Phosphorylation releases constraints to domain motion in ERK2

Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR ¹³C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with k_ex ≈ 300 s⁻¹ (k_AB ≈ 240 s⁻¹/k_BA ≈ 60 s⁻¹) and p_A/p_B ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N- and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of k_ex ≈ 500 s⁻¹ (k_AB ≈ 15 s⁻¹/k_BA ≈ 485 s⁻¹) and p_A/p_B ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling and a model for protein kinase activation mechanisms (1). ERK2 can be activated by MAP kinase kinases 1 and 2 (MKK1 and 2) through dual phosphorylation of Thr and Tyr residues located at the activation loop (Thr183 and Tyr185, numbered in rat ERK2) (1, 2). Phosphorylation at both sites is required for kinase activation, resulting in increased phosphoryl transfer rate and enhanced affinity for ATP and substrate (3).Conformational changes accompanying the activation of ERK2 have been documented by X-ray structures of the inactive, unphosphorylated (0P-ERK2) and the active, dual-phosphorylated (2P-ERK2) forms (4, 5). Phosphorylation rearranges the activation loop, leading to new ion-pair interactions between phospho-Thr and phospho-Tyr residues and basic residues in the N- and C-terminal domains of the kinase core structure. This leads to a repositioning of active site residues surrounding the catalytic base, enabling recognition of the Ser/Thr-Pro sequence motif at phosphorylation sites and exposing a recognition site for interactions with docking sequences in substrates and scaffolds (6).Less is known about how changes in internal motions contribute to kinase activation. Previous studies using hydrogen-exchange mass spectrometry (HX-MS) and electron paramagnetic resonance spectroscopy (7–9) led to a model where conformational mobility at the hinge linking the N- and C-terminal domains is increased by phosphorylation, therefore releasing constraints needed for activation. Such a model differs from other types of autoinhibitory mechanisms in protein kinases, which involve interactions with domains outside the kinase core (10, 11). However, how hinge flexibility regulates ERK2 is unknown.NMR relaxation dispersion methods enable protein dynamics to be monitored by measuring exchange between conformational states (12). In particular, Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiments report on motions on slow (100–2,000 s⁻¹) timescales (13), which are often important for enzymatic function (13–16). In the CPMG experiment, exchange between different conformational states is probed with varying times between “refocusing” pulses. Conformational exchange leads to imperfect refocusing, thus decreasing the intensity of the NMR signal. Increasing the pulse frequency allows less chance for conformational exchange, and therefore increased NMR signal intensity. For a given pulse frequency, analysis of the signal intensity yields the effective relaxation rate for the resonance, R_2,eff. This is typically plotted as a relaxation dispersion curve, which can be fit to a two-state conformational exchange process (e.g., A ⇌ B interconversion). Fitting extracts the populations and the exchange rates between states, thus reflecting the thermodynamics and kinetics of the system (17, 18).Here we performed CPMG relaxation dispersion experiments at multiple field strengths to compare the dynamic properties of [¹³C]methyl-labeled ERK2 in its phosphorylated and unphosphorylated states. The results demonstrate that phosphorylation causes a significant change in exchange dynamics throughout the kinase core, consistent with a global domain motion. Increasing hinge mobility by introducing mutations at the hinge also promotes domain motion within the core but with differing kinetics and populations. Taken together, the results show that large changes in dynamics accompany ERK2 phosphorylation, which are influenced by conformational mobility at the hinge. We propose that the activation of ERK2 involves removing inhibitory constraints to domain motion, which are conferred by the internal architecture of the kinase.     

Social disadvantage,genetic sensitivity,and children’s telomere length

Disadvantaged social environments are associated with adverse health outcomes. This has been attributed, in part, to chronic stress. Telomere length (TL) has been used as a biomarker of chronic stress: TL is shorter in adults in a variety of contexts, including disadvantaged social standing and depression. We use data from 40, 9-y-old boys participating in the Fragile Families and Child Wellbeing Study to extend this observation to African American children. We report that exposure to disadvantaged environments is associated with reduced TL by age 9 y. We document significant associations between low income, low maternal education, unstable family structure, and harsh parenting and TL. These effects were moderated by genetic variants in serotonergic and dopaminergic pathways. Consistent with the differential susceptibility hypothesis, subjects with the highest genetic sensitivity scores had the shortest TL when exposed to disadvantaged social environments and the longest TL when exposed to advantaged environments.A large body of research has documented a positive association between exposure to disadvantaged social environments and morbidity and mortality (1, 2). A key mechanism for explaining this association is chronic stress, which is believed to degrade physiological functioning (3–6), thus “weathering” the individual and making him or her less resistant to disease (7, 8). We examined (i) whether the association between exposure to a disadvantaged environment and stress biomarkers was evident in childhood and (ii) whether the association was more pronounced for children carrying specific genetic variants. To measure the effect of stress on children’s physiological state, we used telomere length (TL) as a biomarker. A recent line of research suggests that accelerated shortening of the telomere, the (TTAGGG)_n sequence repeat at the end of each chromosome, is a good biomarker of exposure to lifetime stress (3, 6, 8–11). To measure the social environment, we used an index based on four dimensions: economic conditions, parenting quality, family structure/stability, and maternal depression.Our analysis extends the existing literature on social environments and TL in several ways. First, we focus on children, whereas prior research has focused primarily on adults, with few exceptions (12). Second, we examine a sample of African American boys whereas prior research has used mostly samples of whites. Third, we examine for the first time, to our knowledge, whether the association between the social environment and TL is moderated by variations in selected genes. Although recent research suggests that some people are genetically more sensitive than others to their social environments (13–15), research on TL has not taken account of this potential moderating factor. Finally, we use a saliva DNA source of TL, which we show to be highly correlated with blood leukocyte TL (see Figs. S2 and S3). Our study validates other work showing that multiple tissues, including saliva, can be used to measure TL (16, 17).     

α-Ketoglutaramate: an overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle

Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L?‐?Glutamine?+?α?‐?keto acid?+?H₂O → α?‐?ketoglutarate?+?L?‐?amino acid?+?ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance.     

Growth of carbon dioxide whiskers

We report the growth of carbon dioxide (CO₂) whiskers at low temperatures (−70 °C to −65 °C) and moderate pressure (4.4 to 1.0 bar). Their axial growth was assessed by optical video analysis. The identities of these whiskers were confirmed as CO₂ solids by Raman spectroscopy. A vapor–solid growth mechanism was proposed based on the influence of the relative humidity on the growth.

Carbon dioxide (CO₂) whiskers were reported to grow at low temperatures (−70 °C to −65 °C) and moderate pressure (4.4 to 1.0 bar).     

Visit-to-visit blood pressure variability as a prognostic marker in patients with cardiovascular and cerebrovascular diseases – Relationships and comparisons with vascular markers of atherosclerosis

Background

Visit-to-visit blood pressure variability (BPV) is a simple surrogate marker for the development of atherosclerotic diseases, cardiovascular and all-cause mortality. Nevertheless, the relative prognostic value of BPV in comparison with other established vascular assessments remain uncertain.

Methods

We prospectively followed-up 656 high-risk patients with diabetes or established cardiovascular or cerebrovascular diseases for the occurrence of major adverse cardiovascular events (MACEs). Baseline brachial endothelial function, carotid intima-media thickness (IMT) and plaque burden, ankle-brachial index and arterial stiffness were determined. Visit-to-visit BPV were recorded during a mean 18 ± 9 outpatient clinic visits.

Results

After a mean 81 ± 12 month's follow-up, 123 patients (19%) developed MACEs. Patients who developed a MACE had significantly higher systolic BPV, more severe endothelial function, arterial stiffness and systemic atherosclerotic burden compared to patients who did not develop a MACE (all P < 0.01). BPV significantly correlated with all of the vascular assessments (P < 0.01). A high carotid IMT had the greatest prognostic value in predicting development of a MACE (area under receiver operating characteristic curve (AUC) 0.69 ± 0.03, P < 0.01). A high BPV also had moderate prognostic value in prediction of MACE (AUC 0.65 ± 0.03, P < 0.01). After adjustment of confounding factors, a high BPV remained a significant independent predictor of MACE (hazards ratio 1.67, 95% confidence interval 1.14-2.43, P < 0.01).

Conclusions

Compared with established surrogate markers of atherosclerosis, visit-to-visit BPV provides similar prognostic information and may represent a new and simple marker for adverse outcomes in patients with vascular diseases.     

Egg consumption and carotid atherosclerosis in the Northern Manhattan Study

Background

The evidence supporting recommendations to limit intake of cholesterol rich foods is inconclusive. We aimed to examine the association between egg consumption and carotid atherosclerosis phenotypes, and the association with clinical vascular events in a prospective, urban, multi-ethnic population.

Methods and results

The Northern Manhattan Study is a population based cohort to determine stroke incidence, risk factors and prognosis. A sub-cohort of 1429 NOMAS participants with both carotid ultrasounds and comprehensive dietary information was evaluated (mean ± SD age of participants 65.80 ± 8.80, 40% male, 18% white, 20% black, 60% Hispanic). The association between egg consumption and carotid intima media thickness (cIMT) was assessed with linear regression. Logistic and quantile regression was used to examine the association between egg consumption and carotid plaque presence, thickness, and area. The relation between egg consumption and clinical vascular events (N = 2669) was examined with Cox models. The mean total cIMT was 0.91 ± 0.08 mm and 58% had carotid plaque present. Increasing egg consumption was inversely associated with cIMT, plaque presence, thickness, and area, in models adjusted for demographics, vascular risk factors and diet. For every additional egg consumed per week, the risk of plaque decreased by 11% (95% CI 3%–18%). No association was detected between egg consumption and risk of clinical vascular outcomes, over a mean follow up of 11 years and after adjustment for covariates.

Conclusions

Frequency of egg consumption in the low to moderate range was inversely related to several markers of carotid atherosclerosis. No association with clinical vascular events, including stroke, was detected. Our findings do not support current vascular health guidelines suggesting the extreme limitation or avoidance of egg consumption due to its cholesterol content.     

Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS

Amyotrophic lateral sclerosis (ALS; Lou Gehrig’s disease) affects motor neurons (MNs) in the brain and spinal cord. Understanding the pathophysiology of this condition seems crucial for therapeutic design, yet few electrophysiological studies in actively degenerating animal models have been reported. Here, we report a novel preparation of acute slices from adult mouse spinal cord, allowing visualized whole cell patch-clamp recordings of fluorescent lumbar MN cell bodies from ChAT-eGFP or superoxide dismutase 1-yellow fluorescent protein (SOD1YFP) transgenic animals up to 6 mo of age. We examined 11 intrinsic electrophysiologic properties of adult ChAT-eGFP mouse MNs and classified them into four subtypes based on these parameters. The subtypes could be principally correlated with instantaneous (initial) and steady-state firing rates. We used retrograde tracing using fluorescent dye injected into fast or slow twitch lower extremity muscle with slice recordings from the fluorescent-labeled lumbar MN cell bodies to establish that fast and slow firing MNs are connected with fast and slow twitch muscle, respectively. In a G85R SOD1YFP transgenic mouse model of ALS, which becomes paralyzed by 5–6 mo, where MN cell bodies are fluorescent, enabling the same type of recording from spinal cord tissue slices, we observed that all four MN subtypes were present at 2 mo of age. At 4 mo, by which time substantial neuronal SOD1YFP aggregation and cell loss has occurred and symptoms have developed, one of the fast firing subtypes that innvervates fast twitch muscle was lost. These results begin to describe an order of the pathophysiologic events in ALS.Amyotrophic lateral sclerosis (ALS; Lou Gehrig’s disease) is a progressive and usually lethal neurodegenerative condition prominently featuring loss of motor neurons (MNs) and muscle denervation (1–3). Inherited forms of ALS, accounting for ∼10% of cases, potentially inform about disease mechanisms, including: protein folding and quality control [e.g., mutant superoxide dismutase 1 (SOD1), ubiquilin2, and VCP]; RNA binding proteins (e.g., TDP43, FUS, and HNRNPA1); or a DNA expansion (C9ORF72 hexanucleotide expansion). The clinical courses of the various heritable forms and the 90% of cases that are considered sporadic are not distinct, however, reflecting a potentially shared progressive loss of MNs and motor circuit dysfunction (4).ALS has been modeled in mice that are transgenic for a variety of mutant forms of SOD1, allowing for the study of the trajectory of the condition at various time points (5, 6). Among the studies conducted to date are a number addressing electrophysiological changes. From these studies, however, there does not appear to be a clear consensus on the changes that occur in MNs before and during the development of symptoms (7). For example, whereas research on the neuromuscular junction has revealed preferential denervation of fast twitch (type IIb) muscle fibers (8–10), the relationship of this selective susceptibility at the muscle level to pathophysiologic change in the spinal cord is not clear.A major challenge to understanding spinal cord physiology of the mouse models of ALS arises from difficulty in distinguishing the individual features of neurons in the anatomically and physiologically heterogeneous motor system. For example, the usefulness of in vivo recordings requires ensuring adequate sampling of anatomically and functionally heterogeneous spinal cord MNs. For the ex vivo alternative, slice physiology is challenging because most mouse models develop disease after 1 mo of age, a time when spinal cord tissue becomes more sensitive to ischemia (11, 12), making isolation of viable slices difficult. In addition, the spinal cord becomes heavily myelinated in the first few weeks of postnatal life (13), making visualization of individual neurons difficult. Hence, most research regarding cellular electrophysiology in mouse models of ALS has been carried out with primary cultures of embryonic (E13–14) spinal cord MNs (14) or induced pluripotent stem (iPS) cell-derived MNs (15). Although they provide a basis for further study, these models may lack the changes that occur progressively in the context of an intact spinal cord. These models may also lack the diversity of MN physiology present in the mature spinal cord.Here we studied a transgenic strain of ALS mice, G85R SOD1YFP (16), that develops motor symptoms by ∼3 mo of age, associated with progressive accumulation of aggregates in MN cell bodies (from ∼1 mo of age), attended by MN cell loss. The mice paralyze uniformly by 5–6 mo of age. We first developed, using ChAT- EGFP mice that express GFP fluorescence in MNs (17), an acute slice preparation of adult mouse spinal cord that yielded healthy MNs in animals up to and beyond 6 mo of age, readily visualized by their fluorescence, enabling whole cell patch-clamp recordings when coupled with differential interference contrast (DIC) imaging. This preparation allowed extensive characterization of normal MN electrophysiology and enabled grouping of MNs, distinguishing four firing types. We then recorded from MNs in slices from ALS animals at two time points: during the course of aggregation at 2–3 mo of age, before symptoms, and after the onset and initial progression of symptoms at 4 mo of age. At the early time, the four distinct clusters of MNs were present, albeit the fastest firing type (cluster 4) exhibited a significant hyperpolarization. At the later time point, this firing type was no longer detectable, with only the other three types observable, the fastest of which (cluster 3) was now hyperpolarized. Retrograde tracing from fast and slow twitch muscles of the lower extremity revealed that aggregates form preferentially in MN cell bodies attached to fast twitch muscle. These observations suggest a possible sequence of events in which hyperpolarization of the cluster 4 MNs, innervating fast twitch muscle, is associated with aggregate formation in these neurons, which then die, denervating fast twitch muscle.     

Remote ischaemic preconditioning modifies serum apolipoprotein D,met‐enkephalin,adenosine, and nitric oxide in healthy young adults

Remote ischaemic preconditioning (RIPC) has been employed as a non‐invasive protective intervention against myocardial ischaemia–reperfusion injury in animal studies. However, the underlying mechanisms are incompletely defined in humans and its clinical efficacy has been inconclusive. As advanced age, disease, and drugs may confound RIPC mechanisms in patients, our aim is to measure whether RIPC evokes release of adenosine, bradykinin, met‐enkephalin, nitric oxide, and apolipoproteins in healthy young adults. Healthy subjects (n = 18, 9 males, 23 ± 1.5 years old; 9 females, 23 ± 1.8 years old) participated after informed consent. RIPC was applied using a blood pressure cuff to the dominant arms for four cycles of 5‐minute cuff inflation (ischaemia) and 5‐minute cuff deflation (reperfusion). Blood was sampled at baseline and immediately after the final cuff deflation (Post‐RIPC). Baseline and Post‐RIPC plasma levels of adenosine, bradykinin, met‐enkephalin, apolipoprotein A‐1 (ApoA‐1), apolipoprotein D (ApoD), and nitric oxide (as nitrite) were measured via ELISA and high‐performance liquid chromatography. Mean (±SD) baseline levels of adenosine, bradykinin, met‐enkephalin, ApoA‐1, ApoD, and nitrite in healthy young adults were 13.8 ± 6.5 ng/mL, 2.6 ± 1.9 μg/mL, 594.1 ± 197.4 pg/mL, 3.0 ± 0.7 mg/mL, 22.2 ± 4.0 μg/mL, and 49.8 ± 13.4 nmol/L, respectively. Post‐RIPC adenosine and nitrite levels increased (59.5 ± 37.9%, P < .0001; 32.2 ± 19.5%, P < .0001), whereas met‐enkephalin and ApoD levels marginally decreased (5.3 ± 14.0%, P = .04; 10.8 ± 20.5%, P = .04). Post‐RIPC levels were not influenced by sex, age, blood pressure, waist circumference, or BMI. RIPC produces increased levels of adenosine and nitrites, and decreased met‐enkephalin and ApoD in the plasma of young healthy adults.