A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network.

Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments

Cardioprotection by an anti-MASP-2 antibody in a murine model of myocardial infarction

Background Myocardial ischaemia-reperfusion injury is a major cause of mortality and morbidity in the developed world. Many approaches have been investigated to counteract the pathological consequences associated with acute myocardial infarction (AMI) and cardiac remodelling. It is accepted that inflammation, and therefore activation of the complement pathway, is a crucial step in the pathogenesis of this injury, and many attempts have been made to ameliorate the infarction and consequent dysfunction using anticomplement therapy, with mixed success. Recently, the lectin complement activation pathway involving the mannose-binding lectin-associated serine protease 2 (MASP-2) has been shown to be an important mediator of the inflammatory response in ischaemia/reperfusion injury in the heart. In this study, therefore, we aimed to investigate the feasibility of using monoclonal antibodies raised against MASP-2 in a murine model of AMI. Methods Mice were injected with anti-MASP-2 antibody or control 18 hours prior to experimental infarction by ligation of the left anterior descending coronary artery for 30 min followed by 120 min reperfusion. The developed infarct was measured, and blood was collected for analysis of lectin pathway functional activity. Results and conclusions We found that mice treated with anti-MASP-2 antibody had smaller infarcts than those treated with control antibody. We believe this may represent a valuable step forward in the protection of the myocardium against ischaemia-reperfusion injury.</p

Conserved metabolite regulation of stress granule assembly via AdoMet

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Begovich, K., Vu, A. Q., Yeo, G., & Wilhelm, J. E. Conserved metabolite regulation of stress granule assembly via AdoMet. Journal of Cell Biology, 219(8), (2020): e201904141, doi:10.1083/jcb.201904141.Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress. Furthermore, the product of one of these enzymes, AdoMet, is a regulator of SG assembly and composition. Decreases in AdoMet levels increase SG formation, while chronic elevation of AdoMet produces SG remnants lacking proteins associated with the 5′ end of transcripts. Interestingly, acute elevation of AdoMet blocks SG formation in yeast and motor neurons. Treatment of ALS-derived motor neurons with AdoMet also suppresses the formation of TDP-43–positive SGs, a hallmark of ALS. Together, these results argue that AdoMet is an evolutionarily conserved regulator of SG composition and assembly with therapeutic potential in neurodegeneration.Work from the Wilhelm laboratory was supported by a grant to J.E. Wilhelm from the Collaborative Innovation Awards program of Howard Hughes Medical Institute and the James Wilhelm Memorial Fund. K. Begovich is a Howard Hughes Medical Institute Gilliam Fellow. Work from the Yeo laboratory was supported by grants to G. Yeo from the National Institutes of Health (HG004659), Target ALS (20193440), and the ALS Association (272 and 438)

Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end–directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation

A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Noree, C., Begovich, K., Samilo, D., Broyer, R., Monfort, E., & Wilhelm, J. E. A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network. Molecular Biology of the Cell, 30(21), (2019): 2721-2736, doi:10.1091/mbc.E19-04-0224.Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments.We thank Douglass Forbes for comments on the manuscript, Susanne Rafelski for the gift of the pVTU-mito-dsRed plasmid, and Brian Zid for the gift of the pKT-mNeonGreen plasmid. Work at the Wilhelm lab was supported by a grant from the Hughes Collaborative Innovation Award program of the Howard Hughes Medical Institute and the James Wilhelm Memorial Fund. Kyle Begovich is a Howard Hughes Medical Institute Gilliam Fellow

Organic Entrainment and Preservation in Volcanic Glasses

Unaltered pyroclastic deposits have previously been deemed to have "low" potential for the formation, concentration and preservation of organic material on the Martian surface. Yet volcanic glasses that have solidified very quickly after an eruption may be good candidates for containment and preservation of refractory organic material that existed in a biologic system pre-eruption due to their impermeability and ability to attenuate UV radiation. Analysis using NanoSIMS of volcanic glass could then be performed to both deduce carbon isotope ratios that indicate biologic origin and confirm entrainment during eruption. Terrestrial contamination is one of the biggest barriers to definitive Martian organic identification in soil and rock samples. While there is a greater potential to concentrate organics in sedimentary strata, volcanic glasses may better encapsulate and preserve organics over long time scales, and are widespread on Mars. If volcanic glass from many sites on Earth could be shown to contain biologically derived organics from the original environment, there could be significant implications for the search for biomarkers in ancient Martian environments

Use of Remote-Sensing Imagery to Estimate Corn Grain Yield

Remote sensing—the process of acquiring information about objects from remote platforms such as ground-based booms, aircraft, or satellites—is a potentially important source of data for site-specific crop management, providing both spatial and temporal information. Our objective was to use remotely sensed imagery to compare different vegetation indices as a means of assessing canopy variation and its resultant impact on corn (Zea mays L.) grain yield. Treatments consisted of five N rates and four hybrids, which were grown under irrigation near Shelton, NE on a Hord silt loam in 1997 and 1998. Imagery data with 0.5-m spatial resolution were collected from aircraft on several dates during both seasons using a multispectral, four-band [blue, green, red, and near-infrared reflectance] digital camera system. Imagery was imported into a geographical information system (GIS) and then geo-registered, converted into reflectance, and used to compute three vegetation indices. Grain yield for each plot was determined at maturity. Results showed that green normalized difference vegetation index (GNDVI) values derived from images acquired during midgrain filling were the most highly correlated with grain yield; maximum correlations were 0.7 and 0.92 in 1997 and 1998, respectively. Normalizing GNDVI and grain yield variability within hybrids improved the correlations in both years, but more dramatic increases were observed in 1997 (0.7 to 0.82) than in 1998 (0.92 to 0.95). This suggested GNDVI acquired during midgrain filling could be used to produce relative yield maps depicting spatial variability in fields, offering a potentially attractive alternative to use of a combine yield monitor

Abundant Lipid and Protein Components of Drusen

Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)(2) analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5+/-13.7, 36.9+/-12.9, and 43.0+/-11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37-44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit beta, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion

Orbital evidence for more widespread carbonate-bearing rocks on Mars

Carbonates are key minerals for understanding ancient Martian environments because they are indicators of potentially habitable, neutral-to-alkaline water and may be an important reservoir for paleoatmospheric CO_2. Previous remote sensing studies have identified mostly Mg-rich carbonates, both in Martian dust and in a Late Noachian rock unit circumferential to the Isidis basin. Here we report evidence for older Fe- and/or Ca-rich carbonates exposed from the subsurface by impact craters and troughs. These carbonates are found in and around the Huygens basin northwest of Hellas, in western Noachis Terra between the Argyre basin and Valles Marineris, and in other isolated locations spread widely across the planet. In all cases they cooccur with or near phyllosilicates, and in Huygens basin specifically they occupy layered rocks exhumed from up to ~5 km depth. We discuss factors that might explain their observed regional distribution, arguments for why carbonates may be even more widespread in Noachian materials than presently appreciated and what could be gained by targeting these carbonates for further study with future orbital or landed missions to Mars