The causes of reduced proton-pumping efficiency in type B and C respiratory heme-copper oxidases,and in some mutated variants of type A

The heme-copper oxidases may be divided into three categories, A, B, and C, which include cytochrome c and quinol-oxidising enzymes. All three types are known to be proton pumps and are found in prokaryotes, whereas eukaryotes only contain A-type cytochrome c oxidase in their inner mitochondrial membrane. However, the bacterial B- and C-type enzymes have often been reported to pump protons with an H⁺/e^- ratio of only one half of the unit stoichiometry in the A-type enzyme. We will show here that these observations are likely to be the result of difficulties with the measuring technique together with a higher sensitivity of the B- and C-type enzymes to the protonmotive force that opposes pumping. We find that under optimal conditions the H⁺/e^- ratio is close to unity in all the three heme-copper oxidase subfamilies. A higher tendency for proton leak in the B- and C-type enzymes may result from less efficient gating of a proton pump mechanism that we suggest evolved before the so-called D-channel of proton transfer. There is also a discrepancy between results using whole bacterial cells vs. phospholipid vesicles inlaid with oxidase with respect to the observed proton pumping after modification of the D-channel residue asparagine-139 (Rhodobacter sphaeroides numbering) to aspartate in A-type cytochrome c oxidase. This discrepancy might also be explained by a higher sensitivity of proton pumping to protonmotive force in the mutated variant. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

The Metabolome in Finnish Carriers of the MYBPC3-Q1061X Mutation for Hypertrophic Cardiomyopathy

Aims

Mutations in the cardiac myosin-binding protein C gene (MYBPC3) are the most common genetic cause of hypertrophic cardiomyopathy (HCM) worldwide. The molecular mechanisms leading to HCM are poorly understood. We investigated the metabolic profiles of mutation carriers with the HCM-causing MYBPC3-Q1061X mutation with and without left ventricular hypertrophy (LVH) and non-affected relatives, and the association of the metabolome to the echocardiographic parameters.

Methods and Results

34 hypertrophic subjects carrying the MYBPC3-Q1061X mutation, 19 non-hypertrophic mutation carriers and 20 relatives with neither mutation nor hypertrophy were examined using comprehensive echocardiography. Plasma was analyzed for molecular lipids and polar metabolites using two metabolomics platforms. Concentrations of branched chain amino acids, triglycerides and ether phospholipids were increased in mutation carriers with hypertrophy as compared to controls and non-hypertrophic mutation carriers, and correlated with echocardiographic LVH and signs of diastolic and systolic dysfunction in subjects with the MYBPC3-Q1061X mutation.

Conclusions

Our study implicates the potential role of branched chain amino acids, triglycerides and ether phospholipids in HCM, as well as suggests an association of these metabolites with remodeling and dysfunction of the left ventricle.     

Improved Fermentation Performance of a Lager Yeast after Repair of Its AGT1 Maltose and Maltotriose Transporter Genes

The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer''s worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer''s yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. α-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer''s ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous α-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24° Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.The main fermentable sugars in brewer''s wort are maltose (ca. 60% of the total), maltotriose (ca. 25%), and glucose (ca. 15%). In traditional brewery fermentations, worts of about 11° Plato (°P) are used, corresponding to a total fermentable sugar concentration of about 80 g · liter⁻¹. Many modern breweries ferment high-gravity worts (15 to 17°P), and there are efforts to raise the concentration to 25°P, corresponding to a total sugar concentration of about 200 g · liter⁻¹. Industrial use of such very-high-gravity (VHG) worts is attractive because it offers increased production capacity from the same-size brew house and fermentation facilities, decreased energy consumption, and decreased labor, cleaning, and effluent costs (34, 35).Whereas glucose, which is used first, is transported into yeast cells by facilitated diffusion, the α-glucosides maltose and maltotriose are carried by proton symporters (2, 26, 39). Maltose transport seems to have a high level of control over the fermentation rate. Thus, during the early and middle stages of fermentation of brewer''s wort by a lager yeast, the specific rate of maltose consumption was the same as the specific zero-trans maltose uptake rate measured off line with each day''s yeast in each day''s wort spiked with [¹⁴C]maltose (27). Furthermore, introducing a constitutive MAL61 (maltose transporter) gene into a brewer''s yeast on a multicopy plasmid accelerated the fermentation of high-gravity worts (17). Maltotriose is the last sugar to be used in brewing fermentations, and significant amounts of residual maltotriose sometimes remain in beer, causing economic losses (lower yield of ethanol on wort carbohydrate) and possibly undesirable organoleptic effects. The problem of residual sugars in beer is more serious when high-gravity and VHG worts are used. Some, but not all, maltose transporters can also carry maltotriose. The MALx1 genes (x = 1 to 4 and 6) encode transporters that carry maltose efficiently but are generally believed to have little or no activity toward maltotriose (1, 3, 13, 30), although substantial activity toward maltotriose was reported by Day et al. (4). Some yeast strains contain a gene 57% identical to MAL11 that is usually known as AGT1 but is recorded in the Saccharomyces Genome Database (SGDB) as MAL11. The Agt1 transporter has relatively high activity toward maltotriose, as well as maltose (13), and similar K_m values (4 to 5 mM) for these two substrates (4). Alves et al. (1) found that the specific deletion of AGT1 from several Saccharomyces cerevisiae strains also containing at least one MALx1 gene (MAL21, MAL31, and/or MAL41) abolished their ability to transport maltotriose but did not decrease their maltose transport activity. These results supported the belief that the Mal21, Mal31, and Mal41 transporters cannot carry maltotriose, though it remains possible that there are differences between Malx1 transporters from different strains. The same group has also shown (33) that overexpression of AGT1 on a multicopy plasmid in an industrial yeast strain with a very limited ability to ferment maltotriose provided the strain with increased maltotriose uptake activity and the ability to ferment maltotriose efficiently. In 2005, a novel kind of α-glucoside transporter was independently found by two groups (6, 30) in some industrial strains of brewer''s, baker''s, and distiller''s yeasts. These transporters are coded by MTT1 (also called MTY1) genes, which are 90 and 54% identical to the MAL31 and AGT1 genes, respectively. The Mtt1 transporters have high activity toward maltotriose and are the only known α-glucoside transporters with lower K_m values for maltotriose than for maltose (30).Before the discovery of the MTT1 genes, Vidgren et al. (36) sequenced AGT1 genes from two apparently unrelated lager strains and two apparently unrelated ale strains of brewer''s yeast. Surprisingly, at that time (because other maltotriose transporters were not known), the AGT1 genes from the lager strains contained an insertion of one nucleotide, resulting in a premature stop codon, and encoded a truncated, nonfunctional 394-amino-acid polypeptide, whereas those from the ale strains encoded full-length 616-amino-acid transporters. This premature stop codon was later shown (37) to be present in AGT1 genes from all eight of the lager strains tested but was not in any of the four ale strains tested, whereas MTT1 genes were present in all of the lager strains tested but in none of the ale strains tested.In the present work, we have tested whether lager fermentations can be accelerated and residual maltotriose levels decreased by repairing the defective AGT1 genes of lager strains with appropriate DNA sequences from ale strains. Furthermore, the MALx1 and AGT1 genes are repressed by glucose and induced by α-glucosides (9, 16, 19, 25), so that replacing the native AGT1 promoter with a constitutive S. cerevisiae promoter might also increase α-glucoside transport activity and accelerate wort fermentations. The objectives of the present work were to confirm that α-glucoside transport has a high level of control over the rate and extent of wort fermentation and to create a genetically modified lager yeast strain that has improved fermentation performance but contains only Saccharomyces DNA.     

Neurotransmitter responsiveness during early maturation of neural progenitor cells

Neurotransmitters are potential regulators of proliferation and differentiation of neural progenitor cells (NPC). To gain insight into the dynamics of neurotransmitter responsiveness, neurospheres were prepared from the lateral ventricles of postnatal day 6/7 mice. Individual NPCs migrating out from spheres were simultaneously monitored using Ca²⁺ imaging, during the initial 8 days of differentiation, at an area between the inner edge of the sphere and outer periphery of the area of migration. At the first day of differentiation most cells showed metabotropic responses (Ca²⁺ discharge from stores) to glutamate (pharmacologically identified as metabotropic glutamate receptor 5, mGluR 5), norepinephrine (NE), acetylcholine (Ach) and ATP, and a smaller proportion of cells also responded to substance P (SP). When outside the neurosphere, many of mGluR5 responding cells gained immunostaining for markers of neuronal lineage (Tuj-1 and NeuN). The number of cells responding through mGluR5 (and responses to Ach, NE and SP) showed during subsequent days of differentiation (day 2–3 onwards) a decline with time and progressively disappeared at the outer periphery of the area of migration. Conversely the number ionotropic glutamate responses as well as responses to depolarization increased in this area. After 5–8 days of differentiation mGluR5 responses could only be observed at the very inner edge of the neurosphere. At 8 days the migrated cells showed very robust ionotropic responses to glutamate, NMDA and depolarization comparable to mature neurons. Taken together, the data presented here suggest that differentiation of NPCs is a dynamic process triggered by cell migration, which leads to a loss of regulatory influences imposed by the inner milieu of the neurosphere. The subsequent switch or loss of metabotropic responses to glutamate, SP, NE, Ach and ATP with the gain of excitable characteristics such as ionotropic responses appears to be a key event in the final differentiation process.