The structural basis of water permeation and proton exclusion in aquaporins (Review)

Aquaporins (AQPs) represent a ubiquitous class of integral membrane proteins that play critical roles in cellular osmoregulations in microbes, plants and mammals. AQPs primarily function as water-conducting channels, whereas members of a sub-class of AQPs, termed aquaglyceroporins, are permeable to small neutral solutes such as glycerol. While AQPs facilitate transmembrane permeation of water and/or small neutral solutes, they preclude the conduction of protons. Consequently, openings of AQP channels allow rapid water diffusion down an osmotic gradient without dissipating electrochemical potentials. Molecular structures of AQPs portray unique features that define the two central functions of AQP channels: effective water permeation and strict proton exclusion. This review describes AQP structures known to date and discusses the mechanisms underlying water permeation, proton exclusion and water permeability regulation.     

A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density

The A kinase anchor protein AKAP150 recruits the cAMP-dependent protein kinase (PKA) to dendritic spines. Here we show that in AKAP150 (AKAP5) knock-out (KO) mice frequency of miniature excitatory post-synaptic currents (mEPSC) and inhibitory post-synaptic currents (mIPSC) are elevated at 2 weeks and, more modestly, 4 weeks of age in the hippocampal CA1 area versus litter mate WT mice. Linear spine density and ratio of AMPAR to NMDAR EPSC amplitudes were also increased. Amplitude and decay time of mEPSCs, decay time of mIPSCs, and spine size were unaltered. Mice in which the PKA anchoring C-terminal 36 residues of AKAP150 are deleted (D36) showed similar changes. Furthermore, whereas acute stimulation of PKA (2-4 h) increases spine density, prolonged PKA stimulation (48 h) reduces spine density in apical dendrites of CA1 pyramidal neurons in organotypic slice cultures. The data from the AKAP150 mutant mice show that AKAP150-anchored PKA chronically limits the number of spines with functional AMPARs at 2-4 weeks of age. However, synaptic transmission and spine density was normal at 8 weeks in KO and D36 mice. Thus AKAP150-independent mechanisms correct the aberrantly high number of active spines in juvenile AKAP150 KO and D36 mice during development.     

Intact Functional Fourteen-subunit Respiratory Membrane-bound [NiFe]-Hydrogenase Complex of the Hyperthermophilic Archaeon Pyrococcus furiosus

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO₂, and H₂, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H₂ production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na⁺/H⁺ antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H₂ with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O₂ sensitivities of the two forms were similar (half-lives of ∼15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H₂ and pump Na⁺ ions.     

Role of Focal Adhesion Tyrosine Kinases in GPVI-Dependent Platelet Activation and Reactive Oxygen Species Formation

Background

We have previously shown the presence of a TRAF4/p47^phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.

Aims

To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.

Methods and Results

Human and mouse washed platelets (from WT or Pyk2 KO mice) were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively) and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P) surface expression) and integrin α_IIbβ₃ activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk), PI3-K and Bruton''s tyrosine kinase (Btk) and upstream of Rac1, PLCγ2, Ca²⁺ release, PKC, Hic-5, NOX1 and α_IIbβ₃ activation.

Conclusion

Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.     

Calmodulin regulates the non-amyloidogenic metabolism of amyloid precursor protein in platelets

A balance between the proteolytic processing of amyloid precursor protein APP through the amyloidogenic and the non-amyloidogenic pathways controls the production and release of amyloid β-protein, whose accumulation in the brain is associated to the onset of Alzheimer Disease. APP is also expressed on circulating platelets. The regulation of APP processing in these cells is poorly understood. In this work we show that platelets store considerable amounts of APP fragments, including sAPPα, that can be released upon stimulation of platelets. Moreover, platelet stimulation also promotes the proteolysis of intact APP expressed on the cell surface. This process is supported by an ADAM metalloproteinase, and causes the release of sAPPα. Processing of intact platelet APP is promoted also by treatment with calmodulin antagonist W7. W7-induced APP proteolysis occurs through the non-amyloidogenic pathway, is mediated by a metalloproteinase, and causes the release of sAPPα. Co-immunoprecipitation and pull-down experiments revealed a physical association between calmodulin and APP. These results document a novel role of calmodulin in the regulation of non-amyloidogenic processing of APP.     

Common genetic variants of MUTYH are not associated with cutaneous malignant melanoma: application of molecular screening by means of high-resolution melting technique in a pilot case-control study

MUTYH glycosylase recognizes the 8-oxoG:A mismatch and is able to excise the adenine base using proofreading mechanisms. Some papers have reported a strong association between cancer development or aggressiveness and MUTYH gene mutations. The aim of this study was to find a possible association between the most frequent MUTYH mutations and melanoma in the context of a case-control pilot study. One hundred ninety-five melanoma patients and 195 healthy controls were matched for sex and age. Clinical and laboratory data were collected in a specific database and all individuals were analyzed for MUTYH mutations by high-resolution melting and direct sequencing techniques. Men and women had significantly different distributions of tumor sites and phototypes. No significant associations were observed between the Y165C, G382D and V479F MUTYH mutations and risk of melanoma development or aggressiveness. Our preliminary findings therefore do not confirm a role for MUTYH gene mutations in the melanoma risk. Further studies are necessary for the assessment of MUTYH not only in melanoma but also other cancer types with the same embryonic origin, in the context of larger arrays studies of genes involved in DNA stability or integrity.     

Photographed rapid HIV test results pilot novel quality assessment and training schemes

HIV rapid diagnostic tests (RDTs) are now used widely in non-laboratory settings by non-laboratory-trained operators. Quality assurance programmes are essential in ensuring the quality of HIV RDT outcomes. However, there is no cost-effective means of supplying the many operators of RDTs with suitable quality assurance schemes. Therefore, it was examined whether photograph-based RDT results could be used and correctly interpreted in the non-laboratory setting. Further it was investigated if a single training session improved the interpretation skills of RDT operators. The photographs were interpreted, a 10-minute tutorial given and then a second interpretation session was held. It was established that the results could be read with accuracy. The participants (n=75) with a range of skills interpreted results (>80% concordance with reference results) from a panel of 10 samples (three negative and seven positive) using four RDTs. Differences in accuracy of interpretation before and after the tutorial were marked in some cases. Training was more effective for improving the accurate interpretation of more complex results, e.g. results with faint test lines or for multiple test lines, and especially for improving interpretation skills of inexperienced participants. It was demonstrated that interpretation of RDTs was improved using photographed results allied to a 10-minute training session. It is anticipated that this method could be used for training but also for quality assessment of RDT operators without access to conventional quality assurance or training schemes requiring wet samples.     

Impact of detectable human cytomegalovirus DNAemia on viro-immunological effectiveness of HAART in HIV-infected patients naive to antiretroviral therapy

Our objective was to explore whether positive human cytomegalovirus (HCMV) DNAemia at baseline impaired CD4+ T-cell increase after 1 year of HAART. A sub-study of a randomized clinical trial in selected patients with <200 cell/mm CD4+ at baseline was conducted. Six out of 30 patients had detectable HCMV DNAemia at baseline, all reaching HCMV suppression at week 52 after HAART (only 1 of them was treated with valgancyclovir). No significant differences were found between patients with detectable or undetectable HCMV DNAemia in terms of CD4+ T-cell increase and HIV RNA response to HAART. Although some data may favor HCVM pre-emptive therapy to decrease immune activation, our results do not indicate that this practice may increase CD4+ T-cell count after HAART. At the same time, HAART proved effective in reducing HCMV DNAemia without the need for a specific therapy.     

Aberrant Dendritic Excitability: A Common Pathophysiology in CNS Disorders Affecting Memory?

Discovering the etiology of pathophysiologies and aberrant behavior in many central nervous system (CNS) disorders has proven elusive because susceptibility to these diseases can be a product of multiple factors such as genetics, epigenetics, and environment. Advances in molecular biology and wide-scale genomics have shown that a large heterogeneity of genetic mutations are potentially responsible for the neuronal pathologies and dysfunctional behaviors seen in CNS disorders. Despite this seemingly complex array of genetic and physiological factors, many disorders of the CNS converge on common dysfunctions in memory. In this review, we propose that mechanisms underlying the development of many CNS disorders may share an underlying cause involving abnormal dendritic integration of synaptic signals. Through understanding the relationship between molecular genetics and dendritic computation, future research may uncover important links between neuronal physiology at the cellular level and higher-order circuit and network abnormalities observed in CNS disorders, and their subsequent affect on memory.