An alcohol and anesthetic binding pocket is hypothesized to exist among transmembrane domains of the α1 glycine receptor (GlyR). Prior work has shown that amino acid residue serine-267 plays a significant role in the enhancing effects of alcohol and anesthetics and is theorized to form part of an alcohol and anesthetic binding cavity among subunit transmembrane domains. Propyl methanethiosulfonate (PMTS), an alcohol-like thiol, was previously shown to bind to a cysteine residue introduced at position 267 (S267C) and this resulted in permanent enhancement of GlyR function. If ethanol is binding to residue 267 in wildtype GlyR to potentiate receptor function then we hypothesized that covalent thiol labeling would produce receptor enhancement by the same mechanisms as ethanol. Using outside-out patch single channel electrophysiology we determined the open and closed dwell-times and burst properties of S267C GlyR in the absence and presence of PMTS. The primary consequence of PMTS binding to S267C GlyR was an increase in the lengths of burst durations, paralleling the main effect of ethanol on wildtype GlyR. Our findings thus provide a new line of evidence suggesting that ethanol is exerting its enhancing effects on the GlyR through its interactions with amino acid residue 267 in the second transmembrane domain. / text

A poly-leucine transmembrane domain library was randomized at positions corresponding to contact surfaces for a right-handed crossing of two helices to determine the significance of small residues, GxxxG motifs, and hydrogen bonding residues in driving helix-helix interactions within membranes. About 10000 sequences, which include the interfaces of tightly interacting biological transmembrane domains, were subjected to increasing selection strength in the membrane interaction assay TOXCAT and surviving clones were sequenced to identify single site and pairwise amino acid trends. Statistical analysis identified a central glycine to be essential to strong dimerization. The next strongest statistical preference was for a phenylalanine three positions before the key glycine. Secondary to these residues, polar histidine and asparagine residues are also favored in strongly dimerizing sequences, but not to the exclusion of hydrophobic leucine and isoleucine. The analysis identifies novel pairwise combinations that contribute to or are excluded from strong dimerization, the most striking of which is that the biologically important GxxxGxxxG/A pattern is under-represented in the most strongly associating BNIP3-like transmembrane dimers. The variety of residue combinations that support strong dimerization indicates that not only key 'motif' residues, but also the residues that flank them, are important for strong dimerization. Because favorable pairwise combinations of flanking residues occur between both proximal positions and residues separated by two or more turns of helix, the complexity of how sequence context influences motif-driven dimerization is very high.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.

No description available.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.

ATP-Binding-Cassette (ABC) transporters are primary active pumps that typically couple the binding and hydrolysis of ATP to the translocation of compounds across cellular membranes. Some, like ABCB1, ABCC1 and ABCC3, are polyspecific and can efflux clinically important drugs which may contribute to their therapeutic failure. In this study I have investigated (1) the mechanism of ABC transporter function, (2) studied the potential for regulation by ubiquitin ligases (both using ABCB1 as a model), and (3) tested the involvement of ABCC1 and ABCC3 in autocrine signalling in cancer. (1) In 1966, Jardetzky et. al [1] proposed that membrane pumps function by exposing their ligand-binding pocket alternately on different sides of the membrane. For ABC transporters, this coupling of the aspect and affinity of the ligand-binding cavities of the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism but is poorly understood at the molecular level. Structure data suggest signals are transduced through intracellular loops of the TMDs which slot into grooves on the top surface of the NBDs. At the base of these grooves is the Q-loop. By analysing the function of Q-loop mutants in combination with ligand binding cavity mutants I have discovered that the Q-loops are crucial to the transport cycle and that they are required to couple ligand binding to conformational changes at the NBDs necessary to drive the transporter into an inward closed state. 4 (2) ABCB1 is known to be a key component of chemical barrier separating the circulation from the cerebrospinal fluid. It has also been reported to transport β-amyloid across the lumenal membrane and into the circulation. Loss of ABCB1 from the barrier with age has therefore been suggested to play a role in Alzheimer’s Disease. The ubiquitin ligase Nedd4-1 has been implicated in the post-translational regulation of ABCB1 abundance in cells. Here, I report that ABCB1 can be ubiquitinated by Nedd4-1 in vitro and identify the residues modified (by mass spectrometry). (3) Lysophosphatidylinositol (LPI) is an autocrine metabolite produced by cancer cells that binds to the G-protein coupled transmembrane receptor GPR55 on the surface of cells. Stimulation of GPR55 activates a signalling cascade that induces proliferation and metastases of the cancer cells. How LPI is released from the cells was not known. In this study I show that ABCC1 and ABCC3, which are known to be expressed in ovarian and pancreatic cancers, can transport LPI into inside-out vesicles suggesting a new role for these “drug resistance” transporters in cancer biology.

No description available.

Les protéines membranaires représentent environ le tiers des gènes dans les différents génomes séquencés. La prépondérance de ce type de protéines en terme de cibles thérapeutiques (50 % des médicaments) ainsi que leur implication dans beaucoup de phénomènes cellulaires tel que la transduction d'énergie, le transport de nutriments et la signalisation reflètent leur importance. Les interactions entre protéines membranaires jouent un rôle primordial dans leur structure, leurs fonctions et leur assemblage en complexes. La fonction de la plupart des protéines membranaires est liée à l'assemblage de leurs segments transmembranaires TMs dans la bicouche lipidique. Les segments TMs sont des morceaux de séquences majoritairement hydrophobes d'environ 20 résidus adoptant une structure en hélice alpha. En fait, les interactions entre hélices TMs sont essentielles pour le repliement des protéines membranaires et leur organisation dans la membrane. Pour cette raison, des interactions qualitatives entre domaines TMs de différentes protéines bitopiques ont été caractérisé en utilisant le système du double hybride bactérien (BACTH) basé sur une complémentation protéique de type adénylate cyclase. Ce système a révélé des interactions homo- et hétérologues entre des domaines TMs appartenant à deux familles de récepteurs humains, la famille des récepteurs du facteur de croissance épidermique à activité tyrosine kinase (EGFRs) et les Neuropilines. / Many cellular and biochemical processes/activities are actually carried out by the complexome, which is defined as a set of protein complexes. Identification and characterization of the complexome are essential for a comprehensive understanding and global visioning of cell functions since protein-protein interactions are the core of an entire interactomics system of any living cell. Membrane proteins make up to 30% of proteomes in eukaryotes and prokaryotes. They form a major class of proteins that are essentially involved in vital processes including bioenergetics, signal transduction, cell adhesion, catalysis and so on. Thus, they also represent more than 50% of all currently available drug targets. The function of most membrane proteins is inextricably linked to the proper packing and assembly of their transmembrane (TM) segments in the lipid bilayer. So, deciphering the contribution of TM domains interaction in the assembly of protein complexes will help to understand the dynamic assembly of membrane proteins complexes which are most important in cell signaling. For this reason, qualitative interactions between the TM domains of different bitopic proteins have been characterized using the bacterial adenylate cyclase complementation assay (BACTH). This system has been successfully adapted in the lab to study the homo- and heteromeric associations of selected TM sequences, using well characterized interactions as controls. Moreover, BACTH has revealed TM interactions of two major classes of mammalian membrane receptors, the family of epidermal growth factor receptors (EGFRs) which belongs to receptor tyrosine kinases (RTKs) superfamily and the neuropilins.