The NTPase activity of the double FYVE domain-containing protein 1 regulates lipid droplet metabolism.

Lipid droplets (LDs) are transient lipid storage organelles that can be readily tapped to resupply cells with energy or lipid building blocks and therefore play a central role in cellular metabolism. However, the molecular factors and underlying mechanisms that regulate the growth and degradation of LDs are poorly understood. It has emerged that proteins that establish contacts between LDs and the endoplasmic reticulum play a critical role in regulating LD metabolism. Recently, the autophagy-related protein, double FYVE domain-containing protein 1 (DFCP1/ZFYVE1) was shown to reside at the interface of the endoplasmic reticulum and LDs, however, little is known about the involvement of DFCP1 in autophagy and LD metabolism. Here, we show that DFCP1 is a novel NTPase that regulates free fatty acid metabolism. Specifically, we show that DFPC1-knockdown, particularly during starvation, increases cellular free fatty acids and decreases the levels of cellular TAGs, resulting in accumulated small LDs. Using selective truncations, we demonstrate that DFCP1 accumulation on LDs in cells and in vitro is regulated by a previously unknown NTPase domain. Using spectroscopic approaches, we show that this NTPase domain can dimerize and can hydrolyze both ATP and GTP. Furthermore, mutations in DFCP1 that either impact nucleotide hydrolysis or dimerization result in changes in the accumulation of DFCP1 on LDs, changes in LD density and size, and colocalization of LDs to autophagosomes. Collectively, our findings suggest that DFCP1 is an NTPase that modulates the metabolism of LDs in cells

Diroximel fumarate in patients with relapsing-remitting multiple sclerosis: Final safety and efficacy results from the phase 3 EVOLVE-MS-1 study

BACKGROUND: Diroximel fumarate (DRF) is approved for adults with relapsing-remitting multiple sclerosis (RRMS) in Europe and for relapsing forms of MS in the United States. DRF and dimethyl fumarate (DMF) yield bioequivalent exposure of the active metabolite monomethyl fumarate. Prior studies indicated fewer gastrointestinal (GI)-related adverse events (AEs) with DRF compared with DMF. OBJECTIVE: To report final outcomes from EVOLVE-MS-1. METHODS: EVOLVE-MS-1 was an open-label, 96-week, phase 3 study assessing DRF safety, tolerability, and efficacy in patients with RRMS. The primary endpoint was safety and tolerability; efficacy endpoints were exploratory. RESULTS: Overall, 75.7% (800/1057) of patients completed the study; median exposure was 1.8 (range: 0.0-2.0) years. AEs occurred in 938 (88.7%) patients, mostly of mild (28.9%) or moderate (50.3%) severity. DRF was discontinued due to AEs in 85 (8.0%) patients, with \u3c 2% discontinuing due to GI or flushing/flushing-related AEs. At Week 96, mean number of gadolinium-enhancing lesions was significantly reduced from baseline (72.7%; CONCLUSION: DRF was generally well tolerated over 2 years, with few discontinuations due to AEs; radiological measures indicated decreased disease activity from baseline. These outcomes support DRF as a treatment option in patients with RRMS

Neuropsychological and functional outcomes in recent-onset major depression, bipolar disorder and schizophrenia-spectrum disorders: a longitudinal cohort study

Functional disability is the lead contributor to burden of mental illness. Cognitive deficits frequently limit functional recovery, although whether changes in cognition and disability are longitudinally associated in recent-onset individuals remains unclear. Using a prospective, cohort design, 311 patients were recruited and assessed at baseline. One hundred and sixty-seven patients met eligibility criteria (M = 21.5 years old, s.d. = 4.8) and returned for follow-up (M = 20.6 months later, s.d. = 7.8). Two-hundred and thirty participants were included in the final analysis, comprising clinically stable patients with major depression (n = 71), bipolar disorder (BD; n = 61), schizophrenia-spectrum disorders (n = 35) and 63 healthy controls. Neuropsychological functioning and self-rated functional disability were examined using mixed-design, repeated-measures analysis, across diagnoses and cognitive clusters, covarying for relevant confounds. Clinical, neuropsychological and functional changes did not differ between diagnoses (all P40.05). Three reliable neuropsychological subgroups emerged through cluster analysis, characterized by psychomotor slowing, improved sustained attention, and improved verbal memory. Controlling for diagnosis and changes in residual symptoms, clusters with improved neuropsychological functioning observed greater reductions in functional disability than the psychomotor slowing cluster, which instead demonstrated a worsening in disability (Po0.01). Improved sustained attention was independently associated with greater likelihood of follow-up employment (Po0.01). Diagnosis of BD uniquely predicted both follow-up employment and independent living. Neuropsychological course appears to be independently predictive of subjective and objective functional outcomes. Importantly, cognitive phenotypes may reflect distinct pathophysiologies shared across major psychiatric conditions, and be ideal targets for personalized early intervention

Understanding and manipulating non-templated peptide bond formation by macrocyclase enzymes

Peptide macrocycles are attractive molecules because they are drug-like, protease resistant, cell permeable, and possess a rigid structure. They have been shown to possess various biological activities and to be able to inhibit protein-protein interactions and other complex targets. Although several macrocyclases have been characterized to date, only two can catalyze the formation of cyclic peptides containing less than 9 amino acids in their core. PatGmac, from the biosynthesis of cyanobactins, is a versatile catalyst with very broad substrate specificity. It can utilize varied peptide sequences, incorporate unnatural amino acids, including substrates that are peptide “chimeras” containing triazoles, peg linkers and sugars (Figure 1A, bottom). Despite its remarkable substrate promiscuity, PatGmac is extremely slow, with turnover rates in the vicinity of once per day. In search for a more efficient macrocyclase we studied GmPOPB, a prolyl oligopeptidase from the mushroom Galerina marginata. GmPOPB (fast macrocyclase) participates in the biosynthesis of the toxic amanitins, catalyzing both peptide bond hydrolysis and peptide bond formation with equal efficiency (Figure 1A, top). We determined crystal structures of apoGmPOPB and GmPOPB mutants bound to a peptidase and a macrocyclase substrate unveiling a mechanism by which the enzyme controls which reaction will be catalyzed. We have also performed an extensive kinetic analysis of this enzyme in comparison to the slow PatGmac. Crucial differences exist between the fast and the slow macrocyclases. Substrate positioning plays an important role towards catalytic efficiency. For the fast macrocyclase GmPOPB there is product inhibition and the rate-limiting step for the reaction is product release. For the slow macrocyclase PatGmac product release is not rate determining for the majority of the substrates tested, and the rate-limiting step is coupled to chemistry. Guided by our kinetic studies, we have designed modified peptide substrates, which eliminate the requirement for a long peptide substrate from 25 amino acids to 13 amino acids for the fast macrocyclase. We are currently designing enzyme variants to improve the catalytic efficiency of the slow macrocyclase and to broaden the substrate scope of the fast macrocyclase. We hope our findings will result in a better, more efficient and substrate permissible macrocyclase that can be used for the biocatalytic generation of cyclic peptide libraries to be tested for biological function. Please click Additional Files below to see the full abstract

TarO : a target optimisation system for structural biology

This work was funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) Structural Proteomics of Rational Targets (SPoRT) initiative, (Grant BBS/B/14434). Funding to pay the Open Access publication charges for this article was provided by BBSRC.TarO (http://www.compbio.dundee.ac.uk/taro) offers a single point of reference for key bioinformatics analyses relevant to selecting proteins or domains for study by structural biology techniques. The protein sequence is analysed by 17 algorithms and compared to 8 databases. TarO gathers putative homologues, including orthologues, and then obtains predictions of properties for these sequences including crystallisation propensity, protein disorder and post-translational modifications. Analyses are run on a high-performance computing cluster, the results integrated, stored in a database and accessed through a web-based user interface. Output is in tabulated format and in the form of an annotated multiple sequence alignment (MSA) that may be edited interactively in the program Jalview. TarO also simplifies the gathering of additional annotations via the Distributed Annotation System, both from the MSA in Jalview and through links to Dasty2. Routes to other information gateways are included, for example to relevant pages from UniProt, COG and the Conserved Domains Database. Open access to TarO is available from a guest account with private accounts for academic use available on request. Future development of TarO will include further analysis steps and integration with the Protein Information Management System (PIMS), a sister project in the BBSRC Structural Proteomics of Rational Targets initiative.Publisher PDFPeer reviewe

Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

H.L. is funded by the George & Stella Lee Scholarship and Criticat EPSRC. This project was also funded by the European Research Council project 339367 NCB-TNT and by the BBSRC (K015508/1). JHN is 1000 talent scholar of the Chinese Academy of Sciences at the University of Sichuan.Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable and capable of modulating protein-protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic Amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the amino acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal 8 residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.Publisher PDFPeer reviewe

Circadian rhythmicity in emerging mood disorders: state or trait marker?

Background: Circadian rhythm disturbances overlap with the symptoms of mood episodes and may trigger or prolong mood symptoms. There is limited research on the role of circadian disturbances in mood disorders in young people and/or first episode cases of unipolar and bipolar disorders. Methods: Actigraphy was undertaken for about 14 days in 63 post-pubertal individuals aged 13–25 years with a recent onset of a mood disorder meeting recognised diagnostic criteria. We examined associations between three easily interpretable markers of circadian rhythm activity (amplitude, acrophase and rhythmicity index) and demography and clinical characteristics. Then, circadian markers were compared between diagnostic groups, controlling for potential confounders. Results: Longer duration of illness was correlated with reduced circadian rhythmicity and lower levels of activity over 24 h. A delay in the timing of maximum activity was associated with the level of manic but not depressive symptoms. The circadian rhythmicity index differentiated unipolar from bipolar cases, and in bipolar but not unipolar disorder, the rhythmicity was less robust in those with more severe manic or depressive symptoms. Conclusions: Less robust circadian rhythmicity, especially associated with increasing symptom severity, may represent a more specific or a trait marker of young people with mood disorders who are at higher risk of a bipolar course of illness

Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters

Biotin is an essential vitamin in plants and mammals, functioning as the carbon dioxide carrier within central lipid metabolism. Bacterial pimeloyl-CoA synthetase (BioW) acts as a highly specific substrate-selection gate, ensuring the integrity of the carbon chain in biotin synthesis. BioW catalyzes the condensation of pimelic acid (C7 dicarboxylic acid) with CoASH in an ATP-dependent manner to form pimeloyl-CoA, the first dedicated biotin building block. Multiple structures of Bacillus subtilis BioW together capture all three substrates, as well as the intermediate pimeloyl-adenylate and product pyrophosphate (PPi), indicating that the enzyme uses an internal ruler to select the correct dicarboxylic acid substrate. Both the catalytic mechanism and the surprising stability of the adenylate intermediate were rationalized through site-directed mutagenesis. Building on this understanding, BioW was engineered to synthesize high-value heptanoyl (C7) and octanoyl (C8) monocarboxylic acid-CoA and C8 dicarboxylic-CoA products, highlighting the enzyme's synthetic potential

Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer mem- brane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we inves- tigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consec- utive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitte- rionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electro- physiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific ori- entation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an addi- tional barrier by “trapping” the antibiotic in an unfavorable ori- entation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Iden- tification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new anti- biotics with improved permeation properties in Gram-negative bacteria

Using TanDEM-X to measure pyroclastic flow source location, thickness and volume: Application to the 3rd June 2018 eruption of Fuego volcano, Guatemala

The estimation of the volume of volcanic flows during an ongoing eruption is challenging but this information is crucial for improving risk assessment and for forecasting future events. Although previous studies have shown the ability of TanDEM-X satellite data to derive the thickness and the volume of lava flow fields during effusive eruptions, the method has not been explored yet for pyroclastic flows. Using bi-static interferometry, we produce TanDEM-X DEM on Fuego volcano (Guatemala) to measure the significant topographic changes caused by the 3rd June 2018 eruption, which destroyed the town of San Miguel Los Lotes. We estimate the volume of the Pyroclastic Density Currents (PDCs) to be 15.1 ± 4.2 × 106 m3. The deposits are likely to be the source of lahars during future rainy seasons. We identify the main channel of deposition (positive elevation changes) and the source region of pyroclastic material, areas of significant substrate erosion, and vegetation destruction (negative elevation changes). Our results show that the June 3rd 2018 pyroclastic flow was predominantly composed of material which had gravitationally collapsed from a location close to the vent. The eroded material increased the volume of the flow (bulking) and likely caused the run-out distance of the 2018 PDC to be larger than previous eruptions (1999–2017). This study highlights the potential of remote sensing techniques for actively monitoring topography changes in inaccessible locations and to rapidly derive deposit volumes