An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Small-molecule inhibition of the archetypal UbiB protein COQ8.

Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

The effects of sustained, low- and high-intensity isometric tasks on performance fatigability and the perceived responses that contributed to task termination.

PURPOSE: The present study examined the effects of sustained, isometric low- versus high-intensity tasks on time to task failure (TTF), performance fatigability (PF), ratings of perceived exertion (RPE), and the perceived causes of task termination from a post-test questionnaire (PTQ). METHODS: Ten men (mean ± SD: age = 21.1 ± 2.3 years; height = 180.2 ± 5.7 cm; body mass = 79.5 ± 8.8 kg) performed maximal voluntary isometric contractions (MVICs) before and after fatiguing, isometric forearm flexion tasks anchored to the torque corresponding to RPE values of 2 (TRQ2FT = 23.8 ± 7.1 N·m) and 8 (TRQ8FT = 60.9 ± 11.4 N·m). In addition, the subjects completed a PTQ which surveyed whether the perceived sensations of fatigue or pain, and/or the psychological factors of loss of focus and motivation contributed to the decision to terminate the task. Repeated measures ANOVAs, Wilcoxon-Signed Rank tests, and Spearman's Rank-Order Correlations were used to analyze the data. RESULTS: Across the fatiguing tasks, there were similar decreases in MVIC torque (95.2 ± 20.3 vs. 68.9 ± 15.6 N·m; p < 0.001) and RPE values (p = 0.122) at task failure for TRQ2FT (7.4 ± 2.7) and TRQ8FT (8.9 ± 1.0), but a longer (p = 0.005) TTF for the TRQ2FT (245.0 ± 177.0 s) than TRQ8FT (36.8 ± 11.1 s). CONCLUSIONS: Despite reaching task failure, the subjects were able to perform MVICs that were 100-300% greater than the target torque values within seconds of terminating the tasks. Thus, we hypothesized that task failure was not caused by an inability to produce sufficient torque to sustain the tasks, but rather an unwillingness to continue the task.

The Effects of Anchoring a Fatiguing Forearm Flexion Task to a High vs. Low Rating of Perceived Exertion on Torque and Neuromuscular Responses.

ABSTRACT: Ortega, DG, Housh, TJ, Smith, RW, Arnett, JE, Neltner, TJ, Schmidt, RJ, and Johnson, GO. The effects of anchoring a fatiguing forearm flexion task to a high versus low rating of perceived exertion on torque and neuromuscular responses. J Strength Cond Res 38(5): e219-e225, 2024-This study examined the torque and neuromuscular responses following sustained, isometric, forearm flexion tasks anchored to 2 ratings of perceived exertion (RPE). Nine men (mean ± SD: age = 21.0 ± 2.4 years; height = 179.5 ± 5.1 cm; body mass = 79.6 ± 11.4 kg) completed maximal voluntary isometric contractions (MVIC) before and after sustained, isometric, forearm flexion tasks to failure anchored to RPE = 2 and RPE = 8. The amplitude (AMP) and mean power frequency (MPF) of the electromyographic (EMG) signal were recorded from the biceps brachii. Normalized torque was divided by normalized EMG AMP to calculate neuromuscular efficiency (NME). A dependent t-test was used to assess the mean difference for time to task failure (TTF). Repeated-measures analysis of variances was used to compare mean differences for MVIC and normalized neuromuscular parameters. There was no significant difference in TTF between RPE = 2 and RPE = 8 (p = 0.713). The MVIC decreased from pretest to posttest at RPE = 2 (p = 0.009) and RPE = 8 (p = 0.003), and posttest MVIC at RPE = 8 was less than that at RPE = 2 (p < 0.001). In addition, NME decreased from pretest to posttest (p = 0.008). There was no change in normalized EMG AMP or EMG MPF (p > 0.05). The current findings indicated that torque responses were intensity specific, but TTF and neuromuscular responses were not. Furthermore, normalized EMG AMP and EMG MPF remained unchanged but NME decreased, likely due to peripheral fatigue and excitation-contraction coupling failure. Thus, this study provides information regarding the neuromuscular responses and mechanisms of fatigue associated with tasks anchored to RPE, which adds to the foundational understanding of the relationship between resistance exercise and the perception of fatigue.

Effects of a Sustained, Isometric Forearm Flexion Task to Failure on Torque and Neuromuscular Responses at 3 Elbow Joint Angles.

ABSTRACT: Ortega, DG, Housh, TJ, Smith, RW, Arnett, JE, Neltner, TJ, Anders, JPV, Schmidt, RJ, and Johnson, GO. The effects of a sustained, isometric forearm flexion task to failure on torque and neuromuscular responses at 3 elbow joint angles. J Strength Cond Res 38(1): e25-e33, 2024-This study examined the effects of a sustained, isometric forearm flexion task anchored to torque to task failure on maximal voluntary isometric contraction (MVIC) and neuromuscular responses at 3 elbow joint angles. Eleven women (mean ± SD: age = 20.8 ± 2.7 years, height = 169.3 ± 7.4 cm, body mass = 67.7 ± 6.9 kg) performed two 3s forearm flexion MVICs at elbow joint angles (JAs) of 75°, 100°, and 125° before and after a sustained, isometric forearm flexion task to failure at a fatiguing joint angle of 100° anchored to a torque value that corresponded to a rating of perceived exertion of 8 (RPE = 8). The amplitude (AMP) and mean power frequency (MPF) of the electromyographic (EMG) and mechanomyographic (MMG) signals were recorded from the biceps brachii. Repeated-measures ANOVAs were used to compare mean differences for MVIC and neuromuscular parameters. Collapsed across JAs, MVIC (p < 0.001) and EMG MPF (p = 0.006) pretest values were greater than posttest values. Collapsed across time, EMG MPF at JA75 was greater than JA100 (p < 0.001) and JA125 (p < 0.001), and JA100 was greater (p = 0.007) than JA125. For EMG AMP, there was a fatigue-induced decrease at JA75 (p = 0.003). For neuromuscular efficiency (NME = normalized torque/normalized EMG AMP), there were decreases from pretest to posttest at JA100 (p = 0.002) and JA125 (p = 0.008). There were no significant interactions or main effects for MMG AMP and MMG MPF. From these findings, it was hypothesized that the decline in MVICs at JA75, JA100, and JA125 was due to fatigue-induced metabolic perturbations that resulted in JA-specific neuromuscular responses. Thus, neuromuscular parameters may provide insight into the JA-specific mechanisms of fatigue.

Benchmarking Social Isolation, Loneliness, and Smoking: Challenges and Opportunities for Public Health.

Over the past decade, the health implications of social isolation and loneliness garnered global attention due in part to a widely cited meta-analysis that benchmarked associations between cigarette smoking and mortality with associations between several social-relationship measures and mortality. Leaders in health systems, research, government, and popular media have since claimed that the harms of social isolation and loneliness are comparable to that of cigarette smoking. Our commentary examines the basis of this comparison. We suggest that comparisons between social isolation, loneliness, and smoking have been helpful for raising awareness of robust evidence linking social relationships and health. However, the analogy often oversimplifies the evidence and may overemphasize treating social isolation or loneliness at the individual level without sufficient attention on population-level prevention. As communities, governments, and health and social sector practitioners navigate opportunities for change, we believe now is time to focus greater attention on the structures and environments that promote and constrain healthy relationships.

Performance Fatigability and Neuromuscular Responses Are Not Joint Angle Specific Following a Sustained Isometric Forearm Flexion Task Anchored to a High Perceptual Intensity in Women.

OBJECTIVES: To examine the effects of joint angle (JA) on maximal voluntary isometric contractions (MVIC) and neuromuscular responses following a sustained, isometric forearm flexion task anchored to a rating of perceived exertion (RPE) of 8 (RPE=8). METHODS: Nine women (age: 20.7±2.9 yrs; height: 168.8±7.2 cm; body mass: 66.3±6.8 kg) performed 2,3s forearm flexion MVICs at JAs of 75°, 100°, and 125° prior to and following a sustained, isometric forearm flexion task anchored to RPE=8 to task failure (torque reduced to zero) at JA100. Electromyographic (EMG) and mechanomyographic (MMG) signals were recorded from the biceps brachii. RESULTS: The MVIC at JA100 (collapsed across Time) was significantly greater (p<0.05) than JA75 and JA125. The pre-test MVIC was significantly greater (p<0.001) than the post-test. For EMG amplitude (AMP) and EMG mean power frequency (MPF), pre-test values were significantly greater (p<0.05) than the post-test values, with no differences between JAs. For MMG AMP and MMG MPF, there were no significant (p>0.05) differences between Time or JAs. Pre-test neuromuscular efficiency (normalized MVIC/normalized EMG AMP) was significantly greater (p=0.005) than post-test. CONCLUSION: Following a sustained, isometric forearm flexion task anchored to RPE=8 at JA100, the fatigue-induced MVIC and neuromuscular responses were not affected by JA.

Utilizing the RPE-Clamp model to examine interactions among factors associated with perceived fatigability and performance fatigability in women and men.

PURPOSE: The purpose of the present study was to examine the interactions between perceived fatigability and performance fatigability in women and men by utilizing the RPE-Clamp model to assess the fatigue-induced effects of a sustained, isometric forearm flexion task anchored to RPE = 8 on time to task failure (TTF), torque, and neuromuscular responses. METHODS: Twenty adults (10 men and 10 women) performed two, 3 s forearm flexion maximal voluntary isometric contractions (MVICs) followed by a sustained, isometric forearm flexion task anchored to RPE = 8 using the OMNI-RES (0-10) scale at an elbow joint angle of 100°. Electromyographic amplitude (EMG AMP) was recorded from the biceps brachii. Torque and EMG AMP values resulting from the sustained task were normalized to the pretest MVIC. Neuromuscular efficiency was defined as NME = normalized torque/normalized EMG AMP. Mixed factorial ANOVAs and Bonferroni corrected dependent t tests and independent t tests were used to examine differences across time and between sex for torque and neuromuscular parameters. RESULTS: There were no differences between the women and men for the fatigue-induced decreases in torque, EMG AMP, or NME, and the mean decreases (collapsed across sex) were 50.3 ± 8.6 to 2.8 ± 2.9% MVIC, 54.7 ± 12.0 to 19.6 ± 5.3% MVIC, and 0.94 ± 0.19 to 0.34 ± 0.16, respectively. Furthermore, there were no differences between the women and men for TTF (251.8 ± 74.1 vs. 258.7 ± 77.9 s). CONCLUSION: The results suggested that the voluntary reductions in torque to maintain RPE and the decreases in NME were likely due to group III/IV afferent feedback from peripheral fatigue that resulted in excitation-contraction coupling failure.

Effects of High-Intensity, Eccentric-Only Muscle Actions on Serum Biomarkers of Collagen Degradation and Synthesis.

ABSTRACT: Neltner, TJ, Sahoo, PK, Smith, RW, Anders, JPV, Arnett, JE, Ortega, DG, Schmidt, RJ, Johnson, GO, Natarajan, SK, and Housh, TJ. Effects of high-intensity, eccentric-only muscle actions on serum biomarkers of collagen degradation and synthesis. J Strength Cond Res 37(9): 1729-1737, 2023-The purpose of this study was to examine the effects of high-intensity, eccentric-only muscle actions of the leg extensors on (a) serum biomarkers of collagen degradation (hydroxyproline [HYP] and C-terminal telopeptide of type I collagen [C1M]) and synthesis (pro-c1α1) and (b) the time course of changes in maximal voluntary isometric contraction (MVIC) and ratings of muscle soreness after the eccentric-only exercise bout. Twenty-five recreationally active men (mean ± SD: age = 21.2 ± 2.0 years) completed 5 sets of 10 bilateral, eccentric-only dynamic constant external resistance muscle actions of the leg extensors at a load of 110% of their concentric leg extension 1 repetition maximum. Analysis of variances (p < 0.05) and a priori planned pairwise comparisons using Bonferroni corrected (p < 0.0167) paired t tests were used to examine mean changes in blood biomarkers from baseline to 48 hours postexercise as well as in MVIC and soreness ratings immediately, 24 hours, and 48 hours postexercise. There were increases in HYP (3.41 ± 2.37 to 12.37 ± 8.11 µg·ml-1; p < 0.001) and C1M (2.50 ± 1.05 to 5.64 ± 4.89 µg·L-1; p = 0.003) from preexercise to 48 hours postexercise, but no change in pro-c1α1. Maximal voluntary isometric contraction declined immediately after the exercise bout (450.44 ± 72.80 to 424.48 ± 66.67 N·m; p = 0.002) but recovered 24 hours later, whereas soreness was elevated immediately (6.56 ± 1.58; p < 0.001), 24 hours (3.52 ± 1.53; p < 0.001), and 48 hours (2.60 ± 1.32; p = 0.001) postexercise. The eccentric-only exercise bout induced increases in collagen degradation but had no effect on collagen synthesis. These findings provide information for clinicians to consider when prescribing exercise after an acute injury or surgery.

Tracking changes in body composition: comparison of methods and influence of pre-assessment standardisation.

The present study reports the validity of multiple assessment methods for tracking changes in body composition over time and quantifies the influence of unstandardised pre-assessment procedures. Resistance-trained males underwent 6 weeks of structured resistance training alongside a hyperenergetic diet, with four total body composition evaluations. Pre-intervention, body composition was estimated in standardised (i.e. overnight fasted and rested) and unstandardised (i.e. no control over pre-assessment activities) conditions within a single day. The same assessments were repeated post-intervention, and body composition changes were estimated from all possible combinations of pre-intervention and post-intervention data. Assessment methods included dual-energy X-ray absorptiometry (DXA), air displacement plethysmography, three-dimensional optical imaging, single- and multi-frequency bioelectrical impedance analysis, bioimpedance spectroscopy and multi-component models. Data were analysed using equivalence testing, Bland-Altman analysis, Friedman tests and validity metrics. Most methods demonstrated meaningful errors when unstandardised conditions were present pre- and/or post-intervention, resulting in blunted or exaggerated changes relative to true body composition changes. However, some methods - particularly DXA and select digital anthropometry techniques - were more robust to a lack of standardisation. In standardised conditions, methods exhibiting the highest overall agreement with the four-component model were other multi-component models, select bioimpedance technologies, DXA and select digital anthropometry techniques. Although specific methods varied, the present study broadly demonstrates the importance of controlling and documenting standardisation procedures prior to body composition assessments across distinct assessment technologies, particularly for longitudinal investigations. Additionally, there are meaningful differences in the ability of common methods to track longitudinal body composition changes.

Time course of changes in torque and neuromuscular parameters during a sustained isometric forearm flexion task to fatigue anchored to a constant rating of perceived exertion.

OBJECTIVE: This study examined the time course of changes in torque and electromyographic (EMG) and mechanomyographic (MMG) responses during a sustained isometric task anchored to a constant perception of exertion (RPE). METHODS: Twelve college-aged men performed an isometric forearm flexion task to failure anchored to RPE=7 (OMNI-RES scale). The amplitude (AMP) and frequency (MPF) of the EMG and MMG signals from the biceps brachii were recorded. Repeated measures ANOVAs were used to examine differences for the normalized (%MVIC) torque and neuromuscular parameters. RESULTS: The time to task failure (TTF) was 678.0±468.1s. Torque decreased significantly (p<0.001, ηp2=0.774) across time and all subjects reduced torque to zero. Post-hoc comparisons indicated that the torque values from 20-100% TTF were less than the value at 10% TTF. There were no significant (p>0.05) changes from 10-100% TTF for the EMG and MMG parameters. CONCLUSION: We hypothesize that RPE was maintained by various mechanisms throughout the task: group III/IV afferent neurons, adequate blood flow, and a combination of reduced contractile efficiency, collective afferent feedback (group III/IV afferents) from muscles involved with forearm flexion, and motivation that resulted in an initial decrease, plateau, and final decline in torque to zero, respectively.

Comparison of Indirect Calorimetry and Common Prediction Equations for Evaluating Changes in Resting Metabolic Rate Induced by Resistance Training and a Hypercaloric Diet.

ABSTRACT: Rodriguez, C, Harty, PS, Stratton, MT, Siedler, MR, Smith, RW, Johnson, BA, Dellinger, JR, Williams, AD, White, SJ, Benavides, ML, and Tinsley, GM. Comparison of indirect calorimetry and common prediction equations for evaluating changes in resting metabolic rate induced by resistance training and a hypercaloric diet. J Strength Cond Res 36(11): 3093-3104, 2022-The ability to accurately identify resting metabolic rate (RMR) changes over time allows practitioners to prescribe appropriate adjustments to nutritional intake. However, there is a lack of data concerning the longitudinal utility of commonly used RMR prediction equations. The purpose of this study was to evaluate the validity of several commonly used prediction equations to track RMR changes during a hypercaloric nutritional intervention and supervised resistance exercise training program. Twenty resistance-trained men completed the study. The protocol lasted 6 weeks, and subjects underwent RMR assessments by indirect calorimetry (IC) preintervention and postintervention to obtain reference values. Existing RMR prediction equations based on body mass (BM) or dual-energy X-ray absorptiometry fat-free mass (FFM) were also evaluated. Equivalence testing was used to evaluate whether each prediction equation demonstrated equivalence with IC. Null hypothesis significance testing was also performed, and Bland-Altman analysis was used alongside linear regression to assess the degree of proportional bias. Body mass and FFM increased by 3.6 ± 1.7 kg and 2.4 ± 1.6 kg, respectively. Indirect calorimetry RMR increased by 165 ± 97 kcal·d -1 , and RMR:FFM increased by 5.6 ± 5.2%. All prediction equations underestimated mean RMR changes relative to IC, with magnitudes ranging from 75 to 155 kcal·d -1 , while also displaying unacceptable levels of negative proportional bias. In addition, no equation demonstrated equivalence with IC. Common RMR prediction equations based on BM or FFM did not fully detect the increase in RMR observed with resistance training plus a hypercaloric diet. Overall, the evaluated prediction equations are unsuitable for estimating RMR changes in the context of this study.

Early Impacts of the COVID-19 Pandemic on Public Health Systems and Practice in 3 Canadian Provinces From the Perspective of Public Health Leaders: A Qualitative Study.

CONTEXT: The COVID-19 pandemic has impacted health systems worldwide. Studies to date have largely focused on the health care system with less attention to the impact on public health systems and practice. OBJECTIVE: To describe the early impacts of COVID-19 on public health systems and practice in 3 Canadian provinces from the perspective of public health system leaders and synthesize lessons learned. DESIGN: A qualitative study using semistructured virtual interviews with public health leaders between October 2020 and April 2021. The World Health Organization's essential public health operations framework guided data collection and analysis. SETTING: This study involved the Canadian provinces of Alberta, Ontario, and Québec. These provinces were chosen for their large populations, relatively high COVID-19 burden, and variation in public health systems. PARTICIPANTS: Public health leaders from Alberta (n = 21), Ontario (n = 18), and Québec (n = 19) in organizations with a primary mandate of stewardship and/or administration of essential public health operations (total n = 58). RESULTS: We found that the COVID-19 pandemic led to intensified collaboration in public health systems and a change in workforce capacity to respond to the pandemic. This came with opportunities but also challenges of burnout and disruption of non-COVID-19 services. Information systems and digital technologies were increasingly used and there was greater proximity between public health leaders and other health system leaders. A renewed recognition for public health work was also highlighted. CONCLUSIONS: The COVID-19 pandemic impacted several aspects of public health systems in the provinces studied. Our findings can help public health leaders and policy makers identify areas for further investment (eg, intersectoral collaboration, information systems) and develop plans to address challenges (eg, disrupted services, workforce burnout) that have surfaced.

The Crystal Structure of Cysteamine Dioxygenase Reveals the Origin of the Large Substrate Scope of This Vital Mammalian Enzyme.

We report the crystal structure of the mammalian non-heme iron enzyme cysteamine dioxygenase (ADO) at 1.9 Šresolution, which shows an Fe and three-histidine (3-His) active site situated at the end of a wide substrate access channel. The open approach to the active site is consistent with the recent discovery that ADO catalyzes not only the conversion of cysteamine to hypotaurine but also the oxidation of N-terminal cysteine (Nt-Cys) peptides to their corresponding sulfinic acids as part of the eukaryotic N-degron pathway. Whole-protein models of ADO in complex with either cysteamine or an Nt-Cys peptide, generated using molecular dynamics and quantum mechanics/molecular mechanics calculations, suggest occlusion of access to the active site by peptide substrate binding. This finding highlights the importance of a small tunnel that leads from the opposite face of the enzyme into the active site, providing a path through which co-substrate O2 could access the Fe center. Intriguingly, the entrance to this tunnel is guarded by two Cys residues that may form a disulfide bond to regulate O2 delivery in response to changes in the intracellular redox potential. Notably, the Cys and tyrosine residues shown to be capable of forming a cross-link in human ADO reside ∼7 Šfrom the iron center. As such, cross-link formation may not be structurally or functionally significant in ADO.

Addressing social determinants of noncommunicable diseases in primary care: a systematic review.

OBJECTIVE: To explore how primary care organizations assess and subsequently act upon the social determinants of noncommunicable diseases in their local populations. METHODS: For this systematic review we searched the online databases of PubMed®, MEDLINE®, Embase® and the Health Management Information Consortium from inception to 28 June 2019, along with hand-searching of references. Studies of any design that examined a primary care organization assessing social determinants of noncommunicable diseases were included. For quality assessment we used Cochrane's tool for assessing risk of bias in non-randomized studies of interventions. We used narrative data synthesis to appraise the extent to which the assessments gathered data on the domains of the World Health Organization social determinants of health framework. FINDINGS: We identified 666 studies of which 17 were included in the review. All studies used descriptive study designs. Clinic-based and household surveys and interviews were more commonly used to assess local social determinants than population-level data. We found no examples of organizations that assessed sociopolitical drivers of noncommunicable diseases; all focused on sociodemographic factors or circumstances of daily living. Nevertheless, the resulting actions to address social determinants ranged from individual-level interventions to population-wide measures and introducing representation of primary care organizations on system-level policy and planning committees. CONCLUSION: Our findings may help policy-makers to consider suitable approaches for assessing and addressing social determinants of health in their domestic context. More rigorous observational and experimental evidence is needed to ascertain whether measuring social determinants leads to interventions which mitigate unmet social needs and reduce health disparities.

Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the ß-aryl ether bond of lignin.

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the ß-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the ß-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-ß-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate ß-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr51, Tyr166, and Tyr224) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the ß-aryl ether bond) and Escherichia coli (which cannot break the ß-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases.

Phytochrome B dynamics departs from photoequilibrium in the field.

Vegetation shade is characterized by marked decreases in the red/far-red ratio and photosynthetic irradiance. The activity of phytochrome in the field has typically been described by its photoequilibrium, defined by the photochemical properties of the pigment in combination with the spectral distribution of the light. This approach represents an oversimplification because phytochrome B (phyB) activity depends not only on its photochemical reactions but also on its rates of synthesis, degradation, translocation to the nucleus, and thermal reversion. To account for these complex cellular reactions, we used a model to simulate phyB activity under a range of field conditions. The model provided values of phyB activity that in turn predicted hypocotyl growth in the field with reasonable accuracy. On the basis of these observations, we define two scenarios, one is under shade, in cloudy weather, at the extremes of the photoperiod or in the presence of rapid fluctuations of the light environment caused by wind-induced movements of the foliage, where phyB activity departs from photoequilibrium and becomes affected by irradiance and temperature in addition to the spectral distribution. The other scenario is under full sunlight, where phyB activity responds mainly to the spectral distribution of the light.

Insight into nuclear body formation of phytochromes through stochastic modelling and experiment.

Spatial relocalization of proteins is crucial for the correct functioning of living cells. An interesting example of spatial ordering is the light-induced clustering of plant photoreceptor proteins. Upon irradiation by white or red light, the red light-active phytochrome, phytochrome B, enters the nucleus and accumulates in large nuclear bodies (NBs). The underlying physical process of nuclear body formation remains unclear, but phytochrome B is thought to coagulate via a simple protein-protein binding process. We measure, for the first time, the distribution of the number of phytochrome B-containing NBs as well as their volume distribution. We show that the experimental data cannot be explained by a stochastic model of nuclear body formation via simple protein-protein binding processes using physically meaningful parameter values. Rather modelling suggests that the data is consistent with a two step process: a fast nucleation step leading to macroparticles followed by a subsequent slow step in which the macroparticles bind to form the nuclear body. An alternative explanation for the observed nuclear body distribution is that the phytochromes bind to a so far unknown molecular structure. We believe it is likely this result holds more generally for other nuclear body-forming plant photoreceptors and proteins.