evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the only known transporter capable of binding phosphate ions in human plasma and may become a new predictor of or a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.     

Micro-algae come of age as a platform for recombinant protein production

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins.     

High-yield production of a human therapeutic protein in tobacco chloroplasts

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.     

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

Background  

Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct.     

Unraveling regulation and new components of human P-bodies through a protein interaction framework and experimental validation

The cellular factors involved in mRNA degradation and translation repression can aggregate into cytoplasmic domains known as GW bodies or mRNA processing bodies (P-bodies). However, current understanding of P-bodies, especially the regulatory aspect, remains relatively fragmentary. To provide a framework for studying the mechanisms and regulation of P-body formation, maintenance, and disassembly, we compiled a list of P-body proteins found in various species and further grouped both reported and predicted human P-body proteins according to their functions. By analyzing protein-protein interactions of human P-body components, we found that many P-body proteins form complex interaction networks with each other and with other cellular proteins that are not recognized as P-body components. The observation suggests that these other cellular proteins may play important roles in regulating P-body dynamics and functions. We further used siRNA-mediated gene knockdown and immunofluorescence microscopy to demonstrate the validity of our in silico analyses. Our combined approach identifies new P-body components and suggests that protein ubiquitination and protein phosphorylation involving 14-3-3 proteins may play critical roles for post-translational modifications of P-body components in regulating P-body dynamics. Our analyses provide not only a global view of human P-body components and their physical interactions but also a wealth of hypotheses to help guide future research on the regulation and function of human P-bodies.     

The DING family of proteins: ubiquitous in eukaryotes,but where are the genes?

PstS and DING proteins are members of a superfamily of secreted, high‐affinity phosphate‐binding proteins. Whereas microbial PstS have a well‐defined role in phosphate ABC transporters, the physiological function of DING proteins, named after their DINGGG N termini, still needs to be determined. PstS and DING proteins co‐exist in some Pseudomonas strains, to which they confer a highly adhesive and virulent phenotype. More than 30 DING proteins have now been purified, mostly from eukaryotes. They are often associated with infections or with dysregulation of cell proliferation. Consequently, eukaryotic DING proteins could also be involved in cell–cell communication or adherence. The ubiquitous presence in eukaryotes of proteins structurally and functionally related to bacterial virulence factors is intriguing, as is the absence of eukaryotic genes encoding DING proteins in databases. DING proteins in eukaryotes could originate from unidentified commensal or symbiotic bacteria and could contribute to essential functions. Alternatively, DING proteins could be encoded by eukaryotic genes sharing special features that prevent their cloning. Both hypotheses are discussed.     

Urokinase-targeted recombinant bacterial protein toxins-a rationally designed and engineered anticancer agent for cancer therapy

Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy.Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons.On the other hand,it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR),making up the uPA system,are overexpressed in a variety of human tumors and tumor cell lines.The expression of uPA system is highly correlated with tumor invasion and metastasis.To exploit these characteristics in the design of tumor cell-selective cytotoxins,two prominent bacterial protein toxins,i.e.,the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins.These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA systemexpressing tumor cells,thereby killing these cells.This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents.It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.