The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation,membrane binding,lipid-specific domains and channel activities

SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23–25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23–25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.     

Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR^−/−) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR^−/− mice fed MCD diet (FXR^−/−/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR^−/−/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR^−/−/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR^−/−/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.     

SecAAA trimer is fully functional as SecAA dimer in the membrane: Existence of higher oligomers?

SecA is an essential ATPase in bacterial Sec-dependent protein translocation pathway, and equilibrates between monomers and dimers in solution. The question of whether SecA functions as monomers or dimers in membranes during the protein translocation is controversial. We previously constructed a tail-to-head SecAA tandem dimer, and showed it is fully functional by complementation in vivo and protein translocation in vitro, indicating that SecA can function at least as a dimer in the membrane without dissociating into monomers. In this study, we further constructed genetically a tail-to-head SecAAA trimer, which is functional in complementing a temperature-sensitive secA mutant. The purified SecAAA trimer per protomer is fully active as SecAA tandem dimers in ATPase activity, in protein translocation in vitro and in ion channel activities in the oocytes. With these functional tail-to-head trimer SecAAA and tandem SecAA, we examined their surface topology in the presence of liposomes using AFM. As expected, the soluble SecAAA without lipids are larger than SecAA. However, the ring/pore structures of SecAAA trimers were, surprisingly, almost identical to the SecA 2-monomers and SecAA dimers, raising the intriguing possibility that the SecA may exist and function as hexamer ring-structures in membranes. Cross-linking with formaldehyde showed that SecA, SecAA and SecAAA could form larger oligomers, including the hexamers. The molecular modeling simulation shows that both tail-to-head and tail-to-tail hexamers in the membranes are possible.     

Identification of a SecM segment required for export-coupled release from elongation arrest

SecM in Escherichia coli has two functionally crucial regions. The arrest motif near the C-terminus interacts with the ribosomal exit tunnel to arrest its own translational elongation. The signal sequence at the N-terminus directs the SecM nascent polypeptide to the Sec-mediated export pathway to release the arrested state of translation. Here, we addressed the importance of the central region of SecM. Characterization of internal substitution and deletion mutants revealed that a segment from residue 100 to residue 109 is required for the export-coupled release of the SecM nascent chain from the elongation-arrested state. Thus, the central region of SecM is not just a geometric linker but it participates actively in the regulation of translation arrest.     

Discovery of the first SecA inhibitors using structure-based virtual screening

Bacterial protein secretion is a critical and complex process. The Sec machinery provides a major pathway for protein translocation across and integration into the cellular membrane in bacteria. Small molecule probes that perturb the functions of individual member proteins within the Sec machinery will be very important research tools as well as leads for future antimicrobial agent development. Herein we describe the discovery of inhibitors, through virtual screening, that specifically act on SecA ATPase, which is a critical member of the Sec system. These are the very first inhibitors reported for intrinsic SecA ATPase.     

Mim1 functions in an oligomeric form to facilitate the integration of Tom20 into the mitochondrial outer membrane

The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Despite its central role in the biogenesis of mitochondria, the assembly process of this complex is not completely understood. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein with an undefined role in the assembly of the TOM complex. The protein is composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space. Here we show that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. We further investigated what the structural characteristics allowing Mim1 to fulfil its function are. The N- and C-terminal domains of Mim1 are crucial neither for the function of the protein nor for its biogenesis. Thus, the TMS of Mim1 is the minimal functional domain of the protein. We show that Mim1 forms homo-oligomeric structures via its TMS, which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs did not form oligomeric structures and was inactive. With all these data taken together, we propose that the homo-oligomerization of Mim1 allows it to fulfil its function in promoting the integration of Tom20 into the mitochondrial outer membrane.     

Plasmodium falciparum: functional mitochondrial ADP/ATP transporter in Escherichia coli plasmic membrane as a tool for selective drug screening

Plasmodium falciparum mitochondrial ADP/ATP transporter or adenylate translocase (PfAdT) was previously characterised at the molecular level and intracellularly located by immuno-electromicroscopy. Inhibition of this transporter blocks parasite development in erythrocytes. In this study, PfAdT was expressed in C43 (DE3) Escherichia coli strain under isopropyl beta-d-thiogalacto-pyranoside (IPTG) induction to screen inhibitory molecules. PfAdT was integrated directly into the bacterial cytoplasmic membrane. Whereas IPTG-induced bacterial cells imported radioactively labelled ATP, non-induced cells did not. The transporter bound specifically ADP and ATP, but not AMP. IPTG-induced cells preloaded with labelled ATP exported ATP after exogenous addition of unlabelled ADP or ATP, indicating a counter exchange transport mechanism. Bongrekic acid and atractyloside, two well-known specific inhibitors of mitochondrial ADP/ATP transporter, were tested. This experimental model was evaluated using three Malagasy crude plants extracts which have shown antiplasmodial activity on in vitro parasite cultures.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> possesses two <Emphasis Type="Italic">secA</Emphasis> homologous genes that are essential for viability

SecA is a central component of the bacterial Sec preprotein translocase. Besides the housekeeping SecA (SecA1), some mostly pathogenic Gram-positive bacteria possess an accessory SecA (SecA2) that is involved in the export of a few substrates only. Here we show that neither of the two secA homologous genes present in the genome of the non-pathogenic bacterium Corynebacterium glutamicum can be deleted, unless a copy of the respective gene is provided in trans on a plasmid. This finding is in marked contrast to all other cases examined so far making C. glutamicum the first reported bacterium possessing two essential SecA proteins.     

Titration of cardiolipin by either 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.     

The conserved regulation of mitochondrial uncoupling proteins: From unicellular eukaryotes to mammals

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier protein family and mediate regulated proton leak across the inner mitochondrial membrane. Free fatty acids, aldehydes such as hydroxynonenal, and retinoids activate UCPs. However, there are some controversies about the effective action of retinoids and aldehydes alone; thus, only free fatty acids are commonly accepted positive effectors of UCPs. Purine nucleotides such as GTP inhibit UCP-mediated mitochondrial proton leak. In turn, membranous coenzyme Q may play a role as a redox state-dependent metabolic sensor that modulates the complete activation/inhibition of UCPs. Such regulation has been observed for UCPs in microorganisms, plant and animal UCP1 homologues, and UCP1 in mammalian brown adipose tissue. The origin of UCPs is still under debate, but UCP homologues have been identified in all systematic groups of eukaryotes. Despite the differing levels of amino acid/DNA sequence similarities, functional studies in unicellular and multicellular organisms, from amoebae to mammals, suggest that the mechanistic regulation of UCP activity is evolutionarily well conserved. This review focuses on the regulatory feedback loops of UCPs involving free fatty acids, aldehydes, retinoids, purine nucleotides, and coenzyme Q (particularly its reduction level), which may derive from the early stages of evolution as UCP first emerged.