Multi-protein Interactions Research Articles

The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum

Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.

The Binding of GATA-1 to a β-Globin LCR HS Core Element in Nuclear Chromatin Requires Multiple Associated Factor Binding Sites.

The GATA-1 transcription factor has been shown to interact in vitro with many gene regulatory elements including promoters, enhancers, silencers and locus control region elements. The GATA-1 consensus binding sequence is generally considered to be WGATAR, yet it can also bind to other related sequences that do not perfectly fit this consensus. This raises the question of whether all potential GATA-1 binding sites identified by in vitro DNA binding assays or sequence analysis are actually bound by GATA-1 in nuclear chromatin. We hypothesized that it was unlikely that all, or even most, predicted GATA-1 sites were bound in vivo. If correct, this would imply that additional mechanisms, beyond DNA sequence, are required to target GATA-1 to specific genetic regulatory elements. To address this hypothesis we first performed GATA-1 ChIP assays in MEL cells at 10 WGATAR sites which spanned the mouse β-globin locus but were not associated with known regulatory elements. Positive controls for GATA-1 binding were the core region of LCR HS2 and the -2.8 mGATA-1 gene upstream enhancer. Negative controls included two regions from the β-globin locus which do not contain WGATAR elements and a WGATAR element from the proximal mGATA-1 gene promoter previously shown by the Bresnick lab to not bind GATA-1. While enrichment of the positive control sites using anti-GATA-1 antibody was 10-20-fold more than with IgG (p<0.001), binding at none of the 10 test WGATAR sites was enriched. This result implied that conditions other than the presence of the WGATAR consensus sequence are required to specify GATA-1 binding in nuclear chromatin. To better understand this process we decided to more closely evaluate the binding of GATA-1 to β-globin LCR HS core elements. We first tested the idea that GATA-1 was able to bind these sites due to high-level affinity based on the presence of tandem-inverted WGATAR elements and the known ability of GATA-1 molecules to self-interact. However, when tested in gel shift assays, GATA-1 binding to the LCR HS core elements had a very rapid off-rate which was much faster than a known high-affinity control site from the murine α-globin promoter. This led us to next test the idea that surrounding factor binding sites were required for GATA-1 binding to the LCR HS cores. Using human LCR HS4 as a model, we systematically assembled combinations of binding sites (3 KLF sites and 1 NF-E2 site) known to flank the tandem-inverted WGATAR elements and tested these artificial HS cores for GATA-1 binding after integration into MEL cell chromatin. The wild-type HS4 core served as a positive control and a fragment of luciferase cDNA served as a negative control for GATA-1 binding. Results for each cell line were normalized to the internal positive control -2.8 mGATA-1 gene site. Our experiments showed that only when all known flanking sites were present was GATA-1 binding detected. Similarly, DNase HS formation and histone H3 acetylation only occurred when all of the surrounding sites were present. Finally, we used ChIP to show that the assembled complex, with all sites present, includes BRG1 a component of the swi/snf nucleosome remodeling complex. These results indicate that GATA-1 targeting to the LCR HS cores involves complex, multi-protein interactions including histone acetylation and nucleosome displacement. Similar mechanisms are likely to be involved in targeting this and other transcription factors to functionally important sites in the genome.

A quantitative determination of multi-protein interactions by the analysis of confocal images using a pixel-by-pixel assessment algorithm

Recent advances in confocal microscopy have allowed scientists to assess the expression, and to some extent, the interaction/colocalization of multiple molecules within cells and tissues. In some instances, accurately quantifying the colocalization of two or more proteins may be critical. This can require the acquisition of multiple Z plane images (Z stacks) throughout a specimen and, as such, we report here the successful development of a freeware, open-source image analysis tool, IMAJIN_COLOC, developed in PERL (v. 5.8, build 806), using the PERLMagick libraries (ImageMagick). Using a pixel-by-pixel analysis algorithm, IMAJIN_COLOC can analyze images for antigen expression (any number of colors) and can measure all possible combinations of colocalization for up to three colors by analyzing a Z stack gallery acquired for each sample. The simultaneous (i.e. in a single pass) analysis of three-color colocalization, and batch analysis capabilities are distinctive features of this program. A control image, containing known individual and colocalized pixel counts, was used to validate the accuracy of IMAJIN_COLOC. As further validation, pixel counts and colocalization values from the control image were compared to those obtained with the software packaged with the Zeiss laser-scanning microscope (LSM AIM, version 3.2). The values from both programs were found to be identical. To demonstrate the applicability of this program in addressing novel biological questions, we examined the role of neurons in eliciting an immune reaction in response to viral infection. Specifically, we successfully examined expression of the chemokine RANTES in measles virus (MV) infected hippocampal neurons and quantified changes in RANTES production throughout the disease period. The resultant quantitative data were also evaluated visually, using a gif image created during the analysis. PERL (ActivePerl, version 5.8) is available at activestate.com; the PERLMagick libraries are available at imagemagick.org, and IMAJIN_COLOC, the source code and user documentation can be downloaded from http://www.fda.gov/cber/research/imaging/imageanalysis.htm.

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.

Structure of the P22 att site. Conservation and divergence in the lambda motif of recombinogenic complexes.

We have defined the bacterial and viral DNA targets (att sites) of P22 site-specific recombination and characterized their interaction with integrase (Int) protein. The bacterial DNA target, attB, is approximately 27 base pairs and consists of two core type Int binding sites as inverted repeats. The top and bottom Int cleavage sites fall within the core type Int binding sites and are separated by a 7-base pair overlap region. A similar core region is found in the viral DNA target, attP, which is approximately 260 base pairs long and contains two IHF binding sites and five arm type binding sites for Int. The results suggest that P22 Int, like lambda Int, is a heterobivalent DNA-binding protein that is capable of forming complex higher order structures with recombinogenic function. Although P22 and lambda recombination involve very similar multiprotein interactions and core region structures, there are significant differences in the arrangements of distal protein binding sites. These differences are discussed in terms of the possible flexibility of the Int protein and the specificity with which the higher order complexes assemble and/or function.

Multiprotein Interactions in Strand Cleavage of DNA Damaged by UV and Chemicals

The chapter explains the characterization of the products of genes uvrA, uvrB, and uvrC purified by means of the complementation assay. It has long been assumed that the initial step of excision repair of UV-irradiated DNA in E.coli is similar to that characterized in vitro by the small dimer-specific glycosylases from M.luteus and T4-infected cells. This assumption has prevented major efforts to elucidate the uvr+-dependent incision enzyme in E coli. However, recent studies of the uvr+ gene products in vitro show that these proteins are basically different from the small DNA glycosylases characterized so far. The interactions between the uvrA+, uvrB+, and uvrC+ gene products resulting in the appearance of strand breaks in DNA damaged by UV and various chemical agents are now being subjected to extensive studies in several laboratories, and it is to be expected that a more detailed knowledge about the excision mechanism in E.coli.