Primer protein of bacteriophage M2 exposes the RGD receptor site upon linking the first deoxynucleotide

Summary Using electroporation with the phage PRD1 genome, we set up a high-frequency DNA transfer system for a linear dsDNA molecule with 5-covalently linked terminal proteins. The transfer was saturated when more than 100 ng of PRD1 genome was used. Electroporation efficiency was about four orders of magnitude higher than that obtained with transfection. Removal of the terminal protein abolished plaque formation, which could not be rescued by supplying the terminal protein or phage DNA polymerase or both in trans.     

Plakins in development and disease

Plakins are large multi-domain molecules that have various functions to link cytoskeletal elements together and to connect them to junctional complexes. Plakins were first identified in epithelial cells where they were found to connect the intermediate filaments to desmosomes and hemidesmosomes [Ruhrberg, C., and Watt, F.M. (1997). The plakin family: versatile organizers of cytoskeletal architecture. Curr Opin Genet Dev 7, 392-397.]. They were subsequently found to be important for the integrity of muscle cells. Most recently, they have been found in the nervous system, where their functions appear to be more complex, including cross-linking of microtubules (MTs) and actin filaments [Leung, C.L., Zheng, M., Prater, S.M., and Liem, R.K. (2001). The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles. J Cell Biol 154, 691-697., Leung, C.L., Sun, D., Zheng, M., Knowles, D.R., and Liem, R.K. (1999). Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J Cell Biol 147, 1275-1286.]. These plakins have also indicated their relationship to the spectrin superfamily of proteins and the plakins appear to be evolutionarily related to the spectrins, but have diverged to perform different specialized functions. In invertebrates, a single plakin is present in both Drosophila melanogaster and Caenorhabditis elegans, which resemble the more complex plakins found in mammals [Roper, K., Gregory, S.L., and Brown, N.H. (2002). The 'spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families. J Cell Sci 115, 4215-4225.]. In contrast, there are seven plakins found in mammals and most of them have alternatively spliced forms leading to a very complex group of proteins with potential tissue specific functions [Jefferson, J.J., Leung, C.L., and Liem, R.K. (2004). Plakins: goliaths that link cell junctions and the cytoskeleton. Nat Rev Mol Cell Biol 5, 542-553.]. In this review, we will first describe the plakins, desmoplakin, plectin, envoplakin and periplakin and then describe two other mammalian plakins, Bullous pemphigoid antigen 1 (BPAG1) and microtubule actin cross-linking factor 1 (MACF1), that are expressed in multiple isoforms in different tissues. We will also describe the relationship of these two proteins to the invertebrate plakins, shortstop (shot) in Drosophila and VAB-10 in C. elegans. Finally, we will describe an unusual mammalian plakin, called epiplakin.     

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.     

PACSIN proteins bind tubulin and promote microtubule assembly

PACSINs are intracellular adapter proteins involved in vesicle transport, membrane dynamics and actin reorganisation. In this study, we report a novel role for PACSIN proteins as components of the centrosome involved in microtubule dynamics. Glutathione S-transferase (GST)-tagged PACSIN proteins interacted with protein complexes containing α- and γ-tubulin in brain homogenate. Analysis of cell lysates showed that all three endogenous PACSINs co-immunoprecipitated dynamin, α-tubulin and γ-tubulin. Furthermore, PACSINs bound only to unpolymerised tubulin, not to microtubules purified from brain. In agreement, the cellular localisation of endogenous PACSIN 2 was not affected by the microtubule depolymerising reagent nocodazole. By light microscopy, endogenous PACSIN 2 localised next to γ-tubulin at purified centrosomes from NIH 3T3 cells. Finally, reduction of PACSIN 2 protein levels with small-interfering RNA (siRNA) resulted in impaired microtubule nucleation from centrosomes, whereas microtubule centrosome splitting was not affected, suggesting a role for PACSIN 2 in the regulation of tubulin polymerisation. These findings suggest a novel function for PACSIN proteins in dynamic microtubuli nucleation.     

Comparative aspects of polyglutamine binding domain in PQBP-1 among Vertebrata

We investigated the evolutionary conservation of polyglutamine binding protein-1 (PQBP-1) among Vertebrata. PQBP-1s were highly conserved and shared the same domain features including a WW domain, a polar amino acid rich domain (PRD), a nuclear localization signal (NLS), and a C-terminal domain (CTD) among Eutheria, but not always among Vertebrata. PQBP-1s of Vertebrata contained a variable region in the middle portion corresponding to the position of PRD. The full form of PRD including both 7aa and DR/ER repeats was specific to Eutheria. PRD of non-eutherian Amniota was minimal. Amphibia had no PRD. The DR/ER repeat was solo in fishes. Agnatha PRD was also rich in polar amino acids, but contained no repetitive sequence. We investigated 3 polyQ-containing proteins known to interact with PQBP-1: BRN-2, Huntingtin, and ATAXIN-1, and showed a diverse nature of protein-protein interaction in Vertebrata. There appears to be no interaction between PQBP-1 and BRN-2, Huntingtin, or ATAXIN-1 in Amphibia, while the interaction between PQBP-1 and BRN-2 is expected to be conserved among Mammalia, and the interaction between PQBP-1 and Huntingtin or ATAXIN-1 depends on the lineage in Eutheria.     

Isoform-Selective Interaction of the Adaptor Protein Tks5/FISH with Sos1 and Dynamins

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.     

Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump which converts atmospheric oxygen to water and couples the oxygen reduction reaction to the creation of a membrane proton gradient. The structure of the enzyme has been solved; however, the mechanism of proton pumping is still poorly understood. Recent calculations from this group indicate that one of the histidine ligands of enzyme's Cu_B center, His291, may play the role of the pumping element. In this paper, we report on the results of calculations that combined first principles DFT and continuum electrostatics to evaluate the energetics of the key energy generating step of the model—the transfer of the chemical proton to the binuclear center of the enzyme, where the hydroxyl group is converted to water, and the concerted expulsion of the proton from δ-nitrogen of His291 ligand of Cu_B center. We show that the energy generated in this step is sufficient to push a proton against an electrochemical membrane gradient of about 200 mV. We have also re-calculated the pK_a of His291 for an extended model in which the whole Fe_a3-Cu_B center with their ligands is treated by DFT. Two different DFT functionals (B3LYP and PBE0), and various dielectric models of the protein have been used in an attempt to estimate potential errors of the calculations. Although current methods of calculations do not allow unambiguous predictions of energetics in proteins within few pK_a units, as required in this case, the present calculation provides further support for the proposed His291 model of CcO pump and makes a specific prediction that could be targeted in the experimental test.     

New function of the proline rich domain in dynamin-2 to negatively regulate its interaction with microtubules in mammalian cells

Microtubule reorganization is necessary for many cellular functions such as cell migration, cell polarity and cell division. Dynamin was originally identified as a microtubule-binding protein. Previous limited digestion experiment revealed that C-terminal 100-amino acids proline rich domain (PRD) of dynamin is responsible for microtubule binding in vitro. However, as obvious localization of dynamin along microtubules is only observed at the spindle midzone during mitosis but not in interphase cells, it remains unclear how dynamin interacts with microtubules in vivo. Here, we report that GFP-dynamin-2-(1-786), a truncated mutant lacking a C-terminal portion of the PRD, localized along microtubules in interphase HeLa cells. GFP-dynamin-2-wild type (WT) and GFP-dynamin-2-(1-745), a construct that was further truncated to remove the entire PRD, localized in discrete punctate structures but not along microtubules. These data suggest that the N-terminal (residues 746-786) but not the entire PRD is necessary for the interaction of dynamin-2 with microtubules in the cell and that the C-terminus of PRD (787-870) negatively regulate this interaction. Microtubules in cells expressing GFP-dynamin-2-(1-786) were stabilized against exposure to cold. These results provide a first evidence for a regulated interaction of dynamin-2 with microtubules in cultured mammalian cells.     

Soil inactivation of DNA viruses in septic seepage

AIMS: To generate field-relevant inactivation data for incorporation into models to predict the likelihood of viral contamination of surface waters by septic seepage. METHODS AND RESULTS: Inactivation rates were determined for PRD1 bacteriophage and Adenovirus 2 in two catchment soils under a range of temperature, moisture and biotic status regimes. Inactivation rates presented for both viruses were significantly different at different temperatures and in different soil types (alpha = 0.05). Soil moisture generally did not significantly affect virus inactivation rate. Biotic status significantly affected inactivation rates of PRD1 in the loam soil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly in the loam soil than PRD1 bacteriophage. CONCLUSIONS: Virus inactivation rates incorporated into models should be appropriate for the climate/catchment in question with particular regard to soil type and temperature. Given that PRD1 is similar in size to adenoviruses, yet more conservative with regard to inactivation in soil, it may be a useful surrogate in studies of Adenovirus fate and transport. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the factors that govern virus fate and transport in catchments would facilitate the design of barrier measures to prevent viral contamination of surface waters by septic seepage.