Biochemical Intermediates Research Articles

Metabolic and Microbial Profiling of Soil Microbial Community under Per- and Polyfluoroalkyl Substance (PFAS) Stress.

Per- and polyfluoroalkyl substances (PFAS) represent significant stress to organisms and are known to disrupt microbial community structure and function. Nevertheless, a detailed knowledge of the soil microbial community responding to PFAS stress at the metabolism level is required. Here we integrated UPLC-HRMS-based metabolomics data with 16S rRNA and ITS amplicon data across soil samples collected adjacent to a fluoropolymer production facility to directly identify the biochemical intermediates in microbial metabolic pathways and the interactions with microbial community structure under PFAS stress. A strong correlation between metabolite and microbial diversity was observed, which demonstrated significant variations in soil metabolite profiles and microbial community structures along with the sampling locations relative to the facility. Certain key metabolites were identified in the metabolite-PFAS co-occurrence network, functioning on microbial metabolisms including lipid metabolism, amino acid metabolism, and secondary metabolite biosynthesis. These results provide novel insights into the impacts of PFAS contamination on soil metabolomes and microbiomes. We suggest that soil metabolomics is an informative and useful tool that could be applied to reinforce the chemical evidence on the disruption of microbial ecological traits.

428 Awardee Talk: Can the Ruminal Microbiome Ever Answer All the Questions That Nutritionists have?

Abstract The rumen microbial consortium has long been viewed as a “black box”, where we recognize what goes in, and most of what comes out, but the ruminal catabolic and anabolic activity remains shrouded in uncertainty. Microbiologists from the 1940s through the early 2000s identified microbes responsible for ruminal feedstuff degradation, highlighted animal-important fermentation endproducts (such as volatile fatty acids, VFA), and examined how microbes interacted with each other (e.g., interspecies hydrogen transfer), and with the host animal. However, our knowledge was dependent on our ability to grow anaerobic microbes in pure or mixed cultures, but it became apparent that our limiting factor was that we could culture <10% of the ruminal microbes. In the past 15 years, there has been a proliferation of new techniques to identify what is occurring in the rumen of live ruminants such as, culturable and unculturable microorganism populations, microbial biochemical pathways and important metabolic intermediates, potential interactions between microbes, and biologically relevant endproducts that impact host animal physiology, health, and production efficiency. As Next Generation Sequencing (NGS) has become increasingly affordable, there has been a surge in research examining the “microbiome” with a resultant influx of massive datasets. However, without the ability to connect microbial populations to biochemical degradative pathways, endproducts, microbial interactions or animal production parameters, this information is of limited impact in ruminant nutrition. Ruminant nutrition is on a precipice where we can finally fully integrate microbiology with metabolomics and animal performance and nutrition. Advancements in ruminal microbiology will allow us to fully grasp the functional nature of the microbial ecosystem and where it fits within ruminant nutrition, which will improve animal productivity and ruminant-derived protein food production sustainability.

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.

NAD+ enhances the activity and thermostability of S-adenosyl-L-homocysteine hydrolase from Pyrococcus horikoshii OT3.

S-Adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important biochemical intermediates. SAM is the major methyl donor for diverse methylation reactions in vivo. The SAM to SAH ratio serves as a marker of methylation capacity. Stable isotope-labeled SAM and SAH are used to measure this ratio with high sensitivity. SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of adenosine and L-homocysteine to SAH, is used to produce labeled SAH. To produce labeled SAH with high efficiency, we focused on the SAHH of Pyrococcus horikoshii OT3, a thermophilic archaeon. We prepared recombinant P. horikoshii SAHH using Escherichia coli and investigated its enzymatic properties. Unexpectedly, the optimum temperature and thermostability of P. horikoshii SAHH were much lower than its optimum growth temperature. However, addition of NAD+ to the reaction mixture shifted the optimum temperature of P. horikoshii SAHH to a higher temperature, suggesting that NAD+ stabilizes the structure of the enzyme.

HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization.

HP1 proteins traverse a complex and crowded chromatin landscape to bind with low affinity but high specificity to histone H3K9 methylation (H3K9me) and form transcriptionally inactive genomic compartments called heterochromatin. Here, we visualize single-molecule dynamics of an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. By tracking single Swi6 molecules, we identify mobility states that map to discrete biochemical intermediates. Using Swi6 mutants that perturb H3K9me recognition, oligomerization, or nucleic acid binding, we determine how each biochemical property affects protein dynamics. We estimate that Swi6 recognizes H3K9me3 with ~94-fold specificity relative to unmodified nucleosomes in living cells. While nucleic acid binding competes with Swi6 oligomerization, as few as four tandem chromodomains can overcome these inhibitory effects to facilitate Swi6 localization at heterochromatin formation sites. Our studies indicate that HP1 oligomerization is essential to form dynamic, higher-order complexes that outcompete nucleic acid binding to enable specific H3K9me recognition.

The Role of Metabolic Dysfunction in T-Cell Exhaustion During Chronic Viral Infection.

T cells are important components of adaptive immunity that protect the host against invading pathogens during infection. Upon recognizing the activation signals, naïve and/or memory T cells will initiate clonal expansion, trigger differentiation into effector populations and traffic to the inflamed sites to eliminate pathogens. However, in chronic viral infections, such as those caused by human immunodeficiency virus (HIV), hepatitis B and C (HBV and HCV), T cells exhibit impaired function and become difficult to clear pathogens in a state known as T-cell exhaustion. The activation and function persistence of T cells demand for dynamic changes in cellular metabolism to meet their bioenergetic and biosynthetic demands, especially the augmentation of aerobic glycolysis, which not only provide efficient energy generation, but also fuel multiple biochemical intermediates that are essential for nucleotide, amino acid, fatty acid synthesis and mitochondria function. Changes in cellular metabolism also affect the function of effectors T cells through modifying epigenetic signatures. It is widely accepted that the dysfunction of T cell metabolism contributes greatly to T-cell exhaustion. Here, we reviewed recent findings on T cells metabolism under chronic viral infection, seeking to reveal the role of metabolic dysfunction played in T-cell exhaustion.

EP004: Screening for metabolic abnormalities in a cohort of patients with hypermobility

We created a special focus clinic for patients presenting with the constellation of findings centered on hypermobility. There has been much speculation over the role of metabolism and mitochondrial function in this patient population but few studies examining biochemical intermediates in this group. We decided to systematically test for metabolic disruption in this group to evaluate the mostly anecdotal evidence around biochemical disruption.

Herbo-metallic ethnomedicine ‘Malla Sindoor’ ameliorates lung inflammation in murine model of allergic asthma by modulating cytokines status and oxidative stress

Ethnopharmacological relevanceAsthma is the leading inflammatory disease of the airways with inadequate therapeutic options. ‘Malla Sindoor’ (MS) is a metal-based ethnomedicinal formulation that has been prescribed in the ancient traditional medicinal system for treating chronic inflammations. Aim of the studyHere, we validated the anti-inflammatory and anti-asthmatic properties of traditional metallic medicine MS in asthmatic mice model and in LPS stimulated human monocytic THP-1 cells, by examining the relevant cellular, biochemical and molecular intermediates. Materials and methodsScanning Electron Microscope (SEM), Electron Dispersive X-ray (EDX), and X-Ray Diffraction (XRD) were performed to characterize MS particles. Allergic asthma was induced in Balb/c mice through intraperitoneal ovalbumin (OVA) injection. Experimental groups include, normal control, disease control, Dexamethasone (2 mg/kg) and three MS treated groups: 4.3 mg/kg, 13 mg/kg, and 39 mg/kg. Quantitative PCR, inflammatory cytokines and anti-oxidant enzymes, and histological analysis were performed, in the treated mice and LPS stimulated human monocytic THP-1 cells for determining the MS efficacy. ResultsSEM image analysis showed the MS to be heterogenous in shape with a particle size distribution between 100 nm–1 μm. Elemental composition showed the presence of mercury (Hg), arsenic (As), and sulphur (S) along with other elements in the forms of mercury sulfide, arsenic trioxide, and their alloy crystals. OVA-challenge of the Balb/c mice resulted in the development of overt pathological features for allergic asthma including smooth muscle thickening and collagen deposition. Mice receiving MS-exhibited alleviation of allergic asthma features. BAL fluid analysis showed a decrease in the total cell count and decreases in neutrophils, monocytes, lymphocytes, and eosinophils. Further, the stimulated levels of interleukin (IL)-1β, −6, and TNF-α cytokines and antioxidant levels were also reduced upon MS-treatment. At the molecular level, MS-treatment reduced stimulated mRNA expression levels for IL-4, -5, -10, -13, -33, and IFN-γ cytokines. Histological analysis following MS-treatment of OVA-stimulated mice lungs showed a reduction in mucus accumulation in airways, decreases in peribronchial collagen deposition, bronchial smooth muscle thickening, and attenuation of inflammatory cell infiltration. In addition, under in-vitro conditions, MS-treatment attenuated the LPS induced secretion of IL-1β, −6, and TNF-α from THP-1 cells. ConclusionCollectively, the results suggest that MS acts as an effective anti-asthmatic and anti-inflammatory agent, by regulating various cellular, biochemical and molecular intermediates.

Metabolomics Analysis of Cell-Free Expression Systems Using Gas Chromatography-Mass Spectrometry.

Metabolomics is the systems-scale study of the biochemical intermediates of metabolism. This approach has great potential to uncover how metabolic intermediates are used and generated in cell-free expression systems, something that is to date not well understood. Here, we present a detailed metabolomics protocol for characterization of the small molecules in cell-free systems. We specifically focus on the analysis of Escherichia coli lysate-based cell-free systems using gas chromatography coupled to mass spectrometry. Measuring and monitoring the metabolic changes in cell-free systems can provide insight into the ways that metabolites affect the productivity of cell-free reactions, ultimately allowing for more informed engineering and optimization efforts for cell-free systems.

Metabolomic Profiling of Biological Reference Materials using a Multiplatform High-Resolution Mass Spectrometric Approach.

The number of metabolomics studies have increased dramatically in recent years, spanning from basic/mechanistic research to the identification and validation of clinical biomarkers. Developments in analyte separation techniques and the growth of databases are largely responsible for the rapid growth of metabolomics, although broad differences in analytical workflows can result in difficulty when comparing data across studies. The establishment of baseline metabolomics data for human reference materials using complementary/orthogonal data acquisition strategies can help to alleviate some of these challenges. To this end, we report nontargeted semiquantitative metabolomics data for 22 commercially available materials including plasma (healthy, diabetic, hypertriglyceridemic, African-American), serum (female, male, pregnant, among others), feces (meconium, vegan, omnivore), urine (smokers' and nonsmokers'), breast milk, saliva, and vaginal fluid, using ultrahigh-performance liquid chromatography-tandem mass spectrometry in positive and negative electrospray ionization, as well as gas chromatography-electron ionization-mass spectrometry. Significant differences were observed in the metabolomic fingerprints between all sample types. Post hoc comparisons between relevant sample types support the relevance of these materials and the validity of nontargeted strategies in global metabolomics. As the number and variety of reference materials continues to increase, it is imperative that their adoption is matched. The results of this study may inform future biomedical research by highlighting several metabolites across matrixes and treatments/states that could serve as clinical biomarkers or important biochemical pathway intermediates. Furthermore, our work can serve as a metric for systems suitability, quality assurance, and quality control across the community via the dissemination of high-quality and publicly available annotated metabolomics data.

Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease

Currently there is an unmet need for treatments that can prevent hypertrophic cardiomyopathy (HCM). Using a murine model we previously identified that HCM causing cardiac troponin I mutation Gly203Ser (cTnI-G203S) is associated with increased mitochondrial metabolic activity, consistent with the human condition. These alterations precede development of the cardiomyopathy. Here we examine the efficacy of in vivo treatment of cTnI-G203S mice with a peptide derived against the α-interaction domain of the cardiac L-type calcium channel (AID-TAT) on restoring mitochondrial metabolic activity, and preventing HCM. cTnI-G203S or age-matched wt mice were treated with active or inactive AID-TAT. Following treatment, targeted metabolomics was utilized to evaluate myocardial substrate metabolism. Cardiac myocyte mitochondrial metabolic activity was assessed as alterations in mitochondrial membrane potential and flavoprotein oxidation. Cardiac morphology and function were examined using echocardiography. Cardiac uptake was assessed using an in vivo multispectral imaging system. We identified alterations in six biochemical intermediates in cTnI-G203S hearts consistent with increased anaplerosis. We also reveal that AID-TAT treatment of precardiomyopathic cTnI-G203S mice, but not mice with established cardiomyopathy, restored cardiac myocyte mitochondrial membrane potential and flavoprotein oxidation, and prevented myocardial hypertrophy. Importantly, AID-TAT was rapidly targeted to the heart, and not retained by the liver or kidneys. Overall, we identify biomarkers of HCM resulting from the cTnI mutation Gly203Ser, and present a safe, preventative therapy for associated cardiomyopathy. Utilizing AID-TAT to modulate cardiac metabolic activity may be beneficial in preventing HCM in "at risk" patients with identified Gly203Ser gene mutations.

Kinetic Proofreading using Substrate Gradients and Enzyme Diffusion

Low error rates in many of the enzymatic processes in biology, such as translation or DNA replication, are attained by employing the nonequilibrium error correction mechanism called kinetic proofreading. Kinetic proofreading operates by delaying product formation through the introduction of additional biochemical intermediates, giving the enzyme more than one chance to discard the non-cognate substrate before reaching its catalytically active state. The requirements of having multiple biochemically distinct intermediates and direct coupling to NTP hydrolysis, however, limit the flexibility and performance scope of traditional proofreading schemes. In our work, we propose an alternative conceptual mechanism of enzymatic error correction where the necessary delay between initial substrate binding and product formation events is achieved by having them occur at distinct physical locations. Specifically, if the substrates are localized primarily at one end of the physical compartment and enzyme activation takes place only at the other end, then the time that it takes for the enzyme to diffuse the distance between these ends can be leveraged to perform efficient proofreading. Importantly, this mechanism requires neither multiple biochemical intermediates nor the presence of an ATP binding site on the enzyme, making its performance easily adaptable through the tuning of the system's characteristic length scales. We discuss how the tuning of these length scales changes the fidelity, speed and energy dissipation of the mechanism, and propose biological examples of its possible implementation in the cell.

A simpler method affords evaluation of π stabilization by phenylalanine of several biochemical carbocations.

Carbocations are important intermediates in the biosynthesis of terpenes and steroids, and it is challenging to try to understand how these relatively unstable species survive even transiently during biochemical reactions. Carbocation-π interaction with aromatic amino acid residues is an important factor in helping to stabilize these positively charged species. However, the short lifetimes of these active site carbocations makes experimental evaluation of the stabilization afforded by such interaction impossible. Computational studies, however, have provided some insight into this phenomenon. Herein we report a simple, computationally efficient method to estimate such stabilization energies afforded by phenylalanine to biochemical carbocation intermediates. A model is constructed in which the biochemical carbocation is replaced by an appropriate carbocation mimic (t-butyl or dimethylallyl). This substitute carbocation is then aligned with an ethylbenzene serving as a surrogate for each proximate phenylalanine in a geometry that replicates as closely as possible the orientation of that phenylalanine using measurements made on an X-ray structure of an enzyme active site in which a carbocation surrogate is bound. Density functional theory computations on such models were then used to yield estimates of stabilization energies. Application of this method to the tertiary carbocation formed in the reaction catalyzed by geranyl diphosphate C-methyl transferase gave a stabilization energy (-12.3 kcal mol-1) that was essentially identical to that obtained previously by analysis of a much more computationally demanding model of the active site. As a check on the accuracy of the simpler method, it was applied with similar success to the farnesyl cation formed in the reaction catalyzed by aristolochene synthase that is stabilized by cation-π interaction with two phenylalanines. Application of this method is also described to estimate carbocation-π stabilization, by the same two phenylalanines, of the final carbocation intermediate leading to aristolochene through analysis of the X-ray structure of an inhibitor of that carbocation bound in the active site of aristolochene synthase. Finally, the stabilization, by either of two phenylalanines, of six carbocation intermediates in the oxidosqualene cyclase-catalyzed formation of lanosterol is estimated by comparable analysis of an X-ray structure of that reaction product bound in the enzyme active site.

Investigation into the mechanism of thin filament regulation by transient kinetics and equilibrium binding: Is there a conflict?

Striated muscle contraction occurs when myosin undergoes a lever-type structural change. This process (the power stroke) requires ATP and is governed by the thin filament, a complex of actin, tropomyosin, and troponin. The authors have used a fast-mixing instrument to investigate the mechanism of regulation. Such (pre-steady-state kinetic) experiments allow biochemical intermediates in a working actomyosin cycle to be monitored. The regulatory focal point is demonstrated to be the step that involves the departure of inorganic phosphate (i.e., AM-ADP-Pi → AM-ADP). This part of the cycle, which lies on the main kinetic pathway and coincides with the drive stroke, is maximally accelerated ∼100-fold by the combined association of ligands (Ca[II] and rigor myosin heads) with the thin filament. However, the observed ligand dependencies of the rates of Pi dissociation that are reported herein are at variance with predictions of models derived from experiments where ATP hydrolysis is not taking place (and myosin exists in a nonphysiological form). It is concluded that the principal influence of the thin filament is in setting the rate of Pi dissociation and that physiological levels of regulation are dependent upon the liganded state of the thin filament as well as the conformation of myosin.

Anaerobic Process for Bioenergy Recovery From Dairy Waste: Meta-Analysis and Enumeration of Microbial Community Related to Intermediates Production.

Dairy wastes are widely studied for the hydrogen and methane production, otherwise the changes in microbial communities related to intermediate valuable products was not deeply investigated. Culture independent techniques are useful tools for exploring microbial communities in engineered system having new insights into their structure and function as well as potential industrial application. The deep knowledge of the microbiota involved in the anaerobic process of specific waste and by-products represents an essential step to better understand the entire process and the relation of each microbial population with biochemical intermediates and final products. Therefore, this study investigated the microbial communities involved in the laboratory-scale anaerobic digestion of a mixture of mozzarella cheese whey and buttermilk amended with 5% w/v of industrial animal manure pellets. Culture-independent methods by employing high-throughput sequencing and microbial enumerations highlighted that lactic acid bacteria, such as Lactobacillaceae and Streptococcaceae dominated the beginning of the process until about day 14 when a relevant increase in hydrogen production (more than 10 ml H2 gVS-1 from days 13 to 14) was observed. Furthermore, during incubation a gradual decrease of lactic acid bacteria was detected with a simultaneous increase of Clostridia, such as Clostridiaceae and Tissierellaceae families. Moreover, archaeal populations in the biosystem were strongly related to inoculum since the non-inoculated samples of the dairy waste mixture had a relative abundance of archaea less than 0.1%; whereas, in the inoculated samples of the same mixture several archaeal genera were identified. Among methanogenic archaea, Methanoculleus was the dominant genus during all the process especially when the methane production occurred, and its relative abundance increased up to 99% at the end of the incubation time highlighting that methane was formed from dairy wastes primarily by the hydrogenotrophic pathway in the reactors.

Gas Chromatography-Mass Spectrometry Microbial Metabolomics for Applications in Strain Optimization.

Metabolomics is the systems-scale measurement of biochemical intermediates in biological systems; by virtue of its deep and broad study of metabolism, it has great potential for applications in metabolic engineering. While a number of the analytical techniques used widely in metabolomics are familiar to metabolic engineers performing post hoc analyses of product titers, the requirements for accurately capturing metabolism at a systems scale rather than just measuring a single secreted product are much more complicated. Nonetheless, metabolomics (which is still not widely available as an affordable consumer service like many molecular biology services) is within reach of many properly equipped metabolic engineering groups. To this end, we present a detailed metabolomics protocol with application to strain optimization. Specifically, we focus on characterizing metabolism in the yeast Saccharomyces cerevisiae using gas chromatography coupled to mass spectrometry. The measurement of metabolic intermediates that results from such approaches has the potential to enable more informed and rational efforts towards pathway engineering and strain optimization.

Protein-Amino Acid Metabolism Disarrangements: The Hidden Enemy of Chronic Age-Related Conditions.

Proteins are macro-molecules crucial for cell life, which are made up of amino acids (AAs). In healthy people, protein synthesis and degradation are well balanced. However, in the presence of hypercatabolic stimulation (i.e., inflammation), protein breakdown increases as the resulting AAs are consumed for metabolic proposes. Indeed, AAs are biochemical totipotent molecules which, when deaminated, can be transformed into energy, lipids, carbohydrates, and/or biochemical intermediates of fundamental cycles, such as the Krebs’ cycle. The biochemical consequence of hyper-catabolism is protein disarrangement, clinically evident with signs such as sarcopenia, hypalbuminemia, anaemia, infection, and altered fluid compartmentation, etc. Hypercatabolic protein disarrangement (HPD) is often underestimated by clinicians, despite correlating with increased mortality, hospitalization, and morbidity quite independent of the primary disease. Simple, cheap, repeatable measurements can be used to identify HPD. Therefore, identification and treatment of proteins’ metabolic impairment with appropriate measurements and therapy is a clinical strategy that could improve the prognosis of patients with acute/chronic hypercatabolic inflammatory disease. Here, we describe the metabolism of protein and AAs in hypercatabolic syndrome, illustrating the clinical impact of protein disarrangement. We also illustrate simple, cheap, repeatable, and worldwide available measurements to identify these conditions. Finally, we provide scientific evidence for HPD nutritional treatment.

Switchable Chemoselective Transfer Hydrogenations of Unsaturated Carbonyls Using Copper(I) N-Donor Thiolate Clusters.

Unsaturated alcohols and saturated carbonyls are important chemical, pharmaceutical, and biochemical intermediates. We herein report an efficient transfer hydrogenation protocol in which conversion of unsaturated carbonyl compounds to either unsaturated alcohols or saturated carbonyls was catalyzed by Cu(I) N-donor thiolate clusters along with changing hydrogen source (isopropanol or butanol) and base (NaOH or K2CO3). Mechanistic studies supported by DFT transition state modeling indicate that such a chemoselectivity can be explained by the relative concentrations of Cu(I) monohydride and protonated Cu(I) hydride complexes in each catalytic system.

Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue

The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define “fatigability” in vivo of fast vs. slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin subfragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH, and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a preequilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs. pH 6.2), and Pi (zero, 15, or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1, but for slow S1, KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.

Topological organisation of the phosphatidylinositol 4,5-bisphosphate-phospholipase C resynthesis cycle: PITPs bridge the ER-PM gap.

Phospholipase C (PLC) is a receptor-regulated enzyme that hydrolyses phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) triggering three biochemical consequences, the generation of soluble inositol 1,4,5-trisphosphate (IP3), membrane-associated diacylglycerol (DG) and the consumption of PM PI(4,5)P2 Each of these three signals triggers multiple molecular processes impacting key cellular properties. The activation of PLC also triggers a sequence of biochemical reactions, collectively referred to as the PI(4,5)P2 cycle that culminates in the resynthesis of this lipid. The biochemical intermediates of this cycle and the enzymes that mediate these reactions are topologically distributed across two membrane compartments, the PM and the endoplasmic reticulum (ER). At the PM, the DG formed during PLC activation is rapidly converted into phosphatidic acid (PA) that needs to be transported to the ER where the machinery for its conversion into PI is localised. Conversely, PI from the ER needs to be rapidly transferred to the PM where it can be phosphorylated by lipid kinases to regenerate PI(4,5)P2 Thus, two lipid transport steps between membrane compartments through the cytosol are required for the replenishment of PI(4,5)P2 at the PM. Here, we review the topological constraints in the PI(4,5)P2 cycle and current understanding how these constraints are overcome during PLC signalling. In particular, we discuss the role of lipid transfer proteins in this process. Recent findings on the biochemical properties of a membrane-associated lipid transfer protein of the PITP family, PITPNM proteins (alternative name RdgBα/Nir proteins) that localise to membrane contact sites are discussed. Studies in both Drosophila and mammalian cells converge to provide a resolution to the conundrum of reciprocal transfer of PA and PI during PLC signalling.