Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology

The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like.

Recycling endosome membrane incorporation into the leading edge regulates lamellipodia formation and macrophage migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R-SNARE VAMP3 on the recycling endosome partnering with the surface Q-SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3-mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3-mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.

Multi-wall carbon nanotube coating of fluorine-doped tin oxide as an electrode surface modifier for polymer solar cells

A controlled layer of multi-wall carbon nanotubes (MWCNT) was grown directly on top of fluorine-doped tin oxide (FTO) glass electrodes as a surface modifier for improving the performance of polymer solar cells. By using low-temperature chemical vapor deposition with short synthesis times, very short MWCNTs were grown, these uniformly decorating the FTO surface. The chemical vapor deposition parameters were carefully refined to balance the tube size and density, while minimizing the decrease in conductivity and light harvesting of the electrode. As created FTO/CNT electrodes were applied to bulk-heterojunction polymer solar cells, both in direct and inverted architecture. Thanks to the inclusion of MWCNT and the consequent nano-structuring of the electrode surface, we observe an increase in external quantum efficiency in the wavelength range from 550 to 650 nm. Overall, polymer solar cells realized with these FTO/CNT electrodes attain power conversion efficiency higher than 2%, outclassing reference cells based on standard FTO electrodes.

Superactivation of metal electrode surfaces and its relevance to COads oxidation at fuel cell anodes

The inhibiting effect of COads on platinum-based anodes is a major problem in the development of ambient temperature, polyelectrolyte membrane-type fuel cells. One of the unusual features of the response for the oxidative removal of the species in question is that the response observed for this reaction in the positive sweep is highly dependent on the CO admission potential, for example, when the COads is formed in the Hads region it undergoes oxidation at unusually low potentials. Such behaviour is attributed here to hydrogen activation of the platinum surface, with the result that oxide mediators (and COads oxidation) occurs at an earlier stage of the positive sweep. It is also demonstrated, for both platinum and gold in acid solution, that dramatic premonolayer oxidation responses may be observed following suitable preactivation of the electrode surfaces. It is suggested that the defect state of a solid electrode surface is an important variable whose investigation may yield improved fuel cell anode performance.

Electrical insulation of high voltage inductor with co-axial electrode at floating voltage

Inductive fault current limiters (FCLs) have several advantages, such as significant current limitation, immediate triggering and relatively low losses. Despite these advantages, saturated core FCLs have not been commercialized due to its large size and associated high costs. A major remaining challenge is to reduce the footprint of the device. In this paper, a solution to reduce the overall footprint is proposed and discussed. In arrangements of windings on a core in reactors such as FCLs, the core is conventionally grounded. The electrical insulation distance between high voltage winding and core can be reduced if the core is left at floating potential. This paper shows the results of the investigation carried out on the insulation of such a coil-core assembly. Two experiments were conducted. In the first, the behavior of the apparatus under high voltage conditions was assessed by performing power frequency and lightning impulse tests. In the second experiment, a low voltage test was conducted during which voltages of different frequencies and pulses with varying rise times were applied. A finite element simulation was also carried out for comparison and further investigation

Adsorption thermodynamics and kinetics study for the self-assembly of 1,8,15,22-tetraaminophthalocyanatocobalt(II) on glassy carbon surface

Spontaneous adsorption of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) on glassy carbon (GC) electrode leads to the formation of a stable self-assembled monolayer (SAM). Since the SAM of 4α-CoIITAPc is redox active, its adsorption on GC electrode was followed by cyclic voltammetry. SAM of 4α-CoIITAPc on GC electrode shows two pairs of well-defined redox peaks corresponding to CoIII/CoII and CoIIIPc−1/CoIIIPc−2. The surface coverage (Γ) value, calculated by integrating the charge under CoII oxidation, was used to study the adsorption thermodynamics and kinetics of 4α-CoIITAPc on GC surface. Cyclic voltammetric studies show that the adsorption of 4α-CoIITAPc on GC electrode has reached the saturation coverage (Γs) within 3 h. The Γs value for the SAM of 4α-CoIITAPc on GC electrode was found to be 2.37 × 10−10 mol cm−2. Gibbs free energy (ΔGads) and adsorption rate constant (kad) for the adsorption of 4α-CoIITAPc on GC surface were found to be −16.76 kJ mol−1 and 7.1 M−1 s−1, respectively. The possible mechanism for the self-assembly of 4α-CoIITAPc on GC surface is through the addition of nucleophilic amines to the olefinic bond on the GC surface in addition to a meager contribution from π stacking. The contribution of π stacking was confirmed from the adsorption of unsubstituted phthalocyanatocobalt(II) (CoPc) on GC electrode. Raman spectra for the SAM of 4α-CoIITAPc on carbon surface shows strong stretching and breathing bands of Pc macrocycle, pyrrole ring and isoindole ring. Raman and CV studies suggest that 4α-CoIITAPc is adopting nearly a flat orientation or little bit tilted orientation.

An SECM study on the influence of cationic, membrane-active peptides on a gold-supported self-assembled monolayer

The influence of the membrane active peptides, Tat44–57 (activator in HIV-1) and melittin (active content of bee venom), on self-assembled monolayers of 6-mercaptohexanoic acid (MHA) on gold electrodes has been studied with scanning electrochemical microscopy (SECM). It was found that MHA, when deprotonated at physiological pH, significantly affected the relative rates of electron transfer between the [Fe(CN)6]4− solution based mediator and the underlying gold electrode, predominantly by the electrostatic interaction between the mediator and MHA. Upon the introduction of Tat44–57 ormelittin to the electrolyte, the relative rate of electron transfer through the MHA layer could be increased or decreased depending on the mediator used. However, in all cases it was found that these peptides have the ability to be incorporated into synthetic SAMs, which has implications for future electrochemical studies carried out using cell mimicking membranes immobilised on such layers.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.