Phase separation in lipid bilayers triggered by low pH

Endocytosis involves the capture of membrane from the cell surface in the form of vesicles, which become rapidly acidified to about pH 5. Here we show using atomic force microscopy (AFM) imaging that this degree of acidification triggers phase separation in lipid bilayers containing mixed acyl chains (e.g. palmitoyl/oleoyl) or complex mixtures (e.g. total brain extract) but not in bilayers containing only lipids with unsaturated chains (e.g. dioleoyl). Since mixed-chain lipids are major constituents of the outer leaflet of the plasma membrane, the type of phase separation reported here might support protein clustering and signaling during endocytosis.     

Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore

After a brief review of the early history of mitochondrial electrophysiology, the contribution of this approach to the study of the mitochondrial permeability transition (MPT) is recapitulated. It has for example provided evidence for a dimeric nature of the MPT pore, allowed the distinction between two levels of control of its activity, and underscored the relevance of redox events for the phenomenon. Single-channel recording provides a means to finally solve the riddle of the biochemical entity underlying it by comparing the characteristics of the pore with those of channels formed by candidate molecules or complexes. The possibility that this entity may be the protein import machinery of the inner mitochondrial membrane is emphasized.     

Structural Basis for the Recognition and Cleavage of Polysialic Acid by the Bacteriophage K1F Tailspike Protein EndoNF

An α-2,8-linked polysialic acid (polySia) capsule confers immune tolerance to neuroinvasive, pathogenic prokaryotes such as Escherichia coli K1 and Neisseria meningitidis and supports host infection by means of molecular mimicry. Bacteriophages of the K1 family, infecting E. coli K1, specifically recognize and degrade this polySia capsule utilizing tailspike endosialidases. While the crystal structure for the catalytic domain of the endosialidase of bacteriophage K1F (endoNF) has been solved, there is yet no structural information on the mode of polySia binding and cleavage available. The crystal structure of activity deficient active-site mutants of the homotrimeric endoNF cocrystallized with oligomeric sialic acid identified three independent polySia binding sites in each endoNF monomer. The bound oligomeric sialic acid displays distinct conformations at each site. In the active site, a Sia₃ molecule is bound in an extended conformation representing the enzyme-product complex. Structural and biochemical data supported by molecular modeling enable to propose a reaction mechanism for polySia cleavage by endoNF.     

Surrobodies with Functional Tails

Surrobodies² are a unique type of binding protein based on the pre-B-cell receptor (pre-BCR). The pre-BCR is transiently expressed during development of the antibody repertoire. Unlike heterotetrameric canonical antibodies that are composed of identical pairs of heavy and light chains, the pre-BCR is a heterohexameric complex composed of identical pairs of heavy chains that are each paired with a two-subunit surrogate light chain (SLC). The SLC contains nonimmunoglobulin-like peptide extensions on each of the two SLC components. This arrangement provides unique opportunities for protein engineering by functional derivatization of these nonimmunoglobulin-like tails. Here we report recombinant fusions to these tails with either a fully active cytokine or with single-chain variable fragment (scFv) domains to generate Surrobodies with unique functions or Surrobodies that are bispecific with respect to targeted binding.     

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A₅₁ and the C-terminal cell wall anchor site at residue T₁₀₈₆. The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572 Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A₅₁, were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.     

Land use and environmental factors affecting red-legged partridge (Alectoris rufa) hunting yields in southern Spain

The red-legged partridge is a small game species widely hunted in southern Spain. Its commercial use has important socioeconomic effects in rural areas where other agrarian uses are of marginal importance. The aims of the present work were to identify areas in Andalusia (southern Spain) where game yields for the red-legged partridge reach high values and to establish the environmental and land use factors that determine them. We analysed 32,134 annual hunting reports (HRs) produced by 6,049 game estates during the hunting seasons 1993/1994 to 2001/2002 to estimate the average hunting yields of red-legged partridge in each Andalusian municipality (n=771). We modelled the favourability for obtaining good hunting yields using stepwise logistic regression on a set of climatic, topographical, land use and vegetation variables that were available as digital coverages or tabular data applied to municipalities. Good hunting yields occur mainly in plain areas located in the Guadalquivir valley, at the bottom of Betic Range and in the Betic depressions. Favourable areas are related to highly mechanised, low-elevation areas mainly dedicated to intensive dry crops. The most favourable areas predicted by our model are mainly located in the Guadalquivir valley.     

Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: Implications for oxidative stress-impaired reverse cholesterol transport

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.     

Thermal, mixing, and oxygen regimes of the Salton Sea, California, 1997–1999

The Salton Sea is a shallow (mean depth = 8 m; maximum depth = 15 m), saline (41–45 g l^–1), intermittently mixing, 57 km long, 980 km² lake located in the arid southwestern United States. The Sea is a wind driven system, with predominant winds paralleling the long axis of the lake, being strongest in spring and weakest in summer and fall. The Sea mixed daily or nearly daily between September and January. During this cooling period, moderate to high levels of dissolved oxygen (3–11 mg l^–1) were found throughout the water column. Mean water column temperature ranged from a minimum of 13–14 °C in early January to a maximum of 30–34 °C in July–September. During most of this warming period, the Sea was thermally stratified but subject to periodic wind driven mixing events. Winds were stronger in spring 1998 than in 1997 or 1999, causing more rapid heating of the lake that year and also delaying onset of anoxic conditions in bottom waters. During summer months, mid-lake surface waters were sometimes supersatured with oxygen, and bottom waters were hypoxic or anoxic with sulfide concentrations > 5 mg l^–1. Oxic conditions (> 1 mg O₂ l^–1) often extended a few meters deeper nearshore than they did well offshore as a consequence of greater mixing nearshore. Mixing events in late summer deoxygenated the entire water column for a period of days. Consumption of oxygen by sulfide oxidation likely was the principal mechanism for these deoxygenation events. Sulfide concentrations in surface waters were 0.5–1 mg l^–1 approximately 3 days after one mixing event in mid-August 1999. These mixing events were associated with population crashes of phytoplankters and zooplankters and with large fish kills. In the southern basin, freshwater inflows tended to move out over the surface of the Sea mixing with saline lake water as a function of wind conditions. Salinity gradients often contributed more to water column stability than did thermal gradients in the southeasternmost portion of the lake.     

Algae in the Anthropocene: Managing,conserving, and utilizing algae in an era of rapid environmental change

The Anthropocene is defined as the current period in which humans have had a large influence over the status and trajectory of earth's climate and environment. Human-induced climate change, pollution, and coastal development have caused major changes to algal persistence, distribution, diversity, and function. This has not only brought new challenges for managing and conserving algae, but also new opportunities. This series of perspective pieces will touch on some of these challenges, potential solutions, and knowledge gaps that we must consider in supporting and understanding algae in the Anthropocene.