Journal articles on the topic 'Synergistic Scaffolding'
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Dajkovic, Alex, Sebastien Pichoff, Joe Lutkenhaus, and Denis Wirtz. "ZAPA Controls the Scaffolding Function of FtsZ Through Three Synergistic Activities." Biophysical Journal 98, no. 3 (January 2010): 9a. http://dx.doi.org/10.1016/j.bpj.2009.12.054.
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Matsuoka, Satoshi, Hideaki Yukawa, Masayuki Inui, and Roy H. Doi. "Synergistic Interaction of Clostridium cellulovorans Cellulosomal Cellulases and HbpA." Journal of Bacteriology 189, no. 20 (August 10, 2007): 7190–94. http://dx.doi.org/10.1128/jb.00842-07.
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ABSTRACT Clostridium cellulovorans, an anaerobic bacterium, produces a small nonenzymatic protein called HbpA, which has a surface layer homology domain and a type I cohesin domain similar to those found in the cellulosomal scaffolding protein CbpA. In this study, we demonstrated that HbpA could bind to cell wall fragments from C. cellulovorans and insoluble polysaccharides and form a complex with cellulosomal cellulases endoglucanase B (EngB) and endoglucanase L (EngL). Synergistic degradative action of the cellulosomal cellulase and HbpA complexes was demonstrated on acid-swollen cellulose, Avicel, and corn fiber. We propose that HbpA functions to bind dockerin-containing cellulosomal enzymes to the cell surface and complements the activity of cellulosomes.
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Koukiekolo, Roger, Hee-Yeon Cho, Akihiko Kosugi, Masayuki Inui, Hideaki Yukawa, and Roy H. Doi. "Degradation of Corn Fiber by Clostridium cellulovorans Cellulases and Hemicellulases and Contribution of Scaffolding Protein CbpA." Applied and Environmental Microbiology 71, no. 7 (July 2005): 3504–11. http://dx.doi.org/10.1128/aem.71.7.3504-3511.2005.
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ABSTRACT Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.
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Jiao, Yan, Yao Zheng, Ping Chen, Mietek Jaroniec, and Shi-Zhang Qiao. "Molecular Scaffolding Strategy with Synergistic Active Centers To Facilitate Electrocatalytic CO2 Reduction to Hydrocarbon/Alcohol." Journal of the American Chemical Society 139, no. 49 (December 2017): 18093–100. http://dx.doi.org/10.1021/jacs.7b10817.
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Murashima, Koichiro, Akihiko Kosugi, and Roy H. Doi. "Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases from Clostridium cellulovorans." Journal of Bacteriology 184, no. 18 (September 15, 2002): 5088–95. http://dx.doi.org/10.1128/jb.184.18.5088-5095.2002.
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ABSTRACT Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.
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Jeon, Sang Duck, Ji Eun Lee, Su Jung Kim, Sung Hyun Park, Gi-Wook Choi, and Sung Ok Han. "Unique Contribution of the Cell Wall-Binding Endoglucanase G to the Cellulolytic Complex in Clostridium cellulovorans." Applied and Environmental Microbiology 79, no. 19 (July 19, 2013): 5942–48. http://dx.doi.org/10.1128/aem.01400-13.
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ABSTRACTThe cellulosomes produced byClostridium cellulovoransare organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain inC. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces ofC. cellulovoransusing its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that inC. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.
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Kurenova, Elena V., Sartaj Singh Sanghera, Jianqun Liao, Michael Yemma, and William G. Cance. "Demonstration of the efficacy of compound CFAK-C4 targeting FAK-VEGFR3 protein-protein interaction in gastric cancer." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e22143-e22143. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22143.
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e22143 Background: While the emerging data strongly suggest that FAK is an excellent target for developmental therapeutics of cancer, kinase inhibitors of FAK have shown crossreactivity with other protein kinases and toxicity in preclinical and clinical studies. It is known that FAK acts pleiotropically, as a kinase and as a scaffolding protein, and our goal is to explore targeting the scaffolding function of FAK to inhibit protein-protein interactions important for tumor progression. Previously, we have shown that FAK physically interacts with VEGFR3 and we identified small molecule inhibitor CFAK-C4 that targets this site of interaction. Both of these kinases are overexpressed in gastric cancers and were found to be independent poor prognostic factors. The prognosis of patients with gastric cancer remains unfavorable and molecular based treatments are necessary for a potential breakthrough in the therapy of this disease. We hypothesize that FAK-VEGFR3 interaction provides essential survival signals for gastric tumor growth and that simultaneous inhibition of these signals will inhibit tumor progression. Methods: Effects of CFAK-C4 on gastric cancer cell lines AGS and NCI-N87 were examined by MTT assay (viability), colony formation assay and Western blotting (phosphorylation, apoptosis). Subcutaneous mouse model was used to demonstrate effect of CFAK-C4 in vivo. Results: CFAK-C4 specifically blocked phosphorylation of VEGFR3 and FAK, directly inhibited cell viability (p<0.05), increased cell detachment and inhibited colony formation in a dose-dependent manner (range 1-100µM). CFAK-C4 (50mg/kg, IP) effectively caused tumor regression in vivo, when administered alone and its effects were synergistic (p<0.05) with chemotherapy. In vivo effects of C4 were confirmed by a decrease in tumor FAK and VEGFR3 phosphorylation, and disruption of their complexes. Conclusions: In this study we have shown that CFAK-C4 inhibits FAK-VEGFR3 signaling in gastric cancer cells and affects tumor growth. This result demonstrates that targeting the scaffolding function of FAK is a unique approach of highly-specific molecular-targeted therapy and can be used to develop oral-based cancer therapeutics.
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Mohan, Maradumane L., Arunachal Chatterjee, Swetha Ganapathy, Sromona Mukherjee, Sowmya Srikanthan, George P. Jolly, Rohit S. Anand, and Sathyamangla V. Naga Prasad. "Noncanonical regulation of insulin-mediated ERK activation by phosphoinositide 3-kinase γ." Molecular Biology of the Cell 28, no. 22 (November 2017): 3112–22. http://dx.doi.org/10.1091/mbc.e16-12-0864.
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Classically Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in extracellular signal–regulated kinase (ERK) activation following G-protein coupled receptor (GPCR) activation. Knock-down of PI3Kγ unexpectedly resulted in loss of ERK activation to receptor tyrosine kinase agonists such as epidermal growth factor or insulin. Mouse embryonic fibroblasts (MEFs) or primary adult cardiac fibroblasts isolated from PI3Kγ knock-out mice (PI3KγKO) showed decreased insulin-stimulated ERK activation. However, expression of kinase-dead PI3Kγ resulted in rescue of insulin-stimulated ERK activation. Mechanistically, PI3Kγ sequesters protein phosphatase 2A (PP2A), disrupting ERK–PP2A interaction, as evidenced by increased ERK–PP2A interaction and associated PP2A activity in PI3KγKO MEFs, resulting in decreased ERK activation. Furthermore, β-blocker carvedilol-mediated β-arrestin-dependent ERK activation is significantly reduced in PI3KγKO MEF, suggesting accelerated dephosphorylation. Thus, instead of classically mediating the kinase arm, PI3Kγ inhibits PP2A by scaffolding and sequestering, playing a key parallel synergistic step in sustaining the function of ERK, a nodal enzyme in multiple cellular processes.
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Moses, Joseph Christakiran, Mainak Dey, K. Bavya Devi, Mangal Roy, Samit Kumar Nandi, and Biman B. Mandal. "Synergistic Effects of Silicon/Zinc Doped Brushite and Silk Scaffolding in Augmenting the Osteogenic and Angiogenic Potential of Composite Biomimetic Bone Grafts." ACS Biomaterials Science & Engineering 5, no. 3 (February 15, 2019): 1462–75. http://dx.doi.org/10.1021/acsbiomaterials.8b01350.
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Walthers, Christopher M., and Stephanie K. Seidlits. "Gene Delivery Strategies to Promote Spinal Cord Repair." Biomarker Insights 10s1 (January 2015): BMI.S20063. http://dx.doi.org/10.4137/bmi.s20063.
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Gene therapies hold great promise for the treatment of many neurodegenerative disorders and traumatic injuries in the central nervous system. However, development of effective methods to deliver such therapies in a controlled manner to the spinal cord is a necessity for their translation to the clinic. Although essential progress has been made to improve efficiency of transgene delivery and reduce the immunogenicity of genetic vectors, there is still much work to be done to achieve clinical strategies capable of reversing neurodegeneration and mediating tissue regeneration. In particular, strategies to achieve localized, robust expression of therapeutic transgenes by target cell types, at controlled levels over defined time periods, will be necessary to fully regenerate functional spinal cord tissues. This review summarizes the progress over the last decade toward the development of effective gene therapies in the spinal cord, including identification of appropriate target genes, improvements to design of genetic vectors, advances in delivery methods, and strategies for delivery of multiple transgenes with synergistic actions. The potential of biomaterials to mediate gene delivery while simultaneously providing inductive scaffolding to facilitate tissue regeneration is also discussed.
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Groves, Andrew, Rebecca Poetschke, Hafsa Mire, Eshini Panditharatna, Maria Tarazona Guzman, Jun Qi, and Mariella Filbin. "DIPG-26. Targeted Protein Degradation of LSD1 synergizes with HDAC inhibitors in Diffuse Intrinsic Pontine Glioma." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i24. http://dx.doi.org/10.1093/neuonc/noac079.083.
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Abstract Diffuse intrinsic pontine glioma (DIPG) remains one of the most lethal brain tumors in all of childhood with no effective treatments besides radiation, which only extends survival a few months. Against this backdrop, our lab recently executed a focused CRISPR negative selection screen in DIPG cell lines after treatment with the histone deacetylase (HDAC) inhibitor panobinostat and discovered a strong co-dependence with the histone demethylase LSD1. To further explore the therapeutic potential of this synergistic interaction, we tested a drug library of HDAC- and LSD1- targeting drugs with the goal of identifying a combination with optimal synergy and blood brain barrier (BBB) penetration suitable for clinical translation. We were surprised to find that traditional catalytic LSD1 inhibitors had minimal effect in isolation and did not seem to synergize with HDAC inhibitors, while a recently described CoREST/LSD1 degrader named UM171 phenocopied the effects seen in our CRISPR screen. Degraders are a class of compounds that recruit an E3 ubiquitin ligase to a protein-of-interest and cause target ubiquitination and proteasomal degradation. Given our unexpected finding, we hypothesized that UM171 induces synergy with HDAC inhibitors through elimination of a scaffolding function of LSD1. To prove this, we knocked out LSD1 using CRISPR/Cas9 and subsequently treated with a panel of HDAC inhibitors, which showed a signification sensitization of DIPG cells to HDAC inhibitors compared to standard controls. We also confirmed that UM171 interacts with the CoREST complex (members include LSD1, RCOR1, HDAC1/2) by performing streptavidin bead pull down with a newly synthesized biotin-conjugated UM171 probe. In summary, our results show that targeting LSD1 for degradation in combination with HDAC inhibition is a synergistic strategy in DIPG worthy of further translational study.
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Deņisova, Arina, Māra Pilmane, and Pavlo Fedirko. "Glycosaminoglycan, Antimicrobial Defence Molecule and Cytokine Appearance in Tracheal Hyaline Cartilage of Healthy Humans." Journal of Functional Morphology and Kinesiology 7, no. 3 (July 21, 2022): 55. http://dx.doi.org/10.3390/jfmk7030055.
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Hyaline cartilage is an important tracheal structure, yet little is known about its molecular composition, complicating investigation of pathologies and replacement options. Our aim was to research tracheal hyaline cartilage structure, protective tissue factors and variations in healthy humans. The tissue material was obtained from 10 cadavers obtained from the Riga Stradins University Institute of Anatomy and Anthropology archive. Tissues were stained with Bismarck brown and PAS for glycosaminoglycans, and immunohistochemistry was performed for HBD-2, HBD-3, HBD-4, IL-10 and LL-37. The slides were inspected by light microscopy and Spearman’s rank correlation coefficient was calculated. The extracellular matrix was positive across hyaline cartilage for PAS, yet Bismarck brown marked positive proliferation and growth zones. Numerous positive cells for both factors were found in all zones. All of the antimicrobial defence molecules and cytokines were found in a moderate number of cells, except in the mature cell zone with few positive cells. Spearman’s rank correlation coefficient revealed strong and moderate correlations between studied factors. Hyaline cartilage is a tracheal defence structure with a moderate number of antimicrobial defence protein and cytokine immunoreactive cells as well as numerous glycosaminoglycan positive cells. The extracellular matrix glycosaminoglycans provide structural scaffolding and intercellular signalling. The correlations between the studied factors confirm the synergistic activity of them.
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Saura, Rex Bomvet De Leon, and Natividad Mamaoag. "MICROCLASS." Malaysian Journal of Learning and Instruction 20, no. 1 (January 10, 2023): 33–61. http://dx.doi.org/10.32890/mjli2023.20.1.2.
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Purpose - Innovation is imperative to address a wide array of challenges in higher education. The term “microclass” is coined by the researcher to describe the division of a large class into smaller classes, which are handled by student facilitators who have undergone microteaching and performed similar teacher tasks to facilitate students, assessment, discussion, and feedback. Methodology - This was an explorative study and employed the triangulation method (self-designed questionnaire, observation byteacher peers, and semi-structured interview) as well as thematic and descriptive analysis to evaluate the roles of the teacher and student facilitators, level of engagement, and effectiveness of this innovation in the teaching-learning process for the subject, biology. Findings - Data revealed that the role of the teacher and student facilitators concurred to work responsibly in a synergistic manner asimplementers of microclass. The teacher, conducted microteaching, observed, supervised, consulted, and provided feedback to the student facilitators. The student facilitators acted as discussant, motivator, collaborator, coordinator, and executor of initiatives such as scaffolding and pair method throughout the duration and stages of the microclass, resulting in an organized classroom. Furthermore, the microclass was innovatively effective and improved student engagement in the teaching-learning process, which in turn developed soft skills among the students—leadership, commitment and discipline. Significance - It assists science teachers in constructively engaging their students in learning science including boosting students’ softskills, essential for 21st-century skills education and thus warrants further investigation.
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BOISSET, Claire, Henri CHANZY, Bernard HENRISSAT, Raphael LAMED, Yuval SHOHAM, and Edward A. BAYER. "Digestion of crystalline cellulose substrates by the Clostridium thermocellum cellulosome: structural and morphological aspects." Biochemical Journal 340, no. 3 (June 8, 1999): 829–35. http://dx.doi.org/10.1042/bj3400829.
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The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valoniaventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Iα/Iβ composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its ‘commitment’ towards complete degradation.
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Caspi, Jonathan, Yoav Barak, Rachel Haimovitz, Diana Irwin, Raphael Lamed, David B. Wilson, and Edward A. Bayer. "Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode." Applied and Environmental Microbiology 75, no. 23 (October 9, 2009): 7335–42. http://dx.doi.org/10.1128/aem.01241-09.
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ABSTRACT We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.
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Kurenova, E. V., J. Liao, D. He, D. L. Hunt, A. Chekhau, S. N. Hochwald, D. A. Ostrov, and W. G. Cance. "Effect of a novel FAK inhibitor targeted to the binding site of VEGFR3 on pancreatic cancer proliferation in vitro and in vivo." Journal of Clinical Oncology 29, no. 4_suppl (February 1, 2011): 214. http://dx.doi.org/10.1200/jco.2011.29.4_suppl.214.
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214 Background: Even with successful surgical resection and treatment pancreatic adenocarcinoma has a high incidence of recurrence because of metastatic spread. Vascular endothelial growth factor receptor 3 (VEGFR3) is related to lymphatic metastasis. VEGFR3 binds to the survival protein, focal adhesion kinase (FAK), to promote pancreatic cancer (PC) lymphangiogenesis. In this study we have pinpointed the site of interaction of VEGFR3 and FAK to create drug capable of disrupting their signaling with antitumor effect. Methods: 200,000 small molecules were screened in silico for their abilities to interact with the VEGFR3 /FAK binding site. Effects of the selected compounds on PC cell lines MiaPaCa2 and Panc1 were examined by MTT assay (viability), BrdU incorporation (proliferation), Boyden chamber (motility), TUNEL and flow cytometry (cell cycle, apoptosis), and Western blot (phosphorylation, apoptosis). Results: We selected compound C4 because it disrupted the FAK-VEGFR3 complexes in PC cells, as was shown by immunoprecipitation. It caused a dose-dependent dephosphorylation and inactivation of the VEGFR3. Inhibition of VEGFR3 with 10μM of C4 resulted in 80% reduction in cell viability, decrease in proliferation and a 50% reduction in motility. Treatment led to apoptosis in PC cells, confirmed by PARP cleavage. C4 increased the sensitivity of PC cells to chemotherapy in vitro. MiaPaca2 have shown 40% apoptosis after only 24 h of treatment with combination of C4 and gemcitabine at nanomolar concentrations ineffective alone. C4 reduced tumor growth in vivo in murine models of PC. C4 (60 mg/kg, daily IP) decreased tumor growth after 21 days of treatment to 60% (P=0.017). Lower dose of the C4 (10 mg/kg) still had an effect and inhibited tumor growth by 30%, but concomitant administration with low dose of gemcitabine (4 mg/kg, Q4dx6), had significant synergistic effect and led to 80% of tumor reduction. We also have shown that combination of C4 with gemcitabine had prolonged cytostatic effect on tumor growth after treatment withdrawal. Conclusions: Targeting the scaffolding function of FAK with the small-molecule inhibitors can be effectively used to develop potential oral-based PC therapeutics. [Table: see text]
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Hartman, Amy D., Annique Wilson-Weekes, Attaya Suvannasankha, Gem S. Burgess, Kathryn J. Hincher, Larry D. Cripe, and H. Scott Boswell. "In AML, Cbl Serves as Nexus for Signaling from Flt3, and Is Required for Coupling JNK1 in a Pathway of Survival and Proliferation Involving c-jun/AP-1." Blood 106, no. 11 (November 16, 2005): 1203. http://dx.doi.org/10.1182/blood.v106.11.1203.1203.
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Abstract Here we demonstrate by immunoprecipitation and immunoblot, cbl is among the most heavily tyrosine phosphorylated adaptor proteins in primary AML blasts with Flt3 signaling, in the context of either mutation or overexpression/autocrine mechanisms. The human leukemic cell lines MV-4-11 and THP-1 model primary AML blasts in terms of Flt3 signaling by these respective criteria and demonstrate identical coupling between Flt3 and p85, the PI-3-kinase adaptor, by coimmunoprecipitation/blot experiments. Although cbl has no direct binding site on Flt3, it binds tightly to p85 SH2 by virtue of its tyrosine phosphorylation, also demonstrated by co-IP in cell lines and primary cells. Tyrosine phosphorylated cbl is a docking site for CrkII/L SH2’s and this provides a branch point for signals from Flt3 to PI-3-kinase or JNK, respectively, because CrkII(L) is known to bind JNK1 through SH3: polyproline interaction to serve as scaffolding; and interaction of JNK1 and CrkII/L was also observed by co-IP. In a survey of primary AML cases (n=33) there was a strict relationship between expression levels of (active) Flt3 and phospho-c-jun as readout for JNK activity level (p=0.001, r=0.54). To demonstrate the functional relevance of these interactions, siRNA knockdown of components was pursued in the cell lines and in primary AML blasts. JNK1 knockdown, and, to a much lesser degree, JNK2 knockdown, led to loss of phospho-c-jun expression in MV-4-11 and THP-1. Indeed, Flt3 signaling is required for JNK signaling because knockdown of Flt3 led to total loss of p-jun and c-jun expression in MV-4-11 and patient blast. By contrast, cbl knockdown led to selective loss of JNK signaling to p-jun without significantly affecting Flt3 or its downstream activating phosphorylation of AKT. Thus, despite binding by cbl to p85, cbl is not required for PI-3-kinase signaling because of redundancy supplied by the p85-Flt3 interaction. Further, by use of LY294002 to inhibit PI-3-kinase, PI-3-kinase is also not required for JNK signaling. However, selective inhibition of JNK signaling by the small molecule approach in these cell lines and in primary AML blasts leads to loss of proliferation, induction of apoptosis, and synergistic killing with daunorubicin. These observations form the platform for a phase I trial of JNK inhibition in refractory, multidrug-resistant, and Flt3-driven AML.
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Xu, Qi, Wenchen Gao, Shi-You Ding, Rina Kenig, Yuval Shoham, Edward A. Bayer, and Raphael Lamed. "The Cellulosome System of Acetivibrio cellulolyticus Includes a Novel Type of Adaptor Protein and a Cell Surface Anchoring Protein." Journal of Bacteriology 185, no. 15 (August 1, 2003): 4548–57. http://dx.doi.org/10.1128/jb.185.15.4548-4557.2003.
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ABSTRACT A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin. The gene immediately downstream of cipV was sequenced and designated scaB. The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin. ScaB cohesins 1 and 2 are very closely linked. Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin. The scaC gene downstream of scaB codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. A long, ca. 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times. The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162. The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein. The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins. Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative ScaB cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the ScaB dockerin. The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA. In addition, ScaB is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the ScaB adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture.
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Krauss, Jan, Vladimir V. Zverlov, and Wolfgang H. Schwarz. "In VitroReconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose." Applied and Environmental Microbiology 78, no. 12 (April 20, 2012): 4301–7. http://dx.doi.org/10.1128/aem.07959-11.
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ABSTRACTArtificial cellulase complexes active on crystalline cellulose were reconstitutedin vitrofrom a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of aC. thermocellum cipAmutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. Thein vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities forin vitrocomplex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.
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Driscoll, James, Elias J. Anaissie, and Sajjeev Jagannathan. "Cullin-1 Controls The Clinical and Cellular response to Bortezomib Through NF-Kb Pathway Activation In Myeloma." Blood 122, no. 21 (November 15, 2013): 4445. http://dx.doi.org/10.1182/blood.v122.21.4445.4445.
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The Ubiquitin (Ub)+Proteasome system (UPS) is a highly complex network that maintains cellular homeostasis through the selective turnover of targeted proteins. The proteasome serves as the catalytic core of the UPS to execute the efficient removal of ubiquitin-conjugated proteins. Pharmacologic inhibitors that exploit the pivotal role of the proteasome in cellular metabolism promote tumor cytotoxicity and yield durable clinical responses that have significantly improved survival of patients diagnosed with the invariably fatal plasma cell malignancy multiple myeloma (MM). However, while functional blockade of the proteasome has emerged as a successful anti-cancer strategy, drug resistance inevitably emerges through mechanisms that remain elusive. Since E3 Ub ligases target biologically-relevant proteins for proteasomal degradation and are frequently overexpressed in cancers, we hypothesized that altered E3 Ub ligase expression served as a mechanism of resistance to proteasome inhibitors (PIs). To address the role of individual E3s in myelomagenegesis, gene expression profiles of MM patient samples obtained prior to treatment were analyzed. Results indicated that Cullin-1 was overexpressed in MM patient tumor samples compared to normal or MGUS samples. Cullin-1 is an essential scaffolding component of multiple Skp1-Cullin-1-F-box protein E3 complexes that mediate the ubiquitination of proteins involved in cell cycle progression. Next. bone marrow-derived CD138+ plasma cells were obtained from MM patients that were then treated with the PI bortezomib. Samples were similarly probed to identify differences in E3 expression and to identify features that dictate therapeutic response. Again, Cullin-1 was overexpressed in samples from MM patients that did not respond to bortezomib but Cullin-1 was not overexpressed in samples from those patients that responded to bortezomib. Cullin-1 levels were also higher in bortezomib-resistant myeloma cell lines and drug-resistant cells were re-sensitized to bortezomib after short-hairpin-RNA-mediated Cul-1 knockdown. Cells overexpressing Cullin-1 displayed increased NF-Kappa B activity and a reduced sensitivity to bortezomib-induced apoptosis as demonstrated by staining for annexin-positivity. The effect of Cullin-1 expression level on the sensitivity to bortezomib treatment was determined using MM xenografts in athymic SCID mice. We have used a biologically-supervised, microarray-based approach to identify Cul-1 overexpression in a subset of myeloma patients and correlated expression with clinical resistance to bortezomib. Functional studies demonstrated that Cullin-1 sustains activation of the NF-Kappa B pathway and decreases cellular response to bortezomib in vitro in human cell lines. We conclude that Cullin-1 attenuates bortezomib anti-MM activity by maintaining NF-Kappa B signaling and hence promoting tumor survival. Engineered overexpression or shRNA-mediated inactivation of Cullin-1 modulated the cytotoxic effect of bortezomib to further recapitulate the premise that tumor genomics dictate therapeutic response. Cullin-1 is a novel effector within the UPS that offers promise as an oncologic target either alone or in synergistic combination with proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.
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Fontan, Lorena, Rebecca Goldstein, Gabriella Casalena, Himaly Shinglot, Ilkay Us, Haiying Ju, Yanwen Jiang, Natalia Bilchuk, and Ari Melnick. "RNA Interference Screen Implicates TNFAIP3 and FOXO1 in MALT1 Inhibition Resistance." Blood 128, no. 22 (December 2, 2016): 837. http://dx.doi.org/10.1182/blood.v128.22.837.837.
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Abstract Recent studies have identified small molecule inhibitors of the paracaspase activity of MALT1, a protease and scaffolding protein involved in the B-cell receptor (BCR) signaling pathway, that are effective killing lymphomas in vitro and in vivo in xenograft models of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL). ABC-DLBCL is characterized by constitutive NF-κB activity. This activation has been attributed to mutations in various protein components of the B-cell receptor (BCR) as well as Toll-like receptor (TLR) pathways. However, not all ABC-DLBCL cell lines and primary patient samples were equally sensitive to MALT1 inhibitors in vitro. In order to discover genetic modifiers of response to MALT1 inhibition we used an shRNA library screening approach. MALT1 inhibition sensitive cell line HBL-1 was infected with DECIPHER barcoded shRNA library Module 1 and cells were treated with vehicle or 300 nM of MALT1 inhibitor MI-2 for 22 days. At this time cells were harvested and genomic DNA extracted. PCR was used to amplify barcodes and gel purified bands were extracted and sequenced. Cellecta's Deconvoluter software was used to quantify the number of reads per shRNA, reads were normalized to total number of reads and fold change between vehicle and MI-2 treated cells was calculated. Among the top positively and negatively enriched hairpins, we found a significant number of genes involved in the BCR pathway including: positively regulated shRNAs against TNFAIP3 and FOXO1 and negatively regulated hairpins against BTK, CD79B and PI3K genes PIK3C2A and PIK3C2D. Interestingly, TNFAIP3 and FOXO1 are negative regulators of the BCR pathway while BTK, CD79B and PI3K genes are positive regulators of this pathway. In order to validate these results and given the abundance of inhibitors of different proteins in the BCR pathway, we run a focused combination screen using MALT1 inhibitor MI-2 and inhibitors against other proteins in the pathway in 4 MALT1 sensitive cell lines. Combinations with PI3K inhibitors were most synergistic (combination index (CI) ranging 0.12-0.67), while BTK and PKC inhibitors showed an additive effect (CI ranging 0.7-0.9). These results were confirmed using a second MALT1 inhibitor, mepazine. In order to characterize the molecular mechanism by which MALT1 inhibition cooperates with PI3K, we focused on the FDA approved drug Idelalisib. In vitro treatment of cells with MI-2 and Idelalisib showed that effect on cell growth was a combination of decreased proliferation and increased apoptosis. Moreover, we found a decrease in AKT phosphorylation followed by a decrease in FOXO1 T24 phosphorylation and an accumulation of FOXO1 protein. This result correlates with our finding that FOXO1 knockdown favors MALT1 inhibition resistance. In vivo treatment of TMD8 xenografts with a combination of MI-2 and Idelalisib showed a stronger effect than either drug used as a single agent or vehicle, confirming the increased efficacy of the combination over either drug alone. In summary, we have used an shRNA library screening in order to determine which proteins and pathways cooperate with MALT1 inhibition to kill ABC-DLBCL and to evaluate combinatorial treatments in an unbiased manner. This same approach has pointed out TNFAIP3 and FOXO1 as possible biomarkers of response. This is especially interesting since these two proteins are mutated in a proportion of ABC-DLBCL patients and could affect response to treatment not only against MALT1 inhibitors but potentially any BCR targeted therapy. Disclosures Melnick: Janssen: Research Funding.
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Saha, Manujendra N., Hua Jiang, Yijun Yang, Donna Reece, and Hong Chang. "Small Molecule MIRA-1 Induces p53-Independent Apoptosis in Multiple Myeloma Cells Through Activation of the p38 MAPK Signaling Pathway." Blood 120, no. 21 (November 16, 2012): 2937. http://dx.doi.org/10.1182/blood.v120.21.2937.2937.
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Abstract Abstract 2937 Mutation of p53, a tumor suppressor protein, is relatively rare (∼10% in newly diagnosed patients) in multiple myeloma (MM). However, p53 mutations/deletions are important risk factors for predicting the resistant to chemotherapy and no treatment is currently available for this subgroup of patients. MIRA-1, a novel class of small molecules with the ability to restore wild type conformation and function to mutant p53, induces apoptosis in different types of solid tumors harboring mutant p53. However, its effect on MM cells is not known. In this study we examined the ability of MIRA-1 to induce cytotoxic and apoptotic response in MM cells and inhibit tumor growth in MM mouse xenograft model. In addition, we explored the molecular mechanisms of MIRA-1-induced apoptosis in MM cells. Treatment of MM cells with MIRA-1 resulted in a time- and dose-dependent decrease in survival and increase in apoptosis of MM cells harboring either wild type (MM.1S, H929) or mutant (U266, 8226, and LP1) p53 suggesting that MIRA-induced apoptosis in MM cells is independent of p53 status. The IC50 of MIRA-1 observed in these cells was ranged between 10 and 15 μM. In addition, MIRA-1 elicited a dose-dependent inhibition of myeloma cell growth in seven primary MM samples with an average IC50of 10 μM. Two of the seven patient samples harbors p53 mutations/deletions. In contrast, MIRA-1 did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from three healthy donors at the concentrations (10–20 μM) that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Apoptosis induced by MIRA-1 in MM cells harbouring either wild type or mutant p53 was associated with time- and dose-dependent activation of caspas-8, caspase-3 and PARP with subsequent up-regulation of a pro-apoptotic protein, Noxa and down-regulation of an anti-apoptotic protein, Mcl-1. Interestingly, MIRA-1 did not significantly modulate the level of p53 expression, although immunoprecipitation studies confirmed the restoration of wild type conformation of mutant p53 in LP1 and 8226 cells. Importantly, genetic knockdown of p53 using siRNA against wild type or mutant p53 had only a little effect on apoptosis induction by MIRA-1 in MM.1S or LP1 cells, respectively, confirming that apoptosis induction by MIRA-1 in MM cells is independent of p53. Furthermore, the combination of MIRA-1 with current anti-myeloma agents, dexamethasone or doxorubicin displayed synergistic cytotoxic response in MM.1S or LP1 cells (CI<1; p<0.05). To delineate the molecular mechanisms of apoptosis in MM cells induced by MIRA-1, we performed RT2 profiler PCR array analysis for the differential expression of 84 genes related to mitogen activated protein kinase (MAPK) signaling pathway. A significant number of genes of the MAPK family including MAP3K: MAP3K2 (MEKK2), MAP3K4 (MEKK4), PAK1; MAP2K: MAP2K5 (MEK5); and MAPK: MAPK11 (p38bMAPK) as well as transcription factors such as c-Jun, c-FOS, EGR1, and MKNK1, whose expression is induced by MAPK signaling, were up-regulated by more than 2-fold in MIRA-1-treated 8226 cells. On the other hand, expression of the scaffolding/anchoring genes, MAPK8IP2 (JIP-1) was down-regulated by ∼2-fold. Up-regulations of c-Jun, c-Fos, and EGR1 at their protein levels were further confirmed by Western blot analysis of MM.1S and 8226 cells treated with MIRA-1. Importantly, Western blot analysis revealed that treatment of MIRA-1 resulted in a time- and dose-dependent increase of phosphorylated p38 MAPK level in both MM.1S and 8226 cells. Taken together, our data indicates that activation of the MAPK signaling pathway is, at least in part, associated with MIRA-1-induced apoptosis of MM cells. Finally, we evaluated anti-tumorigenic potential of MIRA-1 in MM xenograft SCID mouse models. 8266 cells were inoculated into SCID mice and the mice received i.p. injections of either 100 μL PBS (control) or 10 mg/kg MIRA-1 once daily for 18 days after tumor formation was evident. Administration of MIRA-1 resulted in significant inhibition of tumor growth (p<0.05) and increase in survival (p=0.007) of the mice with no apparent toxicity. Our study for the first time demonstrates potent in vitro and in vivo anti-myeloma activity of MIRA-1 and thus providing a framework for clinical evaluation of MIRA-1 either alone or in combination with current anti-myeloma agents. Disclosures: Reece: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Millennium Pharmaceuticals: Research Funding.
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Walker, Duncan, Michele Mayo, Christine Klaus, Dapeng Chen, Samyabrata Bhaduri, Kirti Sharma, Scott Rusin, et al. "Ktx-120, a Novel Irakimid Degrader of IRAK4 and IMiD Substrates Shows Preferential Activity and Induces Regressions in MYD88-Mutant DLBCL CDX and PDX Models." Blood 136, Supplement 1 (November 5, 2020): 41. http://dx.doi.org/10.1182/blood-2020-137750.
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An important goal of cancer therapy is to improve patient outcomes by driving to deep and durable tumor responses. The activity of single-agent targeted therapies, such as BTK inhibitors or IMiDs alone, has been modest in relapsed and refractory DLBCL, necessitating the use of combination therapy. Targeted protein degraders are heterobifunctional small molecules that co-opt the endogenous ubiquitin-proteasome system to drive the selective degradation of target proteins. Degraders of IRAK4, a key component of the myddosome complex, show potent and selective IRAK4 degradation and preferential activity in MYD88-mutant (MYD88MT) DLBCL models. Notably, the activity of IRAK4 degradation is superior to IRAK4 kinase inhibition, supporting the essential scaffolding role of IRAK4 in myddosome signaling. We have previously described IRAKIMiDs, novel IRAK4 degraders that utilize an IMiD as a pharmacologically active cereblon binder. These degraders simultaneously degrade both IRAK4 and IMiD substrates and show synergistic antitumor activity over either IRAK4 degraders or IMiDs alone, enabling a therapeutically relevant biological combination within a single small molecule. Here we describe KTX-120, a novel IRAKIMiD development candidate. KTX-120 is an equipotent degrader of both IRAK4 and the IMiD substrates Ikaros and Aiolos in lymphoma model systems with low single-digit nM DC50 for degradation of all substrates. The cell activity of KTX-120 has shown a high dependence on MYD88MT status: across a panel of MYD88MT cell lines, KTX-120 showed consistent and potent cell activity, with IC50 ranging from 7-29nM, whereas in MYD88WT lines, cell activity was poor ranging from 1800-3400nM. In the OCI-Ly10 ABC DLBCL cell line that harbors a MYD88L265P mutation, the cell activity of KTX-120 was associated with degradation of both IRAK4 and Ikaros, supporting the combined IRAK4 and IMiD targeting of this molecule as contributing to cell activity. Notably, the onset of cell death in OCI-Ly10 cells with KTX-120 treatment was rapid, with cells becoming committed to cell death within 72h of exposure, suggesting that continuous exposure to KTX-120 may not be necessary for antitumor activity. We explored the pharmacological activity of KTX-120 in several in vivo model systems. KTX-120 is orally bioavailable and shows dose-proportional exposure in several species. A single oral dose of KTX-120 (10 mg/Kg or 30 mg/Kg) showed significant degradation of both IRAK4 and Ikaros in a dose and time-dependent manner, with degradation of both substrates being sustained for >96h, further supporting the potential for intermittent dosing. To assess this, we have explored the antitumor efficacy of KTX-120 in intermittent dosing schedules. KTX-120 was well tolerated and showed potent antitumor activity in several CDX models of MYD88MT DLBCL, including OCI-Ly10, TMD8 and SUDHL2, achieving regressions in all models in as little as once every 2 weeks. As an example, in OCI-Ly10, a 30mpk dose Q2W drove >80% degradation of both IRAK4 and Ikaros and showed regressions (including CR) by D28. Similar activity and tolerability were seen with both PO and IV dosing at doses that achieve active exposure, enabling the potential for both oral and parenteral dosing. We have further explored the activity of KTX-120 in a collection of DLBCL patient derived xenograft models. KTX-120 shows robust activity (>85% TGI) in 4/5 models of MYD88MT DLBCL and shows no or modest activity in 2/2 models of MYD88WT DLBCL, supporting the preferential activity of this mechanism in patient samples harboring MYD88 activating mutations. Importantly, activity was observed in models with a variety of co-mutations that activate the NFkB pathway, including alterations in, CD79B and TNFAIP3, suggesting that KTX-120 has the potential for activity in MYD88MT lymphoma regardless of other mutations. Collectively, these data support the combined IRAK4 degradation and IMiD activity of KTX-120 has the potential to achieve robust and durable regressions in MYD88MT lymphomas with the increased convenience of an intermittently administered single agent and decreased potential for drug combination challenges. Disclosures Walker: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Klaus:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chen:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhaduri:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.
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Tsai, Shen-Long, Garima Goyal, and Wilfred Chen. "Surface Display of a Functional Minicellulosome by Intracellular Complementation Using a Synthetic Yeast Consortium and Its Application to Cellulose Hydrolysis and Ethanol Production." Applied and Environmental Microbiology 76, no. 22 (October 1, 2010): 7514–20. http://dx.doi.org/10.1128/aem.01777-10.
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ABSTRACT In this paper, we report the surface assembly of a functional minicellulosome by using a synthetic yeast consortium. The basic design of the consortium consisted of four different engineered yeast strains capable of either displaying a trifunctional scaffoldin, Scaf-ctf (SC), carrying three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f), or secreting one of the three corresponding dockerin-tagged cellulases (endoglucanase [AT], exoglucanase [EC/CB], or β-glucosidase [BF]). The secreted cellulases were docked onto the displayed Scaf-ctf in a highly organized manner based on the specific interaction of the three cohesin-dockerin pairs employed, resulting in the assembly of a functional minicellulosome on the yeast surface. By exploiting the modular nature of each population to provide a unique building block for the minicellulosome structure, the overall cellulosome assembly, cellulose hydrolysis, and ethanol production were easily fine-tuned by adjusting the ratio of different populations in the consortium. The optimized consortium consisted of a SC:AT:CB:BF ratio of 7:2:4:2 and produced almost twice the level of ethanol (1.87 g/liter) as a consortium with an equal ratio of the different populations. The final ethanol yield of 0.475 g of ethanol/g of cellulose consumed also corresponded to 93% of the theoretical value. This result confirms the use of a synthetic biology approach for the synergistic saccharification and fermentation of cellulose to ethanol by using a yeast consortium displaying a functional minicellulosome.
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Fierobe, Henri-Pierre, Florence Mingardon, Adva Mechaly, Anne Bélaïch, Marco T. Rincon, Sandrine Pagès, Raphael Lamed, Chantal Tardif, Jean-Pierre Bélaïch, and Edward A. Bayer. "Action of Designer Cellulosomes on HomogeneousVersusComplex Substrates." Journal of Biological Chemistry 280, no. 16 (February 10, 2005): 16325–34. http://dx.doi.org/10.1074/jbc.m414449200.
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In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002)J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined “bifunctional” chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.
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Zverlov, Vladimir V., Martina Klupp, Jan Krauss, and Wolfgang H. Schwarz. "Mutations in the Scaffoldin Gene, cipA, of Clostridium thermocellum with Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS1447, and Implications for Cellulase Synergism on Crystalline Cellulose." Journal of Bacteriology 190, no. 12 (April 11, 2008): 4321–27. http://dx.doi.org/10.1128/jb.00097-08.
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ABSTRACT Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble β-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.
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Bae, Haesol, Kalani Craig, Fangli Xia, Yuxin Chen, and Cindy E. Hmelo-Silver. "Developing Historical Thinking in Large Lecture Classrooms Through PBL Inquiry Supported with Synergistic Scaffolding." Interdisciplinary Journal of Problem-Based Learning 15, no. 2 (December 26, 2021). http://dx.doi.org/10.14434/ijpbl.v15i2.28776.
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As PBL has gained popularity across disciplines, its move from small medical-school inquiry groups into large-class undergraduate inquiry has led to an increasing need to understand the elements of successful PBL implementations in large classrooms. In this study, we investigated how PBL was appropriated among students to develop historical thinking skills in a 96-person introductory undergraduate history survey course. The video analysis demonstrated that it was initially challenging for students to appropriate the routines and norms of PBL, but instructor interaction with both the students and representational tools in a large classroom provided multiple co-occurring and dynamic supports. This synergistic scaffolding structured around representational tools was instrumental in a semester-long intervention in which we supported student learning of historical thinking skills by encouraging appropriation of the activities that govern PBL. Keywords: PBL in large classrooms, history PBL, developing historical thinking skills, synergistic scaffolding, representation tools
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Zakhireh, Solmaz, Jaleh Barar, Younes Beygi-Khosrowshahi, Abolfazl Barzegari, Yadollah Omidi, and Khosro Adibkia. "Hollow pollen grains as scaffolding building blocks in bone tissue engineering." BioImpacts, December 18, 2021. http://dx.doi.org/10.34172/bi.2021.24.
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Introduction: The current study, for the first time, suggests nature-made pollen grains (PGs) of Pistacia vera L. as a potential candidate for using as scaffolding building blocks with encapsulation capability of bioactive compounds, such as bone morphogenetic protein 4 (BMP4). Methods: A modified method using KOH (5%, 25ºC) was developed to produce nonallergic hollow pollen grains (HPGs), confirmed by energy dispersive X-ray (EDX) analysis, field emission scanning electron microscopy (FESEM), and DNA and protein staining techniques. The in-vitro study was conducted on human adipose-derived mesenchymal stem cells (hAD-MSCs) to investigate the applicability of HPGs as bone scaffolding building blocks. Cytocompability was evaluated by FESEM, MTT assay, and gene expression analysis of apoptotic markers (BAX and BCL2). The osteoconductive potential of HPGs was assessed by alkaline phosphatase (ALP) activity measurement and gene expression analysis of osteogenic markers (RUNX2 and osteocalcin). Results: Findings demonstrated that HPGs can be considered as biocompatible compounds increasing the metabolic activities of the cells. Further, the bioactive nature of HPGs resulted in suitable cellular adhesion properties, required for a potent scaffold. The investigation of apoptotic gene expression indicated a reduced BAX/BCL2 ratio reflecting the protective effect of HPGs on hAD-MSCs. The increased ALP activity and expression of osteogenic genes displayed the osteoconductive property of HPGs. Moreover, the incorporation of BMP4 in HPGs initiated a synergistic effect on osteoblast maturation. Conclusion: Owing to the unique compositional and surface nanotopographical features of the Pistacia vera L. HPG, this microscale architecture provides a favorable microenvironment for the bottom-up remodeling of bone.
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de la Cruz, Lizbeth, Raul Riquelme, Oscar Vivas, Andres Barria, and Jill B. Jensen. "Dishevelled coordinates phosphoinositide kinases PI4KIIIα and PIP5KIγ for efficient PtdInsP2 synthesis." Journal of Cell Science 135, no. 5 (February 3, 2022). http://dx.doi.org/10.1242/jcs.259145.
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ABSTRACT Phosphatidylinositol(4,5)-bisphosphate (PtdInsP2) is an important modulator of many cellular processes, and its abundance in the plasma membrane is closely regulated. We examined the hypothesis that members of the Dishevelled scaffolding protein family can bind the lipid kinases phosphatidylinositol 4-kinase (PI4K) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K), facilitating synthesis of PtdInsP2 directly from phosphatidylinositol. We used several assays for PtdInsP2 to examine the cooperative function of phosphoinositide kinases and the Dishevelled protein Dvl3 in the context of two receptor signaling cascades. Simultaneous overexpression of PI4KIIIα (also known as PI4KA) and PIP5KIγ (also known as PIP5K1C) had a synergistic effect on PtdInsP2 synthesis that was recapitulated by overexpression of Dvl3. Increasing the activity of Dvl3 by overexpression increased resting plasma membrane PtdInsP2. Knockdown of Dvl3 reduced resting plasma membrane PtdInsP2 and slowed PtdInsP2 resynthesis following receptor activation. We confirm that Dvl3 promotes coupling of PI4KIIIα and PIP5KIγ and show that this interaction is essential for efficient resynthesis of PtdInsP2 following receptor activation.
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Vergara, Ander, Carmen Llorens-Cebrià, Nerea Martos, Irene Martínez-Díaz, Frank Stein, Pamela Domínguez-Báez, Mireia Molina-Van den Bosch, et al. "The membrane-associated protein 17 (MAP17) is upregulated in response to empagliflozin on top of RAS blockade in experimental diabetic nephropathy." Clinical Science, December 16, 2022. http://dx.doi.org/10.1042/cs20220447.
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Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to delay diabetic kidney disease (DKD) progression on top of the standard of care with the renin-angiotensin system (RAS) blockade. The molecular mechanisms underlying the synergistic effect of SGLT2i and RAS blockers is poorly understood. We gave a SGLT2i (empagliflozin), an angiotensin-converting enzyme inhibitor (ramipril), or a combination of both drugs for 8 weeks to diabetic (db/db) mice. Vehicle-treated db/db and db/m mice were used as controls. At the end of the experiment, mice were euthanized, and the kidneys were saved to perform a differential high-throughput proteomic analysis by mass spectrometry using isobaric tandem mass tags (TMT labelling) that allow relative quantification of the identified proteins. The differential proteomic analysis revealed 203 proteins differentially expressed in one or more experimental groups (False Discovery Rate 2 Fold Change ≥ ±1). Fourteen were differentially expressed in the kidneys from the db/db mice treated with empagliflozin with ramipril. Among them, MAP17 was upregulated. These findings were subsequently validated by western blot. The combined therapy of empagliflozin and ramipril upregulated MAP17 in the kidney of a diabetic mice model. MAP17 is a major scaffolding protein of the proximal tubular cells that places transporters together, namely SGLT2 and NHE3. Our results suggest that SGLT2i on top of RAS blockade may protect the kidney by boosting the inactivation of NHE3 via the upregulation of key scaffolder proteins such as MAP17.
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Qin, Liena, Han Dai, and Junfeng Wang. "Key Considerations in Targeted Protein Degradation Drug Discovery and Development." Frontiers in Chemistry 10 (August 1, 2022). http://dx.doi.org/10.3389/fchem.2022.934337.
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Targeting proteins’ enzymatic functions with small molecule inhibitors, as well as functions of receptor proteins with small-molecule agonists and antagonists, were the major forms of small-molecule drug development. These small-molecule modulators are based on a conventional occupancy-driven pharmacological approach. For proteome space traditionally considered undruggable by small-molecule modulators, such as enzymes with scaffolding functions, transcription factors, and proteins that lack well-defined binding pockets for small molecules, targeted protein degraders offer the opportunity to drug the proteome with an event-driven pharmacological approach. A degrader molecule, either PROTAC or molecular glue, brings the protein of interest (POI) and E3 ubiquitin ligase in close proximity and engages the ubiquitin-proteasome system (UPS), the cellular waste disposal system for the degradation of the POI. For the development of targeted protein degraders to meet therapeutic needs, several aspects will be considered, namely, the selective degradation of disease-causing proteins, the oral bioavailability of degraders beyond Lipinski’s rule of five (bRo5) scope, demands of new E3 ubiquitin ligases and molecular glue degraders, and drug resistance of the new drug modality. This review will illustrate several under-discussed key considerations in targeted protein degradation drug discovery and development: 1) the contributing factors for the selectivity of PROTAC molecules and the design of PROTACs to selectively degrade synergistic pathological proteins; 2) assay development in combination with a multi-omics approach for the identification of new E3 ligases and their corresponding ligands, as well as molecular glue degraders; 3) a molecular design to improve the oral bioavailability of bRo5 PROTACs, and 4) drug resistance of degraders.
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Artzi, Lior, Ely Morag, Yoav Barak, Raphael Lamed, and Edward A. Bayer. "Clostridium clariflavum: Key Cellulosome Players Are Revealed by Proteomic Analysis." mBio 6, no. 3 (May 19, 2015). http://dx.doi.org/10.1128/mbio.00411-15.
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ABSTRACTClostridium clariflavumis an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivatedC. clariflavumon cellobiose-, microcrystalline cellulose-, and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gel filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in theC. clariflavumgenome, 11 were identified, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including the glycoside hydrolase families GH48, GH9, GH5, GH30, GH11, and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which together may degrade the substrate in a synergistic manner. These observations are compatible with our previously published model of cellulosome assemblies in this bacterium.IMPORTANCEBecause the reservoir of unsustainable fossil fuels, such as coal, petroleum, and natural gas, is overutilized and continues to contribute to environmental pollution and CO2emission, the need for appropriate alternative energy sources becomes more crucial. Bioethanol produced from dedicated crops and cellulosic waste can provide a partial answer, yet a cost-effective production method must be developed. The cellulosome system of the anaerobic thermophileC. clariflavumcomprises a large number of cellulolytic and hemicellulolytic enzymes, which self-assemble in a number of different cellulosome architectures for enhanced cellulosic biomass degradation. Identification of the major cellulosomal components expressed during growth of the bacterium and their influence on its catalytic capabilities provide insight into the performance of the remarkable cellulosome of this intriguing bacterium. The findings, together with the thermophilic characteristics of the proteins, renderC. clariflavumof great interest for future use in industrial cellulose conversion processes.
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Moraïs, Sarah, Yoav Barak, Jonathan Caspi, Yitzhak Hadar, Raphael Lamed, Yuval Shoham, David B. Wilson, and Edward A. Bayer. "Cellulase-Xylanase Synergy in Designer Cellulosomes for Enhanced Degradation of a Complex Cellulosic Substrate." mBio 1, no. 5 (December 14, 2010). http://dx.doi.org/10.1128/mbio.00285-10.
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ABSTRACTDesigner cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy amongThermobifida fuscaglycoside hydrolases. TwoT. fuscacellulases, Cel48A exoglucanase and Cel5A endoglucanase, and twoT. fuscaxylanases, endoxylanases Xyn10B and Xyn11A, were selected as enzymatic components of a mixed cellulase/xylanase-containing designer cellulosome. The resultant mixed multienzyme complex was fabricated on a single scaffoldin subunit bearing all four enzymes. Conversion ofT. fuscaenzymes to the cellulosomal mode followed by their subsequent incorporation into a tetravalent cellulosome led to assemblies with enhanced activity (~2.4-fold) on wheat straw as a complex cellulosic substrate. The enhanced synergy was caused by the proximity of the enzymes on the complex compared to the free-enzyme systems. The hydrolytic properties of the tetravalent designer cellulosome were compared with the combined action of two separate divalent cellulase- and xylanase-containing cellulosomes. Significantly, the tetravalent designer cellulosome system exhibited an ~2-fold enhancement in enzymatic activity compared to the activity of the mixture of two distinct divalent scaffoldin-borne enzymes. These results provide additional evidence that close proximity between cellulases and xylanases is key to the observed concerted degradation of the complex cellulosic substrate in which the integrated enzymes complement each other by promoting access to the relevant polysaccharide components of the substrate. The data demonstrate that cooperation among xylanases and cellulases can be augmented by their integration into a single designer cellulosome.IMPORTANCEGlobal efforts towards alternative energy programs are highlighted by processes for converting plant-derived carbohydrates to biofuels. The major barrier in such processes is the inherent recalcitrance to enzymatic degradation of cellulose combined with related associated polysaccharides. The multienzyme cellulosome complexes, produced by anaerobic bacteria, are considered to be the most efficient systems for degradation of plant cell wall biomass. In the present work, we have employed a synthetic biology approach by producing artificial designer cellulosomes of predefined enzyme composition and architecture. The engineered tetravalent cellulosome complexes contain two different types of cellulases and two distinct xylanases. Using this approach, enhanced synergistic activity was observed on wheat straw, a natural recalcitrant substrate. The present work strives to gain insight into the combined action of cellulosomal enzyme components towards the development of advanced systems for improved degradation of cellulosic material.
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Tomazetto, Geizecler, Agnes C. Pimentel, Daniel Wibberg, Neil Dixon, and Fabio M. Squina. "Multi-omic Directed Discovery of Cellulosomes, Polysaccharide Utilization Loci, and Lignocellulases from an Enriched Rumen Anaerobic Consortium." Applied and Environmental Microbiology 86, no. 18 (July 17, 2020). http://dx.doi.org/10.1128/aem.00199-20.
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ABSTRACT Lignocellulose is one of the most abundant renewable carbon sources, representing an alternative to petroleum for the production of fuel and chemicals. Nonetheless, the lignocellulose saccharification process, to release sugars for downstream applications, is one of the most crucial factors economically challenging to its use. The synergism required among the various carbohydrate-active enzymes (CAZymes) for efficient lignocellulose breakdown is often not satisfactorily achieved with an enzyme mixture from a single strain. To overcome this challenge, enrichment strategies can be applied to develop microbial communities with an efficient CAZyme arsenal, incorporating complementary and synergistic properties, to improve lignocellulose deconstruction. We report a comprehensive and deep analysis of an enriched rumen anaerobic consortium (ERAC) established on sugarcane bagasse (SB). The lignocellulolytic abilities of the ERAC were confirmed by analyzing the depolymerization of bagasse by scanning electron microscopy, enzymatic assays, and mass spectrometry. Taxonomic analysis based on 16S rRNA sequencing elucidated the community enrichment process, which was marked by a higher abundance of Firmicutes and Synergistetes species. Shotgun metagenomic sequencing of the ERAC disclosed 41 metagenome-assembled genomes (MAGs) harboring cellulosomes and polysaccharide utilization loci (PULs), along with a high diversity of CAZymes. The amino acid sequences of the majority of the predicted CAZymes (60% of the total) shared less than 90% identity with the sequences found in public databases. Additionally, a clostridial MAG identified in this study produced proteins during consortium development with scaffoldin domains and CAZymes appended to dockerin modules, thus representing a novel cellulosome-producing microorganism. IMPORTANCE The lignocellulolytic ERAC displays a unique set of plant polysaccharide-degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described here represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharide degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied to lignocellulose-based biorefineries.
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Moraïs, Sarah, Ely Morag, Yoav Barak, Dan Goldman, Yitzhak Hadar, Raphael Lamed, Yuval Shoham, David B. Wilson, and Edward A. Bayer. "Deconstruction of Lignocellulose into Soluble Sugars by Native and Designer Cellulosomes." mBio 3, no. 6 (December 11, 2012). http://dx.doi.org/10.1128/mbio.00508-12.
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ABSTRACTLignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium,Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes ofClostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides.IMPORTANCECellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on the efficient cellulose-degrading strategies of cellulosome-producing anaerobic bacteria. Cellulosomes are multienzyme complexes capable of converting the plant cell wall polysaccharides into soluble sugar products en route to biofuels as an alternative to fossil fuels. Using a designer cellulosome approach, we have constructed the largest form of homogeneous artificial cellulosomes reported to date, which bear a total of six different cellulases and xylanases from the highly cellulolytic bacteriumThermobifida fusca. These designer cellulosomes were comparable in size to natural cellulosomes and displayed enhanced synergistic activities compared to their free wild-type enzyme counterparts. Future efforts should be invested to improve these processes to approach or surpass the efficiency of natural cellulosomes for cost-effective production of biofuels.