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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 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

Head and Neck Squamous Cell Carcinomas Do Not Express EGFRvIII

Purpose: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results: None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2016

Nicotinamide Phosphoribosyltransferase Upregulation by Phenylephrine Reduces Radiation Injury in Submandibular Gland

Purpose: Radiation therapy for head and neck cancer commonly leads to radiation sialadenitis. Emerging evidence has indicated that phenylephrine pretreatment reduces radiosensitivity in the salivary gland; however, the underlying cytoprotective mechanism remains unclear. Nicotinamide phosphoribosyltransferase (NAMPT) is not only a key enzyme for the nicotinamide adenine dinucleotide salvage pathway, but also a cytokine participating in cell survival, metabolism, and longevity, with a broad effect on cellular functions in physiology and pathology. However, the regulatory events of NAMPT in response to the irradiated salivary gland are unknown. Methods and Materials: The cell viability of primary cultured submandibular gland cells was determined using the PrestoBlue assay. NAMPT expression was measured using reverse transcriptase polymerase chain reaction and Western blotting in vitro and in vivo. Silent information regulator 1 (SIRT1) and phosphorylated Akt protein levels were examined by Western blotting. The cellular locations of NAMPT and SIRT1 were detected by immunohistochemistry. NAMPT promoter activity was assessed using the luciferase reporter gene assay. Results: NAMPT was mainly distributed in the cytoplasm of granular convoluted tubule cells and ductal cells in normal submandibular glands. mRNA and protein expression of NAMPT was downregulated after radiation but upregulated with phenylephrine pretreatment both in vivo and in vitro. Moreover, the protein expression of phosphorylated Akt and SIRT1 was decreased in irradiated glands, and phenylephrine pretreatment restored the expression of both. SIRT1 was mainly located in the cell nucleus and cytoplasm in the normal submandibular gland. Phenylephrine dramatically enhanced the expression of SIRT1, which was significantly reduced by radiation. Furthermore, phenylephrine induced a marked increase of NAMPT promoter activity. Conclusions: These findings reveal the regulatory mechanisms of NAMPT expression, which help to understand the mechanism of the cytoprotective role of phenylephrine on irradiated tissues.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2017

Ku70, Ku80, and sClusterin: A Cluster of Predicting Factors for Response to Neoadjuvant Chemoradiation Therapy in Patients With Locally Advanced Rectal Cancer

Purpose: The identification of predictive biomarkers for neoadjuvant chemoradiation therapy (CRT) is a current clinical need. The heterodimer Ku70/80 plays a critical role in DNA repair and cell death induction after damage. The aberrant expression and localization of these proteins fail to control DNA repair and apoptosis. sClusterin is the Ku70 partner that sterically inhibits Bax-dependent cell death after damage in some pathologic conditions. This study sought to evaluate the molecular relevance of Ku70-Ku80-Clu as a molecular cluster predicting the response to neoadjuvant CRT in patients with locally advanced rectal cancer (LARC). Methods and Materials: Patients enrolled in this study underwent preoperative CRT followed by surgical excision. A retrospective study based on individual response, evaluated by computed tomography and diffusion-weighted magnetic resonance imaging, identified responder (56%) and no-responder patients (44%). Ku70/80 and Clu expression were observed in biopsy specimens obtained before and after treatment with neoadjuvant CRT from the same LARC patients. In vitro studies before and after irradiation were also performed on radioresistant (SW480) and radiosensitive (SW620) colorectal cancer cell lines, mimicking sensitive or resistant tumor behavior. Results: We found a conventional nuclear localization of Ku70/80 in pretherapeutic tumor biopsies of responder patients, in agreement with their role in DNA repair and regulating apoptosis. By contrast, in the no-responder population we observed an unconventional overexpression of Ku70 in the cytoplasm (P<.001). In this context we also overexpression of sClu in the cytoplasm, which accorded with its role in stabilizing of Bax-Ku70 complex, inhibiting Bax-dependent apoptosis. Strikingly, Ku80 in these tumor tissues was lost (P<.005). In vitro testing of colon cancer cells finally confirmed the results observed in tumor biopsy specimens, proving that Ku70/80-Clu deregulation is extensively involved in the resistance mechanism. Conclusion: These results strongly suggest a potential role of these proteins as a new prognostic tool to predict the response to chemoradiation in LARC.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2019

The Salix psammophila SpRLCK1 involved in drought and salt tolerance

Receptor-like cytoplasmic kinases ( RLCKs ) play critical roles in biotic and abiotic stress responses in plants. However, the functions of RLCKs from the desert shrub willow Salix psammophila have not been characterized. Here, we focused on the biological function of SpRLCK1 , which was previously identified as a potential drought-related gene. Phylogenetic analysis and subcellular localization revealed that SpRLCK1 was a cytoplasmic-localized protein with a protein kinase domain and belonged to the RLCK VIIa subclass. Gene expression profile revealed that SpRLCK1 was predominantly expressed in the root, being consistent with the GUS staining of pSpRLCK1 :GUS transgenic plants. Additionally, the expression of SpRLCK1 was significantly induced by drought and salt stresses. To verify the function of SpRLCK1 , we generated its overexpressing transgenic lines in Arabidopsis thaliana. The SpRLCK1 -overexpressing plants exhibited higher tolerance to drought and salt stresses, as evidenced by the higher survival rate, relative water content and antioxidant enzyme activity than those of wild-type plants. The SpRLCK1 -overexpressing plants enhanced drought and salt tolerance by improving ROS-scavenging activities. A co-expression network for SpRLCK1 was constructed, and the expression analysis indicated that SpRLCK1 regulated the expression of a series of stress-related genes. Ultimately, our results demonstrate that SpRLCK1 confers plant drought and salt tolerance through enhancing the activity of antioxidant enzymes and cooperating with stress-related genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Co-Folding of a FliF-FliG Split Domain Forms the Basis of the MS:C Ring Interface within the Bacterial Flagellar Motor

The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliFC) bound to the N terminal domain of the switch complex protein FliG (FliGN) reveals that FliGN folds around FliFC to produce a topology that closely resembles both the middle and C-terminal domains of FliG. Furthermore, the interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays. Co-folding with FliFC induces substantial conformational changes in FliGN and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliFC:FliGN complex into cryo-electron microscopy rotor density updates the architecture of the middle and upper switch complex and shows how domain shuffling of a conserved interaction module anchors the cytoplasmic rotor to the membrane.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP{sub 3} pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca{sup 2+}-dependent pro-apoptotic pathways inhibit cancer cell growth.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

The influence of N-acetyl-L-cysteine on damage of porcine oocyte exposed to zearalenone in vitro

Zearalenone (ZEA), one of the mycotoxins produced by Fusarium fungi, impacts porcine reproduction by interfering with the estrogen signaling pathway. Previous studies have shown that ZEA inhibits porcine oocyte maturation through the formation of aberrant spindle. To explore the effect of ZEA on porcine oocyte meiotic maturation, the extent of both nuclear and cytoplasmic maturation was examined in this study. Compared with control group, presence of ZEA (3 μM) during oocyte maturation, significantly inhibited the polar body extrusions from 71% to 51%, and significantly increased intracellular reactive oxygen species (ROS) level (12.01 vs. 5.89). Intracellular glutathione (GSH) content in ZEA treatment group was lower than in the control group (1.08 pmol/oocyte vs. 0.18 pmol/oocyte), and cortical granules of cortical area distributed oocytes were reduced (88% vs. 62%). ZEA decreases cumulus expansion in both morphology and mRNA level (HAS2, PTX3, TNFAIP6 and CX43). Addition of N-acetyl-L-cysteine (NAC) to the oocyte maturation media reversed the ZEA-induced inhibition of polar body extrusion (from 69% to 81%), up-regulated ROS (from 7.9 to 6.5), down-regulated GSH content (from 0.16 to 0.82 pmol/oocyte) and recovered cumulus cells expansion in morphology and mRNA level. It is concluded that ZEA affects both oocyte nucleus and cytoplasmic maturation during in vitro maturation, and NAC can reverse these damages to some extent. - Highlights: • ZEA significantly inhibited the polar body extrusions during oocyte maturation. • ZEA significantly increased intracellular ROS level and reduced GSH content. • ZEA disturbed cortical granules of cortical area distributed oocytes. • NAC reversed the ZEA-induced inhibition of oocyte maturation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particles had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Nuclear actin and myosins in adenovirus infection

Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. - Highlights: • Adenovirus infection causes dramatic changes in the host's transcription machinery. • Nuclear actin is recruited to viral centers during the late phase of infection. • Nuclear myosin I is recruited with nuclear actin to viral replication centers. • Nuclear myosin V and VI are recruited to distinctive regions of viral centers. • Cytoplasmic and nuclear actin polymerization are necessary for Ad replication.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Puromycin aminonucleoside increases podocyte permeability by modulating ZO-1 in an oxidative stress-dependent manner

Puromycin aminonucleoside (PAN)-induced nephrosis is a widely studied animal model of human idiopathic nephrotic syndrome because PAN injection into rats results in increased glomerular permeability with the characteristic ultrastructural changes in podocytes similar to human nephrosis. To investigate the role of zonula occludens (ZO)-1 and oxidative stress on PAN-induced podocyte phenotypical changes and hyperpermeability in vitro, we cultured rat and mouse podocytes and treated with various concentrations of PAN. PAN treatment increased oxidative stress level of podocytes significantly with the induction of Nox4. In addition, PAN changed the ultrastructure of podocytes, such as shortening and fusion of microvilli, and the separation of intercellular gaps, which were improved by anti-oxidative vitamin C and Nox4 siRNA. PAN also disrupted the intercellular linear ZO-1 staining and induced inner cytoplasmic re-localization of ZO-1 protein, resulting in increased podocyte intercellular permeability. PAN reduced ZO-1 protein amount and mRNA expression in a dose-dependent manner, which means that PAN could also modulate ZO-1 protein transcriptionally. However, the decreased ZO-1 protein of podocytes by PAN was improved by Nox4 siRNA transfection. Furthermore, vitamin C mitigated the quantitative and distributional disturbances of ZO-1 protein caused by PAN. Our results demonstrate that the phenotypical changes of intercellular ZO-1 by oxidative stress via Nox4 likely contribute to the glomerular hyperpermeability caused by PAN. - Highlights: • PAN increases ROS and induces the ultrastructural changes in podocytes. • Podocyte intercellular hyperpermeability by PAN is via oxidative stress in vitro. • PAN induces the inner cytoplasmic relocalization of intercellular ZO-1 in podocytes. • PAN suppresses the production of ZO-1 protein at the transcriptional level. • The quantitative and distributional changes of ZO-1 by PAN are via oxidative stress.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Functional transferred DNA within extracellular vesicles

Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

CD147-induced cell proliferation is associated with Smad4 signal inhibition

CD147 is a multifunctional trans-membrane glycoprotein, which is highly expressed in many cancers. However, the mechanism by which CD147 modulates cell proliferation is not fully understood. The aim of this study is to investigate the role of CD147 in cell proliferation associated with the TGF-β/Smad4 signaling pathway. Here, we used cell viability and clone formation assays in LNCaP prostate cancer cells to demonstrate that CD147 promotes cell proliferation. The luciferase assay and western blotting show that silencing CD147 using shRNA enhances transcription and expression of p21{sup WAF1}. Using immunofluorescence and nuclear-cytoplasmic separation, we show that this is primarily attributed to transport of Smad4 from the cytoplasm to nucleus. Other assays (GST pull-down, co-immunoprecipitation and immunofluorescence) demonstrate that Smad4 is a new interaction partner of CD147, with the Smad4 MH2 domain and CD147 intracellular domain (CD147-ICD) being involved in the interaction. Furthermore, we report that a phosphoserine (pSer) in CD147 (pSer252) is responsible for this interaction and inhibition of the Smad4/p21{sup WAF1} signal that promotes cell proliferation. Our results provide a novel molecular mechanism for CD147-induced cell proliferation associated with Smad4 signal inhibition. - Highlights: • Smad4 interacts with CD147 via the MH2 domain. • CD147 prevents Smad4 translocation into the nuclear to regulate p21 transcription. • CD147 inhibits p21 transcription and expression independent on p53. • pCD147-S252 is responsible for interaction with Smad4 and p21 down-regulation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in Drosophila nephrocytes

Highlights: • Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin. • Microtubules, Rho-GTPases and endoplasmic reticulum position the actin cluster. • Nephrocyte diaphragm proteins localize to and regulate actin cluster organization. • Perturbation of any of these inter-dependent components impairs ultrafiltration. Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional macromolecular assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the elements that maintain nephrocyte architecture and the ND are not understood. We show that Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin, which is maintained by the microtubule cytoskeleton and Rho-GTPases. A balance of Rac1 and Cdc42 activity as well as proper microtubule organization and endoplasmic reticulum structure, are required to position the actin cluster. Further, ND proteins Sns and Duf also localize to this cluster and regulate organization of the actin and microtubule cytoskeleton. Perturbation of any of these inter-dependent components impairs nephrocyte ultrafiltration. Thus cytoskeletal components, Rho-GTPases and ND proteins work in concert to maintain the specialized nephrocyte architecture and function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Identification of protein/mRNA network involving the PSORS1 locus gene CCHCR1 and the PSORS4 locus gene HAX1

Highlights: • CCHCR1 and HAX1 proteins directly interact. • CCHCR1 is a psoriasis candidate gene located on PSORS1 locus. • HAX1 gene is located on PSORS4 locus and is overexpressed in psoriasis. • CCHCR1 and HAX1 co-localize with and affect specific mRNAs in vesicles. • Novel protein/mRNA networks involving CCHCR1 and HAX1 could play a role in psoriasis. CCHCR1 (Coiled-Coil alpha-Helical Rod 1), maps to chromosomal region 6p21.3, within the major psoriasis susceptibility locus PSORS1. CCHCR1 itself is a plausible psoriasis candidate gene, however its role in psoriasis pathogenesis remains unclear. We previously demonstrated that CCHCR1 protein acts as a cytoplasmic docking site for RNA polymerase II core subunit 3 (RPB3) in cycling cells, suggesting a role for CCHCR1 in vesicular trafficking between cellular compartments. Here, we report a novel interaction between CCHCR1 and the RNA binding protein HAX1. HAX1 maps to chromosomal region 1q21.3 within the PSORS4 locus and is over-expressed in psoriasis. Both CCHCR1 and HAX1 share subcellular co-localization with mitochondria, nuclei and cytoplasmic vesicles as P-bodies. By a series of ribonucleoprotein immunoprecipitation (RIP) assays, we isolated a pool of mRNAs complexed with HAX1 and/or CCHCR1 proteins. Among the mRNAs complexed with both CCHCR1 and HAX1 proteins, there are Vimentin mRNA, previously described to be bound by HAX1, and CAMP/LL37 mRNA, whose gene product is over-expressed in psoriasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Mercury Uptake by Desulfovibrio desulfuricans ND132: Passive or Active?

Recent studies have identified HgcAB proteins as being responsible for mercury [Hg(II)] methylation by certain anaerobic microorganisms. However, it remains controversial whether microbes take up Hg(II) passively or actively. Here, we examine the dynamics of concurrent Hg(II) adsorption, uptake, and methylation by both viable and inactivated cells (heat-killed or starved) or spheroplasts of the sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 in laboratory incubations. We show that, without addition of thiols, >60% of the added Hg(II) (25 nM) was taken up passively in 48 h by live and inactivated cells and also by cells treated with the proton gradient uncoupler, carbonylcyanide-3-chlorophenylhydrazone (CCCP). Inactivation abolished Hg(II) methylation, but the cells continued taking up Hg(II), likely through competitive binding or ligand exchange of Hg(II) by intracellular proteins or thiol-containing cellular components. Similarly, treatment with CCCP impaired the ability of spheroplasts to methylate Hg(II) but did not stop Hg(II) uptake. Spheroplasts showed a greater capacity to adsorb Hg(II) than whole cells, and the level of cytoplasmic membrane-bound Hg(II) correlated well with MeHg production, as Hg(II) methylation is associated with cytoplasmic HgcAB. Our results indicate that active metabolism is not required for cellular Hg(II) uptake, thereby providing an improved understanding of Hg(II) bioavailability for methylation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2015

A common assembly module in injectisome and flagellar type III secretion sorting platforms

Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design.

Science & Technology - Other Topics↗
DOE OSTI2022

Proteolytic processing induces a conformational switch required for antibacterial toxin delivery

Many Gram-negative bacteria use CdiA effector proteins to inhibit the growth of neighboring competitors. CdiA transfers its toxic CdiA-CT region into the periplasm of target cells, where it is released through proteolytic cleavage. The N-terminal cytoplasm-entry domain of the CdiA-CT then mediates translocation across the inner membrane to deliver the C-terminal toxin domain into the cytosol. Here, we show that proteolysis not only liberates the CdiA-CT for delivery, but is also required to activate the entry domain for membrane translocation. Translocation function depends on precise cleavage after a conserved VENN peptide sequence, and the processed ΔVENN entry domain exhibits distinct biophysical and thermodynamic properties. By contrast, imprecisely processed CdiA-CT fragments do not undergo this transition and fail to translocate to the cytoplasm. These findings suggest that CdiA-CT processing induces a critical structural switch that converts the entry domain into a membrane-translocation competent conformation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading

During phloem unloading, multiple cell-to-cell transport events move organic substances to the root meristem. Although the primary unloading event from the sieve elements to the phloem pole pericycle has been characterized to some extent, little is known about post-sieve element unloading. Here, we report a novel gene, PHLOEM UNLOADING MODULATOR (PLM), in the absence of which plasmodesmata-mediated symplastic transport through the phloem pole pericycle-endodermis interface is specifically enhanced. Increased unloading is attributable to a defect in the formation of the endoplasmic reticulum-plasma membrane tethers during plasmodesmal morphogenesis, resulting in the majority of pores lacking a visible cytoplasmic sleeve. PLM encodes a putative enzyme required for the biosynthesis of sphingolipids with very-long-chain fatty acid. Taken together, our results indicate that post-sieve element unloading involves sphingolipid metabolism, which affects plasmodesmal ultrastructure. They also raise the question of how and why plasmodesmata with no cytoplasmic sleeve facilitate molecular trafficking.

Yan, Dawei (ORCID:0000000182560279)↗