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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 37 records · Page 2
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 OSTI2020

Exosomes From Induced Pluripotent Stem Cell–Derived Cardiomyocytes Promote Autophagy for Myocardial Repair

Background. Induced pluripotent stem cells and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for ischemic cardiomyopathy following myocardial infarctions, but difficulties in viable transplantation limit clinical translation. Exosomes secreted from iCMs (iCM-Ex) can be robustly collected in vitro and injected in lieu of live iCMs as a cell-free therapy for myocardial infarction. Methods and Results. iCM-Ex were precipitated from iCM supernatant and characterized by protein marker expression, nanoparticle tracking analysis, and functionalized nanogold transmission electron microscopy. iCM-Ex were then used in in vitro and in vivo models of ischemic injuries. Cardiac function in vivo was evaluated by left ventricular ejection fraction and myocardial viability measurements by magnetic resonance imaging. Cardioprotective mechanisms were studied by JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) assay, immunohistochemistry, quantitative real-time polymerase chain reaction, transmission electron microscopy, and immunoblotting. iCM-Ex measured ≈140 nm and expressed CD63 and CD9. iCM and iCM-Ex microRNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. Mice treated with iCM-Ex demonstrated significant cardiac improvement post–myocardial infarction, with significantly reduced apoptosis and fibrosis. In vitro iCM apoptosis was significantly reduced by hypoxia and exosome biogenesis inhibition and restored by treatment with iCM-Ex or rapamycin. Autophagosome production and autophagy flux was upregulated in iCM-Ex groups in vivo and in vitro. Conclusions. iCM-Ex improve post–myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyoytes, enabling a cell-free, patient-specific therapy for ischemic cardiomyopathy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts

The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Submicron immunoglobulin particles exhibit FcγRII-dependent toxicity linked to autophagy in TNFα-stimulated endothelial cells

In intravenous immunoglobulins (IVIG), and some other immunoglobulin products, protein particles have been implicated in adverse events. Role and mechanisms of immunoglobulin particles in vascular adverse effects of blood components and manufactured biologics have not been elucidated. We have developed a model of spherical silica microparticles (SiMPs) of distinct sizes 200–2000 nm coated with different IVIG- or albumin (HSA)-coronas and investigated their effects on cultured human umbilical vein endothelial cells (HUVEC). IVIG products (1–20 mg/mL), bare SiMPs or SiMPs with IVIG-corona, did not display significant toxicity to unstimulated HUVEC. In contrast, in TNFα-stimulated HUVEC, IVIG-SiMPs induced decrease of HUVEC viability compared to HSA-SiMPs, while no toxicity of soluble IVIG was observed. 200 nm IVIG-SiMPs after 24 h treatment further increased ICAM1 (intercellular adhesion molecule 1) and tissue factor surface expression, apoptosis, mammalian target of rapamacin (mTOR)-dependent activation of autophagy, and release of extracellular vesicles, positive for mitophagy markers. Toxic effects of IVIG-SiMPs were most prominent for 200 nm SiMPs and decreased with larger SiMP size. Using blocking antibodies, toxicity of IVIG-SiMPs was found dependent on FcγRII receptor expression on HUVEC, which increased after TNFα-stimulation. Similar results were observed with different IVIG products and research grade IgG preparations. In conclusion, submicron particles with immunoglobulin corona induced size-dependent toxicity in TNFα-stimulated HUVEC via FcγRII receptors, associated with apoptosis and mTOR-dependent activation of autophagy. Testing of IVIG toxicity in endothelial cells prestimulated with proinflammatory cytokines is relevant to clinical conditions. Our results warrant further studies on endothelial toxicity of sub-visible immunoglobulin particles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Isoform-specific functions of synaptopodin-2 variants in cytoskeleton stabilization and autophagy regulation in muscle under mechanical stress

Highlights: • Muscle cells express different synaptopodin-2 isoforms with individual functions. • Reduced SYNPO2 expression results in enhanced vulnerability of myofibrils. • SYNPO2e recruites BAG3 to mechanically stressed myofibrillar areas. • SYNPO2 is an organizer and regulator of BAG3-containing proteostasis systems. • BAG3 forms functionally distinct protein complexes with different SYNPO2 isoforms. Protein homeostasis (proteostasis) in multicellular organisms depends on the maintenance of force-bearing and force-generating cellular structures. Within myofibrillar Z-discs of striated muscle, isoforms of synaptopodin-2 (SYNPO2/myopodin) act as adapter proteins that are engaged in proteostasis of the actin-crosslinking protein filamin C (FLNc) under mechanical stress. SYNPO2 directly binds F-actin, FLNc and α-actinin and thus contributes to the architectural features of the actin cytoskeleton. By its association with autophagy mediating proteins, i.e. BAG3 and VPS18, SYNPO2 is also engaged in protein quality control and helps to target mechanical unfolded and damaged FLNc for degradation. Here we show that deficiency of all SYNPO2-isoforms in myotubes leads to decreased myofibrillar stability and deregulated autophagy under mechanical stress. In addition, isoform-specific proteostasis functions were revealed. The PDZ-domain containing variant SYNPO2b and the shorter, PDZ-less isoform SYNPO2e both localize to Z-discs. Yet, SYNPO2e is less stably associated with the Z-disc than SYNPO2b, and is dynamically transferred into FLNc-containing myofibrillar lesions under mechanical stress. SYNPO2e also recruits BAG3 into these lesions via interaction with the WW domain of BAG3. Our data provide evidence for a role of myofibrillar lesions as a transient quality control compartment essential to prevent and repair contraction-induced myofibril damage in muscle and indicate an important coordinating activity for SYNPO2 therein.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ~20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Subunit of ESCRT-III, MoIst1, Is Involved in Fungal Development, Pathogenicity, and Autophagy in Magnaporthe oryzae

The culprit of rice blast, Magnaporthe oryzae , is a filamentous fungus that seriously affects the yield and quality of rice worldwide. MoIst1, a subunit of ESCRT-III, is involved in identified ubiquitinated proteins and transports them into the intraluminal vesicles of multivesicular bodies (MVBs) for degradation in lysosomes. Here, we identify and characterize MoIst1 in M. oryzae . Disruption of MoIst1 leads to a significant decrease in sporulation and formation of appressoria, defects in response to oxidative stress, cell wall stress, hyperosmotic stress, and reduced pathogenicity. Deletion of MoIst1 also caused the decreased Pmk1 phosphorylation levels, appressorium formation, the delayed translocation and degradation of lipid droplets and glycogen, resulting in a decreased appressorium turgor. In addition, deletion of MoIst1 leads to an abnormal autophagy. In summary, our results indicate that MoIst1 is involved in sporulation, appressorium development, plant penetration, pathogenicity, and autophagy in M. oryzae .

Sun, Lixiao↗
DOE OSTI2022

Genome of Paspalum vaginatum and the role of trehalose mediated autophagy in increasing maize biomass

Abstract A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum ( Paspalum vaginatum ) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession of P. vaginatum . Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

PLK1 Inhibition Sensitizes Breast Cancer Cells to Radiation via Suppressing Autophagy

Polo-like kinase 1 (PLK1) is a protein kinase that is overexpressed in breast cancer and may represent an attractive target for breast cancer treatment. However, few studies have investigated the relationship between PLK1 and radiosensitivity in breast cancer. Here, we attempted to explore whether PLK1 inhibition could sensitize breast cancer cells to radiation.

62 RADIOLOGY AND NUCLEAR MEDICINE↗