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At least 1,333 records · Page 74
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Structural basis for autophagy inhibition by the human Rubicon–Rab7 complex

Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7–GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7–GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab–effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Science & Technology - Other Topics↗
DOE OSTI2024

Cation valency in water-in-salt electrolytes alters the short- and long-range structure of the electrical double layer

Highly concentrated aqueous electrolytes (termed water-in-salt electrolytes, WiSEs) at solid-liquid interfaces are ubiquitous in myriad applications including biological signaling, electrosynthesis, and energy storage. This interface, known as the electrical double layer (EDL), has a different structure in WiSEs than in dilute electrolytes. Here, we investigate how divalent salts [zinc bis(trifluoromethylsulfonyl)imide, Zn(TFSI) 2 ], as well as mixtures of mono- and divalent salts [lithium bis(trifluoromethylsulfonyl)imide (LiTFSI) mixed with Zn(TFSI) 2 ], affect the short- and long-range structure of the EDL under confinement using a multimodal combination of scattering, spectroscopy, and surface forces measurements. Raman spectroscopy of bulk electrolytes suggests that the cation is closely associated with the anion regardless of valency. Wide-angle X-ray scattering reveals that all bulk electrolytes form ion clusters; however, the clusters are suppressed with increasing concentration of the divalent ion. To probe the EDL under confinement, we use a Surface Forces Apparatus and demonstrate that the thickness of the adsorbed layer of ions at the interface grows with increasing divalent ion concentration. Multiple interfacial layers form following this adlayer; their thicknesses appear dependent on anion size, rather than cation. Importantly, all electrolytes exhibit very long electrostatic decay lengths that are insensitive to valency. It is likely that in the WiSE regime, electrostatic screening is mediated by the formation of ion clusters rather than individual well-solvated ions. This work contributes to understanding the structure and charge-neutralization mechanism in this class of electrolytes and the interfacial behavior of mixed-electrolyte systems encountered in electrochemistry and biology.

Science & Technology - Other Topics↗
DOE OSTI2025

The chromatin remodeler ADNP regulates neurodevelopmental disorder risk genes and neocortical neurogenesis

Although chromatin remodelers are among the most important risk genes associated with neurodevelopmental disorders (NDDs), the roles of these complexes during brain development are in many cases unclear. Here, we focused on the recently discovered ChAHP chromatin remodeling complex. The zinc finger and homeodomain transcription factor ADNP is a core subunit of this complex, and de novoADNPmutations lead to intellectual disability and autism spectrum disorder. However, germlineAdnpknockout mice were previously shown to exhibit early embryonic lethality, obscuring subsequent roles for the ChAHP complex in neurogenesis. To circumvent this early developmental arrest, we generated a conditionalAdnpmutant allele. Using single-cell transcriptomics, cut&run-seq, and histological approaches, we show that during neocortical development, Adnp orchestrates the production of late-born, upper-layer neurons through a two-step process. First, Adnp is required to sustain progenitor proliferation specifically during the developmental window for upper-layer cortical neurogenesis. Accordingly, we found that Adnp recruits the ChAHP subunit Chd4 to genes associated with progenitor proliferation. Second, in postmitotic differentiated neurons, we define a network of risk genes linked to NDDs that are regulated by Adnp and Chd4. Taken together, these data demonstrate that ChAHP is critical for driving the expansion of upper-layer cortical neurons and for regulating neuronal gene expression programs, suggesting that these processes may potentially contribute to NDD etiology.

Science & Technology - Other Topics↗
DOE OSTI2021

Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion

Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1' recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

High temperature and pressure regime soot: Physical, optical and chemical signatures from high explosive detonations

Submicron particles are formed during the detonation and deflagration of carbon-rich energetic materials known as high explosives (HE) that can be lofted and transported long distances in the atmosphere. Particles include refractory species like soot, a.k.a. black carbon (BC), and have impacts to climate and human health at local, regional, and global scales. Here, we contrast detonation soot aerosol physical, optical and chemical properties that are formed more efficiently in terms of emission factors and ratios with traditional combustion sources from biomass burning and fossil fuels. We utilize ambient aerosol techniques for the first time to identify soot from two well-known HE's (1) Comp B-3: 40% trinitrotoluene (TNT) and 60% 1,3,5-trinitro-1,3,5-triazinane (RDX) and (2) PBX 9501: 95% 1,3,5,7-tetranitro-1,3,5,7-tetrazoctane (HMX) with 2.5% Estane and a 2.5% mixture of bis(2,2-dinitropropyl)-acetal (BDNPA) and bis(2,2-dinitropropyl)-formal (BDNPF). Size distributions, optical and chemical properties are presented. Aggregate soot had small mobility diameters (<100 nm) and unique optical and chemical signatures depending on the explosive composite composition, detonation atmosphere (e.g., air, argon) and pressure. Single-scattering albedos (SSA) from 0.3 to 0.8 and absorption Angstrom exponents (AAE) from 0.5 to 2.0 measured across 405–870 nm wavelengths depended on the explosive composite composition and detonation conditions. Comp B-3 detonation soot was similar to other combustion soot sources, whereas PBX 9501 soot had high scattering relative to absorption. Carbonaceous chemical signatures included the presence of fullerenes that were distinct from ambient and laboratory-generated soot surrogates and the detection of trace metals, including aluminum, copper, iron, and zinc.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Synthesis of Zn2NbN3 ternary nitride semiconductor with wurtzite-derived crystal structure

Binary III-N nitride semiconductors with wurtzite crystal structure such as GaN and AlN have been long used in many practical applications ranging from optoelectronics to telecommunication. The structurally related ZnGeN2 or ZnSnN2 derived from the parent binary compounds by cation mutation (elemental substitution) have recently attracted attention, but such ternary nitride materials are mostly limited to II-IV-N2 compositions. This paper demonstrates synthesis and characterization of zinc niobium nitride (Zn2NbN3) – a previously unreported II2-V-N3 ternary nitride semiconductor. The Zn2NbN3 thin films are synthesized using a one-step adsorption-controlled growth, and a two-step deposition/annealing method that suppresses the loss of Zn and N. Measurements indicate that this sputtered Zn2NbN3 crystalizes in cation-disordered wurtzite-derived structure, in contrast to chemically related rocksalt-derived Mg2NbN3 compound, also synthesized here for comparison using the two-step method. The estimated wurtzite lattice parameter ratio of Zn2NbN3 is 1.55, and the optical absorption onset is at 2.1 eV. Both of these values are lower compared to published Zn2NbN3 computational values of c/a = 1.62 and Eg = 3.5 - 3.6 eV. Additional theoretical calculations indicate that this difference is due to cation disorder in experimental samples, suggesting a way to tune the structural parameters and the resulting properties of heterovalent ternary nitride materials. Overall, this work expands the wurtzite family of nitride semiconductors to include Zn2NbN3, and suggests that related II2-V-N3 and other ternary nitrides should be possible to synthesize.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2023

Large-area epitaxial growth of InAs nanowires and thin films on hexagonal boron nitride by metal organic chemical vapor deposition

Large-area epitaxial growth of III–V nanowires and thin films on van der Waals substrates is key to developing flexible optoelectronic devices. In our study, large-area InAs nanowires and planar structures are grown on hexagonal boron nitride templates using metal organic chemical vapor deposition method without any catalyst or pre-treatments. The effect of basic growth parameters on nanowire yield and thin film morphology is investigated. Under optimised growth conditions, a high nanowire density of 2.1 x 10 9 cm -2 is achieved. A novel growth strategy to achieve uniform InAs thin film on h-BN/SiO 2 /Si substrate is introduced. The approach involves controlling the growth process to suppress the nucleation and growth of InAs nanowires, while promoting the radial growth of nano-islands formed on the h-BN surface. A uniform polycrystalline InAs thin film is thus obtained over a large area with a dominant zinc-blende phase. The film exhibits near-band-edge emission at room temperature and a relatively high Hall mobility of 399 cm -2 /(Vs). This work suggests a promising path for the direct growth of large-area, low-temperature III–V thin films on van der Waals substrates.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2022

Multi-channel front-end ASIC for a 3D position-sensitive detector

Arrays of 3D position-sensitive detectors (3DPSD), operating at room temperature and using cadmium zinc telluride (CZT) and thallium bromide (TIBr) sensors, are suitable for gamma-ray spectrometry in many applications. One detector configuration, the 3D position-sensitive Virtual Frisch-Grid detector (VFG), is particularly advantageous for integrating into large area arrays. The signals generated inside each detector of the array are captured with the anode, cathode and four pads that enable the reconstruction of the position and energy of the ionizing interaction by measurements of amplitude and timing of the signals. For these applications, a low-noise front-end ASIC has been developed, capable of processing bipolar signals (needed because of AC-coupling of certain electrodes). The ASIC can be coupled to an ADC in order to form a compound “waveform digitizer” capable of post-processing the analog signals and determining amplitude and timing information. This paper describes a 32-channel front-end ASIC that is suitable for reading out a 3 × 3 or 4 × 4 element matrix in the VFG configuration. Each channel is composed of a low-noise charge amplifier with an adaptive continuous reset feedback circuit suitable for both positive and negative charge, a first order shaper and a single-to-differential converter output stage. Voltage and current references are all internally generated by 10-bit DACs and the chip is fully controllable with the I 2 C communication protocol. The readout channel response has been verified using the implemented injection circuit. Linear behavior up to ~75 ke ± with the gain of ~80 mV/fC, and up to ~200 ke ± with the gain of ~30 mV/fC was demonstrated. In conclusion, the first test result waveforms using a 137 Cs radioactive source on a 5 × 5 × 12 mm 3 TIBr crystal are reported.

47 OTHER INSTRUMENTATION↗
DOE OSTI2023

Integrating functional scoring and regulatory data to predict the effect of non-coding SNPs in a complex neurological disease

Abstract Most SNPs associated with complex diseases seem to lie in non-coding regions of the genome; however, their contribution to gene expression and disease phenotype remains poorly understood. Here, we established a workflow to provide assistance in prioritising the functional relevance of non-coding SNPs of candidate genes as susceptibility loci in polygenic neurological disorders. To illustrate the applicability of our workflow, we considered the multifactorial disorder migraine as a model to follow our step-by-step approach. We annotated the overlap of selected SNPs with regulatory elements and assessed their potential impact on gene expression based on publicly available prediction algorithms and functional genomics information. Some migraine risk loci have been hypothesised to reside in non-coding regions and to be implicated in the neurotransmission pathway. In this study, we used a set of 22 non-coding SNPs from neurotransmission and synaptic machinery-related genes previously suggested to be involved in migraine susceptibility based on our candidate gene association studies. After prioritising these SNPs, we focused on non-reported ones that demonstrated high regulatory potential: (1) VAMP2_rs1150 (3′ UTR) was predicted as a target of hsa-mir-5010-3p miRNA, possibly disrupting its own gene expression; (2) STX1A_rs6951030 (proximal enhancer) may affect the binding affinity of zinc-finger transcription factors (namely ZNF423) and disturb TBL2 gene expression; and (3) SNAP25_rs2327264 (distal enhancer) expected to be in a binding site of ONECUT2 transcription factor. This study demonstrated the applicability of our practical workflow to facilitate the prioritisation of potentially relevant non-coding SNPs and predict their functional impact in multifactorial neurological diseases.

Felício, Daniela↗
DOE OSTI2022

Zap1 is required for Candida glabrata response to fluconazole

ABSTRACT The increasing prevalence of fluconazole-resistant clinical isolates of Candida spp. strongly hinders the widespread use of the drug. To tackle this problem, great efforts have been made to fully understand the fungal response to fluconazole. In this work, we show that the role of Zap1 in Candida glabrata goes beyond regulating yeast adaptation to zinc deficiency. In line with our previous observation that deletion of ZAP1 makes yeast cells more sensitive to fluconazole, we found that the mutant CgΔzap1 accumulates higher levels of the drug, which correlates well with its lower levels of ergosterol. Surprisingly, Zap1 is a negative regulator of the drug efflux transporter gene CDR1 and of its regulator, PDR1. The apparent paradox of drug accumulation in cells where genes encoding transporters relevant for drug extrusion are being overexpressed led us to postulate that their activity could be impaired. In agreement, Zap1-depleted cells present, in addition to decreased ergosterol levels, an altered composition of membrane phospholipids, which together should impact membrane function and impair the detoxification of fluconazole. Overall, our study brings to light Zap1 as an important hub in Candida glabrata response to fluconazole.

Gaspar-Cordeiro, A. (ORCID:0000000202488965)↗
DOE OSTI2023

Grf10 regulates the response to copper, iron, and phosphate in Candida albicans

Abstract The pathogenic yeast, Candida albicans, and other microbes must be able to handle drastic changes in nutrient availability within the human host. Copper, iron, and phosphate are essential micronutrients for microbes that are sequestered by the human host as nutritional immunity; yet high copper levels are employed by macrophages to induce toxic oxidative stress. Grf10 is a transcription factor important for regulating genes involved in morphogenesis (filamentation, chlamydospore formation) and metabolism (adenylate biosynthesis, 1-carbon metabolism). The grf10Δ mutant exhibited resistance to excess copper in a gene dosage-dependent manner but grew the same as the wild type in response to other metals (calcium, cobalt, iron, manganese, and zinc). Point mutations in the conserved residues D302 and E305, within a protein interaction region, conferred resistance to high copper and induced hyphal formation similar to strains with the null allele. The grf10Δ mutant misregulated genes involved with copper, iron, and phosphate uptake in YPD medium and mounted a normal transcriptional response to high copper. The mutant accumulated lower levels of magnesium and phosphorus, suggesting that copper resistance is linked to phosphate metabolism. Our results highlight new roles for Grf10 in copper and phosphate homeostasis in C. albicans and underscore the fundamental role of Grf10 in connecting these with cell survival.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Evaluation of digestively resistant or soluble fibers, short- and medium-chain fatty acids, trace minerals, and antibiotics in nonchallenged nursery pigs on performance, digestibility, and intestinal integrity

Abstract Three experiments (EXP) were conducted to determine the effect of feed additives on performance, intestinal integrity, gastrointestinal volatile fatty acids (VFA), and energy and nutrient digestion in nonchallenged nursery pigs. In EXP 1, 480 pigs (6.36-kg body weight, BW) were placed into 96 pens with 5 pigs/pen, and allotted to 1 of 10 dietary treatments: 1) negative control containing no feed additive (NC), 2) NC + 44 mg chlortetracycline and 38.5 mg tiamulin/kg diet (CTsb), 3) NC + 5% resistant potato starch (RSpo), 4) NC + 5% soluble corn fiber (SCF), 5) NC + 5% sugar beet pulp (SBP), 6) NC + 0.30% fatty acid mix (FAM), 7) NC + 0.10% phytogenic blend of essential oils and flavoring compounds (PHY), 8) NC + 50 mg Cu and 1,600 mg zinc oxide/kg diet (CuZn), 9) NC + 5% resistant corn starch (RScn), and 10) NC + 0.05% β-glucan (BG) for 28 d. There was no impact of dietary treatment on BW gain or feed intake (P ≥ 0.22). Pigs fed diets containing SCF, CTsb, and RSpo resulted in microbial community differences compared to pigs fed the NC (P < 0.05). In EXP 2, 48 barrows (12.8 kg BW) were selected at the end of EXP 1 and fed the same dietary treatments they had previously received: 1) NC, 2) NC + 5% RScn, 3) NC + 5% SCF, and 4) NC + FAM for 8 d. There was no effect of feeding diets containing RScn, SCF, or FAM on in vivo intestinal permeability (P ≤ 0.21). Ileal or colon pH, concentrations of VFA did not differ due to dietary treatment (P ≥ 0.36), but pigs fed diets containing FAM resulted in a greater butyric acid concentration in the cecum compared to pigs fed the NC (P ≤ 0.05). In EXP 3, 156 pigs (6.11 kg BW) were placed into 52 pens with 3 pigs/pen and allotted to 1 of 4 dietary treatments arranged in a factorial manner: 1) NC, 2) NC + 5% RSpo, 3) NC + 0.30% FAM, and 4) NC + 5% RSpo + 0.30% FAM for 24 d. Feeding pigs diets containing RSpo did not affect BW gain (P = 0.91) while pigs fed diets containing FAM grew improved BW gain (P = 0.09). Colonic butyric acid concentrations were greater in pigs fed diets containing RSpo (P = 0.03), while pigs fed diets containing FAM exhibited reduced total VFA concentrations (P = 0.11). The results indicate that supplementing diets with digestively resistant but fermentable fibers, short- and medium-chain fatty acids, or antibiotics do not have a consistent effect, positive or negative, on markers of intestinal integrity or barrier function, intestinal VFA patterns, ATTD of energy and nutrients, or on pig performance.

Agriculture↗
DOE OSTI2021

Imaging the structural organization of chemical elements in growth cones of developing hippocampal neurons

Abstract During neurodevelopment, neurons form growth cones, F-actin rich extensions located at the distal end of the neurites. Growth cones allow dendrites and axons to build synaptic connections through a process of neurite guidance whose mechanisms have not been fully elucidated. Calcium is an important element in this process by inducing F-actin reorganization. We hypothesized that other biologically active elements might be involved in the growth cone-mediated neurite guidance mechanisms. We performed super resolution and confocal microscopy of F-actin, followed by synchrotron X-ray fluorescence microscopy of phosphorous, sulfur, chlorine, potassium, calcium, iron and zinc on growth cones from primary rat hippocampal neurons. We identified two main patterns of element organization. First, active growth cones presenting an asymmetric distribution of Ca co-localized with the cytoskeleton protein F-actin. In active growth cones, we found that the distributions of P, S, Cl, K, and Zn are correlated with Ca. This correlation is lost in the second pattern, quiescent growth cones, exhibiting a spread elemental distribution. These results suggest that Ca is not the only element required in the F-actin rich active regions of growth cones. In addition, highly concentrated Fe spots of submicrometer size were observed in calcium-rich areas of active growth cones. These results reveal the need for biological active elements in growth cones during neural development and may help explain why early life deficiencies of elements, such as Fe or Zn, induce learning and memory deficits in children.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Zn deficiency disrupts Cu and S homeostasis in Chlamydomonas resulting in over accumulation of Cu and Cysteine

Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. Here, we show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding “first responder” proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine, and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ~80-fold, corresponding to ~2.8 × 10 9 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Synchrotron X-rays reveal the modes of Fe binding and trace metal storage in the brown algae Laminaria digitata and Ectocarpus siliculosus

Abstract Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (μXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation–freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization

Abstract Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the green alga Chlamydomonas reinhardtii, Cu import is dependent on a transcription factor, Copper Response Regulator 1 (CRR1), responsible for activating genes in Cu-deficient cells. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulates the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high-affinity Cu(I) uptake. On the other hand, the overaccumulation of Cu(I) (20 times the quota) in zinc (Zn) deficiency depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and consistent with the lower substrate affinity of CTR2. One sentence summary Regulation of Cu uptake and sequestration by members of the CTR family of proteins in Chlamydomonas.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Allosteric autoregulation of DNA binding via a DNA-mimicking protein domain: a biophysical study of ZNF410–DNA interaction using small angle X-ray scattering

ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA binding affinity, compared to that of the isolated DNA binding ZF array, both in vitro and in cells. AlphaFold predicts a partially-folded N-terminal subdomain that includes a 30-residue long helix, preceded by a hairpin loop rich in acidic (aspartate/glutamate) and serine/threonine residues. This hairpin loop is predicted by AlphaFold to lie against the DNA binding interface of the ZF array. In solution, ZNF410 is a monomer and binds to DNA with 1:1 stoichiometry. Surprisingly, the single best-fit model for the experimental small angle X-ray scattering profile, in the absence of DNA, is the original AlphaFold model with the N-terminal long-helix and the hairpin loop occupying the ZF DNA binding surface. For DNA binding, the hairpin loop presumably must be displaced. After combining biophysical, biochemical, bioinformatic and artificial intelligence-based AlphaFold analyses, we suggest that the hairpin loop mimics the structure and electrostatics of DNA, and provides an additional mechanism, supplementary to sequence specificity, of regulating ZNF410 DNA binding.

59 BASIC BIOLOGICAL SCIENCES↗