PhnJ – A novel radical SAM enzyme from the C–P lyase complex

AbstractPhnJ from the C–P lyase complex catalyzes the cleavage of the carbon–phosphorus bond in ribose-1-phosphonate-5-phosphate (PRPn) to produce methane and ribose-1,2-cyclic-phosphate-5-phosphate (PRcP). This protein is a novel radical SAM enzyme that uses glycyl and thiyl radicals as reactive intermediates in the proposed reaction mechanism. The overall reaction is initiated with the reductive cleavage of S-adenosylmethionine (SAM) by a reduced [4Fe–4S]1+-cluster to form an Ado-CH2∙ radical intermediate. This intermediate abstracts the proR hydrogen from Gly-32 of PhnJ to form Ado-CH3 and a glycyl radical. In the next step, there is hydrogen atom transfer from Cys-272 to the Gly-32 radical to generate a thiyl radical. The thiyl radical attacks the phosphorus center of the substrate, PRPn, to form a transient thiophosphonate radical intermediate. This intermediate collapses via homolytic C–P bond cleavage and hydrogen atom transfer from the proS hydrogen of Gly-32 to produce a thiophosphate intermediate, methane, and a radical intermediate at Gly-32. The final product, PRcP, is formed by nucleophilic attack of the C2-hydroxyl on the transient thiophosphate intermediate. This reaction regenerates the free thiol group of Cys-272. After hydrogen atom transfer from Cys-272 to the Gly-32 radical, the entire process is repeated with another substrate molecule without the use of another molecule of SAM or involvement from the [4Fe–4S]-cluster again

GPU CUDA Accelerated Image Inpainting using Fourth Order PDE Equation

This paper describes the technique to accelerate inpainting process using fourth order PDE equation using GPU CUDA. Inpainting is the process of filling in missing parts of damaged images based on information gleaned from surrounding areas. It uses the GPU computation advantage to process PDE equation into parallel process. Fourth order PDE will be solved using parallel computation in GPU. This method can speed up the computation time up to 36x using NVDIA GEFORCE GTX 67

Phagocytosis: A (Sphingo)Lipid Story

Phagocytosis is an evolutionary conserved innate immunological response, critical for fighting off pathogens and/or infections in higher organisms, including humans. During this process, any detrimental foreign particles (e.g. bacteria, virus, dead cells) are engulfed by immune cells called phagocytes (e.g. macrophages, monocytes), and packaged in an intracellular entity (or organelle) called the phagosome. The phagosome then undergoes a well-choreographed sequence of changes in protein and lipid composition termed “phagosomal maturation”, eventually fuses with the lysosome to form the phagolysosome, and thus marks the end of phagocytosis. While a lot is known of the proteomic changes during phagosomal maturation, in comparison, till recently, little remained known of the lipidomic changes during this process. Here, we review the current knowledge of the lipid changes on purified phagosomes, namely early and late phagosomes, during phagosomal maturation, with a special focus on sphingolipid metabolism during this important immune response

The catalytic mechanism for aerobic formation of methane by bacteria. Nature 497, 132–136. doi: 10.1038/nature12061

Methane is a potent greenhouse gas that is produced in significant quantities by aerobic marine organisms 1 . These bacteria apparently catalyse the formation of methane through the cleavage of the highly unreactive carbon-phosphorus bond in methyl phosphonate (MPn), but the biological or terrestrial source of this compound is unclear 2 . However, the ocean-dwelling bacterium Nitrosopumilus maritimus catalyses the biosynthesis of MPn from 2-hydroxyethyl phosphonate 3 and the bacterial C-P lyase complex is known to convert MPn to methane Here we show that PhnJ is a novel radical S-adenosyl-L-methionine enzyme that catalyses C-P bond cleavage through the initial formation of a 59-deoxyadenosyl radical and two protein-based radicals localized at Gly 32 and Cys 272. During this transformation, the pro-R hydrogen from Gly 32 is transferred to the 59-deoxyadenosyl radical to form 59-deoxyadenosine and the pro-S hydrogen is transferred to the radical intermediate that ultimately generates methane. A comprehensive reaction mechanism is proposed for cleavage of the C-P bond by the C-P lyase complex that uses a covalent thiophosphate intermediate for methane and phosphate formation. The glutathione S-transferase (GST) fusion protein of PhnJ from Escherichia coli was purified under anaerobic conditions 8 . The isolated protein was dark brown in colour, had an absorbance maximum at a wavelength of 410 nm and was EPR silent (produced no electron paramagnetic resonance signal) It was shown previously that 59-deoxyadenosine (Ado-CH 3 ) and L-methionine are formed from the utilization of SAM during the reaction catalysed by PhnJ and that approximately one enzyme equivalent of SAM is consumed under single or multiple turnover

Impact of natalizumab on patient-reported outcomes in multiple sclerosis: a longitudinal study

Abstract Background Natalizumab (Tysabri, Biogen Idec and Elan Pharmaceuticals) significantly reduces the relapse rate and disability progression, and improves health-related quality of life (HRQoL), in patients with relapsing-remitting multiple sclerosis. We investigated the impact of natalizumab on patient-reported outcomes (PROs) in a real-world setting. Methods PRO data were collected from patients enrolled in a longitudinal real-world study using validated measures administered as surveys before the patients initiated natalizumab treatment and after the 3rd, 6th, and 12th monthly infusion. HRQoL, ability to carry out daily activities, disability level, and impact on cognitive functioning and fatigue were assessed. Results A total of 333 patients completed 12 months of assessments. After 12 months of natalizumab treatment, 69% to 88% of patients reported a positive outcome (either an improvement or no further decline) in all PRO measures assessed. Significant improvements in general and disease-specific HRQoL were observed after three infusions, both with physical (p  Conclusions PRO measures were improved with natalizumab in a real-world setting. The improvements were observed as early as after 3 months and sustained over a 12-month period. The improvements in PROs show that, in clinical practice, the clinical benefits of natalizumab are translated into patient-reported benefits.</p

Chemoproteomics of an Indole-Based Quinone Epoxide Identifies Druggable Vulnerabilities in Vancomycin-Resistant Staphylococcus aureus

Publisher's version (útgefin grein)The alarming global rise in fatalities from multidrug-resistant Staphylococcus aureus (S. aureus) infections has underscored a need to develop new therapies to address this epidemic. Chemoproteomics is valuable in identifying targets for new drugs in different human diseases including bacterial infections. Targeting functional cysteines is particularly attractive, as they serve critical catalytic functions that enable bacterial survival. Here, we report an indole-based quinone epoxide scaffold with a unique boat-like conformation that allows steric control in modulating thiol reactivity. We extensively characterize a lead compound (4a), which potently inhibits clinically derived vancomycin-resistant S. aureus. Leveraging diverse chemoproteomic platforms, we identify and biochemically validate important transcriptional factors as potent targets of 4a. Interestingly, each identified transcriptional factor has a conserved catalytic cysteine residue that confers antibiotic tolerance to these bacteria. Thus, the chemical tools and biological targets that we describe here prospect new therapeutic paradigms in combatting S. aureus infections.The authors thank the Department of Biotechnology (DBT), Government of India (BT/PR15848/MED/29/1025/2016 to H.C. and S.C.), a Wellcome Trust DBT India Alliance Intermediate Fellowship (IA/I/15/2/502058 to S.S.K.) and a DST-FIST Infrastructure Development Grant (to IISER Pune Biology) for the financial support for our research. The Council for Scientific and Industrial Research (CSIR) and the Department of Science and Technology—Innovation in Science Pursuit for Inspired Research (DST-INSPIRE) for graduate student fellowships.Peer Reviewe

Metabolic regulation of CTCF expression and chromatin association dictates starvation response in mice and flies

Summary: Coordinated temporal control of gene expression is essential for physiological homeostasis, especially during metabolic transitions. However, the interplay between chromatin architectural proteins and metabolism in regulating transcription is less understood. Here, we demonstrate a conserved bidirectional interplay between CTCF (CCCTC-binding factor) expression/function and metabolic inputs during feed-fast cycles. Our results indicate that its loci-specific functional diversity is associated with physiological plasticity in mouse hepatocytes. CTCF differential expression and long non-coding RNA-Jpx mediated changes in chromatin occupancy, unraveled its paradoxical yet tuneable functions, which are governed by metabolic inputs. We illustrate the key role of CTCF in controlling temporal cascade of transcriptional response, with effects on hepatic mitochondrial energetics and lipidome. Underscoring the evolutionary conservation of CTCF-dependent metabolic homeostasis, CTCF knockdown in flies abrogated starvation resistance. In summary, we demonstrate the interplay between CTCF and metabolic inputs that highlights the coupled plasticity of physiological responses and chromatin function

AIG1 and ADTRP are Atypical Integral Membrane Hydrolases that Degrade Bioactive FAHFAs

Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty-acid esters of hydroxy-fatty acids (FAHFAs), but not other major classes of lipids. We discover multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent/parallel protein evolution that defy conventional sequence- and structure-based predictions