Crucial role for LKB1 to AMPKα2 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytes

Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKα1 versus AMPKα2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKα2-kinase-dead, AMPKα2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKα2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKα2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKα2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKα2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.     

The C. elegans P4-ATPase TAT-1 regulates lysosome biogenesis and endocytosis

P-type adenosine triphosphatases (ATPases) of the Drs2p family (P4-ATPases) are multipass transmembrane proteins required to generate and maintain phospholipid asymmetry in membrane bilayers. In Saccharomyces cerevisiae , several members of this family control distinct transport events within the endosomal and secretory pathways. Comparatively, little is known about the functions of P4-ATPases in multicellular organisms. In this study, we analyzed the role of the Caenorhabditis elegans Drs2p homologue transbilayer amphipath transporter (TAT)-1 in intracellular trafficking. tat-1 is expressed in many tissues including the intestine, the epidermis and the nervous system. In intestinal cells, tat-1 loss-of-function mutants accumulate large vacuoles of mixed endolysosomal identity positive for the lysosomal protein LMP-1. In addition, they lack the same class of storage granules as lmp-1 mutants, suggesting that part of the tat-1 phenotype might result from LMP-1 sequestration in an aberrant compartment. Epidermal cells mutant for tat-1 contain acidified giant hybrid multivesicular bodies probably corresponding to endolysosomal intermediate compartments or deficient lysosomes. Finally, TAT-1 is required for yolk uptake in oocytes and an early step of fluid-phase endocytosis in the intestine. Hence, TAT-1 is required at multiple steps of the endolysosomal pathway, at least in part by ensuring proper trafficking of cell-specific effector proteins.     

The role of the mitochondrial permeability transition pore in heart disease

Like Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty.     

Contributions of the Transmembrane Domain and a Key Acidic Motif to Assembly and Function of the TatA Complex

The twin-arginine translocase (Tat) pathway transports folded proteins across bacterial and thylakoid membranes. In Escherichia coli, a membrane-bound TatA complex, which oligomerizes to form complexes of less than 100 to more than 500 kDa, is considered essential for translocation. We have studied the contributions of various TatA domains to the assembly and function of this heterogeneous TatA complex. The TOXCAT assay was used to analyze the potential contribution of the TatA transmembrane (TM) domain. We observed relatively weak interactions between TatA TM domains, suggesting that the TM domain is not the sole driving force behind oligomerization. A potential hydrogen-bonding role for a TM domain glutamine was also investigated, and it was found that mutation blocks transport at low expression levels, while assembly is unaffected at higher expression levels. Analysis of truncated TatA proteins instead highlighted an acidic motif directly following the TatA amphipathic helix. Mutating these negatively charged residues to apolar uncharged residues completely blocks activity, even at high levels of TatA, and appears to disrupt ordered complex formation.     

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.     

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.     

Contraction-induced skeletal muscle FAT/CD36 trafficking and FA uptake is AMPK independent

The aim of this study was to investigate the molecular mechanisms regulating FA translocase CD36 (FAT/CD36) translocation and FA uptake in skeletal muscle during contractions. In one model, wild-type (WT) and AMP-dependent protein kinase kinase dead (AMPK KD) mice were exercised or extensor digitorum longus (EDL) and soleus (SOL) muscles were contracted, ex vivo. In separate studies, FAT/CD36 translocation and FA uptake in response to muscle contractions were investigated in the perfused rat hindlimb. Exercise induced a similar increase in skeletal muscle cell surface membrane FAT/CD36 content in WT (+34%) and AMPK KD (+37%) mice. In contrast, 5-aminoimidazole-4-carboxamide ribonucleoside only induced an increase in cell surface FAT/CD36 content in WT (+29%) mice. Furthermore, in the perfused rat hindlimb, muscle contraction induced a rapid (1 min, +15%) and sustained (10 min, +24%) FAT/CD36 relocation to cell surface membranes. The increase in cell surface FAT/CD36 protein content with muscle contractions was associated with increased FA uptake, both in EDL and SOL muscle from WT and AMPK KD mice and in the perfused rat hindlimb. This suggests that AMPK is not essential in regulation of FAT/CD36 translocation and FA uptake in skeletal muscle during contractions. However, AMPK could be important in regulation of FAT/CD36 distribution in other physiological situations.     

A mitochondrial-associated link between an effector caspase and autophagic flux

It has become evident that caspases function in nonapoptotic cellular processes in addition to the canonical role for caspases in apoptotic cell death. We recently demonstrated that the Drosophila effector caspase Dcp-1 localizes to the mitochondria and positively regulates starvation-induced autophagic flux during mid-oogenesis. Loss of Dcp-1 leads to elongation of the mitochondrial network, increased levels of the adenine nucleotide translocase sesB, increased ATP levels, and a reduction in autophagy. We found that sesB is a negative regulator of autophagic flux, and Dcp-1 interacts with sesB in a nonproteolytic manner to regulate its stability, uncovering a novel mechanism of mitochondrial associated, caspase-mediated regulation of autophagy in vivo.     

Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase

The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K_m's were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism.Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches.