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Minchul Yoon - One of the best experts on this subject based on the ideXlab platform.
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179 Conjugation process of a freshwater green alga spirogyra varians zygnematales charophyceae examined by fitc lectins
Journal of Phycology, 2003Co-Authors: Minchul YoonAbstract:The Conjugation processes of filamentous freshwater green alga Spirogyra varians were examined by the use of FITC-lectins. Conjugation comprised of five steps: 1) array with adjacent filaments, 2) formation of Conjugation protrusion (papilla), 3) fusion of the protrusions, 4) formation of Conjugation tube, and 5) formation of zygotes. Three lectins, ConA, RCA and UEA, showed considerable labeling during the progression of Conjugation. FITC-ConA labeled the surfaces of filaments throughout the whole Conjugation processes. No labeling of FITC-RCA was detected on the surface of vegetative filaments. FITC-RCA labeling was observed at the Conjugation protrusions only after the papilla formation. Strong labeling continued until formation of zygotes even in hollow area between the Conjugation tube. The labeling decreased gradually over time and disappeared when zygotes were formed. FITC-UEA showed similar labeling pattern with FITC-RCA except that the labeling did not disappear even after zygote formation. Inhibition experiments using D-galactose, L-fucose and D-mannose, which are complementary carbohydrates for the above lectins, showed considerable decrease of Conjugation (<50% vs. 83% in control). Hamagglutination experiment using crude extract of Spyrogyra varians revealed existence of lectins specific for the above carbohydrates. These results suggested that the lectin-carbohydrate recognition system might be involved in the Conjugation of Spirogyra varians.
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179 Conjugation Process of a Freshwater Green Alga, Spirogyra Varians (Zygnematales, Charophyceae Examined by Fitc‐Lectins)
Journal of Phycology, 2003Co-Authors: Minchul YoonAbstract:The Conjugation processes of filamentous freshwater green alga Spirogyra varians were examined by the use of FITC-lectins. Conjugation comprised of five steps: 1) array with adjacent filaments, 2) formation of Conjugation protrusion (papilla), 3) fusion of the protrusions, 4) formation of Conjugation tube, and 5) formation of zygotes. Three lectins, ConA, RCA and UEA, showed considerable labeling during the progression of Conjugation. FITC-ConA labeled the surfaces of filaments throughout the whole Conjugation processes. No labeling of FITC-RCA was detected on the surface of vegetative filaments. FITC-RCA labeling was observed at the Conjugation protrusions only after the papilla formation. Strong labeling continued until formation of zygotes even in hollow area between the Conjugation tube. The labeling decreased gradually over time and disappeared when zygotes were formed. FITC-UEA showed similar labeling pattern with FITC-RCA except that the labeling did not disappear even after zygote formation. Inhibition experiments using D-galactose, L-fucose and D-mannose, which are complementary carbohydrates for the above lectins, showed considerable decrease of Conjugation (
Inchan Kwon - One of the best experts on this subject based on the ideXlab platform.
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Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid Conjugation
Scientific Reports, 2017Co-Authors: Byung Seop Yang, Young S. Hahn, Inchan KwonAbstract:Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-Conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in Conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye Conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-Conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the Conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the Conjugation yield likely due to the enhanced solubility of palmitic acid analog.
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Expansion of bioorthogonal chemistries towards site-specific polymer–protein Conjugation
Polymer Chemistry, 2016Co-Authors: Secheon Jung, Inchan KwonAbstract:Polymer Conjugation to proteins has been widely used to improve or expand protein properties. However, polymer Conjugation to random sites of a target protein often led to a significant loss of critical protein properties. In order to overcome this, polymer Conjugation to specific sites of a protein was developed using the site-specific introduction of non-natural amino acids and bioorthogonal chemistries. This review summarizes the recent advances in bioorthogonal chemistries. As the repertoire of bioorthogonal chemistries available for polymer Conjugation is expanding, the site-specific polymer Conjugation technique would be a valuable platform technique to design novel protein–polymer conjugates.
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expansion of bioorthogonal chemistries towards site specific polymer protein Conjugation
Polymer Chemistry, 2016Co-Authors: Secheon Jung, Inchan KwonAbstract:Polymer Conjugation to proteins has been widely used to improve or expand protein properties. However, polymer Conjugation to random sites of a target protein often led to a significant loss of critical protein properties. In order to overcome this, polymer Conjugation to specific sites of a protein was developed using the site-specific introduction of non-natural amino acids and bioorthogonal chemistries. This review summarizes the recent advances in bioorthogonal chemistries. As the repertoire of bioorthogonal chemistries available for polymer Conjugation is expanding, the site-specific polymer Conjugation technique would be a valuable platform technique to design novel protein–polymer conjugates.
Secheon Jung - One of the best experts on this subject based on the ideXlab platform.
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Expansion of bioorthogonal chemistries towards site-specific polymer–protein Conjugation
Polymer Chemistry, 2016Co-Authors: Secheon Jung, Inchan KwonAbstract:Polymer Conjugation to proteins has been widely used to improve or expand protein properties. However, polymer Conjugation to random sites of a target protein often led to a significant loss of critical protein properties. In order to overcome this, polymer Conjugation to specific sites of a protein was developed using the site-specific introduction of non-natural amino acids and bioorthogonal chemistries. This review summarizes the recent advances in bioorthogonal chemistries. As the repertoire of bioorthogonal chemistries available for polymer Conjugation is expanding, the site-specific polymer Conjugation technique would be a valuable platform technique to design novel protein–polymer conjugates.
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expansion of bioorthogonal chemistries towards site specific polymer protein Conjugation
Polymer Chemistry, 2016Co-Authors: Secheon Jung, Inchan KwonAbstract:Polymer Conjugation to proteins has been widely used to improve or expand protein properties. However, polymer Conjugation to random sites of a target protein often led to a significant loss of critical protein properties. In order to overcome this, polymer Conjugation to specific sites of a protein was developed using the site-specific introduction of non-natural amino acids and bioorthogonal chemistries. This review summarizes the recent advances in bioorthogonal chemistries. As the repertoire of bioorthogonal chemistries available for polymer Conjugation is expanding, the site-specific polymer Conjugation technique would be a valuable platform technique to design novel protein–polymer conjugates.
Jayanta Debnath - One of the best experts on this subject based on the ideXlab platform.
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atg12 atg3 and mitochondria
Autophagy, 2011Co-Authors: Lilliana Radoshevich, Jayanta DebnathAbstract:Ubiquitin-like (UBL) protein Conjugations, such as ubiquitination and sumoylation, alter protein fate, localization, function and transcriptional activity. Two UBL protein Conjugation systems, the ATG12 and ATG8 systems, are required for macroautophagy. The early steps of autophagosome formation require that these two Conjugation systems covalently attach: (1) the UBL ATG12 to the target protein ATG5 (Fig. 1A); and (2) the UBL ATG8 to the lipid phosphotidylethanolamine (PE) (Fig. 1B). Interestingly, since the initial discovery of the ATG12 Conjugation system, ATG5 has remained as the only known target of ATG12. Recently, we demonstrated that ATG12 can modify multiple protein targets in mammalian cells, and identified ATG3, the E2-like enzyme that conjugates ATG8 to PE, as a second ATG12 Conjugation target. Figure 1 ATG12 Conjugation to ATG3. (A) ATG 12 Conjugation system. The E1 enzyme ATG7 activates the C-terminal glycine of ATG12. Subsequently, ATG12 is transferred to the E2 conjugating enzyme ATG10, resulting in the formation of the ATG12-ATG5 complex (top). ... Although ATG12 and ATG3 have been previously reported to physically interact, to our knowledge, we provide the first evidence for a covalent complex between these two essential components of the autophagy Conjugation machineries. Similar to ATG12-ATG5, formation of the ATG12-ATG3 complex requires activation of ATG12 by the E1-like enzyme ATG7. Subsequently, ATG3 autocatalyzes the Conjugation of ATG12 onto a single conserved lysine residue (K243) of this E2-like enzyme (Fig. 1A, see gray box). Based on immunoprecipitation studies, both free ATG3 and the ATG12-ATG3 complex are concomitantly detected in mammalian cells; notably, only a fraction of the total pool of endogenous ATG3 is in a complex with ATG12. This contrasts with ATG5, which appears to be completely conjugated to ATG12. To gain insight into the cellular function of the ATG12-ATG3 complex, we complemented atg3-deficient mouse fibroblasts with either wild-type ATG3 (WTATG3) or a mutant version of ATG3 that is unable to be conjugated to ATG12 due to loss of a single lysine required for isopeptide bond linkage (ATG3KR). Because ATG12, in complex with ATG5, and ATG3 both play critical roles in the biogenesis and elongation of the autophagosomal membrane, we hypothesized that the ATG12-ATG3 complex would likewise regulate the early steps of autophagy. However, upon disrupting ATG12 Conjugation to ATG3, we observed no effects on the lipidation of LC3 or other mammalian ATG8 orthologues. We also found no significant changes in nonselective autophagy in response to multiple stresses, including nutrient starvation, rapamycin-mediated mTOR inhibition and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondrial depolarization. Although nonselective autophagy appears intact, our results indicate that the disruption of ATG12 Conjugation to ATG3 in cells does have profound effects on mitochondria. ATG3KR cells possess increased mitochondrial mass relative to WTATG3 cells, both at baseline and in response to mitochondrial depolarization with CCCP. Upon mitochondrial depolarization, mitochondria targeting to autophagosomes in ATG3KR cells is significantly reduced relative to WTATG3 cells, suggesting a defect in mitochondrial autophagy or mitophagy. To date, the prevailing view has been that specific adapter molecules target mitochondria for autophagic degradation in mammalian cells. For example, the two related BH3 family proteins, BNIP3 and NIX/BNIP3L, promote mitochondrial clearance during reticulocyte development, whereas the PINK1-Parkin pathway directs mitophagy in response to depolarization. In contrast, the ATGs comprising the Conjugation systems have not been previously implicated in the selective targeting of mitochondria; rather, they are proposed to contribute more generally by forming the autophagosome. Our results now broach the hypothesis that this newly identified complex between two Conjugation ATGs mediates a unique function for selectively targeting mitochondria for autophagic degradation. In addition, we discovered that the mitochondrial network of ATG3KR cells is highly fragmented. Mitochondrial fragmentation is similarly present in atg3-deficient cells, whereas cells reconstituted with WTATG3 exhibit tubular mitochondrial networks. Mitochondria undergo extensive remodeling through continuous cycles of mitochondrial fusion and fission; appropriate balance of these two opposing processes is necessary for maintenance of overall mitochondrial structure and function. Mitochondrial fragmentation often indicates an imbalance in mitochondrial fission and fusion; in support of this prediction, cells lacking the ATG12-ATG3 complex display reduced mitochondrial fusion activity. Nonetheless, further studies are required to clarify if ATG12-ATG3 serves as a novel link between autophagy and the mitochondrial fusion machinery. Importantly, one can alternatively hypothesize that the effects of ATG12-ATG3 on mitochondrial fusion are secondary to its effects on mitophagy, since previous work indicates that fragmented mitochondria are preferentially degraded by mitophagy. In other words, due to reduced mitophagy, disruption of ATG12 Conjugation to ATG3 results in the build-up of fusion defective mitochondria. Finally, ATG12 Conjugation to ATG3 sensitizes cells to apoptosis mediated by mitochondrial pathways, but has no effect on death-receptor mediated apoptosis. The resistance to intrinsic apoptosis in ATG3KR cells correlates with an increase in the basal protein levels of antiapoptotic Bcl-2 proteins, namely Bcl-xL. Accordingly, specific antagonism of antiapoptotic Bcl-2 family members with obatoclax, a pharmacological BH3 mimetic, causes equivalent levels of cell death between WTATG3 and ATG3KR cells. An important question for future study is whether these increases in Bcl-XL also contribute functionally to the changes in mitochondrial mass and morphology found in ATG3KR cells. Dissecting the exact mechanisms through which the ATG12-ATG3 complex mediates these diverse effects on mitochondrial homeostasis, morphology and cell death remains an important topic for future investigation. Since a fraction of total ATG3 is in complex with ATG12, this motivates the hypothesis that ATG12 Conjugation to ATG3 promotes the relocation of this complex to mitochondria. Alternatively, ATG12 Conjugation may change ATG3 substrate specificity, alter its enzymatic activity, or facilitate new protein-protein interactions. Importantly, our results support the idea that the effects of the ATG12-ATG3 complex on mitochondrial homeostasis and cell death are unique functions that can be completely separated from the established roles of each individual ATG in autophagosome formation. Although this is a novel finding for ATG12 and ATG3, other studies demonstrate that ATGs may control cellular functions distinct from autophagy. For example, during phagocytosis, LC3 becomes recruited to, and incorporated into, the plasma membrane by a macroautophagy-independent mechanism that requires both ATG5 and Beclin 1. Hence, we propose that ATG12, like ubiquitin and SUMO, may serve as a broadbased protein modification that mediates diverse cellular functions in addition to autophagy.
Xuejun Jiang - One of the best experts on this subject based on the ideXlab platform.
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stimulation of atg12 atg5 Conjugation by ribonucleic acid
Autophagy, 2007Co-Authors: Yufang Shao, Taya Feldman, Xuejun JiangAbstract:The ubiquitin-like Conjugation reactions, ATG8/microtubule-associated protein 1 light chain 3/MAP1LC3 (LC3) to phosphatidylethanolamine (PE) and ATG12 to ATG5, are biochemical hallmarks for autophagy, a cellular process that degrades bulk cellular proteins and organelles. The two Conjugation reactions share the same E1-like enzyme ATG7 but have different E2-like enzymes, ATG3 for LC3-PE and ATG10 for ATG12-ATG5. In cells, ATG12-ATG5 Conjugation appears to be required for LC3-PE Conjugation. Previously, in vitro reconstitution of LC3-PE Conjugation, but not the upstream ATG12-ATG5 Conjugation, was reported. In this study, we describe for the first time the de novo reconstitution of mammalian ATG12-ATG5 Conjugation by using purified recombinant proteins. We show that ATG7, ATG10 and ATP as an energy source are all essential for ATG12-ATG5 Conjugation, and mutation of the specific lysine residue of ATG5 for ATG12 Conjugation abrogates the reaction. Furthermore, a potent stimulating activity for ATG12-ATG5 co...
