New insights of membrane environment effects on MscL channel mechanics from theoretical approaches

The prokaryotic mechanosensitive channel of large conductance (MscL) is a remarkable integral membrane protein. During hypo-osmotic shock, it responses to membrane tension through large conformational changes, that lead to an open state of the pore. The structure of the channel from Mycobacterium tuberculosis has been resolved in the closed state. Numerous experiments have attempted to trap the channel in its open state but they did not succeed in obtaining a structure. A gating mechanism has been proposed based on different experimental data but there is no experimental technique available to follow this process in atomic details. In addition, it has been shown that a decrease of the lipid bilayer thickness lowered MscL activation energy and stabilized a structurally distinct closed channel intermediate. Here, we use atomistic molecular dynamics simulations to investigate the effect of the lipid bilayer thinning on our model of the structure of the Escherichia coli. We thoroughly analyze simulations of the channel embedded in two pre-equilibrated membranes differing by their hydrophobic tail length (DMPE and POPE). The MscL structure remains stable in POPE, whereas a distinct structural state is obtained in DMPE in response to hydrophobic mismatch. This latter is obtained by tilts and kinks of the transmembrane helices, leading to a widening and a diminution of the channel height. Part of these motions is guided by a competition between solvent and lipids for the interaction with the periplasmic loops. We finally conduct a principal component analysis of the simulation and compare anharmonic motions with harmonic ones, previously obtained from a coarse-grained normal mode analysis performed on the same structural model. Significant similarities exist between low-frequency harmonic motions and those observed with essential dynamics in DMPE. In summary, change in membrane thickness permits to accelerate the conformational changes involved in the mechanics of the E. coli channel, providing a closed structural intermediate en route to the open state. These results give clues for better understanding why the channel activation energy is lowered in a thinner membrane.     

LAT displacement from lipid rafts as a molecular mechanism for the inhibition of T cell signaling by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) suppress immune responses and inhibit T cell activation through largely unknown mechanisms. The displacement of signaling proteins from membrane lipid rafts has recently been suggested as underlying PUFA-mediated T cell inhibition. We show here that PUFA treatment specifically interferes with T cell signal transduction by blocking tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase Cgamma1. A significant fraction of LAT was displaced from rafts by PUFA treatment along with other signaling proteins. However, retaining LAT alone in lipid rafts effectively restored phospholipase Cgamma1/calcium signaling in PUFA-treated T cells. These data reveal LAT displacement from lipid rafts as a molecular mechanism by which PUFAs inhibit T cell signaling and underline the predominant importance of LAT localization in rafts for efficient T cell activation.     

Nonneutral admixture of immigrant genotypes in African Drosophila melanogaster populations from Zimbabwe

Drosophila melanogaster originated in Africa and colonized the rest of the world only recently (approximately 10,000 to 15,000 years ago). Using 151 microsatellite loci, we investigated patterns of gene flow between African D. melanogaster populations representing presumptive ancestral variation and recently colonized European populations. Although we detected almost no evidence for alleles of non-African ancestry in a rural D. melanogaster population from Zimbabwe, an urban population from Zimbabwe showed evidence for admixture. Interestingly, the degree of admixture differed among chromosomes. X chromosomes of both rural and urban populations showed almost no non-African ancestry, but the third chromosome in the urban population showed up to 70% of non-African alleles. When chromosomes were broken into contingent microsatellite blocks, even higher estimates of admixture and significant heterogeneity in admixture was observed among these blocks. The discrepancy between the X chromosome and the third chromosome is not consistent with a neutral admixture hypothesis. The higher number of European alleles on the third chromosome could be due to stronger selection against foreign alleles on the X chromosome or to more introgression of (beneficial) alleles on the third chromosome.     

<Emphasis Type="Italic">SONIC HEDGEHOG</Emphasis> mutations causing human holoprosencephaly impair neural patterning activity

Holoprosencephaly (HPE) is a common forebrain malformation associated with mental retardation and craniofacial anomalies. Multiple lines of evidence indicate that loss of ventral neurons is associated with HPE. The condition is etiologically heterogeneous, and abnormalities in any of several genes can cause human HPE. Among these genes, mutations in SONIC HEDGEHOG ( SHH) are the most commonly identified single gene defect causing human HPE. SHH mediates a number of processes in central nervous system development and is required for the normal induction of ventral cell types in the brain and spinal cord. Although a number of missense mutations in SHH have been identified in patients with HPE, the functional significance of these mutations has not yet been determined. We demonstrate that two SHH mutations that cause human HPE result in decreased in vivo activity of SHH in the developing nervous system. These mutant forms of SHH fail to regulate genes properly that are normally responsive to SHH signaling and do not induce ventrally expressed genes. In addition, the immunoreactivity of the mutant proteins is altered, suggesting that the conformation of the SHH protein has been disrupted. These studies are the first demonstration that mutations in SHH associated with human HPE perturb the in vivo patterning function of SHH in the developing nervous system.     

T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression

Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.     

A large Ca2+-dependent channel formed by recombinant ADP/ATP carrier from Neurospora crassa resembles the mitochondrial permeability transition pore

Strong support for the central role of the ADP/ATP carrier (AAC) in the mitochondrial permeability transition (mPT) is provided by the single-channel current measurements in patch-clamp experiments with the isolated reconstituted AAC. In previous work [Brustovetsky, N., and Klingenberg, M. (1996) Biochemistry 35, 8483-8488], this technique was applied to the AAC isolated from bovine heart mitochondria. Here we used recombinant AAC (rAAC) from Neurospora crassa expressed in E. coli, since AAC from mammalian sources cannot be expresssed in E. coli. The rAAC is free from residual mitochondrial components which might associate with the AAC in preparation from bovine heart. Ca(2+)-dependent channels with up to 600 pS are obtained, which are gated at >150 mV. The channel corresponds to a preferential matrix-outside orientation of rAAC in the patch membrane as shown with carboxyatractylate and a polar gating asymmetry. The channel is inhibited by ADP and bongkrekate, not by carboxyatractylate. Cyclophilin, isolated from Neurospora crassa, suppresses the gating, thus increasing conductivity at high positive voltage. Cyclosporin A abolishes the cyclophilin effect. ADP does not eliminate the cyclophilin effect but produces fast large-amplitude flickering of the channel without a stable decrease of the channel conductance. Also the pro-oxidant tert-butyl hydroperoxide reversibly suppresses voltage gating of the channel. The results show that the AAC can be a conducting component of the mPT pore, exhibiting similar characteristics as the mPT pore (response to Ca(2+), BKA, ADP), with a cyclophilin and pro-oxidant-sensitive gating at high voltage.     

Cycle number and waveform of fluid flow affect bovine articular chondrocytes

Many studies have shown that a loading-induced (bio)physical signal regulates chondrocyte behavior. In a recent study our group has demonstrated the shear stress level- and frequency-dependent effect of sinusoidal oscillatory fluid flow on bovine articular chondrocyte (BAC) cytosolic calcium concentration ([Ca(2+)](i)), neglecting the fact that chondrocytes are not likely to see these ideal waveform in vivo or in vitro. Furthermore, possible overload of articular cartilage or excessive shear stress in chondrocyte cultures are more likely to be of a short nature. Therefore, in this study we choose to investigate a saw-tooth waveform oscillating fluid flow at varying exposure times in comparison to the established sinusoidal oscillatory waveform. [Ca(2+)](i), as an early signaling molecule, was quantified using the fluorescent dye fura-2. BAC were exposed to 1 Hz sinusoidal or saw-tooth waveform oscillating fluid flow at 2.2 Pa flow rates in a parallel plate flow chamber for 8 different loading times. As little as 5 cycles of oscillatory fluid flow were sufficient to increase [Ca(2+)](i) significantly over baseline. The number of responding cells could not be increased any further after a sufficient number of cycles (11), regardless of the waveform. Furthermore, a saw-tooth waveform appeared to be more stimulatory than regular sinusoidal oscillating flow at higher cycle numbers. BAC appear to be able to respond to these biophysical stimuli in a differentiated manner. This ability might give every single chondrocyte the capability to maintain its territory autonomously, since chondrocytes distributed in articular cartilage without the possibility to interact, e.g., via cell processes.     

Neurospora crassa CyPBP37: a cytosolic stress protein that is able to replace yeast Thi4p function in the synthesis of vitamin B1

Recently, we identified CyPBP37 of Neurospora crassa as a binding partner of cyclophilin41. CyPBP37 function had not yet been described, although orthologs in other organisms have been implicated in the biosynthesis of the thiazole moiety of thiamine (vitamin B1) and/or stress-related pathways. Here, CyPBP37 is characterized as an abundant cytosolic protein with a functional NAD-binding site. Saccharomyces cerevisiae mutants lacking Thi4p (the CyPBP37 ortholog) are auxotrophic for vitamin B1 (thiamine) but can grow in the presence of the thiazole moiety of thiamine, suggesting a role for Thi4p in the biosynthesis of thiazole. N.crassa CyPBP37 is able to functionally replace Thi4p in yeast thiazole synthesis. Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. This implies that thiamine synthesis takes place in the cytosol of both organisms and not in the mitochondria, as suggested. The expression of CyPBP37 and Thi4p is repressed by thiamine but not by thiazole in the growth medium. In addition to its function in thiazole synthesis, CyPBP37 is a stress-inducible protein. N.crassa cyclophilin41 can chaperone the folding of CyPBP37, its own binding partner.     

FISH diagnosis of the common 57-kb deletion in <Emphasis Type="Italic">CTNS</Emphasis> causing cystinosis

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation, a 57-kb deletion, occurs in ~60% of patients in the United States and northern Europe and removes exons 1–9, most of exon 10, the CTNS promoter region, and all of an adjacent gene of unknown function called CARKL. CTNS codes for the lysosomal cystine transporter, whose absence leads to intracellular cystine accumulation, widespread cellular destruction, renal Fanconi syndrome in infancy, renal glomerular failure in later childhood, and other systemic complications. Because treatment with oral cysteamine can prevent or delay these complications significantly, early and accurate diagnosis is critical. This study describes the generation of fluorescence in situ hybridization (FISH) probes for the 57-kb deletion in CTNS, enabling cytogenetics laboratories to test for this common mutation. The probes would also be able to detect a less frequent 11.7-kb deletion. A blinded study was performed using multiplex PCR analysis as the gold standard to determine the presence or absence of the 57-kb deletion. The FISH probes, evaluated on 12 lymphoblastoid cell lines from singly deleted, doubly deleted, and nondeleted patients, made the correct diagnosis in every case. This appears to be the first FISH-based diagnostic method described for any lysosomal storage disorder. It can assist in the antenatal and perinatal diagnosis of cystinosis and promote earlier salutary therapy with cysteamine.