New paradigms in actomyosin energy transduction: Critical evaluation of non-traditional models for orthophosphate release.

Release of the ATP hydrolysis product ortophosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of force production by myosin in health and disease and our understanding of myosin-active drugs. Since the 1990s and up to today, models that incorporate the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze three influential alternative models proposed previously. These are either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we suggest critical tests of the models aiming for a unified picture.

Live Cell Imaging of Enzymatic Turnover of an Adenosine 5'-Tetraphosphate Analog.

The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5'-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.

Invited review: Quantifying proton exchange from chemical reactions - Implications for the biochemistry of metabolic acidosis.

Given that the chemistry of lactate production disproves the existence of a lactic acidosis, there is a need to further reveal and explain the importance of the organic and computational chemistry of pH dependent competitive cation fractional (~) proton (H+) exchange (~H+e). An additional importance of this knowledge is that it could potentially contradict the assumption of the Stewart approach to the physico-chemical theory of acid-base balance. For example, Stewart proposed that chemical reaction and pH dependent H+ dissociation and association do not directly influence the pH of cellular and systemic body fluids. Yet at the time of Stewart's work, there were no data that quantified the H+ exchange during chemical reactions, or from pH dependent metabolite H+ association or dissociation. Consequently, the purpose of this review and commentary was three-fold; 1) to provide explanation of pH dependent competitive cation ~H+e exchange; 2) develop a model of and calculate new data of substrate flux in skeletal muscle during intense exercise; and 3) then combine substrate flux data with the now known ~H+e from chemical reactions of non-mitochondrial energy catabolism to quantify chemical reaction and metabolic pathway ~H+e. The results of purpose 3 were that ~H+ release for the totality of cytosolic energy catabolism = -187.2 mmol·L-1, where total glycolytic ~H+te = -85.0 mmol·L-1. ATP hydrolysis had a ~H+te = -43.1 mmol·L-1. Lactate production provided the largest metabolic ~H+ buffering with a ~H+te = 44.5 mmol·L-1. The total ~H+ release to La ratio = 4.25. The review content and research results of this manuscript should direct science towards new approaches to understanding the cause and source of H+e during metabolic acidosis and alkalosis.

Mitochondrial Activity and Skeletal Muscle Insulin Resistance in Kidney Disease.

Insulin resistance is a key feature of the metabolic syndrome, a cluster of medical disorders that together increase the chance of developing type 2 diabetes and cardiovascular disease. In turn, type 2 diabetes may cause complications such as diabetic kidney disease (DKD). Obesity is a major risk factor for developing systemic insulin resistance, and skeletal muscle is the first tissue in susceptible individuals to lose its insulin responsiveness. Interestingly, lean individuals are not immune to insulin resistance either. Non-obese, non-diabetic subjects with chronic kidney disease (CKD), for example, exhibit insulin resistance at the very onset of CKD, even before clinical symptoms of renal failure are clear. This uraemic insulin resistance contributes to the muscle weakness and muscle wasting that many CKD patients face, especially during the later stages of the disease. Bioenergetic failure has been associated with the loss of skeletal muscle insulin sensitivity in obesity and uraemia, as well as in the development of kidney disease and its sarcopenic complications. In this mini review, we evaluate how mitochondrial activity of different renal cell types changes during DKD progression, and discuss the controversial role of oxidative stress and mitochondrial reactive oxygen species in DKD. We also compare the involvement of skeletal muscle mitochondria in uraemic and obesity-related muscle insulin resistance.

Palmitate-induced changes in energy demand cause reallocation of ATP supply in rat and human skeletal muscle cells.

Mitochondrial dysfunction has been associated with obesity-related muscle insulin resistance, but the causality of this association is controversial. The notion that mitochondrial oxidative capacity may be insufficient to deal appropriately with excessive nutrient loads is for example disputed. Effective mitochondrial capacity is indirectly, but largely determined by ATP-consuming processes because skeletal muscle energy metabolism is mostly controlled by ATP demand. Probing the bioenergetics of rat and human myoblasts in real time we show here that the saturated fatty acid palmitate lowers the rate and coupling efficiency of oxidative phosphorylation under conditions it causes insulin resistance. Stearate affects the bioenergetic parameters similarly, whereas oleate and linoleate tend to decrease the rate but not the efficiency of ATP synthesis. Importantly, we reveal that palmitate influences how oxidative ATP supply is used to fuel ATP-consuming processes. Direct measurement of newly made protein demonstrates that palmitate lowers the rate of de novo protein synthesis by more than 30%. The anticipated decrease of energy demand linked to protein synthesis is confirmed by attenuated cycloheximide-sensitivity of mitochondrial respiratory activity used to make ATP. This indirect measure of ATP turnover indicates that palmitate lowers ATP supply reserved for protein synthesis by at least 40%. This decrease is also provoked by stearate, oleate and linoleate, albeit to a lesser extent. Moreover, palmitate lowers ATP supply for sodium pump activity by 60-70% and, in human cells, decreases ATP supply for DNA/RNA synthesis by almost three-quarters. These novel fatty acid effects on energy expenditure inform the 'mitochondrial insufficiency' debate.

Mitochondrial involvement in skeletal muscle insulin resistance: A case of imbalanced bioenergetics.

Skeletal muscle insulin resistance in obesity associates with mitochondrial dysfunction, but the causality of this association is controversial. This review evaluates mitochondrial models of nutrient-induced muscle insulin resistance. It transpires that all models predict that insulin resistance arises as a result of imbalanced cellular bioenergetics. The nature and precise origin of the proposed insulin-numbing molecules differ between models but all species only accumulate when metabolic fuel supply outweighs energy demand. This observation suggests that mitochondrial deficiency in muscle insulin resistance is not merely owing to intrinsic functional defects, but could instead be an adaptation to nutrient-induced changes in energy expenditure. Such adaptive effects are likely because muscle ATP supply is fully driven by energy demand. This market-economic control of myocellular bioenergetics offers a mechanism by which insulin-signalling deficiency can cause apparent mitochondrial dysfunction, as insulin resistance lowers skeletal muscle anabolism and thus dampens ATP demand and, consequently, oxidative ATP synthesis.

Energy demand and supply in human skeletal muscle.

The energy required for muscle contraction is provided by the breakdown of ATP but the amount of ATP in muscles cells is sufficient to power only a short duration of contraction. Buffering of ATP by phosphocreatine, a reaction catalysed by creatine kinase, extends the duration of activity possible but sustained activity depends on continual regeneration of PCr. This is achieved using ATP generated by oxidative processes and, during intense activity, by anaerobic glycolysis. The rate of ATP breakdown ranges from 70 to 140 mM min-1 during isometric contractions of various intensity to as much as 400 mM min-1 during intense, dynamic activity. The maximum rate of oxidative energy supply in untrained people is ~50 mM min-1 which, if the contraction duty cycle is 0.5 as is often the case in cyclic activity, is sufficient to match an ATP breakdown rate during contraction of 100 mM min-1. During brief, intense activity the rate of ATP turnover can exceed the rates of PCr regeneration by combined oxidative and glycolytic energy supply, resulting in a net decrease in PCr concentration. Glycolysis has the capacity to produce between 30 and 50 mM of ATP so that, for example, anaerobic glycolysis could provide ATP at an average of 100 mM min-1 over 30 s of exhausting activity. The creatine kinase reaction plays an important role not only in buffering ATP but also in communicating energy demand from sites of ATP breakdown to the mitochondria. In that role, creatine kinases acts to slow and attenuate the response of mitochondria to changes in energy demand.

ATP turnover by individual myosin molecules hints at two conformers of the myosin active site.

Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.

Purification and Handling of the Chaperonin GroEL.

GroEL is an important model molecular chaperone. Despite being extensively studied, several critical aspects of its functionality are still in dispute due partly to difficulties in obtaining protein samples of consistent purity. Here I describe an easy-to-carry-out purification protocol that can reliably produce highly purified and fully functional GroEL protein in large quantities. The method takes advantage of the remarkable stability of the GroEL tetradecamer in 45% acetone which efficiently extracts and removes tightly bound substrate proteins that cannot be separated from GroEL by the usual chromatographic methods. The efficiency of the purification method can be assessed by the amount of residual tryptophan fluorescence associated with the purified GroEL sample. The functionality of the thus obtained GroEL sample is demonstrated by measuring its ATPase turnover both in the presence and absence of the GroEL model substrate protein α-lactalbumin.

A synthetic ancestral kinesin-13 depolymerizes microtubules faster than any natural depolymerizing kinesin.

The activity of a kinesin is largely determined by the approximately 350 residue motor domain, and this region alone is sufficient to classify a kinesin as a member of a particular family. The kinesin-13 family are a group of microtubule depolymerizing kinesins and are vital regulators of microtubule length. Kinesin-13s are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerization activity, we created a synthetic ancestral kinesin-13 motor domain. This phylogenetically inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the kinesin-13 family. Here we show that the ancestral kinesin-13 motor depolymerizes stabilized microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied kinesin-13, MCAK and allows the ancestral kinesin-13 to depolymerize doubly stabilized microtubules and cause internal breaks within microtubules. These data suggest that the ancestor of the kinesin-13 family was a 'super depolymerizer' and that members of the kinesin-13 family have evolved away from this extreme depolymerizing activity to provide more controlled microtubule depolymerization activity in extant cells.

How expensive is the astrocyte?

The energy cost of information processing is thought to be chiefly neuronal, with a minor fraction attributed to glial cells. However, there is compelling evidence that astrocytes capture synaptic K+ using their Na+/K+ ATPase, and not solely through Kir4.1 channels as was once thought. When this active buffering is taken into account, the cost of astrocytes rises by >200%. Gram-per-gram, astrocytes turn out to be as expensive as neurons. This conclusion is supported by 3D reconstruction of the neuropil showing similar mitochondrial densities in neurons and astrocytes, by cell-specific transcriptomics and proteomics, and by the rates of the tricarboxylic acid cycle. Possible consequences for reactive astrogliosis and brain disease are discussed.

Direct Spectroscopic Detection of ATP Turnover Reveals Mechanistic Divergence of ABC Exporters.

We have applied high-field (W-band) pulse electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (EDNMR) to characterize the coordination sphere of the Mn2+ co-factor in the nucleotide binding sites (NBSs) of ABC transporters. MsbA and BmrCD are two efflux transporters hypothesized to represent divergent catalytic mechanisms. Our results reveal distinct coordination of Mn2+ to ATP and transporter residues in the consensus and degenerate NBSs of BmrCD. In contrast, the coordination of Mn2+ at the two NBSs of MsbA is similar, which provides a mechanistic rationale for its higher rate constant of ATP hydrolysis relative to BmrCD. Direct detection of vanadate ion, trapped in a high-energy post-hydrolysis intermediate, further supports the notion of asymmetric hydrolysis by the two NBSs of BmrCD. The integrated spectroscopic approach presented here, which link energy input to conformational dynamics, can be applied to a variety of systems powered by ATP turnover.

The family-specific α4-helix of the kinesin-13, MCAK, is critical to microtubule end recognition.

Kinesins that influence the dynamics of microtubule growth and shrinkage require the ability to distinguish between the microtubule end and the microtubule lattice. The microtubule depolymerizing kinesin MCAK has been shown to specifically recognize the microtubule end. This ability is key to the action of MCAK in regulating microtubule dynamics. We show that the α4-helix of the motor domain is crucial to microtubule end recognition. Mutation of the residues K524, E525 and R528, which are located in the C-terminal half of the α4-helix, specifically disrupts the ability of MCAK to recognize the microtubule end. Mutation of these residues, which are conserved in the kinesin-13 family and discriminate members of this family from translocating kinesins, impairs the ability of MCAK to discriminate between the microtubule lattice and the microtubule end.

On the mechanism by which dietary nitrate improves human skeletal muscle function.

Inorganic nitrate is present at high levels in beetroot and celery, and in green leafy vegetables such as spinach and lettuce. Though long believed inert, nitrate can be reduced to nitrite in the human mouth and, further, under hypoxia and/or low pH, to nitric oxide. Dietary nitrate has thus been associated favorably with nitric-oxide-regulated processes including blood flow and energy metabolism. Indeed, the therapeutic potential of dietary nitrate in cardiovascular disease and metabolic syndrome-both aging-related medical disorders-has attracted considerable recent research interest. We and others have shown that dietary nitrate supplementation lowers the oxygen cost of human exercise, as less respiratory activity appears to be required for a set rate of skeletal muscle work. This striking observation predicts that nitrate benefits the energy metabolism of human muscle, increasing the efficiency of either mitochondrial ATP synthesis and/or of cellular ATP-consuming processes. In this mini-review, we evaluate experimental support for the dietary nitrate effects on muscle bioenergetics and we critically discuss the likelihood of nitric oxide as the molecular mediator of such effects.