Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

The complex relationship between late-onset caloric restriction and synaptic plasticity in aged Wistar rats.

Age-related reduction in spine density, synaptic marker expression, and synaptic efficiency are frequently reported. These changes provide the cellular and molecular basis for the cognitive decline characteristic for old age. Nevertheless, there are several approaches that have the potential to ameliorate these processes and improve cognition, caloric restriction being one of the most promising and widely studied. While lifelong caloric restriction is known for its numerous beneficial effects, including improved cognitive abilities and increased expression of proteins essential for synaptic structure and function, the effects of late-onset and/or short-term CR on synaptic plasticity have yet to be investigated. We have previously documented that the effects of CR are strongly dependent on whether CR is initiated in young or old subjects. With this in mind, we conducted a long-term study in aging Wistar rats to examine changes in the expression of several key synaptic markers under the regimen of CR started at different time points in life. We found a significant increase in the expression of both presynaptic and postsynaptic markers. However, taking into account previously reported changes in the behavior detected in these animals, we consider that this increase cannot represent beneficial effect of CR.

Spectroelectrochemistry for determination of the redox potential in heme enzymes: Dye-decolorizing peroxidases.

Dye-decolorizing peroxidases (DyPs) are heme-containing enzymes that are structurally unrelated to other peroxidases. Some DyPs show high potential for applications in biotechnology, which critically depends on the stability and redox potential (E°') of the enzyme. Here we provide a comparative analysis of UV-Vis- and surface-enhanced resonance Raman-based spectroelectrochemical methods for determination of the E°' of DyPs from two different organisms, and their variants generated targeting E°' upshift. We show that substituting the highly conserved Arginine in the distal side of the heme pocket by hydrophobic amino acid residues impacts the heme architecture and redox potential of DyPs from the two organisms in a very distinct manner. We demonstrate the advantages and drawbacks of the used spectroelectrochemical approaches, which is relevant for other heme proteins that contain multiple heme centers or spin populations.

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

Dietary restriction alters insulin signaling pathway in the brain.

Insulin is known to be a key hormone in the regulation of peripheral glucose homeostasis, but beyond that, its effects on the brain are now undisputed. Impairments in insulin signaling in the brain, including changes in insulin levels, are thought to contribute significantly to declines in cognitive performance, especially during aging. As one of the most widely studied experimental interventions, dietary restriction (DR) is considered to delay the neurodegenerative processes associated with aging. Recently, however, data began to suggest that the onset and duration of a restrictive diet play a critical role in the putative beneficial outcome. Because the effects of DR on insulin signaling in the brain have been poorly studied, we decided to examine the effects of DR that differed in onset and duration: long-term DR (LTDR), medium-term DR (MTDR), and short-term DR (STDR) on the expression of proteins involved in insulin signaling in the hippocampus of 18- and 24-month-old male Wistar rats. We found that DR-induced changes in insulin levels in the brain may be independent of what happens in the periphery after restricted feeding. Significantly changed insulin content in the hippocampus, together with altered insulin signaling were found under the influence of DR, but the outcome was highly dependent on the onset and duration of DR.

Exploring substrate interaction in respiratory alternative complex III from Rhodothermus marinus.

Rhodothermus marinus is a thermohalophilic organism that has optimized its microaerobic metabolism at 65 °C. We have been exploring its respiratory chain and observed the existence of a quinone:cytochrome c oxidoreductase complex, named Alternative Complex III, structurally different from the bc1 complex. In the present work, we took profit from nanodiscs and liposomes technology to investigate ACIII activity in membrane-mimicking systems. In addition, we studied the interaction of ACIII with menaquinone, its potential electron acceptors (HiPIP and cytochrome c) and the caa3 oxygen reductase.

Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER.

Bacterial nucleotide excision repair (NER), mediated by the UvrA, UvrB and UvrC proteins is a multistep, ATP-dependent process, that is responsible for the removal of a very wide range of chemically and structurally diverse DNA lesions. DNA damage removal is performed by UvrC, an enzyme possessing a dual endonuclease activity, capable of incising the DNA on either side of the damaged site to release a short single-stranded DNA fragment containing the lesion. Using biochemical and biophysical approaches, we have probed the oligomeric state, UvrB- and DNA-binding abilities and incision activities of wild-type and mutant constructs of UvrC from the radiation resistant bacterium, Deinococcus radiodurans. Moreover, by combining the power of new structure prediction algorithms and experimental crystallographic data, we have assembled the first model of a complete UvrC, revealing several unexpected structural motifs and in particular, a central inactive RNase H domain acting as a platform for the surrounding domains. In this configuration, UvrC is maintained in a 'closed' inactive state that needs to undergo a major rearrangement to adopt an 'open' active state capable of performing the dual incision reaction. Taken together, this study provides important insight into the mechanism of recruitment and activation of UvrC during NER.

Substrate-Dependent Conformational Switch of the Noncubane [4Fe-4S] Cluster in Heterodisulfide Reductase HdrB.

The noncubane [4Fe-4S] cluster identified in the active site of heterodisulfide reductase (HdrB) displays a unique geometry among Fe-S cofactors found in metalloproteins. Here we employ resonance Raman (RR) spectroscopy and density functional theory (DFT) calculations to probe structural, electronic, and vibrational properties of the noncubane cluster in HdrB from a non-methanogenic Desulfovibrio vulgaris (Dv) Hildenborough organism. The immediate protein environment of the two neighboring clusters in DvHdrB is predicted using homology modeling. We demonstrate that in the absence of substrate, the oxidized [4Fe-4S]3+ cluster adopts a "closed" conformation. Upon substrate coordination at the "special" iron center, the cluster core translates to an "open" structure, facilitated by the "supernumerary" cysteine ligand switch from iron-bridging to iron-terminal mode. The observed RR fingerprint of the noncubane cluster, supported by Fe-S vibrational mode analysis, will advance future studies of enzymes containing this unusual cofactor.

Human endonuclease III/NTH1: focusing on the [4Fe-4S] cluster and the N-terminal domain.

Human Endonuclease III (EndoIII), hNTH1, is an FeS containing enzyme which repairs oxidation damaged bases in DNA. We report here the first comparative biophysical study of full-length and an N-terminally truncated hNTH1, with a domain architecture homologous to bacterial EndoIII. Vibrational spectroscopy, spectroelectrochemistry and SAXS experiments reveal distinct properties of the two enzyme forms, and indicate that the N-terminal domain is important for DNA binding at the onset of damage recognition.

Disentangling Unusual Catalytic Properties and the Role of the [4Fe-4S] Cluster of Three Endonuclease III from the Extremophile D. radiodurans.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities.

Calorie restriction changes the anxiety-like behaviour of ageing male Wistar rats in an onset- and duration-dependent manner.

Although initially recognized as a universally beneficial approach for the prevention of age-related impairments, the outcome of calorie restriction (CR) is now known to depend on several factors, most notably the age of the subject at the CR commencement, and CR duration. We aimed to examine if and how CR affects anxiety-like behaviour when it is introduced at middle age and late middle age. In addition, as the dopaminergic system is one of the main neurotransmitter systems involved in controlling anxiety, we examined the expression of dopamine receptors (D1R, D2R) in the cortex, striatum, and mesencephalon of male Wistar rats of varying ages. The study was performed on rats fed ad libitum (AL) or exposed to calorie restriction (60% of AL intake). Open field and light-dark tests were used to study anxiety-like behaviour, while PCR and Western blot were used to examine the expression of dopamine receptors. Calorie restriction implemented at middle-age led to variable outcomes on anxiety-like behaviour, while CR implemented at late middle age increased anxiety and decreased the availability of D2R levels in the cortex and mesencephalon. Taken together, these results advise caution when implementing calorie restriction late in life.

Late-Onset Calorie Restriction Worsens Cognitive Performances and Increases Frailty Level in Female Wistar Rats.

The current study aims to determine the potential benefits of calorie restriction (CR), one of the most promising paradigms for life span and healthspan extension, on cognitive performances in female Wistar rats during aging. As a measure of a healthspan, we evaluated the effects of different onset and duration of CR on frailty level. Female Wistar rats were exposed to either ad libitum (AL) or CR (60% of AL daily intake) food intake during aging. Two different CR protocols were used, life-long CR with an early-onset that started at the adult stage (6 months) and 3-month-long CR, started at the middle (15 months) and late-middle (21 months) age, thus defined as a late-onset CR. The effects of CR were evaluated using open-field, Y-maze, and novel object recognition tests. We broadened 2 tools for frailty assessment currently in use for experimental animals, and in alignment with our previous study, we created a physical-cognitive frailty tool that combines both physical and cognitive performances. Our results clearly showed that CR effects are highly dependent on CR duration and onset. While a life-long restriction with an early-onset has been proven as protective and beneficial, short-term restriction introduced at late age significantly worsens an animal's behavior and frailty. These results complement our previous study conducted in males and contribute to the understanding of sex differences in a response to CR during aging.

Neuroprotective effects of food restriction in a rat model of traumatic brain injury - the role of glucocorticoid signaling.

OBJECTIVE: Traumatic brain injury (TBI) is one of the most common causes of neurological damage in young and middle aged people. Food restriction (FR) has been shown to act neuroprotectively in animal models of stroke and TBI. Indeed, our previous studies showed that FR attenuates inflammation, through suppression of microglial activation and TNF-α production, suppresses caspase-3-induced neuronal cell death and enhances neuroplasticity in the rat model of TBI. Glucocorticoids (GCs) play a central role in mediating both molecular and behavioral responses to food restriction. However, the exact mechanisms of FR neuroprotection in TBI are still unclear. The goal of the present study was to examine whether FR exerts its beneficial effects by altering the glucocorticoid receptor (GR) signaling alone and/or together with other protective factors. METHODS: To this end, we examined the effects of FR (50% of regular daily food intake for 3 months prior to TBI) on the protein levels of total GR, GR phosphoisoform Ser232 (p-GR) and its transcriptional activity, as well as 11ß-HSD1, NFκB (p65) and HSP70 as factors related to the GR signaling. RESULTS: Our results demonstrate that FR applied prior to TBI significantly changes p-GR levels, and it's transcriptional activity during the recovery period after TBI. Moreover, as a pretreatment, FR modulates other protective factors in response to TBI, such as 11ß-HSD1, NF-κB (p65) and HSP70 that act in parallel with GR in it's anti-inflammatory and neuroprotective effects in the rat model of brain injury. CONCLUSION: Our results suggest that prophylactic FR represents a potent non-invasive approach capable of changing GR signalling, together with other factors related to the GR signaling in the model of TBI.

Unique Biradical Intermediate in the Mechanism of the Heme Enzyme Chlorite Dismutase.

The heme enzyme chlorite dismutase (Cld) catalyzes O-O bond formation as part of the conversion of the toxic chlorite (ClO2 -) to chloride (Cl-) and molecular oxygen (O2). Enzymatic O-O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae (AoCld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. AoCld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, AoCld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at k obs = 2-5 × 104 s-1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of AoCld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety's reorganization and thereby maximize the enzyme's catalytic efficiency.

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices.

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.

Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies.

Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a3 in caa3-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN- ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.

Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. DATABASE: Structural data are available in the PDB database under the accession number 7ANV.

Pharmacological intervention in a transgenic mouse model improves Alzheimer's-associated pathological phenotype: Involvement of proteasome activation.

Alzheimer's disease (AD) is the most common form of dementia worldwide, characterized by a progressive decline in a variety of cognitive and non-cognitive functions. The amyloid beta protein cascade hypothesis places the formation of amyloid beta protein aggregates on the first position in the complex pathological cascade leading to neurodegeneration, and therefore AD might be considered to be a protein-misfolding disease. The Ubiquitin Proteasome System (UPS), being the primary protein degradation mechanism with a fundamental role in the maintenance of proteostasis, has been identified as a putative therapeutic target to delay and/or to decelerate the progression of neurodegenerative disorders that are characterized by accumulated/aggregated proteins. The purpose of this study was to test if the activation of proteasome in vivo can alleviate AD pathology. Specifically by using two compounds with complementary modes of proteasome activation and documented antioxidant and redox regulating properties in the 5xFAD transgenic mice model of AD, we ameliorated a number of AD related deficits. Shortly after proteasome activation we detected significantly reduced amyloid-beta load correlated with improved motor functions, reduced anxiety and frailty level. Essentially, to our knowledge this is the first report to demonstrate a dual activation of the proteasome and its downstream effects. In conclusion, these findings open up new directions for future therapeutic potential of proteasome-mediated proteolysis enhancement.

Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing.

Immobilized dye-decolorizing peroxidase from Pseudomonas putida MET94 (PpDyP) and three variants generated by directed evolution (DE) are studied aiming at the design of a biosensor for H2O2 detection. Structural properties of the enzymes in solution and immobilized state are addressed by resonance Raman (RR) and surface enhanced RR (SERR) spectroscopy, and the electrocatalytic properties are analyzed by electrochemistry. The wild-type (wt) and 29E4 variant (with E188K and H125Y mutations) represent excellent candidates for development of H2O2 biosensors, since they exhibit a good dynamic response range (1-200 µM H2O2), short response times (2 s) and a superior sensitivity (1.3-1.4 A⋅M-1⋅cm-2) for H2O2, as well as selectivity and long term stability. In contrast to the solution state, 6E10 (with E188K, A142V and H125Y mutations) and 25F6 (with E188K, A142V, H125Y and G129D mutations) variants display much lower activity and are inhibited by high concentrations of H2O2 upon adsorption on an electrode. In terms of sensitivity, the bioelectrodes employing wt PpDyP and 29E4 variant outperform HRP based counterparts reported in the literature by 1-4 orders of magnitude. We propose the development of wt or 29E4 PpDyP based biosensor as a valuable alternative to devices that rely on peroxidases.