Cell-based arrays for the identification of interacting polypeptide domains or epitopes.

The specific regions on proteins which are responsible for protein-protein interaction are called interacting domains, or epitopes in case of antigen-antibody binding. These domains are one feature to characterize proteins and are important in clinical diagnostics and research. For the mapping of such domains the use of protein/peptide arrays has become popular. Regardless of which kind of array, the major requirements are a high number of candidates arranged in the array, high quality, ease of use, and cost-effectiveness. Here, the authors describe a general protocol for mapping the interacting domains of proteins demonstrated by a high affinity protein interaction, the interaction of an antibody to an antigen. The chapter describes a stepwise protocol from library production to the verification of the domain by the use of an automated cell-based polypeptide array, which comprises the named requirements of a good array.

Deciphering the calcitriol-induced transcriptomic response in keratinocytes: presentation of novel target genes.

Numerous studies to date have been aimed at unraveling the large suite of calcitriol (1α,25-dihydroxyvitamin D(3)) response genes in diverse tissues including skin, where this hormone is involved in regulating keratinocyte proliferation, differentiation, permeability barrier formation, innate immunity promotion, antimicrobial peptide production, and wound healing. However, the various approaches differ considerably in probed cell types, scale, throughput, and statistical reliability and do, of note, not reveal much overlap. To further expand our knowledge on presently elusive targets and characterize the extent of fragmentation of existing datasets, we have performed whole-transcriptome microarray examinations of calcitriol-treated human primary keratinocytes. Out of 28,869 genes investigated, we uncovered 86 differentially expressed (67 upregulated and 19 downregulated) candidates that were functionally clustered into five annotation categories: response to wounding, protease inhibition, secondary metabolite biosynthesis, cellular migration, and amine biosynthetic processes. A complementary RTq-PCR study of 78 nominees selected thereof demonstrated significant differential expression of 55 genes (48 upregulated and seven downregulated) within biological replicates. Our hit list contains nine previously authenticated targets (16.36%, proof of concept) and 46 novel genes (83.6%) that have not yet been explicitly described as being differentially regulated within human primary keratinocytes. Direct vitamin D receptor response element predictions within the regulatory promoter regions of 50 of the RTq-PCR-validated targets agreed with known biological functionality and corroborated our stringent data validation pipeline. Altogether, our results indicate the value of continuing these kinds of gene expression studies, which contribute to an enhanced comprehension of calcitriol-mediated processes that may be dysregulated in human skin pathophysiology.

Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae.

BACKGROUND: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. RESULTS: Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. CONCLUSION: This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.

Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction.

PIM-1 kinase interacts with the DNA binding domain of the vitamin D receptor: a further kinase implicated in 1,25-(OH)2D3 signaling.

BACKGROUND: The vitamin D3 receptor (VDR) is responsible for mediating the pleiotropic and, in part, cell-type-specific effects of 1,25-dihydroxyvitamin D3 (calcitriol) on the cardiovascular and the muscle system, on the bone development and maintenance, mineral homeostasis, cell proliferation, cell differentiation, vitamin D metabolism, and immune response modulation. RESULTS: Based on data obtained from genome-wide yeast two-hybrid screenings, domain mapping studies, intracellular co-localization approaches as well as reporter transcription assay measurements, we show here that the C-terminus of human PIM-1 kinase isoform2 (amino acid residues 135-313), a serine/threonine kinase of the calcium/calmodulin-regulated kinase family, directly interacts with VDR through the receptor's DNA-binding domain. We further demonstrate that PIM-1 modulates calcitriol signaling in HaCaT keratinocytes by enhancing both endogenous calcitriol response gene transcription (osteopontin) and an extrachromosomal DR3 reporter response. CONCLUSION: These results, taken together with previous reports of involvement of kinase pathways in VDR transactivation, underscore the biological relevance of this novel protein-protein interaction.

DDX5 is a multifunctional co-activator of steroid hormone receptors.

The vitamin D receptor (VDR), an evolutionarily conserved member of the nuclear receptor superfamily, links the metabolically activated vitamin D ligand, calcitriol, with its vitamin D-responsive target genes that are implicated in diverse physiological processes. By genome-wide protein-protein interaction screening of a keratinocyte cDNA library using VDR as bait, we found that the DEAD box RNA helicase p68, also referred to as DDX5, directly interacts with VDR. Domain analysis reveals that the ligand-binding domain of VDR is responsible for the binding, an interaction typical of NR co-activators. Interestingly, the VDR interacting domain of DDX5 lacks a LXXLL-motif and interaction analysis of helix 12 VDR mutants E420K, E420Q and L417S, known to decrease binding affinity of LxxLL motif-containing co-activators showed no change in their interactions. As further support that this novel interactor might be involved in vitamin D-stimulated transcriptional regulation, we demonstrate that VDR and DDX5 co-localize within the nuclei of HaCaT keratinocytes and sub-cellular protein fractions. In vivo validation studies demonstrate, that overexpression of DDX5 has the capability to enhance both, calcitriol-dependent transcription of known response genes and an extrachromosomal DR3-type reporter response. In agreement with this, shRNA based knock-down of DDX5 in keratinocytes compensates for this particular response. Finally, our findings reveal parallels between the VDR-DDX5 interaction and the well-characterized interaction between DDX5 and human estrogen receptor α and the androgen receptor, thus underscoring the physiological significance of the novel protein-protein interaction.

Construction of improved Yeast Two-Hybrid libraries.

The Yeast Two-Hybrid (Y2H) system is the most frequently used method for identifying protein-protein interactions. The use of recombination-amenable Y2H vectors would reduce time and effort for cloning prey or bait vectors, and increase the quality of Y2H screenings due to the production of improved screening libraries. These libraries can heighten the amount of new candidates in Y2H screenings significantly by representing more correct candidate genes in frame and outperform a classical Y2H library. The described vectors can be used for the construction of genomic, peptide, or cDNA-based Y2H libraries. Furthermore, the compatibility to newer ORFeome libraries is also given. Here, we describe a vector system for site-specific recombination and for the construction of high-content Y2H libraries. In summary, we will describe the construction of these vectors and the production of Y2H screening libraries.

Coupled yeast 2-hybrid-mammalian 2-hybrid reading-frame-independent and site-specific recombinational cloning vector system.

The yeast 2-hybrid (Y2H) system is a powerful method for identifying protein-protein interactions (PPIs), requiring minimal prior information of the putative interactors. Currently available automated versions of the Y2H system are sufficiently developed to allow facile genome-wide PPI screening to compile extensive inter-actome data. A limitation of the Y2H approach, however, is that all primary hits have to be technically verified and biologically evaluated by complementary methods, which is time-consuming, costly, and laborious. Furthermore, the yeast intracellular environment can lead to spurious results for proteins of other organisms, for example because of differences in post-translational modifications or the presence/absence of bridging proteins. Many researchers now confirm PPIs found in the Y2H system by retesting the candidates in the mammalian 2-hybrid system (M2H). However, although such combined Y2H-M2H testing is desirable and perhaps necessary, recloning of Y2H candidates into M2H vectors, especially on a large scale, is time-consuming and costly. To address this shortcoming, we introduce here a new site-specific recombination-capable M2H vector system that is fully compatible with the site-specific Y2H system that we recently described in Biotechniques [2008;45(3):235-244]. The results show that the new vectors are: (a) Gateway®, compatible and suitable for fast cloning; (b) fully functional in the M2H system without influencing the capacity of the selection system or creating autoactivators; and (c) directly compatible with the existing site-specific Y2H vector system, as demonstrated by confirmation of Y2H PPI candidates in the M2H system.

Epitope mapping of antibodies using a cell array-based polypeptide library.

The authors describe a technique for mapping the epitopes of protein antigens recognized by mono- or polyclonal antibodies. This method is based on a recombinant polypeptide library, expressed in a bacterial expression system, arrayed at high density, and tested on a membrane with automated procedures. The authors analyzed the epitope of a commercially available monoclonal antibody to vitamin D receptor (VDR). About 2300 overlapping VDR peptides were screened on a test array, and a contiguous stretch of 37 amino acids was identified as the epitope. Its authenticity was confirmed by Western blotting and an immunofluorescence competition assay on human skin tissue samples. The authors define the proposed method as a cell-based protein or peptide array that is adaptable to many applications, including epitope mapping of antibodies and autoantibodies, autoantigen detection from patient sera, whole-proteome approaches such as protein-peptide interactions, or selection of monoclonal antibodies from polyclonal sera. The advantages of this method are (a) its ease of protein array production based on well-established bacterial protein/peptide expression procedures; (b) the large number of printable colonies (as many as approximately 25,000) that can be arrayed per membrane; (c) there is no need for protein purification of recombinantly expressed proteins; (d) DNA, rather than protein, is the starting material to generate the arrays; and (e) its high-throughput and automatable format.