Different mechanisms of mitochondrial proton leak in ischaemia/reperfusion injury and preconditioning: implications for pathology and cardioprotection

The mechanisms of mitochondrial proton (H+) leak under various pathophysiological conditions are poorly understood. In the present study it was hypothesized that different mechanisms underlie H+ leak in cardiac IR (ischaemia/reperfusion) injury and IPC (ischaemic preconditioning). Potential H(+) leak mechanisms examined were UCPs (uncoupling proteins), allosteric activation of the ANT (adenine nucleotide translocase) by AMP, or the PT (permeability transition) pore. Mitochondria isolated from perfused rat hearts that were subjected to IPC exhibited a greater H+ leak than did controls (202+/-27%, P<0.005), and this increased leakage was completely abolished by the UCP inhibitor, GDP, or the ANT inhibitor, CAT (carboxyattractyloside). Mitochondria from hearts subjected to IR injury exhibited a much greater amount of H+ leak than did controls (411+/-28%, P<0.001). The increased leakage after IR was weakly inhibited by GDP, but was inhibited, >50%, by carboxyattractyloside. In addition, it was inhibited by cardioprotective treatment strategies including pre-IR perfusion with the PT pore inhibitors cyclosporin A or sanglifehrin A, the adenylate kinase inhibitor, AP5A (diadenosine pentaphosphate), or IPC. Together these data suggest that the small increase in H+ leak in IPC is mediated by UCPs, while the large increase in H+ leak in IR is mediated by the ANT. Furthermore, under all conditions studied, in situ myocardial O2 efficiency was correlated with isolated mitochondrial H+ leak (r2=0.71). In conclusion, these data suggest that the modulation of H+ leak may have important implications for the outcome of IR injury.     

Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy

Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.Influenza A viruses can infect a wide range of avian and mammalian species (49). Most avian strains of influenza virus infect wild waterfowl and domestic poultry but usually do not spread to humans. However, adaptation of pathogenic avian viruses to humans can occur either by mutation or reassortment, leading to potentially very serious pandemics, as was the case in 1918 when the “Spanish” flu caused 20 to 40 million deaths worldwide (33). Due to this ability to cross the species barrier, influenza A viruses are a permanent threat to human health. Since 2005 the spread of highly pathogenic H5N1 avian strains in Asia, Europe, and Africa has raised serious concern about the potential of this strain to cause an influenza pandemic (50). Since early 2009, an ongoing new, rapidly evolving pandemic threat has arisen from the emergence of a highly contagious, interhuman-transmissible “quadruple reassortant” swine H1N1 virus to which the world population is antigenically naïve (6).Influenza A viruses are enveloped viruses of the orthomyxovirus family whose genomes comprise eight negative-strand RNA segments (2). In contrast to many RNA viruses, the influenza virus genome is transcribed and replicated by the trimeric viral RNA polymerase (PA, PB1, and PB2) in the nuclei of the infected cells. Therefore, the polymerase subunits, which are produced in the cytoplasm, have to be imported into the nucleus and assembled into a functional trimer (2, 18). Many studies have demonstrated that the viral polymerase plays a major role in host specificity, probably due to the necessity for the polymerase subunits to adapt to host cell-interacting partners such as nuclear import factors (13, 16, 25, 37, 46). Due to the lack of in vivo data concerning the interactions between the polymerase subunits in the nucleus and the cytoplasm of the host cells, the mechanisms of polymerase assembly and nuclear import, as well as their spatial and temporal relationships, are still not completely understood. Putative nuclear localization signals (NLSs) have been identified on PB1 (31), PB2 (29), and PA (32), suggesting that each subunit could be imported separately. However, based on in vitro assembly observations and cellular localization studies (8, 9, 12), it has been proposed that PB1 and PA are imported into the nucleus as a subcomplex by import factor RanBP5 (a member of the importin β superfamily). PB2 is thought to enter the nucleus separately, probably via the canonical importin α/importin β pathway (46), and then associates with the PB1/PA heterodimer in the nucleus to form the functional trimeric polymerase. Nevertheless, alternative pathways have also been proposed. Naito et al. (30) suggested that the nuclear import of PB1 requires the formation of a PB2/PB1 heterodimer, stabilized by Hsp90, in the cytoplasm, while PA is transported in the nucleus separately. More recently, a pathway in which the PA/PB2 heterodimer would be formed in the cytoplasm and then imported into the nucleus has been proposed (17). It has also been recently shown that efficient assembly of the trimeric polymerase could be a major limiting factor in the viability of reassortant influenza viruses (26). Since gene reassortment is an evolutionary mechanism of influenza virus which can lead to new strains with pandemic potential, a precise understanding of the processes leading to the formation of an active viral polymerase in the nuclei of infected cells is of great importance.Recent publications have demonstrated that fluorescence cross-correlation spectroscopy (FCCS) is a method of choice to study protein-protein interactions in vivo (23, 27, 42). FCCS is the dual-color extension of fluorescence correlation spectroscopy (FCS), a technique based on the analysis of the temporal fluorescence fluctuations arising from single fluorescently labeled molecules diffusing in and out of the femtoliter-scale detection volume commonly obtained with a confocal microscope. From the autocorrelation of the fluctuating signal, it is possible to extract the local concentrations and mobilities of the molecules of interest (10, 28, 39). In the case of FCCS, signals from two spectrally separated dyes labeling two different molecules are recorded. If the two molecules interact with each other, they diffuse synchronously through the detection volume, resulting in correlated fluctuations in the fluorescence signals acquired in the two channels. The cross-correlation between the two signals is then a direct and quantitative readout of the interactions between the molecular species studied (22, 38, 40). To our knowledge, this study is the first application of FCCS to viral protein interactions and thus provides a general methodological framework to analyze the effects of different host range mutations and the interactions of viral proteins and host factors in the future.In this study, we applied FCCS to monitor the interactions between the subunits of influenza A virus RNA polymerase in live cells. Based both on the study of these interactions in the cytoplasm and nucleus and on the quantitative analysis of the intracellular localization of the subunits, we show that PB1 and PA form a heterodimer in the cytoplasm while PB2 remains a monomer in this compartment. Association of PB1/PA with PB2 to form the trimeric polymerase was detected only in the nucleus, arguing that the PB1/PA heterodimer is normally imported separately from PB2. Interestingly, when we impaired the nuclear import of PB2 by mutating its nuclear localization signal, this induced the aberrant presence of the trimeric polymerase in the cytoplasm and led to the retention of PB1 and PA outside the nucleus. Finally, by comparing the molecular brightnesses of the single polymerase subunits with that of the trimeric complex, we show that trimeric polymerase complexes can interact with each other in the nucleus to form higher-order oligomers.     

Prediction of amyloidogenic and disordered regions in protein chains

The determination of factors that influence protein conformational changes is very important for the identification of potentially amyloidogenic and disordered regions in polypeptide chains. In our work we introduce a new parameter, mean packing density, to detect both amyloidogenic and disordered regions in a protein sequence. It has been shown that regions with strong expected packing density are responsible for amyloid formation. Our predictions are consistent with known disease-related amyloidogenic regions for eight of 12 amyloid-forming proteins and peptides in which the positions of amyloidogenic regions have been revealed experimentally. Our findings support the concept that the mechanism of amyloid fibril formation is similar for different peptides and proteins. Moreover, we have demonstrated that regions with weak expected packing density are responsible for the appearance of disordered regions. Our method has been tested on datasets of globular proteins and long disordered protein segments, and it shows improved performance over other widely used methods. Thus, we demonstrate that the expected packing density is a useful value with which one can predict both intrinsically disordered and amyloidogenic regions of a protein based on sequence alone. Our results are important for understanding the structural characteristics of protein folding and misfolding.     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I

Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues. Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively. Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed. However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).     

Quaternary structure of alpha-crystallin is necessary for the binding of unfolded proteins: a surface plasmon resonance study

The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein. Our data indicate that quaternary structure of alpha-crystallin is necessary for its chaperone-like activity.     

Involvement of mtDNA damage in free fatty acid-induced apoptosis

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.     

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44‐expressing cancer cells

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44⁺ human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide‐field and low phototoxic hyperspectral imaging system has been successful for performing spectral three‐dimensional (3D) localization and spectroscopic identification of CD44‐targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic‐based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub‐cellular activity with the use of NPs as spectral sensors. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)