Formation of Palmitic Acid/Ca2+ Complexes in the Mitochondrial Membrane: A Possible Role in the Cyclosporin-Insensitive Permeability Transition

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.     

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.     

Crystal structure of the YffB protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> suggests a glutathione-dependent thiol reductase function

Background  

The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein.     

AMPK beta subunit targets metabolic stress sensing to glycogen

AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients, as well as hormones, including adiponectin and leptin. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK. Mammalian AMPK is an alphabetagamma heterotrimer with multiple isoforms of each subunit comprising alpha1, alpha2, beta1, beta2, gamma1, gamma2, and gamma3, which have varying tissue and subcellular expression. Mutations in the AMPK gamma subunit cause glycogen storage disease in humans, but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK beta subunit contains a functional glycogen binding domain (beta-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished beta-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK beta1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.     

Sorsby fundus dystrophy mutation Timp3(S156C) affects the morphological and biochemical phenotype but not metalloproteinase homeostasis

The tissue inhibitor of metalloproteinases-3 (TIMP3) is a multifunctional protein tightly associated with the extracellular matrix (ECM). A specific type of mutation in TIMP3 which results in potentially unpaired cysteine residues at the C-terminus of the protein has been shown to cause Sorsby fundus dystrophy (SFD), an autosomal dominant retinopathy of late onset. An early finding in SFD is a striking accumulation of protein and lipid material in Bruch's membrane, a multilayered ECM structure located between the choroid and the RPE. To study the molecular mechanisms underlying SFD pathology, we recently generated two mouse lines, one deficient in Timp3 (Timp3(-/-)) and one carrying an SFD-related mutation in the orthologous murine Timp3 gene (Timp3(S156C/S156C)). We now established immortalized fibroblast cells from the mutant mouse strains and provide evidence that the various cell lines display distinct morphological and physiological features that are dependent on the mutational status of the Timp3 protein in the secreted ECM. We show that matrix metalloproteinase (MMP) activity and inhibitory properties of Timp3 are not affected by the SFD-associated mutation. We further demonstrate that Timp3(S156C) protein accumulates in the ECM of the mutant fibroblast cells and that this accumulation is not due to a prolonged turnover rate of mutant vs. normal Timp3. We also show that the relative abundance of mutant and normal Timp3 in the ECM has no measurable effects on cellular phenotypes. Together, these findings suggest (i) a functional role of normal Timp3 in pathways determining cellular morphology and (ii) a loss of this particular function as a consequence of the Ser156Cys mutation. We therefore hypothesize that SFD pathogenesis is due to a loss-of-function mutation in TIMP3.     

Spectral tuning of obelin bioluminescence by mutations of Trp92

The Ca(2+)-regulated photoprotein obelin was substituted at Trp92 by His, Lys, Glu, and Arg. All mutants fold into stable conformations and produce bimodal bioluminescence spectra with enhanced contribution from a violet emission. The W92R mutant has an almost monomodal bioluminescence (lambdamax=390 nm) and monomodal fluorescence (lambdamax=425 nm) of the product. Results are interpreted by an excited state proton transfer mechanism involving the substituent side group and His22 in the binding cavity.     

Crystal structure of the YchF protein reveals binding sites for GTP and nucleic acid

The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 A resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced alpha-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an alpha-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.