Intermuscular adipose tissue in patients with systemic lupus erythematosus.

OBJECTIVE: Patients with SLE frequently have debilitating fatigue and reduced physical activity. Intermuscular adipose tissue (IMAT) accumulation is associated with reduced physical exercise capacity. We hypothesised that IMAT is increased in patients with SLE and associated with increased fatigue, reduced physical activity and increased inflammation. METHODS: In a cross-sectional study, 23 patients with SLE and 28 control participants were evaluated. IMAT was measured in the calf muscles using sequential T 1-weighted MRI. Patient-reported physical activity and fatigue were measured and a multiplex proteomic assay was used to measure markers and mediators of inflammation. RESULTS: IMAT accumulation (percentage of IMAT area to muscle area) was significantly higher in SLE versus control participants (7.92%, 4.51%-13.39% vs 2.65%, 1.15%-4.61%, median, IQR, p<0.001) and remained significant after adjustment for age, sex, race and body mass index (p<0.001). In patients with SLE, IMAT accumulation did not differ significantly among corticosteroid users and non-users (p=0.48). In the study cohort (patients and controls), IMAT was positively correlated with self-reported fatigue score (rho=0.52, p<0.001) and inversely correlated with self-reported walking distance (rho=-0.60, p<0.001). Several markers of inflammation were associated with IMAT accumulation in patients with SLE, and gene ontology analysis showed significant enrichment for pathways associated with macrophage migration and activation in relation to IMAT. CONCLUSION: Patients with SLE have greater IMAT accumulation than controls in the calf muscles. Increased IMAT is associated with greater fatigue and lower physical activity. Future studies should evaluate the effectiveness of interventions that improve muscle quality to alleviate fatigue in patients with SLE.

EET Analog Treatment Improves Insulin Signaling in a Genetic Mouse Model of Insulin Resistance.

We previously showed that global deletion of the cytochrome P450 epoxygenase Cyp2c44, a major epoxyeicosatrienoic acid (EET) producing enzyme in mice, leads to impaired hepatic insulin signaling resulting in insulin resistance. This finding led us to investigate whether administration of a water soluble EET analog restores insulin signaling in vivo in Cyp2c44(-/-) mice and investigated the underlying mechanisms by which this effect is exerted. Cyp2c44(-/-) mice treated with the analog EET-A for 4 weeks improved fasting glucose and glucose tolerance compared to Cyp2c44(-/-) mice treated with vehicle alone. This beneficial effect was accompanied by enhanced hepatic insulin signaling, decreased expression of gluconeogenic genes and increased expression of glycogenic genes. Mechanistically, we show that insulin-stimulated phosphorylation of insulin receptor ß (IRß) is impaired in primary Cyp2c44(-/-) hepatocytes and this can be restored by cotreatment with EET-A and insulin. Plasma membrane fractionations of livers indicated that EET-A enhances the retention of IRß in membrane rich fractions, thus potentiating its activation. Altogether, EET analogs ameliorate insulin signaling in a genetic model of hepatic insulin resistance by stabilizing membrane-associated IRß and potentiating insulin signaling.

EET Analog Treatment Improves Insulin Signaling in a Genetic Mouse Model of Insulin Resistance.

We previously showed that global deletion of the cytochrome P450 epoxygenase Cyp2c44, a major epoxyeicosatrienoic acid (EET) producing enzyme in mice, leads to impaired hepatic insulin signaling resulting in insulin resistance. This finding led us to investigate whether administration of a water soluble EET analog restores insulin signaling in vivo in Cyp2c44(-/-) mice and investigated the underlying mechanisms by which this effect is exerted. Cyp2c44(-/-) mice treated with the analog EET-A for 4 weeks improved fasting glucose and glucose tolerance compared to Cyp2c44(-/-) mice treated with vehicle alone. This beneficial effect was accompanied by enhanced hepatic insulin signaling, decreased expression of gluconeogenic genes and increased expression of glycogenic genes. Mechanistically, we show that insulin-stimulated phosphorylation of insulin receptor ß (IRß) is impaired in primary Cyp2c44(-/-) hepatocytes and this can be restored by cotreatment with EET-A and insulin. Plasma membrane fractionations of livers indicated that EET-A enhances the retention of IRß in membrane rich fractions, thus potentiating its activation. Altogether, EET analogs ameliorate insulin signaling in a genetic model of hepatic insulin resistance by stabilizing membrane-associated IRß and potentiating insulin signaling.

GSK2256294 Decreases sEH (Soluble Epoxide Hydrolase) Activity in Plasma, Muscle, and Adipose and Reduces F2-Isoprostanes but Does Not Alter Insulin Sensitivity in Humans.

[Figure: see text].

Treatment of Primary Aldosteronism Increases Plasma Epoxyeicosatrienoic Acids.

[Figure: see text].

Primary Aldosteronism Decreases Insulin Secretion and Increases Insulin Clearance in Humans.

Primary aldosteronism is a frequent cause of resistant hypertension and is associated with an increased risk of developing diabetes mellitus. Aldosterone impairs insulin secretion in isolated islets, and insulin secretion is increased in aldosterone synthase-deficient mice. We hypothesized that treatment for primary aldosteronism increases insulin secretion and insulin sensitivity in humans. We conducted a prospective cohort study in patients with primary aldosteronism, with assessment of glucose metabolism before and 3 to 12 months after treatment. Participants underwent treatment for primary aldosteronism with adrenalectomy or a mineralocorticoid receptor antagonist at the discretion of their treating physician. We assessed insulin secretion and insulin sensitivity by hyperglycemic and hyperinsulinemic-euglycemic clamps, respectively, on 2 study days after a 5-day standardized diet. After treatment, the C-peptide and insulin response during the hyperglycemic clamp increased compared with pretreatment (ΔC-peptide at 90-120 minutes +530.5±384.1 pmol/L, P=0.004; Δinsulin 90-120 minutes +183.0±122.6, P=0.004). During hyperinsulinemic-euglycemic clamps, insulin sensitivity decreased after treatment (insulin sensitivity index 30.7±6.2 versus 18.5±4.7 nmol·kg-1·min-1·pmol-1·L; P=0.02). Insulin clearance decreased after treatment (872.8±207.6 versus 632.3±178.6 mL/min; P=0.03), and disposition index was unchanged. We conclude that the insulin response to glucose increases and insulin clearance decreases after treatment for primary aldosteronism, and these effects were not due to alterations in creatinine clearance or plasma cortisol. These studies may provide further insight into the mechanism of increased diabetes mellitus risk in primary aldosteronism.

Structure and physiological function of the human KCNQ1 channel voltage sensor intermediate state.

Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevisoocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

Early urine electrolyte patterns in patients with acute heart failure.

AIMS: We conducted a prospective study of emergency department (ED) patients with acute heart failure (AHF) to determine if worsening HF (WHF) could be predicted based on urinary electrolytes during the first 1-2 h of ED care. Loop diuretics are standard therapy for AHF patients. A subset of patients hospitalized for AHF will develop a blunted natriuretic response to loop diuretics, termed diuretic resistance, which often leads to WHF. Early detection of diuretic resistance could facilitate escalation of therapy and prevention of WHF. METHODS AND RESULTS: Patients were eligible if they had an ED AHF diagnosis, had not yet received intravenous diuretics, had a systolic blood pressure > 90 mmHg, and were not on dialysis. Urine electrolytes and urine output were collected at 1, 2, 4, and 6 h after diuretic administration. Worsening HF was defined as clinically persistent or WHF requiring escalation of diuretics or administration of intravenous vasoactives after the ED stay. Of the 61 patients who qualified in this pilot study, there were 10 (16.3%) patients who fulfilled our definition of WHF. At 1 h after diuretic administration, patients who developed WHF were more likely to have low urinary sodium (9.5 vs. 43.0 mmol; P < 0.001) and decreased urine sodium concentration (48 vs. 80 mmol/L; P = 0.004) than patients without WHF. All patients with WHF had a total urine sodium of <35.4 mmol at 1 h (100% sensitivity and 60% specificity). CONCLUSIONS: One hour after diuretic administration, a urine sodium excretion of <35.4 mmol was highly suggestive of the development of WHF. These relationships require further testing to determine if early intervention with alternative agents can prevent WHF.

A pH-Mediated Topological Switch within the N-Terminal Domain of Human Caveolin-3.

Caveolins mediate the formation of caveolae, which are small omega-shaped membrane invaginations involved in a variety of cellular processes. There are three caveolin isoforms, the third of which (Cav3) is expressed in smooth and skeletal muscles. Mutations in Cav3 cause a variety of human muscular diseases. In this work, we characterize the secondary structure, dynamics, and topology of the monomeric form of the full-length lipidated protein. Cav3 consists of a series of membrane-embedded or surface-associated helical elements connected by extramembrane connecting loops or disordered domains. Our results also reveal that the N-terminal domain undergoes a large scale pH-mediated topological rearrangement between soluble and membrane-anchored forms. Considering that roughly one-third of pathogenic mutations in Cav3 influence charged residues located in this domain, we hypothesize that this transition is likely to be relevant to the molecular basis of Cav3-linked diseases. These results provide insight into the structure of Cav3 and set the stage for mechanistic investigations of the effects of pathogenic mutations.

Biophysical characterization of interactions between the C-termini of peripheral nerve claudins and the PDZ1 domain of zonula occludens.

Our recent study has shown that cellular junctions in myelin and in the epi-/perineruium that encase nerve fibers regulate the permeability of the peripheral nerves. This permeability may affect propagation of the action potential. Direct interactions between the PDZ1 domain of zonula occludens (ZO1 or ZO2) and the C-termini of claudins are known to be crucial for the formation of tight junctions. Using the purified PDZ1 domain of ZO2 and a variety of C-terminal mutants of peripheral nerve claudins (claudin-1, claudin-2, claudin-3, claudin-5 in epi-/perineurium; claudin-19 in myelin), we have utilized NMR spectroscopy to determine specific roles of the 3 C-terminal claudin residues (position -2, -1, 0) for their interactions with PDZ1 of ZO2. In contrast to the canonical model that emphasizes the importance of residues at the -2 and 0 positions, our results demonstrate that, for peripheral nerve claudins, the residue at position -1 plays a critical role in association with PDZ1, while the side-chain of residue 0 plays a significant but lesser role. Surprisingly, claudin-19, the most abundant claudin in myelin, exhibited no binding to ZO2. These findings reveal that the binding mechanism of claudin/ZO in epi-/perineurium is distinct from the canonical interactions between non-ZO PDZ-containing proteins with their ligands. This observation provides the molecular basis for a strategy to develop drugs that target tight junctions in the epi-/perineurium of peripheral nerves.

Structural and functional insights into the N-terminus of Schizosaccharomyces pombe Cdc5.

The spliceosome is a dynamic macromolecular machine composed of five small nuclear ribonucleoparticles (snRNPs), the NineTeen Complex (NTC), and other proteins that catalyze the removal of introns mature to form the mature message. The NTC, named after its founding member Saccharomyces cerevisiae Prp19, is a conserved spliceosome subcomplex composed of at least nine proteins. During spliceosome assembly, the transition to an active spliceosome correlates with stable binding of the NTC, although the mechanism of NTC function is not understood. Schizosaccharomyces pombe Cdc5, a core subunit of the NTC, is an essential protein required for pre-mRNA splicing. The highly conserved Cdc5 N-terminus contains two canonical Myb (myeloblastosis) repeats (R1 and R2) and a third domain (D3) that was previously classified as a Myb-like repeat. Although the N-terminus of Cdc5 is required for its function, how R1, R2, and D3 each contribute to functionality is unclear. Using a combination of yeast genetics, structural approaches, and RNA binding assays, we show that R1, R2, and D3 are all required for the function of Cdc5 in cells. We also show that the N-terminus of Cdc5 binds RNA in vitro. Structural and functional analyses of Cdc5-D3 show that, while this domain does not adopt a Myb fold, Cdc5-D3 preferentially binds double-stranded RNA. Our data suggest that the Cdc5 N-terminus interacts with RNA structures proposed to be near the catalytic core of the spliceosome.

Modest effects of lipid modifications on the structure of caveolin-3.

Caveolin-3 (Cav3) is an unconventional membrane protein that serves as a critical scaffolding hub in caveolae and is genetically linked to various muscle disorders. In this work, we report the expression, purification, and characterization of full-length human Cav3. To mimic the palmitoylation of endogenous Cav3, we developed a generally applicable approach to covalently attached thioalkyl chains at natively modified cysteine residues. Nuclear magnetic resonance measurements indicate that lipidation exerts only a modest and local effect on the Cav3 structure, with little impact on the structures of the N-terminal domain, the scaffolding domain, and the extreme C-terminus.

Purification and structural study of the voltage-sensor domain of the human KCNQ1 potassium ion channel.

KCNQ1 (also known as KV7.1 or KVLQT1) is a voltage-gated potassium channel modulated by members of the KCNE protein family. Among multiple functions, KCNQ1 plays a critical role in the cardiac action potential. This channel is also subject to inherited mutations that cause certain cardiac arrhythmias and deafness. In this study, we report the overexpression, purification, and preliminary structural characterization of the voltage-sensor domain (VSD) of human KCNQ1 (Q1-VSD). Q1-VSD was expressed in Escherichia coli and purified into lyso-palmitoylphosphatidylglycerol micelles, conditions under which this tetraspan membrane protein yields excellent nuclear magnetic resonance (NMR) spectra. NMR studies reveal that Q1-VSD shares a common overall topology with other channel VSDs, with an S0 helix followed by transmembrane helices S1-S4. The exact sequential locations of the helical spans do, however, show significant variations from those of the homologous segments of previously characterized VSDs. The S4 segment of Q1-VSD was seen to be α-helical (with no 310 component) and underwent rapid backbone amide H-D exchange over most of its length. These results lay the foundation for more advanced structural studies and can be used to generate testable hypotheses for future structure-function experiments.

Structure and expression of a novel compact myelin protein - small VCP-interacting protein (SVIP).

SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

Reversible folding of human peripheral myelin protein 22, a tetraspan membrane protein.

Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22.

Solution NMR characterization of magnetic/electronic properties of azide and cyanide-inhibited substrate complexes of human heme oxygenase: implications for steric ligand tilt.

Solution 2D (1)H NMR was carried out on the azide-ligated substrate complex of human heme oxygenase, hHO, to provide information on the active site molecular structure, chromophore electronic/magnetic properties, and the distal H-bond network linked to the exogenous ligand by catalytically relevant oriented water molecules. While 2D NMR exhibited very similar patterns of two-dimensional nuclear Overhauser spectroscopy cross peaks of residues with substrate and among residues as the previously characterized cyanide complex, significant, broadly distributed chemical shift differences were observed for both labile and non-labile protons. The anisotropy and orientation of the paramagnetic susceptibility tensor, χ, were determined for both the azide and cyanide complexes. The most significant difference observed is the tilt of the major magnetic axes from the heme normal, which is only half as large for the azide than cyanide ligand, with each ligand tilted toward the catalytically cleaved α-meso position. The difference in chemical shifts is quantitatively correlated with differences in dipolar shifts in the respective complexes for all but the distal helix. The necessity of considering dipolar shifts, and hence determination of the orientation/anisotropy of χ, in comparing chemical shifts involving paramagnetic complexes, is emphasized. The analysis shows that the H-bond network cannot detect significant differences in H-bond acceptor properties of cyanide versus azide ligands. Lastly, significant retardation of distal helix labile proton exchange upon replacing cyanide with azide indicates that the dynamic stability of the distal helix is increased upon decreasing the steric interaction of the ligand with the distal helix.

The quiet renaissance of protein nuclear magnetic resonance.

From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 1980s.

Role of propionates in substrate binding to heme oxygenase from Neisseria meningitidis: a nuclear magnetic resonance study.

Heme oxygenase (HO) cleaves hemin into biliverdin, iron, and CO. For mammalian HOs, both native hemin propionates are required for substrate binding and activity. The HO from the pathogenic bacterium Neisseria meningitidis (NmHO) possesses a crystallographically undetected C-terminal fragment that by solution (1)H nuclear magnetic resonance (NMR) is found to fold and interact with the active site. One of the substrate propionates has been proposed to form a salt bridge to the C-terminus rather than to the conventional buried cationic side chain in other HOs. Moreover, the C-terminal dipeptide Arg208His209 cleaves spontaneously over ~24 h at a rate dependent on substituent size. Two-dimensional (1)H NMR of NmHO azide complexes with hemins with selectively deleted or rearranged propionates shows that all bind to NmHO with a structurally conserved active site as reflected in optical spectra and NMR nuclear Overhauser effect spectroscopy cross-peak and hyperfine shift patterns. In contrast to mammalian HOs, NmHO requires only a single propionate interacting with the buried terminus of Lys16 to exhibit full activity and tolerates the existence of a propionate at the exposed 8-position. The structure of the C-terminus is qualitatively retained upon deletion of the 7-propionate, but a dramatic change in the 7-propionate carboxylate (13)C chemical shift upon C-terminal cleavage confirms its role in the interaction with the C-terminus. The stronger hydrophobic contacts between pyrroles A and B with NmHO contribute more substantially to the substrate binding free energy than in mammalian HOs, "liberating" one propionate to stabilize the C-terminus. The functional implications of the C-terminus in product release are discussed.

Impact of heat treatment on size, structure, and bioactivity of elemental selenium nanoparticles.

BACKGROUND: Elemental selenium nanoparticles have emerged as a novel selenium source with the advantage of reduced risk of selenium toxicity. The present work investigated whether heat treatment affects the size, structure, and bioactivity of selenium nanoparticles. METHODS AND RESULTS: After a one-hour incubation of solution containing 80 nm selenium particles in a 90°C water bath, the nanoparticles aggregated into larger 110 nm particles and nanorods (290 nm × 70 nm), leading to significantly reduced bioavailability and phase II enzyme induction in selenium-deficient mice. When a solution containing 40 nm selenium nanoparticles was treated under the same conditions, the nanoparticles aggregated into larger 72 nm particles but did not transform into nanorods, demonstrating that the thermostability of selenium nanoparticles is size-dependent, smaller selenium nanoparticles being more resistant than larger selenium nanoparticles to transformation into nanorods during heat treatment. CONCLUSION: The present results suggest that temperature and duration of the heat process, as well as the original nanoparticle size, should be carefully selected when a solution containing selenium nanoparticles is added to functional foods.