To address some of these issues, we reasoned that a-helical coiled coils might provide suitable scaﬀolds for presenting motifs to disrupt PPIs. In the rst aspect of our concept—which involves graing a motif onto the outer, solvent-exposed face of a de novo homodimeric coiledcoil—two peptide helices eﬀec- tively template and stabilise each other. In the second—which uses a de novo heterodimeric coiledcoil with only one of the peptides decorated with the motif—an element of control is introduced, as the graed peptide is only stabilised and competent to interfere with the PPI with its partner present. Others have reported a similar system based on the natural homodimeric leucine zipper, GCN4, from yeast. 34 Here we
The first examples are of materials composed entirely of coiled coils and the following examples are assemblies that are formed from coiled-coil hybrids, but which nevertheless derive their higher order structure from coiled-coil folding. A well established mode of assembly is fibers and fibrils. 1, 108-112 This field takes inspiration from nature, in which coiled-coil proteins are often in the form of fibers, such as spacer rods or intermediate filaments. The rod-like structure of long native coiled coils is mimicked by using multiple short homo or hetero coiled-coil forming peptides which associate laterally 113 and in a staggered way such that each peptide is involved in two coiled-coil interactions simultaneously, leading to fibers, some up to hundreds of micrometers long. The fibers are generally composed of a bundle of coiled coils due to interactions between the amino acids on the outside of the coiledcoil. To control this higher order structure more thought has to be put into the design of the amino acids in positions b, c, and f of the heptad repeat, an analogue to the decreased sequence variation in buried native coiled coils in comparison to non-buried motifs, e.g. in myosin filaments. Although the native rod structures can be emulated, the functions have by in large not been mimicked yet. With an eye to this current efforts are geared towards controlled design of the fiber morphology and related properties, for instance thinner and more flexible peptide fibers (Figure 13a). 114 The functionality of the fibers has been increased by conjugating additional molecules to the coiled-coil forming peptides, resulting in fibers coated with recruiting agents. These molecules on the surface of the fibers were able to bind to and hence localize proteins from solution (Figure 13b). 115 An additional dimension can be introduced by engineering kinks and branches into the fibers (Figure 13c). 116, 117 These coiled-coil fibers have been used to template silica layers at ambient temperature and physiological pH, which upon removal of the peptide (achieved most effectively by a protease), resulted in hollow silica tubes nanometers wide and microns long that are straight, kinked, or branched depending on the peptide template. 118 Alternatively, the fibrils can be induced to change to spherical objects at neutral pH, 119 or to reversibly dissociate at low pH. 120
Hydrogels have attracted tremendous research interest over many years, in part for fundamental reasons and in part because of the potential for a wide range of applications. Hydrogels have been successfully used in biomedical fields due to their high water content and the consequent biocompatibility. Successful examples include soft contact lenses 5 , wound dressings 6,7 , superabsorbents 8-10 , and drug-delivery systems 11,12 . The most recent and exciting applications of hydrogels are cell-based therapeutics 1,13 and soft tissue engineering 2 . The biomaterial used to grow the first living, tissue-engineered skin product was a collagen hydrogel 14 . Although the success of skin tissue engineering is encouraging, efforts to engineer other soft tissues have not achieved similar success. The progress in large measure is limited by inappropriate properties of the biomaterials currently available. To elicit desired cell response and coax cells to assemble into functional tissues, the materials that support and contact the cells need to be carefully designed 15,16 . For hydrogels used for cell-based therapeutics and soft tissue engineering, critical design parameters include both physical properties (such as appropriate mechanical strength and integrity) and biological properties (such as nontoxicity and ability to incorporate appropriate biological determinants (cell-binding domains, enzyme recognition sites)) 2,17 .
Cells and tissues were incubated 30 min in RIPA buffer containing protein inhibitor cocktail for lysis (Thermo Fisher Scientific). After centrifugation and denaturation, protein (20 μg) was separated by 10% polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore; Shanghai, China). Membranes were blocked with Tris Buffered Saline with Tween 20 (TBST, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20) containing 5% bovine serum albumin (BSA, Thermo Fisher Scientific),and incubated overnight at 4 °C with the following primary antibodies against CCDC109B (1:500), HIF1α (1:1000), MMP2 (1:1000), MMP9 (1:1000) and β-Tubulin (1:1000; Cell Signaling Technology; Danvers, MA, USA). Mem- branes were incubated the next day with secondary anti- body (1:5000; Santa Cruz; Dallas, TX, USA) conjugated to horseradish peroxidase (HRP) for 1 h at room tempera- ture. Proteins were quantified using a system for detecting chemiluminescence (Bio-Rad; Irvine, CA, USA), accord- ing to the manufacturer’s protocol. Representative images and data were obtained from at least three independent biological replicate experiments.
Background: Generation of the amyloid b (Ab) peptide of Alzheimer’s disease (AD) is differentially regulated through the intracellular trafficking of the amyloid b precursor protein (APP) within the secretory and endocytic pathways. Protein kinase C (PKC) and rho-activated coiled-coil kinases (ROCKs) are two “third messenger” signaling molecules that control the relative utilization of these two pathways. Several members of the Vps family of receptors (Vps35, SorL1, SorCS1) play important roles in post-trans-Golgi network (TGN) sorting and generation of APP derivatives, including Ab at the TGN, endosome and the plasma membrane. We now report that Vps10- domain proteins are candidate substrates for PKC and/or ROCK2 and act as phospho-state-sensitive physiological effectors for post-TGN sorting of APP and its derivatives.
furin (21, 40). This proteolytic cleavage event has been shown to lead to major conformational changes in the SV5 F protein and other paramyxovirus F proteins (9, 19, 22, 53). Once the F protein is present on the cellular surface or on the surface of a virion, promotion of membrane fusion can be initiated. While the triggering event for paramyxovirus F protein-mediated fu- sion remains unclear, considerable evidence points to a role for the SV5 attachment protein HN (reviewed in reference 24). The promotion of membrane fusion is hypothesized to occur with a series of conformational changes in the F protein, be- ginning with rearrangement of the protein to allow insertion of the fusion peptide into the target membrane (34), followed by refolding of the protein and finally formation of the six-helix FIG. 5. Reporter gene assays of the wt F protein and the HRA mutants. Vero cells were cotransfected with pCAGGS plasmids en- coding the SV5 HN protein and either the wt F protein or the HRA mutants under the control of the chicken ␤ -actin promoter. In addi- tion, these cells were transfected with the plasmid pINT7 ␤ gal, con- taining the ␤ -galactosidase gene under the control of the T7 polymer- ase promoter. A second set of Vero cells was transfected with the plasmid encoding the T7 polymerase under the control of the ␤ -actin promoter. After ON incubation, the T7 polymerase-expressing cells were overlaid on the cells expressing the F and HN proteins. Subse- quent membrane fusion between the cell populations allowed the T7 polymerase to transcribe the ␤ -galactosidase gene. ␤ -Galactosidase activity was assayed as described in Materials and Methods. Samples were done in triplicate. The experiment presented is representative of five separate experiments.
Decondesation of the highly compacted chromatin architecture is essential for efficient DNA repair, but how this is achieved remains largely unknown. Here, we report that microrchidia family CW-type zinc finger protein 2 (MORC2), a newly identified ATPase-dependent chromatin remodeling enzyme, is required for nucleosome destabilization after DNA damage through loosening the histone-DNA interaction. Depletion of MORC2 attenuates phosphorylated histone H2AX ( γ H2AX) focal formation, compromises the recruitment of DNA repair proteins, BRCA1, 53BP1, and Rad51, to sites of DNA damage, and consequently reduces cell survival following treatment with DNA-damaging chemotherapeutic drug camptothecin (CPT). Furthermore, we demonstrate that MORC2 can form a homodimer through its C-terminal coiled-coil (CC) domain, a process that is enhanced in response to CPT-induced DNA damage. Deletion of the C-terminal CC domain in MORC2 disrupts its homodimer formation and impairs its ability to destabilize histone-DNA interaction after DNA damage. Consistently, expression of dimerization-defective MORC2 mutant results in impaired the recruitment of DNA repair proteins to damaged chromatin and decreased cell survival after CPT treatment. Together, these findings uncover a new mechanism for MORC2 in modulating chromatin dynamics and DDR signaling through its c-terminal dimerization.
Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD), and an accu- mulating dose over 40 pack-years is a major predictable factor (1). Unfortunately, mortality due to COPD has been increasing, and it recently has emerged as the third leading cause of death in the United States (2). Despite the growing public threat from COPD, there has been no effective therapy that significantly reduces the burden of COPD. Insights into COPD pathogenesis suggest a role of persistent DNA damage in the pathobiology of COPD (3, 4). In response to cigarette smoke (CS) exposure, lung cells exhibit an orchestrated signaling process, the DNA damage response (DDR), which senses DNA damage and initi- ates DNA repair to maintain genomic integrity (5). The DDR signaling is mediated by phosphoinositide 3-kinase related protein kinases (PIKKs), including ataxia telangiectasia mutated (ATM). ATM, primarily activated by double-strand breaks (DSBs), regulates cell cycle progression and DNA repair through phos- phorylating multiple key substrates, including histone H2A at serine 139 (γH2AX). There are 2 major DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR) (6).
We next sequenced the mei-P26 gene in su(eas)16 flies to determine the molecular lesion underlying the su(eas)16 phenotypes. Typically EMS induces GC → AT base pair substitutions in DNA. Sequencing of the mei-P26 gene in su(eas)16 flies reveals two different missense mutations caused by two different single-nucleotide substitutions typical of the type produced by EMS (Figure 4D). The first mutation is a C → T switch that results in a serine- to-leucine substitution (S627L) at a nonconserved amino acid in the coiled-coil domain of the protein. The sec- ond mutation is a C → T switch that causes a proline- to-leucine substitution (P1071L) at an important con- served residue of the NHL protein-protein interaction domain. The P1071L mutation probably has an especially detrimental effect on the MEI-P26 protein since the pro- line 1071 residue is highly conserved and thought to be important for the ability of the NHL domain to adopt the proper three-dimensional ␤-propeller conformation (Slack and Ruvkun 1998; Edwards et al. 2003). To en- sure that the identified molecular mutations were a result of EMS mutagenesis and not already present in the genetic background, we sequenced the corresponding regions in su(eas)7 flies as a control since they should share similar Figure 3.—Suppression of seizures in sda flies by su(eas)16.
PCR primers (Table 2) were designed for each proP ho- mologue based on C. sakazakii strain BAA-894 sequence data available from the NCBI database (NC_009778.1). The formation of the chimeric ProP protein (ProPc), which consists of the extended coiledcoil region of ProP1 (amino acid position 422 to 505) fused to the C-terminus of ProP2 (encoded by ESA_01706), was performed using a modified SOEing (Splicing by overlap extension) tech- nique . In silico comparative analysis of the native ProP1 and ProP2 sequences, revealed a point of amino acid homology within the twelfth predicted transmem- brane domain, a leucine/isoleucine/threonine triplet (LIT) at position 422–424 and 437–439 respectively, which was selected as the splice site. Briefly, the fusion was per- formed using three separate PCR reactions: the first PCR (primer set Chimeric-01706) resulted in an ESA_01706 (proP2) amplicon lacking the C-terminal extension but containing a 15-bp 3’overhang corresponding to the LIT triplet of the ProP1 C- terminal extension. The second
Biological membrane fusion is a highly specific and coordinated process, with multiple different vesicular fusion events proceeding simultaneously in the complex environment of a cell. Specific molecular recognition between transport vesicles and target membranes is established by various proteins, mostly belonging to the SNARE family of proteins, with minimal recognition between non-complementary proteins to avoid non-targeted fusion events. Here, specific recognition is introduced to a liposomal fusion model system by using lipidated derivatives of a set of four de novo designed heterodimeric coiled-coil peptide pairs. Content mixing of liposomes was only obtained between liposomes functionalized with complementary peptides, demonstrating the fusogenic activity of various coiled-coil peptides in general. The used peptide fusogens differ in coiled-coil binding affinities and peptide-membrane interactions which allowed us to study the relationship of the peptide characteristics and the efficiency of the fusion process. It was found that peptide-membrane interactions enhanced the fusion process, especially when the affinity between complementary peptides was relatively low. Furthermore, a new coiled-coil pair (E/1 K ) was discovered, which was demonstrated to
TRIM5 ␣ dimerization is dependent on the coiled-coil do- main and is required for capsid binding (12, 37). However, the inability of the squirrel monkey coiled-coil domain to support TRIM5 ␣ -mediated restriction of B- and Mo-MLV was un- likely to be caused by a failure to self-associate, because (i) a TRIM5 ␣ chimera containing the squirrel monkey coiled-coil domain efficiently restricted SIVmac and HIV2 and (ii) over- expression of a truncated mutant bearing the squirrel monkey coiled-coil and lacking the PRYSPRY domain exerted a strong dominant-negative effect over endogenous sqTRIM5 ␣ . Although this latter experiment demonstrated that truncated proteins can hetero-oligomerize with full-length sqTRIM5 ␣ , it does not exclude the possibility that squirrel monkey coiled-coil domain displays subtle impairments in its affinity to mediate hetero- oligomerization and/or homo-oligomerization of full-length proteins. Alternatively, it may be that the formation of higher- order TRIM5 ␣ complexes, previously documented to be de- pendent on the B-box II domain, is additionally influenced by the coiled-coil domain (6, 19). Such higher-order TRIM5 ␣ multimers were shown to contribute to restriction activity par- ticularly when the PRYSPRY domain has a low affinity for the
Generation of mutations in the A27L gene and plasmid vectors. The complete DNA sequence of the 14-kDa protein (encoded by the A27L gene) was obtained by PCR with pT7Nd14K (31) as the template and with the oligonucleotide primers 59-AC TTT CCA TGG ATG GAA CTC TTT TCC C and 59-CCC AAG CTT GGG TTA CTC ATA TGG GCG CCG TCC. The specific sites for the restriction endonucleases NcoI and HindIII, respectively, are underlined. To generate point mutations in the A27L gene, we used PCR as previously described by Higuchi et al. (23). The mutagenic primers used were 59-TA GAA AAG gcT gcT AAA CGC AACG and 59-GCT GAA ACT gcg AGA GCG. The lowercase letters in the first primer indicate the nucleotides that were changed in order to alter the two contiguous cysteine residues at positions 71 and 72 to alanines, and in the second primer, the lowercase letters indicate the change of the leucine residue at position 89 to alanine. D29 and D43 deletions at the N-terminal regions of the 14-kDa protein were generated with the primers 59-CAT GCC ATG GAG GCT AAA CGC GAA GC and 59-CAT GCC ATG GAC AAT GAG GAA ACT CTC AAAC, respectively, flanked by NcoI restriction sites (underlined). The N-terminal deletions were constructed by using as the template mutant forms of the 14-kDa protein with the two cysteine residues replaced by alanines. The fragment corresponding to amino acids 29 to 74 was obtained with the following oligonucleotides flanked by NcoI and HindIII restriction sites (underlined) (59 and 39 ends, respectively): 59-CAT GCC ATG GAG GCT AAA CGC GAA GC and 59-CCC AAG CTT TTA GCG TTT agC agC CTT TTC, where small letters are mutations for alanine residues. PCR products flanked by NcoI and HindIII restriction sites at the 59 and 39 ends, respectively, were cloned in the E. coli expression vector pBAT-4 (39), which was digested with the same restriction endonucleases. The corrected sequences of the mutant forms of the 14-kDa protein were confirmed by automated DNA sequencing analysis using the se- quencing primer T7. All DNA vectors were transformed in BL21(DE3) E. coli cells for expression via IPTG induction. High-level expression was observed at different times postinduction by sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (SDS-PAGE) analyses after Coomassie blue staining (not shown) of all mutant forms. Schematic drawings of the mutant forms are presented in Fig. 1A with the nomenclature of each one on the left. No detectable levels of the VV 14-kDa protein or its mutant forms were observed in the absence of IPTG, while when the inducer was present, proteins were produced and accumulated during a 5-h period. The protein lacking 43 amino acids at the N terminus did not react with monoclonal antibody C3 (MAbC3), although it was produced to a high level (Fig. 1B).
The most abundant capsid protein is VP5, the major capsid protein, which along with VP19C, VP23, and VP26 forms the outer shell of the capsid. The remaining capsid proteins (VP22a, VP21, and VP24), the assembly protein, and the cleavage prod- ucts of the protease occupy the interior of the nascent capsid structure. The virus assembly protein forms the interior core of the assembling capsid and is essential for the appropriate for- mation of the outer capsid shell. Assembly protein-capsid pro- tein interactions are critical for imparting the correct curvature to the nascent capsid shell and for generating closed capsid structures (9, 23, 40, 41). The assembly protein therefore must interact with itself and other capsid components to perform these functions. The inherent capacity of the assembly protein to self-associate is supported by a number of experimental observations. High-level expression of ICP35cd in insect cells, either alone or in the presence of the protease, results in the formation of scaffold-like structures visible by electron micros- copy (18, 32). Moreover, ICP35ef (VP22a) extracted from iso- lated B capsids can reassociate in vitro to form torus-shaped structures 60 nm in diameter, which closely resemble the inte- rior core of native B capsids (26). ICP35cd has also been shown to interact with the major capsid protein, VP5, in in vitro studies (16, 41) and has been implicated in the localization of other capsid components to the nucleus in cellular transfection assays (18, 28). The precise molecular basis for ICP35 self- association and the influence of ICP35 self-association on its ability to associate with other capsid proteins, such as VP5, during the complex process of capsid morphogenesis remain undefined. We have therefore carried out experiments to char- acterize in detail the structural requirements for ICP35 self- association, in both in vivo and in vitro assays, and have de- termined that the interaction of ICP35 with VP5 is positively influenced by its self-association, an observation consistent with the notion that ICP35 and VP5 may assemble into small oligomers, perhaps in the cytoplasm of infected cells, and the notion that these subunits are recruited to the nascent capsid particle in the nucleus.
The scientific and technical core (including engineering design), in engineering education, must therefore incorporate themes such as ecology, ecosystems, and natural resources, to facilitate sustainable solutions to problems of technology. Placing environmental (and sustainability) issues, as ideological cores of engineering educational discourses, is essential to the development of engineering awareness. This has been recognized by the Industry Commission which suggested that an environmental focus of engineering education would improve the technological literacy of professional engineers because it carried with it the implicit understanding of liability and long-term economic viability of engineering practice, and also recognised the fact that technical solutions can only provide numerical answers to a complex situation . Constraints imposed by environmental sustainability allows greater emphasis, in engineering education, to be placed on comparative technologies as tools for choosing most appropriate designs or manufacturing routes which will minimize environmental and social impacts.
Abstract: This paper describes the development of an inclined axis manually operated coiled tube pump using plastic drum as a coil supporting structure. This pump could be used in small scale irrigation where it is difficult to transport water from lowland to highland area. A physical model of the prototype was fabricated using a small cylindrical plastic container to understand the pumping action of the pump. The prototype of the pump was then constructed considering the availability of material and size in the local hardware. The tests were carried out to collect the amount of water discharged at different angles. The maximum discharge was found 9.0 liter per minute at angle of inclination 20 degrees by the discharge pipe. The human power calculated to manually rotate the pump was approximately 11.36 W (0.15 HP) within five minutes of rotation. The maximum human power is 75 W within eight hours of working hours. Therefore the pump could be manually operated to collect 500 Liters of water per hour by using manual power. Further improvement can be done by using large diameter coiled pipe.
The enzymatic moiety of BvgS harbors the same DHp and CA domains as those of canonical TCS sensor kinases, but in many other aspects, BvgS differs from the latter (8). First, instead of extracytoplasmic PAS-like PDC sensor domains, BvgS harbors VFT perception domains of the type found in transport systems (32). Second, each monomer in BvgS harbors a single transmem- brane segment, while two TM segments flank the extracytoplas- mic PDC sensor domains. Third, canonical sensor kinases fre- quently harbor a HAMP domain between the TM and DHp domains, while HAMP domains are not found in the BvgS family (our unpublished observations). Instead, BvgS and its homologs harbor between the TM and DHp domains one or several cyto- plasmic PAS domains or, in rare cases, a GAF domain, followed by one predicted ␣ -helix per monomer or, for those that have no PAS or GAF domain, only one ␣-helix per monomer. All those features and the dynamics of VFT domains, which close by clamshell mo- tions in response to ligand binding, have led us to propose that sensor kinases of the BvgS family regulate their activity along dis- tinct mechanistic principles (9). This study shows that the linker upstream from the kinase moiety of BvgS is organized as a coiledcoil of two parallel ␣-helices. The kinase or phosphatase activity of the protein depends on whether this 2-HCC is flexible and dy- namic or whether it is rigid and adopts the low-energy conforma- tion dictated by its hydrophobic interface.
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding in ﬂ uence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an a-helical construct upon complexation with Cu( II ) and Ni( II ) but not Co( II ) or Zn( II ). The structure,