Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.     

Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2–3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser⁶⁰² lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr⁴⁶ and Ser⁵⁸ are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.     

Deletion of late cornified envelope genes,LCE3C_LCE3B-del,is not associated with psoriatic arthritis in Tunisian patients

A deletion of two genes from the late cornified envelope (LCE), LCE3B and LCE3C within epidermal differentiation complex on chromosome 1 was shown to be associated with both psoriasis and psoriatic arthritis (PsA) in several populations. To assess whether this deletion may contribute to the genetic predisposition to PsA in Tunisia, a total of 73 patients with PsA and 120 healthy matched controls were screened for the deletion, LCE3C_LCE3B-del, and its tag SNP, rs4112788. We also evaluated a possible relationship between PSORS1 and LCE3C_LCE3B-del through genotyping two proxy markers to HLA-C (rs12191877 and rs2073048). Our results did not provide evidence for association between the LCE3C_LCE3B-del nor the rs4112788 and the PsA. Similarly, no significant epistatic effect was observed. Our data suggest that The LCE deletion, previously identified in patients with psoriasis, is not of a major importance in the development of PsA in Tunisian patients supporting the current perception that different genetic risk factors contribute to skin and joint disease. However, these results need to be confirmed by additional large-scale studies of Tunisian PsA patients and controls.     

PRIMSIPLR: Prediction of inner‐membrane situated pore‐lining residues for alpha‐helical transmembrane proteins

Transmembrane proteins such as transporters and channels mediate the passage of inorganic and organic substances across biological membranes through their central pore. Pore‐lining residues (PLRs) that make direct contacts to the substrates have a crucial impact on the function of the protein and, hence, their identification is a key step in mechanistic studies. Here, we established a nonredundant data set containing the three‐dimensional (3D) structures of 90 α‐helical transmembrane proteins and annotated the PLRs of these proteins by a pore identification software. A support vector machine was then trained to distinguish PLRs from other residues based on the protein sequence alone. Using sixfold cross‐validation, our best performing predictor gave a Matthews's correlation coefficient of 0.41 with an accuracy of 0.86, sensitivity of 0.61, and specificity of 0.89, respectively. We provide a novel software tool that will aid biomedical scientists working on transmembrane proteins with unknown 3D structures. Both standalone version and web service are freely available from the URL http://service.bioinformatik.uni-saarland.de/PRIMSIPLR/ . Proteins 2014; 82:1503–1511. © 2014 Wiley Periodicals, Inc.     

Wnt Signaling is Essential for Bone Regeneration

This article has no abstract.     

Reconstitution of steps in the constitutive secretory pathway in permeabilized cells. Secretion of glycosylated tripeptide and truncated sphingomyelin

The constitutive secretory pathway has been reconstituted in mechanically permeabilized Chinese hamster ovary cells using two secretory markers, an acyltripeptide (N-octanoyl-Asn-Tyr-Thr-NH2) that is glycosylated at Asn in the endoplasmic reticulum and a truncated ceramide that is converted to sphingomyelin. Secretion of these bulk phase markers is dependent on cytosolic proteins and ATP. Secretion of both the glycosylated tripeptide and truncated sphingomyelin was inhibited at 15 degrees C. These results are taken as evidence that the vesicle flow to the plasma membrane (rather than artificial lysis of endoplasmic reticulum or Golgi cisternae) is required for the release of markers to the medium. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a nonhydrolyzable analogue of GTP, inhibited secretion, resulting in an accumulation of both the glycosylated tripeptide and truncated sphingomyelin in the semi-intact cell. Inhibition of secretion by GTP gamma S was not observed in the presence of the aminoglycoside antibiotic neomycin.     

Identification of more than 500 RFLPs by screening random genomic clones

As part of our genome-mapping effort, we undertook a large-scale screening study to identify RFLPs useful as genetic markers. Some 1,664 single-copy or repeat-containing phage clones from a Charon 4A genomic library were tested for polymorphism against a panel of DNAs, from five unrelated individuals, digested with eight restriction enzymes. Approximately 30% (515) of the clones revealed polymorphism by Southern hybridization; 67 loci detected had PIC values greater than .5. Restriction enzymes MspI, TaqI, and RsaI were most efficient in detecting polymorphism within the 1-20-kb-fragment size range resolved. With only one exception each of the clones detected polymorphism originating from a single locus.     

Thermodynamics of water mediating protein-ligand interactions in cytochrome P450cam: a molecular dynamics study.

Ordered water molecules are observed by crystallography and nuclear magnetic resonance to mediate protein-ligand interactions. Here, we examine the energetics of hydrating cavities formed at protein-ligand interfaces using molecular dynamics simulations. The free energies of hydrating two cavities in the active site of two liganded complexes of cytochrome P450cam were calculated by multiconfigurational thermodynamic integration. The complex of cytochrome P450cam with 2-phenyl-imidazole contains a crystallographically well defined water molecule mediating hydrogen bonds between the protein and the inhibitor. We calculate that this water molecule is stabilized by a binding free energy of -11.6 +/- kJ/mol. The complex of cytochrome P450cam with its natural substrate, camphor, contains a cavity that is empty in the crystal structure although a water molecule in it could make a hydrogen bond to camphor. Here, solvation of this cavity is calculated to be unfavorable by +15.8 +/- 5.0 kJ/mol. The molecular dynamics simulations can thus distinguish a hydrated interfacial cavity from an empty one. They also provide support for the notion that protein-ligand complexes can accommodate empty interfacial cavities and that such cavities are likely to be unhydrated unless more than one hydrogen bond can be made to a water molecule in the cavity.     

Seasonality and the sudden infant death syndrome during 1987-9 and 1991-3 in Australia and Britain.

OBJECTIVE--To determine whether seasonality of the sudden infant death syndrome persists now that rates have fallen, mostly after widespread adoption of the "face upwards" sleeping position. DESIGN--Monthly data on the sudden infant death syndrome during 1987-9 were compared for seasonality with those of 1991-3; rates were studied as deaths per 1000 live births. SETTING--Australia and Britain (England, Wales, and Scotland). SUBJECTS--Infants under 1 year dying of the syndrome (2401 for Australia and 6630 for Britain). MAIN OUTCOME MEASURE--Extent of seasonal variation (amplitude) was established by cosinor analysis; amplitudes for the earlier and later years were compared. RESULTS--The rate fell in every month, and, though it did so relatively more in winter than summer, seasonality remained a distinctive feature. In the comparison of amplitudes the ratio between the earlier and later years was 1.4 in both Australia and Britain. Some differences between the hemispheres were noted. CONCLUSIONS--Seasonality of the sudden infant death syndrome remains to be explained and continues to be an important aetiological lead. Studies from other countries are needed.