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At least 19 records

The Plasmodium falciparum exported protein PF3D7_0402000 binds to erythrocyte ankyrin and band 4.1

Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through PEXEL-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 70 exported P. falciparum proteins tested, 18 bound to IOVs in 2 or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments and the split-luciferase assay, we identified 31 putative protein-protein interactions between erythrocyte cytoskeletal proteins and exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact PHIST domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites. Finally, we found that PfEPF3 proteins bound to erythrocyte GAPDH, providing a potential link between Maurer’s clefts and the erythrocyte cytoskeleton early during infection.

Shakya, Bikash↗

Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.

60 APPLIED LIFE SCIENCES↗

An Operational Overview of the EXport Processes in the Ocean from RemoTe Sensing (EXPORTS) Northeast Pacific Field Deployment

The goal of the EXport Processes in the Ocean from RemoTe Sensing (EXPORTS) field campaign is to develop a predictive understanding of the export, fate, and carbon cycle impacts of global ocean net primary production. To accomplish this goal, observations of export flux pathways, plankton community composition, food web processes, and optical, physical, and biogeochemical (BGC) properties are needed over a range of ecosystem states. Here we introduce the first EXPORTS field deployment to Ocean Station Papa in the Northeast Pacific Ocean during summer of 2018, providing context for other papers in this special collection. The experiment was conducted with two ships: a Process Ship, focused on ecological rates, BGC fluxes, temporal changes in food web, and BGC and optical properties, that followed an instrumented Lagrangian float; and a Survey Ship that sampled BGC and optical properties in spatial patterns around the Process Ship. An array of autonomous underwater assets provided measurements over a range of spatial and temporal scales, and partnering programs and remote sensing observations provided additional observational context. The oceanographic setting was typical of late-summer conditions at Ocean Station Papa: a shallow mixed layer, strong vertical and weak horizontal gradients in hydrographic properties, sluggish sub-inertial currents, elevated macronutrient concentrations and low phytoplankton abundances. Although nutrient concentrations were consistent with previous observations, mixed layer chlorophyll was lower than typically observed, resulting in a deeper euphotic zone. Analyses of surface layer temperature and salinity found three distinct surface water types, allowing for diagnosis of whether observed changes were spatial or temporal.The 2018 EXPORTS field deployment is among the most comprehensive biological pump studies ever conducted. A second deployment to the North Atlantic Ocean occurred in spring 2021, which will be followed by focused work on data synthesis and modeling using the entire EXPORTS data set.

Biological pump↗

Inhibition of CRM1-mediated nuclear export of influenza A nucleoprotein and nuclear export protein as a novel target for antiviral drug development

An anti-influenza compound, DP2392-E10 based on inhibition of the nuclear export function of the viral nucleoprotein-nuclear export signal 3 (NP-NES3) domain was successfully identified by our previous high-throughput screening system. Here, we demonstrated that DP2392-E10 exerts its antiviral effect by inhibiting replication of a broad range of influenza A subtypes. In regard to the molecular mechanism, we revealed that DP2392-E10 inhibits nuclear export of both viral NP and nuclear export protein (NEP). More specifically, in vitro pull-down assays revealed that DP2392-E10 directly binds cellular CRM1, which mediates nuclear export of NP and NEP. In silico docking suggested that DP2392-E10 binds at a region close to the HEAT9 and HEAT10 domains of CRM1. Together, these results indicate that the CRM1-mediated nuclear export function of influenza virus represents a new potential target for antiviral drug development, and also provide a core structure for a novel class of inhibitors that target this function. - Highlights: •DP2392-E10 inhibits replication of a broad range of influenza A subtypes. •DP2392-E10 inhibits nuclear exports of NP and NEP via their NP-NES3 and NEP-NES2 domains, respectively. •DP2392-E10 is predicted to directly bind CRM1 in the region near the HEAT9 and HEAT10 repeats.

60 APPLIED LIFE SCIENCES↗

Thiol-facilitated cell export and desorption of methylmercury by anaerobic bacteria

Neurotoxic methylmercury (MeHg), formed by anaerobic bacteria, is shown to be rapidly excreted from the cell, but the mechanism of this process is unclear. Using both Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132 strains, we investigated the factors affecting export and distribution of MeHg in mercury methylation and MeHg sorption-desorption assays. Thiols, such as cysteine, were found to greatly facilitate desorption and export of MeHg, particularly by PCA cells. However, in cysteine-free assays (4 h) >90% of the synthesized MeHg was associated with PCA, among which ~73% was sorbed on the cell surface and 19% remained inside the cells. In comparison, a majority of the MeHg (70%) was exported by ND132, leaving ~20% of the MeHg sorbed on the surface and 10% inside the cells. When MeHg was added directly to the cell suspensions, ND132 adsorbed much lower MeHg but took up more MeHg inside cells than PCA did. These results demonstrate that MeHg export is bacteria strain-specific, time dependent, and is influenced by thiols, implicating important roles of ligand complexation in facilitating MeHg production and mobilization in the environment.

59 BASIC BIOLOGICAL SCIENCES↗

Assessment of Oceanographic Conditions during the North Atlantic EXport Processes in the Ocean from RemoTe Sensing (EXPORTS) Field Campaign

This manuscript presents an overview of NASA’s EXport Processes in the Ocean from Remote Sensing 2021 Field Campaign in the North Atlantic (EXPORTS NA) and provides quantitative and dynamical descriptions of the physical processes modulating water transformations during the study. A major programmatic goal was to conduct the sampling in a Lagrangian mode so that ocean ecological and biogeochemical changes can be observed independent from physical advective processes. To accomplish this goal, EXPORTS NA conducted a multi-ship, multi-asset field sampling program within a retentive, anticyclonic mode water eddy. Beneath depths of ∼100 m, Lagrangian sampling assets remained within the eddy core waters (ECWs) throughout the experiment, demonstrating that the ECWs within the mode water eddy were retentive. However, strong westerly winds from four storm events deepened the mixed layer (ML) of the surface core waters (SCWs) above the eddy’s mode water core by 25-40 m and exchanged some of the SCWs with surface waters outside of the eddy via Ekman transport. Estimates of flushing times ranged from 5-8 days, with surface exchange fractions ranging from 20-75% and were consistent with particle tracking advected by combined geostrophic and Ekman velocities. The relative contributions of horizontal and vertical advection on changes in ECW tracers depended on the horizontal and vertical gradients of that tracer. For example, horizontal advection played a large role in ECW salinity fluxes, while vertical entrainment played a larger role in the fluxes of nutrients into SCW ML. Each storm injected nutrients and low oxygen waters into the ML, after which the surface ocean ecosystem responded by reducing nutrient concentrations and increasing %O2 saturation levels. Overall, ECW values of chlorophyll and POC were the largest at the onset of the field program and decreased throughout the campaign. The analysis presented provides a physical oceanographic context for the many measurements made during the EXPORTS NA field campaign while illustrating the many challenges of conducting a production-flux experiment, even in a Lagrangian frame, and the inherent uncertainties of interpreting biological carbon pump observations that were collected in a Eulerian frame of reference.

oceanography↗

Structural determinants of nuclear export signal orientation in binding to exportin CRM1

The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). A comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome.

59 BASIC BIOLOGICAL SCIENCES↗

Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals

Nuclear export receptor CRM1 binds highly variable nuclear export signals (NESs) in hundreds of different cargoes. Previously we have shown that CRM1 binds NESs in both polypeptide orientations. Here, we show crystal structures of CRM1 bound to eight additional NESs which reveal diverse conformations that range from loop-like to all-helix, which occupy different extents of the invariant NES-binding groove. Analysis of all NES structures show 5-6 distinct backbone conformations where the only conserved secondary structural element is one turn of helix that binds the central portion of the CRM1 groove. All NESs also participate in main chain hydrogen bonding with human CRM1 Lys568 side chain, which acts as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs.

59 BASIC BIOLOGICAL SCIENCES↗

Silicate deposition during decomposition of cyanobacteria may promote export of picophytoplankton to the deep ocean

Marine particles transport organic matter through the water column to the sediment where the organic matter can be buried. This pathway is one of the few natural removal mechanisms of CO 2 from the atmosphere over geological time. Picophytoplankton, major primary producers in the ocean, have until recently been thought unimportant regarding particle transport. Here we provide evidence that silicate is deposited on extracellular polymeric substance (EPS) associated with decomposing picophytoplankton. We also find that Si is enriched in a previously unexplored group of marine particles (called micro-blebs) from the deep-water column. The surprising similarity in morphology and composition between EPS-Si and micro-blebs suggests that EPS-Si may be a precursor of micro-blebs observed in the deep ocean. Finally, this previously unexplored source of silicon may be important to silicon cycling and may further enhance export of picophytoplankton to the deep ocean.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Improving bamboo’s fuel and storage properties with a net energy export through torrefaction paired with catalytic oxidation

Using torrefied char, or biocoal, as solid fuel provides an opportunity to introduce a sustainable feedstock into the energy market. The goals of this study were to investigate how torrefaction improves the energy content and the grindability of a Malaysian bamboo along with understanding the potential for integrated energy recovery from torrefaction gases. The feedstock was torrefied at 250–290 °C for 1 h and the combustion characteristics and grindability of the solid products along with the composition of torrefied gas species were measured. The results showed a beneficial increase in elemental carbon increased from 47 to 63 wt% at 290 °C torrefaction, reflecting an increase in higher heating value from 17.8 to 25.6 MJ/kg. The combustion behavior of all the products appeared to show three distinct combustion zones; however, torrefaction severity leading to higher combustion temperature and time as the fixed carbon augmented with the increase of torrefaction temperature. This increase in severity also lead to more friable and grindable material, and the 290 °C condition required a factor of 2.7 less hold-up time in the mill compared to the raw bamboo, and a factor of 8.5 less energy (938 and 111 kWh/tonne respectively). Through analysis of the gas and volatile formation, a case study showed that catalytic oxidation can convert nearly 100% of the embodied chemical energy into usable thermal energy. These experimental findings were scaled to a 100,000 tonne/y capacity torrefaction plant and in the moderate case of 270 °C operating temperature, the plant has 1.25 GJ/tonne excess energy beyond what the process needs.

09 BIOMASS FUELS↗

Identifying Key Environmental Factors Explaining Temporal Patterns of DOC Export From Watersheds in the Conterminous United States

The export of dissolved organic carbon (DOC) from a watershed is a key component of the terrestrial biosphere carbon cycle. There is a need to improve our understanding of how and by how much various environmental factors are driving the temporal patterns of DOC export in order to accurately model and evaluate terrestrial carbon storage and fluxes. In this synthesis, we compiled observational data sets from 14 watersheds in the conterminous United States spanning the time period from 1981 to 2017. We used these data sets to examine the relative impacts of various climate, atmospheric deposition, and land cover factors on the temporal patterns of DOC export across watersheds of different sizes and landscape conditions, as well as the time-series autocorrelation of DOC export. Overall, our results suggest that the dominant factor on an annual scale was the amount of precipitation, which had a positive correlation with DOC export from a watershed. Overall increasing nitrogen deposition was coincident with increasing DOC export, and increasing sulfur deposition was coincident with declining DOC export. The seasonal pattern of DOC export was strongly regulated by air temperature, the long-term trend was negatively influenced by increasing sulfur deposition, and no obvious autocorrelation was detected in DOC export. In addition, higher rates of DOC export were positively correlated with greater area of wetlands within a watershed, but was not found to be strongly related to any of the other land cover types.

54 ENVIRONMENTAL SCIENCES↗

Reports From EXPORTS Modeling and Data-Mining Activities

EXPORTS -EXport Processes in the Ocean from RemoTe Sensing is NASA’s large field campaign focusing on development of a predictive understanding of the export, fate and carbon cycle impacts of the global net primary production. This co-funded program (NSF, private funding1, and international participation) was conceived in 2013, with EXPORTS Science plan published in 2016 (Siegel et al 2016, EXPORTS Writing Team 2015) and its implementation plan finalized in 2016 (EXPORTS Science Definition Team 2016). More details are presented in Siegel et al (2021). EXPORTS campaign is structured as a multiyear effort (Figure 1). It started with “Pre-EXPORTS” modeling and data-mining activity followed by a first phase with two major field programs and a second synthesis and modeling phase. The “Pre-EXPORTS” projects, total of 6 of them (Table 1), funded under A.3 Ocean Biology and Biogeochemistry 2015 call, helped to plan the field campaign (Resplandy et al 2019, Rousseaux & Gregg 2017), and supporting further global synthesis with datasets mined from the literature (e.g. Bisson et al (2020), Bisson et al (2018), Kramer and Siegel (2019)), directly responding to objectives outlined in Science Plan (see section 6 in Team (2015)) and Implementation team (see Figure 1 in Team (2016)). This document presents a compilation of the final reports of the Pre-EXPORTS funded projects, in hope of synthesizing the outcomes, and insuring the legacy of this program. Each of these reports contains a list of published papers, and reader should refer to them to see results in details.

Brandi J. McCarty↗

Anomalous DOC Signatures Reveal Iron Control on Export Dynamics in the Pacific Southern Ocean

Here we shed light on two mechanisms that stimulate deep particle export via upper-ocean iron fertilization in the Southern Ocean: deep frontal mixing and melting of sea ice. We present data collected a decade apart in the Pacific sector of the Southern Ocean when, serendipitously, seasonal Antarctic ice melt was anomalously low (2008) and anomalously high (2017). In 2008, the low ice melt year, we concluded that vertical mixing of iron into the euphotic zone via deep-mixing fronts was the primary stimulant of export that reached depths of ~1500 meters. This process was evidenced by localized enhancements of dissolved organic carbon (DOC) concentrations up to 4 μmol C kg-1 beneath seven branches of fronts embedded within the Antarctic Circumpolar Current (ACC). We used these enhanced DOC concentrations in the bathypelagic as primary indications of the depths and locations of recent export, as it is a logical residue of such. In 2017, the year in which sea ice melt was anomalously high, we concluded that the main driver of a widespread export event to the seafloor was the lateral influx of iron within the melt. Indications of this event included substantial enhancements of DOC concentrations (2 - 31 6 μmol C kg-1), elevated beam attenuation, and enhanced surface iron concentrations associated with a layer of low salinity water at a nearby station. Further, significant deficits of upper ocean silicic acid during the 2017 occupation indicated that deep export was likely stimulated by an iron-fueled diatom bloom. This analysis highlights the impact of iron supplied from frontal vertical mixing and sea ice melt on export and ultimately for long-term carbon sequestration in the Southern Ocean, as well as the utility of deep DOC enrichments as signatures of particle export. Understanding the impact that ice melt events have on carbon export is crucial given that anomalous events are occurring more often as our climate changes.

Dissolved Organic Carbon↗

“The structure of the Type III secretion system export gate with CdsO, an ATPase lever arm”

Type III protein secretion systems (T3SS) deliver effector proteins from the Gram-negative bacterial cytoplasm into a eukaryotic host cell through a syringe-like, multi-protein nanomachine. Cytosolic components of T3SS include a portion of the export apparatus, which traverses the inner membrane and features the opening of the secretion channel, and the sorting complex for substrate recognition and for providing the energetics required for protein secretion. Two components critical for efficient effector export are the export gate protein and the ATPase, which are proposed to be linked by the central stalk protein of the ATPase. We present the structure of the soluble export gate homo-nonamer, CdsV, in complex with the central stalk protein, CdsO, of its cognate ATPase, both derived from Chlamydia pneumoniae. This structure defines the interface between these essential T3S proteins and reveals that CdsO engages the periphery of the export gate that may allow the ATPase to catalyze an opening between export gate subunits to allow cargo to enter the export apparatus. We also demonstrate through structure-based mutagenesis of the homologous export gate in Pseudomonas aeruginosa that mutation of this interface disrupts effector secretion. These results provide novel insights into the molecular mechanisms governing active substrate recognition and translocation through a T3SS.

59 BASIC BIOLOGICAL SCIENCES↗