Redox active motifs in selenoproteins

Selenoproteins use the rare amino acid selenocysteine (Sec) to act as the first line of defense against oxidants, which are linked to aging, cancer, and neurodegenerative diseases. Many selenoproteins are oxidoreductases in which the reactive Sec is connected to a neighboring Cys and able to form a ring. These Sec-containing redox motifs govern much of the reactivity of selenoproteins. To study their fundamental properties, we have used ⁷⁷Se NMR spectroscopy in concert with theoretical calculations to determine the conformational preferences and mobility of representative motifs. This use of ⁷⁷Se as a probe enables the direct recording of the properties of Sec as its environment is systematically changed. We find that all motifs have several ring conformations in their oxidized state. These ring structures are most likely stabilized by weak, nonbonding interactions between the selenium and the amide carbon. To examine how the presence of selenium and ring geometric strain governs the motifs’ reactivity, we measured the redox potentials of Sec-containing motifs and their corresponding Cys-only variants. The comparisons reveal that for C-terminal motifs the redox potentials increased between 20–25 mV when the selenenylsulfide bond was changed to a disulfide bond. Changes of similar magnitude arose when we varied ring size or the motifs’ flanking residues. This suggests that the presence of Sec is not tied to unusually low redox potentials. The unique roles of selenoproteins in human health and their chemical reactivities may therefore not necessarily be explained by lower redox potentials, as has often been claimed.Selenium in biological systems has long been studied for its role in aging, cancer and chronic diseases (1). Selenium is incorporated into proteins via the amino acid selenocysteine (Sec or U), which is structurally similar to cysteine yet more reactive (2). Selenoproteins exploit Sec to catalyze a wide range of electron-transfer reactions (3). Although selenoproteins are rare, they are essential for mammals and have important roles in antioxidant defense and redox regulation (4). The majority of selenoproteins either directly scavenge oxidants (such as the reactive oxygen and nitrogen species that lead to deleterious covalent modification of cellular components), or they may signal the cellular level of these reactive species, thereby activating defense pathways. Selenoproteins are particularly well-suited for the task of combating oxidative stress because they are both sensitive to low levels of oxidants and yet resilient to inactivation by these reactive species (3). Because of their role in longevity and human diseases, there has been sustained interest in deciphering the unique role of Sec in selenoproteins (5). However, it remains largely unclear what sets selenoproteins apart from their cysteine-containing counterparts and why, in mammals, they cannot be replaced by them. Sec commonly forms a selenenylsulfide bond with Cys, resulting in redox motifs analogous to the Cys-containing ones of nonselenoprotein oxidoreductases. Although the latter are well-studied, many of the basic properties of Sec-containing redox motifs are not known. To better understand features modulating the reactivity of selenocysteine, we set out to investigate the redox potential, conformational preferences, and dynamics of Sec-containing redox motifs using mass spectrometry, ⁷⁷Se NMR spectroscopy, and density functional theory (DFT).We have selected four representative redox motifs with different ring sizes and geometric strains. The first and second redox motifs, Ser-Cys-Sec-Ser-COOH (SCUS) and Gly-Cys-Sec-Gly-COOH (GCUG), form a highly constrained eight-membered ring in the oxidized state (6). The GCUG is naturally found in mammalian thioredoxin reductase (TrxR), whereas the more geometrically constrained SCUS motif is related to the SCCS motif in Drosophila melanogaster TrxR (7, 8). Interestingly, similar variations of these TrxR-based motifs have previously been shown to affect kinetic parameters in TrxR isoenzymes (7, 9, 10). The third and fourth motifs were selected to demonstrate effects of increased ring size; the Gly-Cys-Ala-Sec-Gly-COOH (GCAUG) and the Gly-Cys-Gly-Ala-Sec-Gly-COOH (GCGAUG) motifs form less constrained 11- and 14-membered rings, respectively. The size of the ring is directly linked to its chemical reactivity because if the strain is increased, the selenenylsulfide bond is destabilized. This in turn leads to a higher redox potential (more oxidizing), which favors a reduced protein (11, 12). The 14-membered ring is the most ubiquitous motif in the selenoproteome. The motifs were positioned at the C terminus of a redox-inactive stable protein scaffold (see below), noting that many selenoproteins use such C-terminally located active sites (13). Sec and [⁷⁷Se]Sec were incorporated via heterologous expression in Escherichia coli using the native genetic selenium incorporation machinery (14).It should be noted that the overall features of a selenoprotein are dependent upon both the intrinsic physicochemical features of the Sec residue (5, 15), and the interactions of a Sec-containing redox active motif with either substrates or other residues of the active site microenvironment. Context-dependent effects were previously demonstrated with TrxR, where the solely Cys-based motif of the Drosophila enzyme is highly active as part of that enzyme (7, 8) but inactive if placed into the mammalian enzyme (9). Such observations illustrate that the inherent features of Sec- vs. Cys-containing motifs may be difficult to judge within the context of natural selenoproteins and their active sites. Hence, to avoid the contextual difficulties associated with the use of a given specialized active site environment, we fused the four motifs within the flexible C-terminal region of a stable well-folded protein scaffold (here, a redox inactive E. coli thioredoxin mutant), so that we could probe these introduced Sec- or Cys-containing redox active motifs without confounding long-range interactions. Indeed, this approach mirrors the salient features of native selenoproteins with C-terminal redox motifs. Specifically, such native motifs are also located 1–2 aa from the C terminus and use small amino acids as bridging residues (i.e., G, A, V, S, and T). The conformational mobility and influence of the remaining protein skeleton in both model and native proteins are expected to be similar because the C terminus of proteins tends to be unstructured and hence relatively free from geometric constraints placed by secondary structure. In contrast to peptides, the N and C termini of both the native selenoproteins and the model proteins studied here are spatially sufficiently separated, so as to not influence each other (16). Thus, the trends uncovered in this work should be directly applicable to native selenoproteins with C-terminal redox motifs. Furthermore, the methodology used here can be extended to encompass studies of all selenoproteins, including those that do not contain redox motifs.We investigated the conformational preferences and dynamics of the Sec-containing redox active motifs using ⁷⁷Se NMR spectroscopy. This method directly probes the properties of Sec in a given redox motif and is highly sensitive to changes in the electronic environment. ⁷⁷Se has previously been used as a spectroscopic probe of protein structure and function (17–20), but the relation between chemical shifts and the specific location or properties of Sec was not deduced in these earlier studies. Here, DFT calculations of the ⁷⁷Se chemical shift were used to determine conformational preferences of the oxidized selenenylsulfide-containing rings. Such calculations have been shown to correlate well with experimental values and provide an important predictive tool (21, 22). This study is, to our knowledge, the first to combine DFT calculations with experimental selenium NMR spectroscopy to study the environment and molecular properties of selenium in biological systems and lays the foundation for comparable studies of structural and dynamic features of Sec-dependent redox motifs found in native selenoproteins (23).In addition to reporting the NMR parameters and conformational preferences of the selenenylsulfide motifs, we also compared the redox potentials of the Sec-containing proteins with their Cys homologs. This is a critical aspect of the reactivity of many selenoproteins because reduction of the selenenylsulfide liberates the nucleophilic, and catalytically essential, selenolate (R-Se⁻) species. Although it was proposed that the redox potential of selenoproteins might be much lower then their Cys-containing orthologs (15), only a few Sec-containing proteins have been examined experimentally (24–26). Herein, we also provide, to our knowledge, the first quantitative comparisons of redox potentials of Sec-containing to all Cys-containing redox motifs in a range of ring sizes. Surprisingly, these systematic studies reveal a much less dramatic impact of selenium on the redox potential of these centers than was previously believed.     

Cholestasis in active chronic hepatitis

Of 30 patients with active chronic hepatitis studied prospectively over the past 3 years, 14 showed one or more of the following features: prolonged jaundice, pruritus, hypercholesterolemia and elevation of the serum alkaline phosphatase. Six patients had conspicuous clinical and biochemical evidence of cholestasis. Wedge biopsies of the liver were obtained in 3 of these 6 patients, and multiple biopsies in 4 of them, but none of the histologic features characteristic of primary biliary cirrhosis were seen at the time of diagnosis. Antimitochondrial antibodies were absent on repeated testing, except in 1 patient in whom the test became strongly positive 2 years after diagnosis. Repeat biopsy in this patient showed apparent diminution in the number of small bile ducts. In addition, electron microscopic examination of liver biopsies from 2 patients with active chronic hepatitis, without overt evidence of cholestasis, showed ultrastructural evidence of this complication. This study illustrates the overlap which exists between active chronic hepatitis and primary biliary cirrhosis.     

Biologically active polypeptides in milk

Many biologically active polypeptides have been described in the milk of several species. Various functions for these polypeptides in addition to nutrition have been proposed in the maternal body and in the breast-fed infant. These polypeptides are derived from several sources and multiple factors control their secretion into milk as well as their fate in the mother and infant. An increasing body of evidence supports the concept that they may function physiologically.     

Sleep apnea in active acromegaly

Previous case reports have shown an association between acromegaly and the sleep apnea syndrome (SAS). Some of the patients described had central SAS, raising the possibility that an elevation of the growth hormone (GH) level may cause a defect in respiratory drive. We determined the prevalence of SAS in 21 patients with a history of acromegaly. We separated them into two groups based on serum GH concentrations. Ten patients had active acromegaly (mean GH concentration, 62.2 ng/mL; range, 12.6 to 148 ng/mL), while 11 patients had inactive acromegaly (mean GH, 3.2 ng/mL; range, 0.7 to 6.4 ng/mL). Four of the ten patients with active acromegaly had SAS; none of the 11 patients with inactive acromegaly had SAS. Three patients with SAS had the purely obstructive type, and one had the mixed central and obstructive type. The hypercapnic ventilatory response was normal in all patients tested and was not influenced by the GH level. We conclude that SAS is associated with active acromegaly and that the GH level does not affect the hypercapnic ventilatory response. The absence of SAS in successfully treated patients suggests that it may resolve after a normal GH level is restored.     

Chronic active hepatitis in children

Twenty-eight children with chronic active hepatitis are reported. The majority were diagnosed within the last 5 years. Their mode of presentation was usually indistinguishable from acute viral hepatitis (63%). Extrahepatic manifestations were more common in those patients with an insidious onset. All presented sporadic cases without known exposure or secondary spread. The 19 patients tested for hepatitis-associated antigen (HAA) were all negative and SGOT values were higher (mean, 1080 IU/liter) than those reported in adult patients with chronic active hepatitis. Twenty-seven patients had marked elevation of serum gammaglobulin (mean, 4.04 g/100 ml) and 42% had positive LE cells. All patients had histologic features of aggressive hepatitis; in addition, five biopsies also showed areas of submassive lobular necrosis. The initial response to corticosteroid therapy was excellent in all but 3 patients. To date, drug therapy has been suspended in 4 patients for periods of 4 months to 2 years without clinical or biochemical relapse. There have been 4 deaths while on therapy, 2 with hepatic insufficiency and 2 with sepsis. One patient with end-stage liver disease underwent orthotopic liver transplantation and subsequently died.Supported in part by a grant (RR-69) from the General Clinical Research Centers Program of the Division of Research Resources. National Institutes of Health.     

Chemotactic self-caging in active emulsions

A common feature of biological self-organization is how active agents communicate with each other or their environment via chemical signaling. Such communications, mediated by self-generated chemical gradients, have consequences for both individual motility strategies and collective migration patterns. Here, in a purely physicochemical system, we use self-propelling droplets as a model for chemically active particles that modify their environment by leaving chemical footprints, which act as chemorepulsive signals to other droplets. We analyze this communication mechanism quantitatively both on the scale of individual agent–trail collisions as well as on the collective scale where droplets actively remodel their environment while adapting their dynamics to that evolving chemical landscape. We show in experiment and simulation how these interactions cause a transient dynamical arrest in active emulsions where swimmers are caged between each other’s trails of secreted chemicals. Our findings provide insight into the collective dynamics of chemically active particles and yield principles for predicting how negative autochemotaxis shapes their navigation strategy.

Motile microorganisms have evolved to sense their environment and react to external chemical or physical cues via taxis. Specifically, organisms respond to a gradient in the concentration field of a chemical species by chemotaxis (1) or autochemotaxis when the gradient is generated by the organisms themselves (2). In microorganisms, chemotaxis and autochemotaxis guide many collective processes, such as colony migration (3, 4), aggregation (5, 6), or biofilm formation (7), where the emergent complex behavior is governed by the interplay of physical effects and biological processes. Many aggregatory, quorum-sensing (8) behaviors are based on attractive signaling (i.e., positive autochemotaxis). Repulsive signaling (negative autochemotaxis) is of practical importance to efficient space exploration (e.g., when ant colonies forage using mutual avoidance) (9).Complex collective behavior can result from intricate biological mechanisms but also, can be solely caused by nonequilibrium dynamics (refs. 10–14 have such examples), such that there is a need to untangle physics and biology. To this end, current research in artificial active matter aims to design and develop synthetic microswimmers that can mimic strategies like chemotaxis by purely physicochemical means (15). Self-phoretic particles, which propel via a self-generated local chemical gradient (16–18), are widely studied in theory and experiment. Suspensions of these particles exhibit nontrivial dynamics influenced by autochemotaxis (19–24). Specifically, self-propelling droplets (25) provide an experimental model for repulsive chemical signaling (12, 26–28). Along their way, the droplets shed a persistent trail of depleted fuel, which acts as a chemorepellent to other droplets. Hence, the motion of such a droplet is affected by the previous passage of another droplet.In this study, we show that in an active emulsion, droplets remodel their chemical environment while adapting their dynamics to that evolving resource landscape. In spirit, this resembles Pseudomonas aeruginosa organizing their interactions by shedding attractive trails (7, 29), with the difference that droplet trails are chemorepulsive. We start by a quantitative analysis of individual “delayed collision” events in quasi–two-dimensional (quasi-2D) confinement; we directly visualize and map the chemical footprints of droplets and measure the diffusion coefficient of the constituent chemicals. We use these results to fit a generic analytical model in 2D based on time delay, angle of impact, and chemical coupling strength. We show that these parameters determine whether a droplet crosses a chemical trail or rebounds from it. We then proceed to the collective dynamics, comparing experimental data with simulations using our single-event fits. We demonstrate how such individual binary collisions cause autochemotactic arrest in ensembles of chemically active droplets, a kind of “history caging,” where swimmers are transiently trapped in an evolving network of repulsive trails. We finally address the question of whether this type of caging is possible in three dimensions (3D) via experiments in density-matched bulk media.     

Minocycline in active rheumatoid arthritis

Objective. To determine the efficacy of minocycline in the treatment of rheumatoid arthritis (RA). Methods. Minocycline (maximal oral daily dose 200 mg) or placebo was administered in a 26-week, randomized, double-blind study to 80 patients with active RA, who were treated or had previously been treated with at least one disease-modifying antirheumatic drug. Results. There were 15 premature discontinuations: 6 (5 taking minocycline) because of adverse effects, 8 (all taking placebo) because of lack of efficacy, and 1 (taking placebo) because of intercurrent illness. There was a statistically significant improvement in the minocycline group over the placebo group. There was a pronounced improvement in laboratory parameters of disease activity; however, improvement in clinical parameters was less impressive. The observed adverse effects attributable to minocycline were mainly gastrointestinal symptoms and dizziness. Conclusion. The results of the present study suggest that minocycline is beneficial and relatively safe in RA patients.     

Monoclonal gammopathy in chronic active hepatitis

ABSTRACT— Immunoelectrophoresis was performed in 31 of 272 patients with chronic active hepatitis (CAH) because of an M-spike component (seven patients, 2.6%) or hypergammaglobulinemia (24 patients) revealing a monoclonal gammopathy (MG) in 11 patients. In addition, 50 randomly selected patients with CAH and no evidence for an M-spike component were tested by immunoelectrophoresis. In 13 patients (26%), an MG was found. The mean age of the 24 patients with MG was 57.4 years (range: 23–76). HBsAg was present in nine patients (37.5%), no HBV-marker was detected in ten patients (41.7%). The immunoglobulin class of MG was IgG in ten patients (41.7%), IgA in one patient (4.2%) and IgM in 11 patients (45.8%). In two patients, Bence Jones protein was found in either serum or urine. In only one patient was the MG associated with multiple myeloma, whereas none of the other 23 patients developed a malignant lymphoproliferative disease within the median observation period of 6 years. We conclude that there is an unexpectedly high prevalence of benign MG in patients with CAH.     

Chronic active hepatitis in the elderly

Five patients with autoimmune chronic active hepatitis were diagnosed for the first time over the age of 70 years. They tolerated diagnostic and follow-up liver biopsies well and made gratifying responses to conventional immunosuppressive therapy when indicated. Autoimmune chronic active hepatitis certainly occurs in the elderly and management should be similar to younger patients. We see no reason to withhold immunosuppressive agents.     

Frozen steady states in active systems

Even simple active systems can show a plethora of intriguing phenomena and often we find complexity where we would have expected simplicity. One striking example is the occurrence of a quiescent or absorbing state with frozen fluctuations that at first sight seems to be impossible for active matter driven by the incessant input of energy. While such states were reported for externally driven systems through macroscopic shear or agitation, the investigation of frozen active states in inherently active systems like cytoskeletal suspensions or active gels is still at large. Using high-density motility assay experiments, we demonstrate that frozen steady states can arise in active systems if active transport is coupled to growth processes. The experiments are complemented by agent-based simulations which identify the coupling between self-organization, growth, and mechanical properties to be responsible for the pattern formation process.     

Hypercalcemia in active pulmonary tuberculosis.

We ascertained the incidence of hypercalcemia in 79 consecutive patients with active pulmonary tuberculosis and a control group of 79 patients with chronic obstructive pulmonary disease. Twenty-two patients developed hypercalcemia (serum calcium greater than 10.5 mg/dl) within 4 to 16 weeks after initiation of chemotherapy for tuberculosis. The duration of hypercalcemia ranged from 1 to 7 months, and remission occurred spontaneously in all patients. The mean daily vitamin D supplement was greater in hypercalcemic patients than in the normocalcemic group. There was a positive correlation between daily vitamin D supplement and degree and duration of hypercalcemia. Mean serum calcium in patients with tuberculosis was higher than in patients with chronic obstructive pulmonary disease supplemented with the same dose of vitamin D. Hypercalcemia appears to be related to the activity of pulmonary tuberculosis and the intake of vitamin D; the exact mechanism, however, remains unknown.