The genetic basis of a craniofacial disease provides insight into COPII coat assembly

Proteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in SEC23A, a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission.     

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.     

Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor

A growing number of pancreatic-type ribonucleases (RNases) present cytotoxic activity against malignant cells. The cytoxicity of these enzymes is related to their resistance to the ribonuclease protein inhibitor (RI). In particular, bovine seminal ribonuclease (BS-RNase) is toxic to tumor cells both in vitro and in vivo. BS-RNase is a covalent dimer with two intersubunit disulfide bridges between Cys(31) of one chain and Cys(32) of the second and vice versa. The native enzyme is an equilibrium mixture of two isomers, MxM and M=M. In the former the two subunits swap their N-terminal helices. The cytotoxic action is a peculiar property of MxM. In the reducing environment of cytosol, M=M dissociates into monomers, which are strongly inhibited by RI, whereas MxM remains as a non-covalent dimer (NCD), which evades RI. We have solved the crystal structure of NCD, carboxyamidomethylated at residues Cys(31) and Cys(32) (NCD-CAM), in a complex with 2'-deoxycitidylyl(3'-5')-2'-deoxyadenosine. The molecule reveals a quaternary structural organization much closer to MxM than to other N-terminal-swapped non-covalent dimeric forms of RNases. Model building of the complexes between these non-covalent dimers and RI reveals that NCD-CAM is the only dimer equipped with a quaternary organization capable of interfering seriously with the binding of the inhibitor. Moreover, a detailed comparative structural analysis of the dimers has highlighted the residues, which are mostly important in driving the quaternary structure toward that found in NCD-CAM.     

Ordered shotgun sequencing of a 135 kb Xq25 YAC containing ANT2 and four possible genes, including three confirmed by EST matches.

Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate. yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced. The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses. One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches. The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA.     

PCR-based immortalization and screening of hierarchical pools of cDNAs.

Starting from sequences of at least 60 bp, PCR-based screening has been developed to recover cDNAs from libraries without the necessity for hybridization or extensive DNA extraction steps. The method maintains the indefinite availability of even scarce cDNA libraries and provides an estimate of the relative abundance of the mRNA species. Isolation of a cDNA clone can be done in less than a week. cDNAs were isolated that were cognate for fragments of expressed sequences and for an exon predicted from genomic sequence.     

Exercise training and high-fat diet elicit endocannabinoid system modifications in the rat hypothalamus and hippocampus

The purpose of the present study was to examine the effect of chronic exercise on the hypothalamus and hippocampus levels of the endocannabinoids (eCBs) anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and of two AEA congeners and on the expression of genes coding for CB1, CB2 receptors (Cnr1 and Cnr2, respectively), and the enzymes responsible for eCB biosynthesis and degradation, in rats fed with a standard or high-fat diet. Male Wistar rats (n = 28) were placed on a 12-week high-fat (HFD) or standard diet period, followed by 12 weeks of exercise training for half of each group. Tissue levels of eCBs and related lipids were measured by liquid chromatography mass spectrometry, and expression of genes coding for CB1 and CB2 receptors and eCB metabolic enzymes was measured by quantitative real-time polymerase chain reaction (qPCR). HFD induced a significant increase in 2-AG (p < 0.01) in hypothalamus. High-fat diet paired with exercise training had no effect on AEA, 2-AG, and AEA congener levels in the hypothalamus and hippocampus. Cnr1 expression levels were significantly increased in the hippocampus in response to HFD, exercise, and the combination of both (p < 0.05). Our results indicate that eCB signaling in the CNS is sensitive to diet and/or exercise.     

An Order-Disorder Transition Plays a Role in Switching Off the Root Effect in Fish Hemoglobins

The Root effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α₁β₂ and α₂β₁ interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.     

An evolutionary conserved motif is responsible for immunoglobulin heavy chain packing in the B cell membrane

All species of vertebrates synthesize immunoglobulin molecules, which differ in an number of aspects but also share a few common features responsible for their function, such as the presence of a transmembrane domain in the membrane bound form of the immunoglobulin heavy chain (IgTMD) that ensures communication with the signal transducing Igα-Igβ peptides. We have analyzed the gene sequence encoding the IgTMD of different heavy chain isotypes of very distant species, from shark to mammals. The IgTMD sequences show a high degree of sequence identity and their encoding nucleotide sequences were shown to be subject to purifying selection at most sites. We have built molecular models of seven IgTMDs from different vertebrate species and have investigated the formation of homodimer in a palmitoyl oleoyl phosphatidylcholine (POPC) lipid bilayer by molecular dynamics simulations. We found that the conserved FXXXFXXS/TXXXS motif, never observed to date in protein transmembrane chains, is responsible for the two heavy chains association through two pairs of Phe-Phe hydrophobic interactions and two pairs of Ser/Thr-Ser/Ser hydrogen bonds. This interaction pattern, which stabilizes the dimer conformation in the lipid bilayer, was unique, being different from any other pattern identified in transmembrane helices to date.