Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of [?]1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation- velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition.WeseeNADPHbound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination

Structural and Kinetic Studies on the Potent Inhibition of Metallo-β-Lactamases by 6- Phosphonomethylpyridine-2-Carboxylates

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acs.biochem.7b01299There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs), enzymes which hydrolyze β-lactam antibiotics and confer resistance on Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values 0.3 – 7.2 µM; Ki 0.03 – 1.5 µM). Minimum inhibitory concentration assays demonstrated potentiation of β-lactam (meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations where eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of inhibitor binding to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions, but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221/223, respectively (3 Å distance). The potency, low toxicity, cellular activity and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.This work was supported by grants from the U.K. Medical Research Council and Canadian Institutes of Health Research (U.K.-Canada Team Grant G1100135 and FRN114046) to J.S. and G.I.D, Canadian Institutes of Health Research operating grant (FRN106531) to G.I.D., the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health to J.S. (R01AI100560) and the Engineering and Physical Sciences Research Council (EP/M027546/1) to M.B.A. and J.S..K.C. was in receipt of a postgraduate scholarship from SENESCYT, Ecuador

The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells.

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a 'challenge' experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as 'Moderate' in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose

A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates.

We present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level

Storm impacts on phytoplankton community dynamics in lakes

In many regions across the globe, extreme weather events such as storms have increased in frequency, intensity, and duration due to climate change. Ecological theory predicts that such extreme events should have large impacts on ecosystem structure and function. High winds and precipitation associated with storms can affect lakes via short-term runoff events from watersheds and physical mixing of the water column. In addition, lakes connected to rivers and streams will also experience flushing due to high flow rates. Although we have a well-developed understanding of how wind and precipitation events can alter lake physical processes and some aspects of biogeochemical cycling, our mechanistic understanding of the emergent responses of phytoplankton communities is poor. Here we provide a comprehensive synthesis that identifies how storms interact with lake and watershed attributes and their antecedent conditions to generate changes in lake physical and chemical environments. Such changes can restructure phytoplankton communities and their dynamics, as well as result in altered ecological function (e.g., carbon, nutrient and energy cycling) in the short- and long-term. We summarize the current understanding of storm-induced phytoplankton dynamics, identify knowledge gaps with a systematic review of the literature, and suggest future research directions across a gradient of lake types and environmental conditions.Peer reviewe

EarthFinder Probe Mission Concept Study: Characterizing nearby stellar exoplanet systems with Earth-mass analogs for future direct imaging

EarthFinder is a NASA Astrophysics Probe mission concept selected for study as input to the 2020 Astrophysics National Academies Decadal Survey. The EarthFinder concept is based on a dramatic shift in our understanding of how PRV measurements should be made. We propose a new paradigm which brings the high precision, high cadence domain of transit photometry as demonstrated by Kepler and TESS to the challenges of PRV measurements at the cm/s level. This new paradigm takes advantage of: 1) broad wavelength coverage from the UV to NIR which is only possible from space to minimize the effects of stellar activity; 2) extremely compact, highly stable, highly efficient spectrometers (R>150,000) which require the diffraction-limited imaging possible only from space over a broad wavelength range; 3) the revolution in laser-based wavelength standards to ensure cm/s precision over many years; 4) a high cadence observing program which minimizes sampling-induced period aliases; 5) exploiting the absolute flux stability from space for continuum normalization for unprecedented line-by-line analysis not possible from the ground; and 6) focusing on the bright stars which will be the targets of future imaging missions so that EarthFinder can use a ~1.5 m telescope

Storm impacts on phytoplankton community dynamics in lakes

In many regions across the globe, extreme weather events, such as storms, have increased in frequency, intensity and duration. Ecological theory predicts that such extreme events should have large impacts on ecosystem structure and function. For lake ecosystems, high winds and rainfall associated with storms are linked by short term runoff events from catchments and physical mixing of the water column. Although we have a well-developed understanding of how such wind and precipitation events alter lake physical processes, our mechanistic understanding of how these short-term disturbances 48 translate from physical forcing to changes in phytoplankton communities is poor. Here, we provide a conceptual model that identifies how key storm features (i.e., the frequency, intensity, and duration of wind and precipitation) interact with attributes of lakes and their watersheds to generate changes in a lake’s physical and chemical environment and subsequently phytoplankton community structure and dynamics. We summarize the current understanding of storm-phytoplankton dynamics, identify knowledge gaps with a systematic review of the literature, and suggest future research directions by generating testable hypotheses across a global gradient of lake types and environmental conditions.Fil: Stockwell, Jason D.. University of Vermont; Estados UnidosFil: Adrian, Rita. Leibniz Institute of Freshwater Ecology and Inland Fisheries; AlemaniaFil: Andersen, Mikkel. Dundalk Institute of Technology; IrlandaFil: Anneville, Orlane. Institut National de la Recherche Agronomique; FranciaFil: Bhattacharya, Ruchi. University of Missouri; Estados UnidosFil: Burns, Wilton G.. University of Vermont; Estados UnidosFil: Carey, Cayelan C.. Virginia Tech University; Estados UnidosFil: Carvalho, Laurence. Freshwater Restoration & Sustainability Group; Reino UnidoFil: Chang, ChunWei. National Taiwan University; República de ChinaFil: De Senerpont Domis, Lisette N.. Netherlands Institute of Ecology; Países BajosFil: Doubek, Jonathan P.. University of Vermont; Estados UnidosFil: Dur, Gaël. Shizuoka University; JapónFil: Frassl, Marieke A.. Griffith University; AustraliaFil: Gessner, Mark O.. Leibniz Institute of Freshwater Ecology and Inland Fisheries; AlemaniaFil: Hejzlar, Josef. Biology Centre of the Czech Academy of Sciences; República ChecaFil: Ibelings, Bas W.. University of Geneva; SuizaFil: Janatian, Nasim. Estonian University of Life Sciences; EstoniaFil: Kpodonu, Alfred T. N. K.. City University of New York; Estados UnidosFil: Lajeunesse, Marc J.. University of South Florida; Estados UnidosFil: Lewandowska, Aleksandra M.. Tvarminne Zoological Station; FinlandiaFil: Llames, Maria Eugenia del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Matsuzaki, Shin-ichiro S.. National Institute for Environmental Studies; JapónFil: Nodine, Emily R.. Rollins College; Estados UnidosFil: Nõges, Peeter. Estonian University of Life Sciences; EstoniaFil: Park, Ho-Dong. Shinshu University; JapónFil: Patil, Vijay P.. US Geological Survey; Estados UnidosFil: Pomati, Francesco. Swiss Federal Institute of Water Science and Technology; SuizaFil: Rimmer, Alon. Kinneret Limnological Laboratory; IsraelFil: Rinke, Karsten. Helmholtz-Centre for Environmental Research; AlemaniaFil: Rudstam, Lars G.. Cornell University; Estados UnidosFil: Rusak, James A.. Ontario Ministry of the Environment and Climate Change; CanadáFil: Salmaso, Nico. Research and Innovation Centre - Fondazione Mach; ItaliaFil: Schmitt, François. Laboratoire d’Océanologie et de Géosciences; FranciaFil: Seltmann, Christian T.. Dundalk Institute of Technology; IrlandaFil: Souissi, Sami. Universite Lille; FranciaFil: Straile, Dietmar. University of Konstanz; AlemaniaFil: Thackeray, Stephen J.. Lancaster Environment Centre; Reino UnidoFil: Thiery, Wim. Vrije Unviversiteit Brussel; Bélgica. Institute for Atmospheric and Climate Science; SuizaFil: Urrutia Cordero, Pablo. Uppsala University; SueciaFil: Venail, Patrick. Universidad de Ginebra; SuizaFil: Verburg, Piet. 8National Institute of Water and Atmospheric Research; Nueva ZelandaFil: Williamson, Tanner J.. Miami University; Estados UnidosFil: Wilson, Harriet L.. Dundalk Institute of Technology; IrlandaFil: Zohary, Tamar. Israel Oceanographic & Limnological Research; IsraelGLEON 20: All Hands' MeetingRottnest IslandAustraliaUniversity of Western AustraliaUniversity of AdelaideGlobal Lake Ecological Observatory Networ