The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment.

Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2TM22, or a peptide mutated at the central residues G100/C103 (VAMP2TM22VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2TM22, in terms of α-helices and ß-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G100/C103 within the transmembrane domain during lipid rearrangements in membrane fusion.

Loss-of-function mutations in ADCY3 cause monogenic severe obesity.

Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin-melanocortin pathway in controlling energy balance, appetite and body weight 1 . The majority of loss-of-function mutations (mostly recessive or co-dominant) have been identified in genes that are directly involved in leptin-melanocortin signaling. These genes, however, only explain obesity in <5% of cases, predominantly from outbred populations 2 . We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity3-5. These data suggested that new monogenic forms of obesity could also be identified in this population. Here we identify and functionally characterize homozygous mutations in the ADCY3 gene encoding adenylate cyclase 3 in children with severe obesity from consanguineous Pakistani families, as well as compound heterozygous mutations in a severely obese child of European-American descent. These findings highlight ADCY3 as an important mediator of energy homeostasis and an attractive pharmacological target in the treatment of obesity.

A Central Small Amino Acid in the VAMP2 Transmembrane Domain Regulates the Fusion Pore in Exocytosis.

Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial. The TMD of the SNARE protein synaptobrevin2/VAMP2 contains two highly conserved small amino acids, G100 and C103, in its central portion. Substituting G100 and/or C103 with the ß-branched amino acid valine impairs the structural flexibility of the TMD in terms of α-helix/ß-sheet transitions in model membranes (measured by infrared reflection-absorption or evanescent wave spectroscopy) during increase in protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs at fusion sites. This structural change is accompanied by reduced membrane fluidity (measured by infrared ellipsometry). The G100V/C103V mutation nearly abolishes depolarization-evoked exocytosis (measured by membrane capacitance) and hormone secretion (measured biochemically). Single-vesicle optical (by TIRF microscopy) and biophysical measurements of ATP release indicate that G100V/C103V retards initial fusion-pore opening, hinders its expansion and leads to premature closure in most instances. We conclude that the TMD of VAMP2 plays a critical role in membrane fusion and that the structural mobility provided by the central small amino acids is crucial for exocytosis by influencing the molecular re-arrangements of the lipid membrane that are necessary for fusion pore opening and expansion.

Synaptotagmin 11 interacts with components of the RNA-induced silencing complex RISC in clonal pancreatic ß-cells.

Synaptotagmins are two C2 domain-containing transmembrane proteins. The function of calcium-sensitive members in the regulation of post-Golgi traffic has been well established whereas little is known about the calcium-insensitive isoforms constituting half of the protein family. Novel binding partners of synaptotagmin 11 were identified in ß-cells. A number of them had been assigned previously to ER/Golgi derived-vesicles or linked to RNA synthesis, translation and processing. Whereas the C2A domain interacted with the Q-SNARE Vti1a, the C2B domain of syt11 interacted with the SND1, Ago2 and FMRP, components of the RNA-induced silencing complex (RISC). Binding to SND was direct via its N-terminal tandem repeats. Our data indicate that syt11 may provide a link between gene regulation by microRNAs and membrane traffic.

A charged prominence in the linker domain of the cysteine-string protein Cspα mediates its regulated interaction with the calcium sensor synaptotagmin 9 during exocytosis.

The cochaperone cysteine-string protein (Csp) is located on vesicles and participates in the control of neurotransmission and hormone exocytosis. Csp contains several domains, and our previous work demonstrated the requirement of the Csp linker domain in regulated exocytosis of insulin in rodent pancreatic ß cells. We now address the molecular details to gain insight into the sequence of events during exocytosis. According to pulldown experiments and in vitro binding assays, Cspα interacts indirectly with SNAP-25 and directly with the calcium sensor synaptotagmin 9 (Syt9), which could be an intermediate between the chaperone and the t-SNARE. The C(2)A calcium binding domain of Syt9 and the linker domain of Cspα constituted the minimal interacting module. FRET-FLIM experiments confirmed the interaction between Syt9 and Cspα. Moreover, the point mutation E93V in the linker domain of Cspα significantly reduced the interaction between the two proteins. Molecular modeling revealed that this point mutation abolished a charged prominence on the surface of Cspα required for interaction. Strikingly, free calcium in the physiological low micromolar range enhanced the interaction between Syt9 and the linker domain of Cspα in vitro. These data indicate that Cspα interacts with Syt9, and such a complex may be relevant in the calcium-mediated control of a late stage of exocytosis by triggering the specific recruitment of a folding catalyst at the fusion point.

Effect of monolayer lipid charges on the structure and orientation of protein VAMP1 at the air-water interface.

SNARE proteins are implicated in membrane fusion during neurotransmission and peptide hormone secretion. Relatively little is known about the molecular interactions of their trans- and juxtamembrane domains with lipid membranes. Here, we report the structure and the assembling behavior of one of the SNARE proteins, VAMP1/synaptobrevin1 incorporated in a lipid monolayer at an air-water interface which mimics the membrane environment. Our results show that the protein is extremely sensitive to surface pressure as well as the lipid composition. Monolayers of proteins alone or in the presence of the neutral phospholipid DMPC underwent structural transition from alpha-helix to beta-sheet upon surface compression. In contrast, the anionic phospholipid DMPG inhibited this transition in a concentration-dependent manner. Moreover, the orientation of the proteins was highly sensitive to the charge density of the lipid layers. Thus, the structure of VAMP1 is clearly controlled by protein-lipid interactions.

Reversible transition between alpha-helix and beta-sheet conformation of a transmembrane domain.

Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP/synaptobrevin and syntaxin 1 are implicated in membrane fusion. Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold into an alpha-helix inserted at approximately 35 degrees to the normal of the membranes, an observation in line with molecular simulations. Upon an increase in the peptide/lipid ratio, the peptides readily exhibited transition to beta-sheet structure. Moreover, the insertion angle of these beta-sheets increased to 54 degrees and was accompanied by a derangement of lipid acyl chains. For both proteins the transition from alpha-helix to beta-sheet was reversible under certain conditions by increasing the peptide/lipid ratio. This phenomenon was observed in different model systems including multibilayers and small unilamellar vesicles. In addition, differences in peptide structure and transitions were observed when using distinct lipids (DMPC, DPPC or DOPC) thus indicating parameters influencing transmembrane domain structure and conversion from helices to sheets. The putative functional consequences of this unprecedented dynamic behavior of a transmembrane domain are discussed.

Human skin cell stress response to GSM-900 mobile phone signals. In vitro study on isolated primary cells and reconstructed epidermis.

In recent years, possible health hazards due to radiofrequency radiation (RFR) emitted by mobile phones have been investigated. Because several publications have suggested that RFR is stressful, we explored the potential biological effects of Global System for Mobile phone communication at 900 MHz (GSM-900) exposure on cultures of isolated human skin cells and human reconstructed epidermis (hRE) using human keratinocytes. As cell stress markers, we studied Hsc70, Hsp27 and Hsp70 heat shock protein (HSP) expression and epidermis thickness, as well as cell proliferation and apoptosis. Cells were exposed to GSM-900 under optimal culture conditions, for 48 h, using a specific absorption rate (SAR) of 2 W x kg(-1). This SAR level represents the recommended limit for local exposure to a mobile phone. The various biological parameters were analysed immediately after exposure. Apoptosis was not induced in isolated cells and there was no alteration in hRE thickness or proliferation. No change in HSP expression was observed in isolated keratinocytes. By contrast, a slight but significant increase in Hsp70 expression was observed in hREs after 3 and 5 weeks of culture. Moreover, fibroblasts showed a significant decrease in Hsc70, depending on the culture conditions. These results suggest that adaptive cell behaviour in response to RFR exposure, depending on the cell type and culture conditions, is unlikely to have deleterious effects at the skin level.