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Results for “Organelle Targeting”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

Highlights: • ABCD proteins classifies based on with or without NH{sub 2}-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH{sub 2}-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH{sub 2}-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH{sub 2}-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH{sub 2}-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH{sub 2}-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH{sub 2}-terminal H0 motif in organelle targeting is widely conserved in living organisms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Beyond the Western front: Targeted proteomics and organelle abundance profiling

The application of westerns or immunoblotting techniques for assessing the composition, dynamics, and purity of protein extracts from plant material has become common practice. While the approach is reproducible, can be readily applied and is generally considered robust, the field of plant science suffers from a lack of antibody variety against plant proteins. The development of approaches that employ mass spectrometry to enable both relative and absolute quantification of many hundreds of proteins in a single sample from a single analysis provides a mechanism to overcome the expensive impediment in having to develop antibodies in plant science. Here, we consider it an opportune moment to consider and better develop the adoption of multiple reaction monitoring (MRM)-based analyses in plant biochemistry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Targeted protein degradation: from small molecules to complex organelles—a Keystone Symposia report

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome–lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. Additionally, on June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium “Targeting protein degradation: from small molecules to complex organelles.” The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

MTS-VAE

This repository accompanies the work "Design of diverse, functional mitochondrial targeting sequences across eukaryotic organisms using variational autoencoder". It includes the datasets for model training, validation, and downstream analysis.

Mitochondria↗
DOE OSTI2020

Peptoid-Based Programmable 2D Nanomaterial Sensor for Selective and Sensitive Detection of H 2 S in Live Cells

During the past decades, a variety of two-dimensional (2D) nanosheets have been developed through supramolecular self-assembly. Among them, those assembled from sequence-defined molecules have received particularly attention because they enable a precise displaying of functional groups, including fluorescent dyes, within nanosheet surfaces. On the other hand, due to the fluorescence self-quenching, synthesis of organic 2D nanosheets exhibiting high fluorescence quantum yields is a significant challenge. Herein, we report the utilization of H 2 S-responsive probes as peptoid sidechains to design and synthesize crystalline 2D nanomembranes (2DNMs) as selective and sensitive H 2 S sensors. These 2DNM sensors exhibit a high quantum yield as a result of high crystallinity and tunable probe density. Compared with amorphous state of peptoid assembly and pre-assembled peptoid-probe conjugates, these crystalline 2DNM sensors exhibit a significantly strong fluorescence intensity and a high sensitivity (~ 0.36 nM). By sonication-cutting these 2DNM sensors into a colloidal form in aqueous solution, we further demonstrated the use of colloidal 2DNMs for detecting both exogenous and endogenous H 2 S inside cells or targeted cell organelles. As far as we know, this is the first example of using sequence-defined 2D nanomaterials for selective and sensitive detection of H 2 S. Because peptoids are biocompatible and peptoid-based 2DNMs are highly programmable, we expect that this new class of 2DNM sensors offer great potentials for detecting H 2 S and for biological applications.

2D nanomaterials↗
DOE OSTI2022

Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm type IV secretion system. Those effectors are essential for the biogenesis of the Legionella-containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases (ANTs) by ADP-ribosylation and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a novel virulence mechanism for L. pneumophila.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

Abstract The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe.

Wijesooriya, Chamari S.↗
DOE OSTI2018

A Photoactivatable BODIPY Probe for Localization-based Super-Resolution Cellular Imaging

Here, the synthesis and application of a photoactivatable boron–alkylated BODIPY probe for localization–based super–resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously–unknown boron–photodealkylation reaction with a single low–power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization–based microscopy of biological systems. The probe can be easily linked to nucleophile–containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super–resolution imaging of microtubules is demonstrated. This is the first example of single molecule localization–based super–resolution microscopy using a visible–light activated BODIPY compound as a fluorescent probe.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Defining upstream enhancing and inhibiting sequence patterns for plant peroxisome targeting signal type 1 using large–scale in silico and in vivo analyses

Peroxisomes are universal eukaryotic organelles essential to plants and animals. Most peroxisomal matrix proteins carry peroxisome targeting signal type 1 (PTS1), a C-terminal tripeptide. Studies from various kingdoms have revealed influences from sequence upstream of the tripeptide on peroxisome targeting, supporting the view that positive charges in the upstream region are the major enhancing elements. However, a systematic approach to better define the upstream elements influencing PTS1 targeting capability is needed. Here, we used protein sequences from 177 plant genomes to perform large-scale and in-depth analysis of the PTS1 domain, which includes the PTS1 tripeptide and upstream sequence elements. We identified and verified 12 low-frequency PTS1 tripeptides and revealed upstream enhancing and inhibiting sequence patterns for peroxisome targeting, which were subsequently validated in vivo. Follow-up analysis revealed that nonpolar and acidic residues have relatively strong enhancing and inhibiting effects, respectively, on peroxisome targeting. However, in contrast to the previous understanding, positive charges alone do not show the anticipated enhancing effect and that both the position and property of the residues within these patterns are important for peroxisome targeting. We further demonstrated that the three residues immediately upstream of the tripeptide are the core influencers, with a ‘basic-nonpolar-basic’ pattern serving as a strong and universal enhancing pattern for peroxisome targeting. These findings have significantly advanced our knowledge of the PTS1 domain in plants and likely other eukaryotic species as well. The principles and strategies employed in the present study may also be applied to deciphering auxiliary targeting signals for other organelles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria

We report that Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial–fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situhybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial–fungal relationships.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their ability to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of novel enzymatic activity in protein coacervates with a light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of C. elegans LAF-1, we show functionalization of condensates with enzymatic activity in vitro and that localization to protein coacervates enhances the assembly and activity of split enzymes. To build condensates that function as light emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. Furthermore, the resulting condensates concentrated NanoLuc by 10-fold over bulk solution and display significantly increased net reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. By splitting NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. Further, we demonstrate control of the spatial localization of enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity, and that this activity can be spatially and temporally controlled via enzyme reconstitution and design of protein surfactants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Transgenic Plant-Produced Hydrolytic Enzymes and the Potential of Insect Gut-Derived Hydrolases for Biofuels

Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review

enzyme targeting↗
DOE OSTI2017

Repurposing the Saccharomyces cerevisiae peroxisome for compartmentalizing multi-enzyme pathways

The peroxisome of Saccharomyces cerevisiae was targeted for repurposing in order to create a synthetic organelle that provides a generalizable compartment for engineered metabolic pathways. Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk, improving pathway efficiency, and ultimately modifying the chemical environment to be distinct from that of the cytoplasm. We focused on the Saccharomyces cerevisiae peroxisome, as this organelle is not required for viability when grown on conventional media. We identified an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly importing non-native cargo proteins. Additionally, we performed the first systematic in vivo measurements of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay and characterized the size dependency of metabolite trafficking. Finally, we applied these new insights to compartmentalize a two-enzyme pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titer. This work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.

10 SYNTHETIC FUELS↗
DOE OSTI2018

Multimodal LA-ICP-MS and nanoSIMS imaging enables copper mapping within photoreceptor megamitochondria in a zebrafish model of Menkes disease

Here, copper is essential for eukaryotic life, and animals must acquire this nutrient through the diet and distribute it to cells and organelles for proper function of biological targets. Indeed, mutations in the central copper exporter ATP7A contribute to a spectrum of diseases, including Menkes disease, with symptoms ranging from neurodegeneration to lax connective tissue. As such, a better understanding of the fundamental impacts of ATP7A mutations on in vivo copper distributions is of relevance to those affected by these diseases. For this work, we combine metal imaging and optical imaging techniques at a variety of spatial resolutions to identify tissues and structures with altered copper levels in the Calamity gw71 zebrafish model of Menkes disease. Rapid profiling of tissue slices with LA-ICP-MS identified reduced copper levels in the brain, neuroretina, and liver of Menkes fish compared to control specimens. High resolution nanoSIMS imaging of the neuroretina, combined with electron and confocal microscopies, identified the megamitochondria of photoreceptors as loci of copper accumulation in wildtype fish, with lower levels of megamitochondrial copper observed in Calamity gw71 zebrafish. Interestingly, this localized copper decrease does not result in impaired photoreceptor development or altered megamitochondrial morphology, suggesting the prioritization of copper at sufficient levels for maintaining essential mitochondrial functions. Together, these data establish the Calamity gw71 zebrafish as an optically transparent in vivo model for the study of neural copper misregulation, illuminate a role for the ATP7A copper exporter in trafficking copper to the neuroretina, and highlight the utility of combining multiple imaging techniques for studying metals in whole organism settings with spatial resolution.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Neutral rhenium( i ) tricarbonyl complexes with sulfur-donor ligands: anti-proliferative activity and cellular localization

Rhenium(I) tricarbonyl complexes are widely studied for their cell imaging properties and anti-cancer and anti-microbial activities, but the complexes with S-donor ligands remain relatively unexplored. A series of six fac-[Re(NN)(CO) 3 (SR)] complexes, where (NN) is 2,2'-bipyridyl (bipy) or 1,10-phenanthroline (phen), and RSH is a series of thiocarboxylic acid methyl esters, have been synthesized and characterized. Cellular uptake and anti-proliferative activities of these complexes in human breast cancer cell lines (MDA-MB-231 and MCF-7) were generally lower than those of the previously described fac-[Re(NN)(CO) 3 (OH 2 )]+ complexes; however, one of the complexes, fac-[Re(CO) 3 (phen)(SC(Ph)CH 2 C(O)OMe)] (3b), was active (IC 50 ~ 10 μM at 72 h treatment) in thiol-depleted MDA-MB-231 cells. Moreover, unlike fac-[Re(CO) 3 (phen)(OH 2 )]+, this complex did not lose activity in the presence of extracellular glutathione. Taken together these properties show promise for further development of 3b and its analogues as potential anti-cancer drugs for co-treatment with thiol-depleting agents. Conversely, the stable and non-toxic complex, fac-[Re(bipy)(CO) 3 (SC(Me)C(O)OMe)] (1a), predominantly localized in the lysosomes of MDA-MB-231 cells, as shown by live cell confocal microscopy (λ ex = 405 nm, λ em = 470–570 nm). It is strongly localized in a subset of lysosomes (25 μM Re, 4 h treatment), as shown by co-localization with a Lysotracker dye. Longer treatment times with 1a (25 μM Re for 48 h) resulted in partial migration of the probe into the mitochondria, as shown by co-localization with a Mitotracker dye. These properties make complex 1a an attractive target for further development as an organelle probe for multimodal imaging, including phosphorescence, carbonyl tag for vibrational spectroscopy, and Re tag for X-ray fluorescence microscopy.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Neutral rhenium(i) tricarbonyl complexes with sulfur-donor ligands: anti-proliferative activity and cellular localization

Rhenium(I) tricarbonyl complexes are widely studied for their cell imaging properties and anticancer and anti-microbial activities, but the complexes with S-donor ligands remain relatively unexplored. A series of six fac-[Re(NN)(CO) 3 (SR)] complexes, where (NN) is 2,2'-bipyridyl (bipy) or 1,10-phenanthroline (phen), and RSH is a series of thiocarboxylic acid methyl esters, have been synthesized and characterized. Cellular uptake and anti-proliferative activities of these complexes in human breast cancer cell lines (MDA-MB-231 and MCF-7) were generally lower than those of the previously described fac-[Re(NN)(CO) 3 (OH 2 )] + complexes; however, one of the complexes, fac-[Re(CO) 3 (phen)(SC(Ph)CH 2 C(O)OMe))] (3b), was active (IC 50 ~ 10 μM at 72 h treatment) in thiol-depleted MDA-MB-231 cells. Moreover, unlike fac- [Re(CO) 3 (phen)(OH 2 )] + , this complex did not lose activity in the presence of extracellular glutathione. Taken together these properties show promise for further development of 3b and its analogues as potential anti-cancer drugs for co-treatment with thiol-depleting agents. Conversely, the stable and non-toxic complex, fac-[Re(bipy)(CO) 3 (SC(Me)C(O)OMe)] (1a), predominantly localized in the lysosomes of MDA-MB-231 cells, as shown by live cell confocal microscopy (λ ex = 405 nm, λ em = 470-570 nm). It is strongly localized in a subset of lysosomes (25 μM Re, 4 h treatment), as shown by co-localization with a Lysotracker dye. Longer treatment times with 1a (25 μM Re for 48 h) resulted in partial migration of the probe into the mitochondria, as shown by co-localization with a Mitotracker dye. These properties make complex 1a an attractive target for further development as an organelle probe for multimodal imaging, including phosphorescence, carbonyl tag for vibrational spectroscopy, and Re tag for X-ray fluorescence microscopy.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗