Structural insights into the modulation Of SOD1 aggregation By a fungal metabolite Phialomustin-B: Therapeutic potential in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal human motor neuron disease leading to muscle atrophy and paralysis. Mutations in superoxide dismutase 1 (SOD1) are associated with familial ALS (fALS). The SOD1 mutants in ALS have a toxic-gain of function by destabilizing the functional SOD1 homodimer, consequently inducing fibril-like aggregation with a cytotoxic non-native trimer intermediate. Therefore, reducing SOD1 oligomerization via chemical modulators is an optimal therapy in ALS. Here, we report the discovery of Phialomustin-B, an unsaturated secondary metabolite from the endophytic fungus Phialophora mustea, as a modulator of SOD1 aggregation. The crystal structure of the SOD1-Phialomustin complex refined to 1.90 Å resolution demonstrated for the first time that the ligand binds to the dimer interface and the lateral region near the electrostatic loop. The aggregation analyses of SOD1WT and the disease mutant SOD1A4V revealed that Phialomustin-B reduces cytotoxic trimerization. We propose that Phialomustin-B is a potent lead molecule with therapeutic potential in fALS.

Pseudogap in BaPbxBi1‒xO3(x = 0.7, 0.75 and 1.0).

In this work, we have investigated the crystal and electronic structure of the orthorhombic phase of BaPbxBi1-xO3(BPBO) forx = 0.7 (BPBO70), 0.75 (BPBO75) and 1.0 (BPO), using temperature dependent x-ray diffraction measurements, photoemission spectroscopy, and electronic structure calculations. Our results show the importance of particle size and strain in governing superconductivity. Interestingly, the temperature evolution of the structural parameters in the case of BPBO70 is similar to that of BPBO75 but the magnitude of the change is diminished. The BPBO75 and BPO compounds exhibit metallic nature, which is corroborated by the core level studies. The electronic structure calculations in conjunction with the core level studies suggest that oxygen vacancies play an important role for metallicity observed in the end compound. The exponent to the spectral line shape close to the Fermi level suggests the origin of pseudogap to be due to other contributions in addition to disorder in the case of BPBO70 and BPO. The core level studies also show that as one goes fromx = 0.70 to 1.0, there occurs chemical potential shift towards the valence band suggesting hole doping. Our results open the venue to further study these compounds as a function of particle size, nature of carriers for its transport behaviour, electronic structure belowTC, composition at the grain boundaries and microscopic origin of pseudogap in the non-superconducting phase. We believe that our results call for a revision of the temperature-doping phase diagram of BPBO to include the pseudogap phase.

Neuroanatomical zones of human traumatic brain injury reveal significant differences in protein profile and protein oxidation: Implications for secondary injury events.

Traumatic brain injury (TBI) causes significant neurological deficits and long-term degenerative changes. Primary injury in TBI entails distinct neuroanatomical zones, i.e., contusion (Ct) and pericontusion (PC). Their dynamic expansion could contribute to unpredictable neurological deterioration in patients. Molecular characterization of these zones compared with away from contusion (AC) zone is invaluable for TBI management. Using proteomics-based approach, we were able to distinguish Ct, PC and AC zones in human TBI brains. Ct was associated with structural changes (blood-brain barrier (BBB) disruption, neuroinflammation, axonal injury, demyelination and ferroptosis), while PC was associated with initial events of secondary injury (glutamate excitotoxicity, glial activation, accumulation of cytoskeleton proteins, oxidative stress, endocytosis) and AC displayed mitochondrial dysfunction that could contribute to secondary injury events and trigger long-term degenerative changes. Phosphoproteome analysis in these zones revealed that certain differentially phosphorylated proteins synergistically contribute to the injury events along with the differentially expressed proteins. Non-synaptic mitochondria (ns-mito) was associated with relatively more differentially expressed proteins (DEPs) compared to synaptosomes (Syn), while the latter displayed increased protein oxidation including tryptophan (Trp) oxidation. Proteomic analysis of immunocaptured complex I (CI) from Syn revealed increased Trp oxidation in Ct > PC > AC (vs. control). Oxidized W272 in the ND1 subunit of CI, revealed local conformational changes in ND1 and the neighboring subunits, as indicated by molecular dynamics simulation (MDS). Taken together, neuroanatomical zones in TBI show distinct protein profile and protein oxidation representing different primary and secondary injury events with potential implications for TBI pathology and neurological status of the patients.

Mitochondrial Complex I Inhibition in Dopaminergic Neurons Causes Altered Protein Profile and Protein Oxidation: Implications for Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis in a dopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.

Eustachian tube dysfunction in an intensive care unit: A prospective observational study.

Background: Eustachain tube (ET) dysfunction can aggravate the morbidity in ICU patients, but is often ignored. In this prospective observational study we followed a cohort of patients (without pre-existing ET dysfunction) requiring in-patient management, hospitalized either to ICU or to non ICU wards, for any development of ET dysfunction during hospitalization. Methods: Patients requiring hospitalization to ICU or non ICU wards from Dec 2018 to Jun 2019 were included. Those with pre-existing ET dysfunction, disease of ear/nose or sinuses, head neck tumours and history of radiotherapy or glucocorticoid therapy were excluded. All patients were evaluated by serial tympanometry. Type A curve was considered normal while type B, C1 and C2 were considered as ET dysfunction. Results: There were 385 patients, 258 (67.01%) males and 127 (32.99%) females in the ICU group; while non ICU group comprised 129 patients, 86 (66.67%) males and 43 (33.33%) females. ET dysfunction developed in 107 (27.79%) patients in ICU group, but only in 3 (2.3%) in non ICU group (Relative risk 11.95, 95% CI 3.86 to 36.99, P < 0.0001). Within ICU, ET dysfunction showed significant positive association with endotracheal intubation, Ramsay sedation score and number of days on tracheostomy; but not with age, male gender, number of days in ICU, mechanical ventilation or route of feeding. Conclusion: Our study demonstrates high incidence of ET dysfunction in patients admitted to ICU.

Chlorhexidine as a Keap1-Nrf2 inhibitor: a new target for an old drug for Parkinson's disease therapy.

Oxidative stress plays a vital role in the pathophysiology of most neurodegenerative diseases such as Parkinson's disease (PD). The Keap1-Nrf2-ARE pathway, one of the internal defense mechanisms, curbs the reactive oxygen species (ROS) generated in the cellular environment. The pathway leads to the expression of antioxidant genes such as HO-1, GCLC, and NQO1, which act as cellular redox switches and protect the cellular environment. Keap1, the negative regulator of Nrf2, is a potential therapeutic target for treating age-related neurodegenerative diseases. Tecfidera (Dimethyl fumarate), used in the intervention for relapsing multiple sclerosis, is the only commercial drug known to regulate the Nrf2 function. Here, we have identified a repurposing drug, chlorhexidine (LBP125), through ligand-based pharmacophore development and screening against the DrugBank, as a potential inhibitor of the ß-propeller domain of Keap1 (Keap1-DC). Chlorhexidine, an antimicrobial agent, is widely used as a mouthwash, skin cleanser, and intervening bacterial infection during childbirth. The biochemical assay confirmed a significant binding affinity of 30 µM and competitively inhibited the Nrf2 peptide interaction. Moreover, chlorhexidine also exerts cytoprotection in a neurotoxic cell model of PD through Keap1-Nrf2 disruption leading to nuclear translocation of Nrf2 and expression of downstream genes, HO-1, and NQO1. Hence, the chemical scaffold of chlorhexidine is a potential lead to develop new chemical libraries with drug-like properties for treating PD.Communicated by Ramaswamy H. Sarma.

Evidence of lattice strain as a precursor to superconductivity in BaPb0.75Bi0.25O3.

In this work, we have investigated the precursor effects to superconductivity in BaPb0.75Bi0.25O3using temperature dependent resistivity, x-ray diffraction technique and photoemission spectroscopy. The present compound exhibits superconductivity around 11 K (TC). The synthesis procedure adopted is much simpler as compared to the procedure available in the literature. In the temperature range (10 K-25 K) i.e. aboveTC, our results show an increase in both the orthorhombic and tetragonal strain. The well screened features observed in Bi and Pb 4f7/2core levels are indicative of the metallic nature of the sample. The compound exhibits finite intensity at the Fermi level at 300 K and this intensity decreases with decrease in temperature and develops into a pseudogap; the energy dependence of the spectral density of states suggests disordered metallic state. Furthermore, our band structure calculations reveal that the structural transition upon Pb doping results in the closing of the band gap at the Fermi level.

Regional heterogeneity in mitochondrial function underlies region specific vulnerability in human brain ageing: Implications for neurodegeneration.

Selective neuronal vulnerability (SNV) of specific neuroanatomical regions such as frontal cortex (FC) and hippocampus (HC) is characteristic of age-associated neurodegenerative diseases (NDDs), although its pathogenetic basis remains unresolved. We hypothesized that physiological differences in mitochondrial function in neuroanatomical regions could contribute to SNV. To investigate this, we evaluated mitochondrial function in human brains (age range:1-90 y) in FC, striatum (ST), HC, cerebellum (CB) and medulla oblongata (MD), using enzyme assays and quantitative proteomics. Striking differences were noted in resistant regions- MD and CB compared to the vulnerable regions- FC, HC and ST. At younger age (25 ± 5 y), higher activity of electron transport chain enzymes and upregulation of metabolic and antioxidant proteins were noted in MD compared to FC and HC, that was sustained with increasing age (≥65 y). In contrast, the expression of synaptic proteins was higher in FC, HC and ST (vs. MD). In line with this, quantitative phospho-proteomics revealed activation of upstream regulators (ERS, PPARα) of mitochondrial metabolism and inhibition of synaptic pathways in MD. Microtubule Associated Protein Tau (MAPT) showed overexpression in FC, HC and ST both in young and older age (vs. MD). MAPT hyperphosphorylation and the activation of its kinases were noted in FC and HC with age. Our study demonstrates that regional heterogeneity in mitochondrial and other cellular functions contribute to SNV and protect regions such as MD, while rendering FC and HC vulnerable to NDDs. The findings also support the "last in, first out" hypothesis of ageing, wherein regions such as FC, that are the most recent to develop phylogenetically and ontogenetically, are the first to be affected in ageing and NDDs.

Mitochondrial Dysfunction in Rabies Virus-Infected Human and Canine Brains.

Rabies is a fatal encephalitis caused by the Rabies lyssavirus (RABV). The presence of minimal neuropathological changes observed in rabies indicates that neuronal dysfunction, rather than neuronal death contributes to the fatal outcome. The role of mitochondrial changes has been suggested as a possible mechanism for neuronal dysfunction in rabies. However, these findings are mostly based on studies that have employed experimental models and laboratory-adapted virus. Studies on brain tissues from naturally infected human and animal hosts are lacking. The current study investigated the role of mitochondrial changes in rabies by morphological, biochemical and proteomic analysis of RABV-infected human and canine brains. Morphological analysis showed minimal inflammation with preserved neuronal and disrupted mitochondrial structure in both human and canine brains. Proteomic analysis revealed involvement of mitochondrial processes (oxidative phosphorylation, cristae formation, homeostasis and transport), synaptic proteins and autophagic pathways, with over-expression of subunits of mitochondrial respiratory complexes. Consistent with these findings, human and canine brains displayed elevated activities of complexes I (p < 0.05), IV (p < 0.05) and V (p < 0.05). However, this did not result in elevated ATP production (p < 0.0001), probably due to lowered mitochondrial membrane potential as noted in RABV-infected cells in culture. These could lead to mitochondrial dysfunction and mitophagy as indicated by expression of FKBP8 (p < 0.05) and PINK1 (p < 0.001)/PARKIN (p > 0.05) and ensuing autophagy, as shown by the status of LCIII (p < 0.05), LAMP1 (p < 0.001) and pertinent ultrastructural markers. We propose that altered mitochondrial bioenergetics and cristae architecture probably induce mitophagy, leading to autophagy and consequent neuronal dysfunction in rabies.

Effects of early life stress during stress hyporesponsive period (SHRP) on anxiety and curiosity in adolescent rats.

Repeated exposure to adverse experiences in early life, termed Early Life Stress (ELS), can increase anxiety disorders later in life. Anxiety is directly associated with curiosity, a form of intrinsic drive state associated with increased novelty-seeking behaviour and risk taking for challenging opportunities and could probably modulate learning and memory. In humans, elevated curiosity during adolescence tends to elicit increased exploration, novelty seeking, high risk-taking behaviour and heightened emotionality. Such behaviours are beneficial in maintaining social skills and cognitive functions later in life. We investigated whether ELS-induced anxiety impacts curiosity-like behaviour at adolescence in an animal model. ELS was induced by subjecting Sprague Dawley rat pups to maternal separation and isolation (MS) stress during the stress hyporesponsive period (SHRP) from post-natal days (PND) 4-PND 14. This rat model was tested for anxiety, spontaneous exploratory behaviour and curiosity-like behaviour in a custom-designed arena during adolescence (PND 30-45). ELS-induced changes in the stress were confirmed by corticosterone, while, basal dopamine level was estimated to understand the neurochemical basis of MS stress-induced changes in curiosity. We observed an increase in the levels of anxiety and intrinsic drive state such as curiosity-like behaviour, which was associated with elevated plasma corticosterone and dopamine in MS animals during adolescence suggesting the impact of ELS during SHRP on adolescent behaviour.

Low oxygen levels caused by Noctiluca scintillans bloom kills corals in Gulf of Mannar, India.

Coral reefs around the world are undergoing severe decline in the past few decades. Mass coral mortalities have predominantly been reported to be caused by coral bleaching or disease outbreaks. Temporary hypoxic conditions caused by algal blooms can trigger mass coral mortalities though are reported rarely. In this study in Gulf of Mannar (GoM), southeast India, we report a significant coral mortality caused by a bloom of the ciguatoxic dinoflagellate Noctiluca scintillans during September-October 2019. Dissolved oxygen levels declined below 2 mg l-1 during the bloom causing temporary hypoxia and mortality (up to 71.23%) in the fast growing coral genera Acropora, Montipora and Pocillopora. Due to global climate change, more frequent and larger algal blooms are likely in the future. Hence, it is likely that shallow water coral reefs will be affected more frequently by episodic hypoxic conditions driven by algal blooms. More studies are, however, required to understand the mechanism of coral mortality due to algal blooms, impacts on community composition and the potential for subsequent recovery.

Nanocarriers for effective nutraceutical delivery to the brain.

Neurodegenerative disorders are common among aging populations around the globe. Most are characterized by loss of neurons, protein aggregates, oxidative stress, mitochondrial damage, neuroinflammation among others. Although symptomatic treatment using conventional pharmacotherapy has been widely employed, their therapeutic success is limited due to varied reasons. In the need to identify an alternative approach, researchers successfully demonstrated the therapeutic utility of plant-derived nutraceuticals in cell and animal models of neurodegenerative conditions. However, most nutraceuticals failed during clinical trials in humans owing to their poor bioavailability in vivo and limited permeability across the blood brain barrier (BBB). The current emphasis is therefore on the improved delivery of nutraceuticals to the brain. In this regard, development of nanoparticle conjugated nutraceuticals to enhance bioavailability and therapeutic efficacy in the brain has gained attention. Here, we review the research advances in nanoparticles conjugated nutraceuticals applied in neurodegenerative disorders and discuss their advantages and limitations, clinical trials and toxicity concerns.

Exposure to the neurotoxin 3-nitropropionic acid in neuronal cells induces unique histone acetylation pattern: Implications for neurodegeneration.

Mitochondrial dysfunction is critical for neurodegeneration in movement disorders. Neurotoxicological models recapitulating movement disorder involve mitochondrial damage including inhibition of mitochondrial complexes. Previously, we demonstrated that neurotoxic models of Parkinson's disease and Manganism showed distinct morphological, electrophysiological and molecular profile indicating disease-specific characteristics. In a recent study, we demonstrated that the transcriptomic changes triggered by the neurotoxic mitochondrial complex II inhibitor 3-nitropropionic acid (3-NPA), was significantly different from the profile induced by the neurotoxic mitochondrial complex I inhibitor 1-methyl-4- phenylpyridinium (MPP+) and mitochondrial toxin Manganese (Mn). Among the plausible pathways, we surmised that epigenetic mechanisms could contribute to 3-NPA specific transcriptomic profile. To address this, we assessed global and individual lys-specific acetylation profile of Histone H3 and H4 in the 3-NPA neuronal cell model. Our data revealed histone acetylation profile unique to the 3-NPA model that was not noted in the MPP+ and Mn models. Among the individual lys, Histone H3K56 showed robust dose and time-dependent hyperacetylation in the 3-NPA model. Chromatin Immunoprecipitation-sequencing (ChIP-seq) revealed that acetylated H3K56 was associated with 13072 chromatin sites, which showed increased occupancy in the transcription start site-promoter site. Acetylated histone H3K56 was associated with 1747 up-regulated and 263 down-regulated genes in the 3-NPA model, which included many up-regulated autophagy and mitophagy genes. Western analysis validated the involvement of PINK1-Parkin dependent mitophagy in the 3-NPA model. We propose that 3-NPA specific chromatin dynamics could contribute to the unique transcriptomic profile with implications for movement disorders.

Marine debris - An emerging threat to the reef areas of Gulf of Mannar, India.

Underwater survey was conducted to assess the accumulation and impact of marine debris in the reef areas of Gulf of Mannar in southeast India. A combination of roving diver technique and belt transect method was applied for the assessment, which was conducted during the period between February 2018 and March 2019. An estimated total reef area of 1152 m2 has been affected by marine debris. Abandoned fishing nets were found to constitute the major portion of 43.17 ± 5.48% of the marine debris. Live corals were found to be dominant substrates for marine debris with 39.11%. The average prevalence of coral colonies in contact with marine debris was 3.28 ± 0.27%. Prevalence of corals in contact with debris was very high in genus Acropora with 8.23 ± 1.29% followed by Montipora with 4.63 ± 1.29% due to their complex growth form. Of the corals in contact with debris, 47.56% were fragmented and 34% were found with tissue loss.

Human muscle pathology is associated with altered phosphoprotein profile of mitochondrial proteins in the skeletal muscle.

Analysis of human muscle diseases highlights the role of mitochondrial dysfunction in the skeletal muscle. Our previous work revealed that diverse upstream events correlated with altered mitochondrial proteome in human muscle biopsies. However, several proteins showed relatively unchanged expression suggesting that post-translational modifications, mainly protein phosphorylation could influence their activity and regulate mitochondrial processes. We conducted mitochondrial phosphoprotein profiling, by proteomics approach, of healthy human skeletal muscle (n = 10) and three muscle diseases (n = 10 each): Dysferlinopathy, Polymyositis and Distal Myopathy with Rimmed Vacuoles. Healthy human muscle mitochondrial proteins displayed 253 phosphorylation sites (phosphosites), which contributed to metabolic and redox processes and mitochondrial organization etc. Electron transport chain complexes accounted for 84 phosphosites. Muscle pathologies displayed 33 hyperphosphorylated and 14 hypophorphorylated sites with only 5 common proteins, indicating varied phosphorylation profile across muscle pathologies. Molecular modelling revealed altered local structure in the phosphorylated sites of Voltage-Dependent Anion Channel 1 and complex V subunit ATP5B1. Molecular dynamics simulations in complex I subunits NDUFV1, NDUFS1 and NDUFV2 revealed that phosphorylation induced structural alterations thereby influencing electron transfer and potentially altering enzyme activity. We propose that altered phosphorylation at specific sites could regulate mitochondrial protein function in the skeletal muscle during physiological and pathological processes.

Interplay of lattice distortion and electronic structure in BaBiO3.

We investigate room temperature core level and valence band spectra of BaBiO3 using x-ray photoemission spectroscopy and band structure calculations. The features in the valence band spectrum were studied using density functional theory (DFT) under local density approximation (LDA) and Tran Blaha modified Becke Johnson (TB mBJ) exchange potential. The calculations were performed for three different structural parameters; monoclinic, cubic and monoclinic (M[Formula: see text]). Our results of the core level spectrum and DFT calculations rule out charge disproportionation of the Bi ions. The valence band spectrum displays gap at the Fermi edge and fine structures in the region close to the Fermi edge. The DFT calculation under TB mBJ for the monoclinic structure is able to generate gap and match the energy positions of the fine structure in a better way. Our calculation results show that there are holes in the O 2[Formula: see text] states and unequal transfer of electrons to the states of the Bi ions. Such mechanism could lead to bond disproportionation and its association with the fine structures in the valence band. The current results reveal the significance of strong link between the lattice distortion and electronic structure and hence to its physical properties.

Design, synthesis, in-vitro evaluation and molecular docking studies of novel indole derivatives as inhibitors of SIRT1 and SIRT2.

Sirtuins (SIRTs), class III HDAC (Histone deacetylase) family proteins, are associated with cancer, diabetes, and other age-related disorders. SIRT1 and SIRT2 are established therapeutic drug targets by regulating its function either by activators or inhibitors. Compounds containing indole moiety are potential lead molecules inhibiting SIRT1 and SIRT2 activity. In the current study, we have successfully synthesized 22 indole derivatives in association with an additional triazole moiety that provide better anchoring of the ligands in the binding cavity of SIRT1 and SIRT2. In-vitro binding and deacetylation assays were carried out to characterize their inhibitory effects against SIRT1 and SIRT2. We found four derivatives, 6l, 6m, 6n, and 6o to be specific for SIRT1 inhibition; three derivatives, 6a, 6d and 6k, specific for SIRT2 inhibition; and two derivatives, 6s and 6t, which inhibit both SIRT1 and SIRT2. In-silico validation for the selected compounds was carried out to study the nature of binding of the ligands with the neighboring residues in the binding site of SIRT1. These derivatives open up newer avenues to explore specific inhibitors of SIRT1 and SIRT2 with therapeutic implications for human diseases.

Tryptophan Oxidation in the UQCRC1 Subunit of Mitochondrial Complex III (Ubiquinol-Cytochrome C Reductase) in a Mouse Model of Myodegeneration Causes Large Structural Changes in the Complex: A Molecular Dynamics Simulation Study.

Muscle diseases display mitochondrial dysfunction and oxidative damage. Our previous study in a cardiotoxin model of myodegeneration correlated muscle damage with mitochondrial dysfunction, which in turn entailed altered mitochondrial proteome and oxidative damage of mitochondrial proteins. Proteomic identification of oxidized proteins in muscle biopsies from muscular dystrophy patients and cardiotoxin model revealed specific mitochondrial proteins to be targeted for oxidation. These included respiratory complexes which displayed oxidative modification of Trp residues in different subunits. Among these, Ubiquinol-Cytochrome C Reductase Core protein 1 (UQCRC1), a subunit of Ubiquinol-Cytochrome C Reductase Complex or Cytochrome b-c1 Complex or Respiratory Complex III displayed oxidation of Trp395, which could be correlated with the lowered activity of Complex III. We hypothesized that Trp395 oxidation might contribute to altered local conformation and overall structure of Complex III, thereby potentially leading to altered protein activity. To address this, we performed molecular dynamics simulation of Complex III (oxidized at Trp395 of UQCRC1 vs. non-oxidized control). Molecular dynamic simulation analyses revealed local structural changes in the Trp395 site. Intriguingly, oxidized Trp395 contributed to decreased plasticity of Complex III due to significant cross-talk among the subunits in the matrix-facing region and subunits in the intermembrane space, thereby leading to impaired electron flow from cytochrome C.

Mapping the protein phosphorylation sites in human mitochondrial complex I (NADH: Ubiquinone oxidoreductase): A bioinformatics study with implications for brain aging and neurodegeneration.

In eukaryotes, mitochondrial complex I (NADH: ubiquinone oxidoreductase; CI) is central to oxidative phosphorylation (OXPHOS). Mammalian CI is a 45 subunit complex that forms supercomplexes with other OXPHOS complexes. Since CI defects are associated with aging and neurodegeneration, it is pertinent to understand its structure-function relationship. Although genetic mutations could lower CI activity causing mitochondrial dysfunction in several pathologies, post-translational modifications (PTMs) have emerged as a key mechanism contributing to altered CI activity. Among non-oxidative PTMs, protein phosphorylation is the most intricate regulatory mechanism controlling CI structure and function during normal physiology, aging and neurodegeneration. To comprehend this, we carried out a comprehensive bioinformatics analysis of protein phosphorylation of human CI subunits using software-based prediction of phosphorylation (phospho) sites and associated kinases. Phosphorylation was higher among core subunits and active domains of the complex. Among the subunits, NDUFS1 displayed significantly higher number as well as percent phospho sites compared to others. Analysis of the subunits containing iron-sulfur (Fe-S) cluster, NADH and FMN binding sites and quinone binding sites indicated the presence of phospho sites in close proximity to the binding sites of these cofactors with potential functional implications. Phosphoproteomics experiment in rat and human muscle mitochondria identified specific phospho sites in CI subunits, thereby validating the bioinformatic analysis. Molecular modeling of CI subunits indicated structural implications following phosphorylation. We surmise that protein phosphorylation, a transient and regulatory event could influence the structure-function relationship of CI thereby impinging on bioenergetics and ultimately contributing to aging and neurodegeneration.

Rational discovery of a SOD1 tryptophan oxidation inhibitor with therapeutic potential for amyotrophic lateral sclerosis.

Formation of Cu, Zn superoxide dismutase 1 (SOD1) protein inclusions within motor neurons is one of the principal characteristics of SOD1-related amyotrophic lateral sclerosis (ALS). A hypothesis as to the nature of SOD1 aggregation implicates oxidative damage to a solvent-exposed tryptophan as causative. Here, we chart the discovery of a phenanthridinone based compound (Lig9) from the NCI Diversity Set III by rational methods by in silico screening and crystallographic validation. The crystal structure of the complex with SOD1, refined to 2.5 Å, revealed that Lig9 binds the SOD1 ß-barrel in the ß-strand 2 and 3 region which is known to scaffold SOD1 fibrillation. The phenanthridinone moiety makes a substantial π-π interaction with Trp32 of SOD1. The compound possesses a significant binding affinity for SOD1 and inhibits oxidation of Trp32; a critical residue for SOD1 aggregation. Thus, Lig9 is a good candidate from which to develop a new library of SOD1 aggregation inhibitors through protection of Trp32 oxidation. Communicated by Ramaswamy H. Sarma.