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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 1,027 records · Page 57
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Th17 cell-mediated immune responses promote mast cell proliferation by triggering stem cell factor in keratinocytes

Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes, SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL−17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL−17 in vitro. However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti−CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF. - Highlights: • Psoriasis-like skin inflammation increase dermal mast cells. • Keratinocyte produce stem cell factor in psoriasis-like skin inflammation. • Keratinocyte promote mast cell proliferation by stem cell factor dependent manner.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Highly efficient one-step scarless protein tagging by type IIS restriction endonuclease-mediated precision cloning

Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Spatiotemporal control of mitochondrial network dynamics in astroglial cells

Highlights: • Astrocytes display highly complex mitochondrial architectures in vivo. • Mitochondrial trafficking and dynamics in astrocytes are regulated. • Mitochondria critically contribute to astrocyte Ca{sup 2+} handling. • Astrocyte reacting to injury undergo marked changes in mitochondrial dynamics. Mitochondria are increasingly recognized for playing important roles in regulating the evolving metabolic state of mammalian cells. This is particularly true for nerve cells, as dysregulation of mitochondrial dynamics is invariably associated with a number of neuropathies. Accumulating evidence now reveals that changes in mitochondrial dynamics and structure may play equally important roles also in the cell biology of astroglial cells. Astroglial cells display significant heterogeneity in their morphology and specialized functions across different brain regions, however besides fundamental differences they seem to share a surprisingly complex meshwork of mitochondria, which is highly suggestive of tightly regulated mechanisms that contribute to maintain this unique architecture. Here, we summarize recent work performed in astrocytes in situ indicating that this may indeed be the case, with astrocytic mitochondrial networks shown to experience rapid dynamic changes in response to defined external cues. Although the mechanisms underlying this degree of mitochondrial re-shaping are far from being understood, recent data suggest that they may contribute to demarcate astrocyte territories undergoing key signalling and metabolic functions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The regulation of tumor cell physiology by mitochondrial dynamics

Highlights: • Mitochondrial fusion and fission can influence mitochondrial function. • Mitochondrial function underlies a number of physiological changes associated with cancer. • Mitochondrial dynamics represent a potential mechanism cancer cells use to promote tumorigenic growth. Mitochondrial dynamics are increasingly recognized to play an important role in regulating mitochondrial function in response to diverse stimuli. Given the overlap in the physiological processes influenced by mitochondria and the physiological processes disrupted in tumor cells, we speculate that tumor cells alter mitochondrial shape to promote the tumorigenic phenotype. Here, we briefly review the evidence linking changes in mitochondrial fusion and fission to a number of key tumorigenic processes, including metabolic rewiring, inhibition of cell death, cell migration, cell proliferation and self-renewal capacity. The role of mitochondrial dynamics in tumor growth is an important emerging area of research, a better understanding of which may lead to promising new therapeutic options for the treatment of cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Structure insights into the molecular mechanism of the interaction between UHRF2 and PCNA

Highlights: • UHRF2 interacts with PCNA through its PIP box. • Complex structure of PCNA/UHRF2{sup PIP} reveals the molecular mechanism of how PCNA recognizes UHRF2. • UHRF2-PIP box interacts with PCNA mainly via hydrophobic interaction. • The hydrogen bond formed between PCNA-H44 and UHRF2{sup PIP}-D792 is critical for the interaction. UHRF2 (Ubiquitin-like with PHD and ring finger domains 2) is an E3 ubiquitin ligase that plays important roles in DNA methylation, histone modifications and cell cycle regulation by interacting with multiple epigenetic or cell-cycle related proteins. Previous studied have identified PCNA (Proliferating cell nuclear antigen) as an interacting partner of UHRF2 by using the antibody microarray. However, the molecular mechanism and the function of UHRF2-PCNA interaction remains unclear. Here, we report the complex structure of PCNA and the peptide ({sup 784}NEILQTLLDLFFPGYSK{sup 800}) derived from UHRF2 that contains a PIP box. Structural analysis combined with mutagenesis experiments provide the molecular basis for the recognition of UHRF2 by PCNA via PIP-box.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Identification of a selective inhibitor of human monocarboxylate transporter 4

Highlights: • hMCT4 transporter mediates the transport of metabolites such as l-lactate and pyruvate. • 5-ETZ, a bioisostere of propionate, is recognized by hMCT1 and hMCT4. • Fibrates, a group of isobutyrate derivatives, selectively inhibit hMCT4. • Bindarit might be the first-identified potent and selective inhibitor of hMCT4. The human monocarboxylate transporters (hMCTs/SLC16As) mediate the uptake of various monocarboxylates. Several isoforms of hMCTs are expressed in cancerous tissue as well as in normal tissue. In cancerous tissue, hypoxia induces the expression of hMCT4, which transports the energetic metabolite l-lactate across the plasma membrane. Since hMCT4 is involved in pH regulation and the transport of l-lactate in cancer cells, an hMCT4 inhibitor could function as an anticancer agent. Although several non specific hMCT inhibitors have been developed, a selective hMCT4 inhibitor has not yet been identified. The aim of this study was therefore to identify a selective hMCT4 inhibitor for use as a pharmacological tool for studying hMCT4. The heterologous expression system of the Xenopus oocyte was used to assess the effects of test compounds on hMCT4, whereupon isobutyrate derivatives, fibrates, and bindarit (2-[(1-benzyl-1H-indazol-3-yl)methoxy]-2-methylpropanoic acid) were demonstrated to exhibit selective inhibitory effects against this transporter. It is suggested that the structure formed from the joining of an isobutyrate moiety and two aromatic rings by appropriate linkers is important for acquiring the selective hMCT4-inhibiting activity. These findings provide novel insights into the ligand recognition of hMCT4, and contribute to the development of novel anticancer agents.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Building kit for metal cation binding sites in proteins

Starting with conformations of calcium-binding sites in parvalbumin and integrin (representative structures of EF-hand and calcium blade zones, respectively) we introduce four new different local Ca{sup 2+}-recognition units in proteins: a one-residue unit type I (ORI); a three-residue unit type I (TRI); a one-residue unit type II (ORII) and a three-residue unit type II (TRII). Based on the amount and nature of variable atoms, the type I and II units theoretically can have four and twelve variants, respectively. Analysis of known “Ca{sup 2+}-bound functional niches” in proteins revealed presence of almost all possible variants of Ca{sup 2+}-recognition units in actual structures. Parvalbumin, integrin alpha-IIb and sixteen other proteins with different Ca{sup 2+}-bound functional niches contain various consecutively joined combinations of OR(I/II) and TR(I/II) units. Such a OR(I/II)+TR(I/II) joint unit forms a tripeptide, which uses three main-chain atoms for metal binding: nitrogen{sub n} (Donor), oxygen{sub n} (Acceptor) and nitrogen{sub n+2} (Donor). Thus, taken together, the described ORI, TRI, ORII and TRII units can serve as elementary blocks to construct more complex calcium recognizing substructures in a variety of calcium binding sites of unrelated proteins.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Sez6l2 regulates phosphorylation of ADD and neuritogenesis

Highlights: • Sez6l2 interacts with ADD and GluR1. • Extracellular domains of Sez6l2 and GluR1 are required for their interactions. • Sez6l2 may be one of the auxiliary subunits with AMPA receptors. • Sez6l2 may link GluR1 to ADD and be an up-regulator of ADD phosphorylation. Increasing evidence shows that immune-mediated mechanisms may contribute to the pathogenesis of central nervous system disorders including cerebellar ataxias, as indicated by the aberrant production of neuronal surface antibodies. We previously reported a patient with cerebellar ataxia associated with production of a new anti-neuronal antibody, anti-seizure-related 6 homolog like 2 (Sez6l2). Sez6l2 is a type 1 membrane protein that is highly expressed in the hippocampus and cerebellar cortex and mice lacking Sez6l2 protein family members develop ataxia. Here we used a proteomics-based approach to show that serum derived from this patient recognizes the extracellular domain of Sez6l2 and that Sez6l2 protein binds to both adducin (ADD) and glutamate receptor 1 (GluR1). Our results indicate that Sez6l2 is one of the auxiliary subunits of the AMPA receptor and acts as a scaffolding protein to link GluR1 to ADD. Furthermore, Sez6l2 overexpression upregulates ADD phosphorylation, whereas siRNA-mediated downregulation of Sez612 prevents ADD phosphorylation, suggesting that Sez6l2 modulates AMPA-ADD signal transduction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Molecular insights into the specific recognition between the RNA binding domain qRRM2 of hnRNP F and G-tract RNA: A molecular dynamics study

Heterogeneous nuclear ribonucleoprotein F (hnRNP F) controls the expression of various genes through regulating the alternative splicing of pre-mRNAs in the nucleus. It uses three quasi-RNA recognition motifs (qRRMs) to recognize G-tract RNA which contains at least three consecutive guanines. The structures containing qRRMs of hnRNP F in complex with G-tract RNA have been determined by nuclear magnetic resonance (NMR) spectroscopy, shedding light on the recognition mechanism of qRRMs with G-tract RNA. However, knowledge of the recognition details is still lacking. To investigate how qRRMs specifically bind with G-tract RNA and how the mutations of any guanine to an adenine in the G-tract affect the binding, molecular dynamics simulations with binding free energy analysis were performed based on the NMR structure of qRRM2 in complex with G-tract RNA. Simulation results demonstrate that qRRM2 binds strongly with G-tract RNA, but any mutation of the G-tract leads to a drastic reduction of the binding free energy. Further comparisons of the energetic components reveal that van der Waals and non-polar interactions play essential roles in the binding between qRRM2 and G-tract RNA, but the interactions are weakened by the effect of RNA mutations. Structural and dynamical analyses indicate that when qRRM2 binds with G-tract RNA, both qRRM2 and G-tract maintain stabilized structures and dynamics; however, the stability is disrupted by the mutations of the G-tract. These results provide novel insights into the recognition mechanism of qRRM2 with G-tract RNA that are not elucidated by the NMR technique. - Highlights: • qRRM2 has greater binding free energy with G-tract RNA than the mutated RNAs. • Non-polar and van der Waals interactions contribute mainly to the binding free energy. • qRRM2 maintains stabilized structure and dynamics when binding with G-tract RNA. • G-tract adopts stabilized conformation in complex with qRRM2.

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

JAK2V617F influences epigenomic changes in myeloproliferative neoplasms

Highlights: • Ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity of pSTAT3- and EZH2-associated targets. • JAK2V617F can induce downstream epigenomic modifications. • The methylation changes in the JAK2-STAT3 target loci were clustered differently from the control and other mutation genes. Negative valine (V) to phenylalanine (F) switch at the Janus kinase (JAK2) 617 codon (V617F) is the dominant driver mutation in patients with myeloproliferative neoplasms (MPNs). JAK2V617F was proved to be sufficient for cell transformation; however, independent mutations might influence the following epigenomic modifications. To assess the JAK2V617F-induced downstream epigenomic changes without interferences, we profiled the epigenomic changes in ectopically expressed JAK2V617F in Ba/F3 cells. Antibodies against phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and enhancer of zeste homolog 2 (EZH2) were used for chromatin-immunoprecipitation sequencing (ChIP-seq) to detect the downstream epigenomic targets in the JAK2–STAT3 signaling pathway. To confirm the JAK2V617F-induced epigenetic changes in vivo, DNA methylation changes in the target loci in patients with MPNs were detected through methylation-specific polymerase chain reaction and were clustered against the changes within controls. We found that ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity; it was associated with cis-regulatory elements and recognized DNA motifs in both pSTAT3-downstream and EZH2-associated targets. Overlapping target loci between the control and JAK2V617F were FOXH1, HOXC9, and SRF) were clustered independently from the control locus (L1TD1) and other mutation genes (HMGA2 and Lin28A) in the analyzed MPN samples. Therefore, JAK2V617F influences target binding in both pSTAT3 and EZH2. Without mutations in epigenetic regulators, JAK2V617F can induce downstream epigenomic modifications. Thus, epigenetic changes in JAK2 downstream targets might be trackable in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens

Highlights: • We cloned a fat-taste receptor candidate gene, CD36, from chickens. • Fat-taste receptor genes, GPR120 and CD36, were expressed in chicken oral tissues. • Several lipase genes were expressed in chicken oral tissues. • Chicken oral tissues contained functional lipases. It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells

Highlights: • Effects of cancer cell-derived exosomes on stellate cell functions were examined. • Exosomes increased proliferation and migration in stellate cells. • Cancer cells increased miR-1246 and miR-1290 expression in stellate cells. • Exosomes and miR-1290 overexpression increased the expression of profibrogenic genes and ACTA2. • Exosome-mediated cell-to-cell communication regulates stellate cell functions. Pancreatic cancer cells (PCCs) interact with pancreatic stellate cells (PSCs), which play a pivotal role in pancreatic fibrogenesis, to develop the cancer-conditioned tumor microenvironment. Exosomes are membrane-enclosed nanovesicles, and have been increasingly recognized as important mediators of cell-to-cell communications. The aim of this study was to clarify the effects of PCC-derived exosomes on cell functions in PSCs. Exosomes were isolated from the conditioned medium of Panc-1 and SUIT-2 PCCs. Human primary PSCs were treated with PCC-derived exosomes. PCC-derived exosomes stimulated the proliferation, migration, activation of ERK and Akt, the mRNA expression of α-smooth muscle actin (ACTA2) and fibrosis-related genes, and procollagen type I C-peptide production in PSCs. Ingenuity pathway analysis of the microarray data identified transforming growth factor β1 and tumor necrosis factor as top upstream regulators. PCCs increased the expression of miR-1246 and miR-1290, abundantly contained in PCC-derived exosomes, in PSCs. Overexpression of miR-1290 induced the expression of ACTA2 and fibrosis-related genes in PSCs. In conclusion, PCC-derived exosomes stimulate activation and profibrogenic activities in PSCs. Exosome-mediated interactions between PSCs and PCCs might play a role in the development of the tumor microenvironment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Proximity labeling of cis-ligands of CD22/Siglec-2 reveals stepwise α2,6 sialic acid-dependent and -independent interactions

Highlights: • Cis-ligands of CD22 are efficiently labeled by proximity labeling with tyramide. • Cis-ligands in relative proximity of CD22 in the absence of lectin-glycan interaction. • Lectin-glycan interaction further recruits cis-ligands to the close proximity of CD22. • Very low affinity interaction is sufficient for recruitment of cis-ligands to CD22. Lectins expressed on the cell surface are often bound and regulated by the membrane molecules containing the glycan ligands on the same cell (cis-ligands). However, molecular nature and function of cis-ligands are generally poorly understood partly because of weak interaction between lectins and glycan ligands. Cis-ligands are most extensively studied in CD22 (also known as Siglec-2), an inhibitory B lymphocyte receptor specifically recognizing α2,6 sialic acids. CD22, CD45 and IgM are suggested to be ligands of CD22. Here we labeled molecules in the proximity of CD22 in situ on B cell surface using biotin-tyramide. Molecules including CD22, CD45 and IgM were labeled in wild-type but not ST6GalI{sup −/-} B cells that lack α2,6 sialic acids, indicating that these molecules associate with CD22 by lectin-glycan interaction, and are therefore cis-ligands. In ST6GalI{sup −/-} B cells, these cis-ligands are located in a slightly more distance from CD22. Thus, the lectin-glycan interaction recruits cis-ligands already located in the relative proximity of CD22 through non-lectin-glycan interaction to the close proximity. Moreover, cis-ligands are labeled in Cmah{sup −/-} B cells that lack Neu5Gc preferred by mouse CD22 as efficiently as in wild-type B cells, indicating that very low affinity lectin-glycan interaction is sufficient for recruiting cis-ligands, and can be detected by proximity labeling. Thus, proximity labeling with tyramide appears to be a useful method to identify cis-ligands and to analyze their interaction with the lectins.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 enhances macrophages response to LPS in a Ca+2-dependent manner

Highlights: • S-layer protein of L. kefiri CIDCA 8348 (SLP8348) is recognized by ConA. • Uptake of SLP8348 by murine macrophages is inhibited by glucose, mannose and EGTA. • SLP8348 significantly increased LPS-induced response of macrophages. • SLP8348-LPS synergism is abrogated by EGTA, indicating a role of Ca{sup +2} in the effect. • Results suggest the potentiality of SLP8348 for development of new adjuvants. The S-layer is a (glyco)-proteinaceous envelope constituted by self-assembled subunits that form a two-dimensional lattice covering the surface of different species of Bacteria and Archaea. It could be considered as one of the most abundant biopolymers in our planet. Because of their unique self-assembly features, exhibiting repetitive identical physicochemical properties down to the subnanometer scale, as well as their involvement in specific interactions with host cells, the S-layer proteins (SLPs) show a high potential application in different areas of biotechnology, including the development of antigen carriers or new adjuvants. The presence of a glycosylated SLP on potentially probiotic Lactobacillus kefiri strains was previously described by our research group. In this study, we aim to investigate the role of carbohydrates present in the SLP from L. kefiri CIDCA 8348 (SLP-8348) in their internalization by murine macrophages, as well as to analyze their immunomodulatory capacity and their effect on LPS-stimulated macrophages. RAW 264.7 cells internalized the SLP-8348 in a process that was mediated by carbohydrate-receptor interactions since it was inhibited by glucose, mannose or EGTA, a Ca{sup +2} chelating agent. These results correlated with the recognition of SLP-8348 by ConA lectin. We further show that while SLP-8348 was not able to induce the activation of macrophages by itself, it favored the LPS-induced response, since there was a significant increase in the expression of surface cell markers MHC-II, CD86 and CD40, as well as in IL-6 and IL-10 expression at both transcript and protein levels, in comparison with LPS-stimulated cells. The presence of EGTA completely abrogated this synergistic effect. Taken together, these results strongly suggest the involvement of both glycosidic residues and Ca{sup +2} ions in the recognition of SLP-8348 by cellular receptors on murine macrophages. Moreover, these results suggest the potentiality of the SLP-8348 for the development of new adjuvants capable of stimulating antigen presenting cells by interaction with glycan receptors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structural insights into the oligomerization of FtsH periplasmic domain from Thermotoga maritima

Highlights: • We report the 1.5–1.95A resolution crystal structures of the Thermotoga maritima FtsH periplasmic domain (tmPD). • Acidic residues at the pore region of tmPD are highly conserved among only the thermophilic, FtsH homologs. • The negative charge repulsion of tmPD destabilizes its hexamerization. • Dynamical features of the PD in hexameric full-length FtsH may be relevant for the substrate recognition. Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. The transmembrane protein, FtsH, is the only known ATP-dependent protease responsible for this task. The mechanisms by which FtsH recognizes, unfolds, translocates, and proteolyzes its substrates remain unclear. The structure and function of the ATPase and protease domains of FtsH have been previously characterized while the role of the FtsH periplasmic domain has not clearly identified. Here, we report the 1.5–1.95 Å resolution crystal structures of the Thermotoga maritima FtsH periplasmic domain (tmPD) and describe the dynamic features of tmPD oligomerization.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Hypothesis: Hypoxia induces de novo synthesis of NeuGc gangliosides in humans through CMAH domain substitute

Highlights: • In humans, the key enzyme for NeuGc synthesis, CMAH, is non-functional. • The presence of NeuGc gangliosides in human cancer cells represents an enigma. • We tested the effect of hypoxia by stable isotope labeling in cell culture (SILAC). • Subunit B of respiratory complex II (SDHB) was found upregulated. • Could CMAH be substituted by SDHB, another [Fe{sub 2}S{sub 2}]-containing domain? Immunotherapy is a growing field in cancer research. A privileged tumor-associated antigen that has received much attention is N-glycolyl (NeuGc) GM3. This ganglioside is present in several types of cancer, but is almost undetectable in human healthy tissues. However, its non-hydroxylated variant, NeuAc GM3, is abundant in all mammals. Due to a deletion in the human gene encoding the key enzyme for synthesis of NeuGc, humans, in contrast to other mammals, cannot synthesize NeuGc GM3. Therefore the presence of this ganglioside in human cancer cells represents an enigma. It has been shown that hypoxic conditions trigger the expression of NeuGc gangliosides, which not only serve as attractive targets for cancer therapy, but also as diagnostic and prognostic tumor marker. Here, we confirm hypoxia-induced expression of the NeuGc GM3 ganglioside also in HeLa cells and reveal several candidate proteins, in particular GM3 synthase and subunit B of respiratory complex II (SDHB), that may be involved in the generation of NeuGc GM3 by SILAC-based proteome analysis. These findings have the potential to significantly advance our understanding of how this enigmatic tumor-associated antigen is produced in humans, and also suggest a possible mechanism of action of anti-tumor antibodies that recognize hypoxia markers, such as 14F7.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of the chloroplast RNA editing factor MORF2

Highlights: • Crystal structure of MORF2 is determined at 2.4 Å. • MORF2 forms dimer through hydrophobic interaction. • Dimerization interface of MORF2 overlaps with its PPR interacting site. • A dimer-to-monomer transition is implicated for MORF2's role in RNA editing. RNA editing is a post-transcription process that alters the genetic information on RNA molecules. In plastids and mitochondria of flowering plants, the multiple organellar RNA editing factors (MORFs) interact with the PLS-type pentatricopeptide repeat (PPR) proteins and participate in RNA editing of cytidine-to-uridine conversion. The PPR proteins recognize cytidine targets around the editing sites, and the MORF proteins modulate the RNA-binding activity of the PPR proteins. Here, we report the structure of the Arabidopsis thaliana chloroplast MORF2 at 2.4 Å resolution. The structure, adopting typical MORF-box fold as observed in mitochondrial MORF1 and chloroplast MORF9, reveals an MORF1-like dimerization mode. The difference between the two dimerization modes can be attributed to F157 (corresponding F162 in MORF1 and W160 in MORF9), which causes a 60° shift upon dimerization. This observation, together with the PPR–MORF2 model, suggests a dimer-to-monomer transition during RNA editosome formation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Butylphthalide ameliorates experimental autoimmune encephalomyelitis by suppressing PGAM5-induced necroptosis and inflammation in microglia

Highlights: • The Dl-3-n-butylphthalide ameliorates the disease severity of multiple sclerosis. • Necroptosis and inflammation play important roles in the function of NBP on EAE. • PGAM5 functions as a key mediator for the protective role of NBP on the progression of EAE. Multiple sclerosis (MS) is a long-lasting autoimmune disease of the central nervous system. Currently, the etiology of MS is not known. Experimental autoimmune encephalomyelitis (EAE), has been recognized as the most widely used animal models to study the molecular mechanisms underlying MS and the efficacy of potential drugs for treatment of MS. In the present study, we found that Dl-3-n-butylphthalide (NBP), a neuroprotective drug in ischemic brain injury, prevented development of disease in experimental autoimmune encephalomyelitis (EAE) and significantly reduced inflammatory factors and necroptosis-associated genes, including PGAM5 in the spinal cord tissues. Similarly, silence of PGAM5 in spinal cord also ameliorated the disease severity in the mice with EAE. Moreover, re-expression of PGAM5 counteracted the protective effect of NBP on the pathogenesis of EAE. Importantly, we found that both NBP and silence of PGAM5 inhibited cellular necroptosis and inflammation in microglia induced by TNFα plus zVAD-fmk. Meanwhile, overexpression of PGAM5 reactivated cellular necroptosis and inflammation suppressed by NBP in vitro. Taken together, our findings provide evidence that NBP can attenuate the progression of EAE by suppressing PGAM5-induced necroptosis and inflammation in microglia and represents a new therapeutic strategy for treating autoimmune diseases.

60 APPLIED LIFE SCIENCES↗