Fluorescent photoaffinity labeling of cytochrome P450 3A4 by lapachenole: identification of modification sites by mass spectrometry

While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.     

Tissue kallikrein stimulates Ca(2+) reabsorption via PKC-dependent plasma membrane accumulation of TRPV5

The transient receptor potential vanilloid 5 (TRPV5) channel determines urinary Ca(2+) excretion, and is therefore critical for Ca(2+) homeostasis. Interestingly, mice lacking the serine protease tissue kallikrein (TK) exhibit robust hypercalciuria comparable to the Ca(2+) leak in TRPV5 knockout mice. Here, we delineated the molecular mechanism through which TK stimulates Ca(2+) reabsorption. Using TRPV5-expressing primary cultures of renal Ca(2+)-transporting epithelial cells, we showed that TK activates Ca(2+) reabsorption. The stimulatory effect of TK was mimicked by bradykinin (BK) and could be reversed by application of JE049, a BK receptor type 2 antagonist. A cell permeable analog of DAG increased TRPV5 activity within 30 min via protein kinase C activation of the channel since mutation of TRPV5 at the putative PKC phosphorylation sites S299 and S654 prevented the stimulatory effect of TK. Cell surface labeling revealed that TK enhances the amount of wild-type TRPV5 channels, but not of the TRPV5 S299A and S654A mutants, at the plasma membrane by delaying its retrieval. In conclusion, TK stimulates Ca(2+) reabsorption via the BK-activated PLC/DAG/PKC pathway and the subsequent stabilization of the TRPV5 channel at the plasma membrane.     

Dual effect of methylglyoxal on the intracellular Ca2+ signaling and neurite outgrowth in mouse sensory neurons

The formation of advanced glycation end products is one of the major factors involved in diabetic neuropathy, aging, and neurodegenerative diseases. Reactive carbonyl compounds, such as methylglyoxal (MG), play a key role in cross-linking to various proteins in the extracellular matrix, especially in neurons, which have a high rate of oxidative metabolism. The MG effect was tested on dorsal root ganglia primary neurons in cultures from adult male Balb/c mice. Lower MG doses contribute to an increased adherence of neurons on their support and an increased glia proliferation, as proved by MTS assay and bright-field microscopy. Time-lapse fluorescence microscopy by Fura-2 was performed for monitoring the relative fluorescence ratio changes (ΔR/R(0)) upon depolarization and immunofluorescence staining for quantifying the degree of neurites extension. The relative change in fluorescence ratio modifies the amplitude and dispersion depending on the subtype of sensory neurons, the medium-sized neurons are more sensitive to MG treatment when compared to small ones. Low MG concentrations (0-150 μM) increase neuronal viability, excitability, and the capacity of neurite extension, while higher concentrations (250-750 μM) are cytotoxic in a dose-dependent manner. In our opinion, MG could be metabolized by the glyoxalase system inside sensory neurons up to a threshold concentration, afterwards disturbing the cell equilibrium. Our study points out that MG has a dual effect concentration dependent on the neuronal viability, excitability, and neurite outgrowth, but only the excitability changes are soma-sized dependent. In conclusion, our data may partially explain the distinct neuronal modifications in various neurodegenerative pathologies.     

Numerical streamline patterns at swimmer's surface using RANS equations

The objective of this article is to perform a numerical modeling on the flow dynamics around a competitive female swimmer during the underwater swimming phase for a velocity of 2.2 m/s corresponding to national swimming levels. Flow around the swimmer is assumed turbulent and simulated with a computational fluid dynamics method based on a volume control approach. The 3D numerical simulations have been carried out with the code ANSYS FLUENT and are presented using the standard k-ω turbulence model for a Reynolds number of 6.4 × 10(6). To validate the streamline patterns produced by the simulation, experiments were performed in the swimming pools of the National Institute of Sports and Physical Education in Paris (INSEP) by using the tufts method.     

Activation of ERAD pathway by human hepatitis B virus modulates viral and subviral particle production

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. It was previously shown that HBV can induce endoplasmic reticulum (ER) stress and activate the IRE1-XBP1 pathway of the unfolded protein response (UPR), through the expression of the viral regulatory protein X (HBx). However, it remained obscure whether or not this activation had any functional consequences on the target genes of the UPR pathway. Of these targets, the ER degradation-enhancing, mannosidase-like proteins (EDEMs) are thought to play an important role in relieving the ER stress during UPR, by recognizing terminally misfolded glycoproteins and delivering them to the ER-associated degradation (ERAD). In this study, we investigated the role of EDEMs in the HBV life-cycle. We found that synthesis of EDEMs (EDEM1 and its homologues, EDEM2 and EDEM3) is significantly up-regulated in cells with persistent or transient HBV replication. Co-expression of the wild-type HBV envelope proteins with EDEM1 resulted in their massive degradation, a process reversed by EDEM1 silencing. Surprisingly, the autophagy/lysosomes, rather than the proteasome were involved in disposal of the HBV envelope proteins. Importantly, inhibition of the endogenous EDEM1 expression in HBV replicating cells significantly increased secretion of both, enveloped virus and subviral particles. This is the first report showing that HBV activates the ERAD pathway, which, in turn, reduces the amount of envelope proteins, possibly as a mechanism to control the level of virus particles in infected cells and facilitate the establishment of chronic infections.     

Transport at the nanoscale: temperature dependence of ion conductance

Temperature dependent ion conductance in nanopores is measured in a wide range of electrolyte concentrations and compared with molecular modeling. Single outer membrane protein F (OmpF) channels from E. coli are reconstituted into planar lipid bilayers. In qualitative agreement with the experimental data, applied-field molecular dynamics unraveled atomistic details of the ion transport. Comparing the temperature dependence of the channel conductance with that of the bulk conductivity in the range from 0 to 90°C revealed that at low salt concentrations the transport is mainly driven along the pore surface. Increasing the salt concentration saturates the surface charge transport and induces ion transport in the center of the nanopore. The confinement of the nanopore then favors the formation of ion pairs. Stepping up the temperature reduces the life time of the ion pairs and increases the channel conductance more than expected from the bulk behavior.     

Two-Dimensional Nanoscale Structural and Functional Imaging in Individual Collagen Type I Fibrils

The piezoelectric properties of single collagen type I fibrils in fascia were imaged with sub-20 nm spatial resolution using piezoresponse force microscopy. A detailed analysis of the piezoresponse force microscopy signal in controlled tip-fibril geometry revealed shear piezoelectricity parallel to the fibril axis. The direction of the displacement is preserved along the whole fiber length and is independent of the fiber conformation. It is shown that individual fibrils within bundles in skeletal muscle fascia can have opposite polar orientations and are organized into domains, i.e., groups of several fibers having the same polar orientation. We were also able to detect piezoelectric activity of collagen fibrils in the high-frequency range up to 200 kHz, suggesting that the mechanical response time of biomolecules to electrical stimuli can be ∼5 μs.