The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptoti...The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5（KD= 9.48 nmol/L for Bcl-2） and SM-6（KD= 0.08 nmol/L for Bcl-xL）, which exhibited improved binding affinity compared with the lead Bim（KD= 16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively）. These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.展开更多
In recent years, selected cry genes from Bacillus thuringiensis（Bt） encoding the production of Cry proteins（Bt toxins） have been engineered into crop plants（Bt-crops）. Through the cultivation of Bt crops and the...In recent years, selected cry genes from Bacillus thuringiensis（Bt） encoding the production of Cry proteins（Bt toxins） have been engineered into crop plants（Bt-crops）. Through the cultivation of Bt crops and the application of Bt pesticides, Cry proteins could be introduced into arable soils. The interaction between the proteins and soils was analyzed in this study to investigate the affinity of Cry proteins in paddy soil ecosystems. Four Paddy soils were selected to represent different soil textures. Cry proteins were spiked in soils, and the amount of protein adsorbed was measured over 24 h. Desorption of Cry1Ab proteins from paddy soils was performed by washing with sterile Milli-Q water（H_2O_（MQ））, and subsequently extracted with an extraction buffer. The paddy soils had a strong affinity for Cry1Ab proteins. Most of the Cry1Ab proteins added（＆gt; 98%） were rapidly adsorbed on the paddy soils tested. More Cry1Ab proteins were adsorbed on non-sterile soils than on sterile soils. Less than 2% of the adsorbed Cry1Ab proteins were desorbed using H2 OMQ, while a considerable proportion of the adsorbed proteins could be desorbed with the buffer, ranging from 20% to 40%.The amount of proteins desorbed increased with the increases in the initial amount of Cry1Ab proteins added to the paddy soils. The concentration of Cry1Ab proteins desorbed from the paddy soils was higher for sterile soils than non-sterile ones. Our results indicate that Bt toxins released via the cultivation of Bt crops, the application of Bt pesticides can be adsorbed on paddy soils, and soil texture could impose an impact on the adsorption capability.展开更多
Forster resonance energy transfer （FRET） techniques have been widely used in biological studies in vitro andin vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occur...Forster resonance energy transfer （FRET） techniques have been widely used in biological studies in vitro andin vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair. Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters. SUMOylation is an important post-translational protein modification with key roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases （SENP） act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate. Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant, Kd, and protease kinetics parameters, kcat and Kin, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.展开更多
Protein-protein interactions and enzyme-catalyzed reactions are the fundamental processes in life,and the quantification and manipulation,kinetics determination,and ether activation or inhibition of these processes ar...Protein-protein interactions and enzyme-catalyzed reactions are the fundamental processes in life,and the quantification and manipulation,kinetics determination,and ether activation or inhibition of these processes are critical for fully understanding physiological processes and discovering new medicine.Various methodologies and technologies have been developed to determine the parameters of these biological and medical processes.However,due to the extreme complexity of these processes,current methods and technologies can only determine one or a few parameters.The recent development of quantitative Forster resonance energy transfer(qFRET)methodology combined with technology aims to establish a high-throughput assay platform to determine protein interaction affinity,enzymatic kinetics,high-throughput screening,and pharmacological parameters using one assay platform.The FRET assay is widely used in biological and biomedical research in vitro and in vivo and provides high-sensitivity measurement in real time.Extensive efforts have been made to develop the FRET assay into a quantitative assay to determine protein-protein interaction affinity and enzymatic kinetics in the past.However,the progress has been challenging due to complicated FRET signal analysis and translational hurdles.The recent qFRET analysis utilizes cross-wavelength correlation coefficiency to dissect the sensitized FRET signal from the total fluorescence signal,which then is used for various biochemical and pharmacological parameter determination,such as K_(D),K_(cat),K_(M),K_(i),IC_(50),and product inhibition kinetics parameters.The qFRET-based biochemical and pharmacological parameter assays and qFRET-based screenings are conducted in 384-well plates in a high-throughput assay mode.Therefore,the qFRET assay platform can provide a universal high-throughput assay platform for future large-scale protein characterizations and therapeutics development.展开更多
基金financially supported by Postdoctoral Applied Research Project of Qingdao(No.861605040085,to CZ,SW)Grant of Innovation Plan in Biomedical Research of Qingdao City(No.15-10-3-15-(28)-zch,to SW)
文摘The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5（KD= 9.48 nmol/L for Bcl-2） and SM-6（KD= 0.08 nmol/L for Bcl-xL）, which exhibited improved binding affinity compared with the lead Bim（KD= 16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively）. These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.
文摘In recent years, selected cry genes from Bacillus thuringiensis（Bt） encoding the production of Cry proteins（Bt toxins） have been engineered into crop plants（Bt-crops）. Through the cultivation of Bt crops and the application of Bt pesticides, Cry proteins could be introduced into arable soils. The interaction between the proteins and soils was analyzed in this study to investigate the affinity of Cry proteins in paddy soil ecosystems. Four Paddy soils were selected to represent different soil textures. Cry proteins were spiked in soils, and the amount of protein adsorbed was measured over 24 h. Desorption of Cry1Ab proteins from paddy soils was performed by washing with sterile Milli-Q water（H_2O_（MQ））, and subsequently extracted with an extraction buffer. The paddy soils had a strong affinity for Cry1Ab proteins. Most of the Cry1Ab proteins added（＆gt; 98%） were rapidly adsorbed on the paddy soils tested. More Cry1Ab proteins were adsorbed on non-sterile soils than on sterile soils. Less than 2% of the adsorbed Cry1Ab proteins were desorbed using H2 OMQ, while a considerable proportion of the adsorbed proteins could be desorbed with the buffer, ranging from 20% to 40%.The amount of proteins desorbed increased with the increases in the initial amount of Cry1Ab proteins added to the paddy soils. The concentration of Cry1Ab proteins desorbed from the paddy soils was higher for sterile soils than non-sterile ones. Our results indicate that Bt toxins released via the cultivation of Bt crops, the application of Bt pesticides can be adsorbed on paddy soils, and soil texture could impose an impact on the adsorption capability.
文摘Forster resonance energy transfer （FRET） techniques have been widely used in biological studies in vitro andin vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair. Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters. SUMOylation is an important post-translational protein modification with key roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases （SENP） act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate. Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant, Kd, and protease kinetics parameters, kcat and Kin, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.
基金supported by the National Institutes of Health Grant AI076504,the UCR Academic Senate Grant,and the Attaisina gift grant.
文摘Protein-protein interactions and enzyme-catalyzed reactions are the fundamental processes in life,and the quantification and manipulation,kinetics determination,and ether activation or inhibition of these processes are critical for fully understanding physiological processes and discovering new medicine.Various methodologies and technologies have been developed to determine the parameters of these biological and medical processes.However,due to the extreme complexity of these processes,current methods and technologies can only determine one or a few parameters.The recent development of quantitative Forster resonance energy transfer(qFRET)methodology combined with technology aims to establish a high-throughput assay platform to determine protein interaction affinity,enzymatic kinetics,high-throughput screening,and pharmacological parameters using one assay platform.The FRET assay is widely used in biological and biomedical research in vitro and in vivo and provides high-sensitivity measurement in real time.Extensive efforts have been made to develop the FRET assay into a quantitative assay to determine protein-protein interaction affinity and enzymatic kinetics in the past.However,the progress has been challenging due to complicated FRET signal analysis and translational hurdles.The recent qFRET analysis utilizes cross-wavelength correlation coefficiency to dissect the sensitized FRET signal from the total fluorescence signal,which then is used for various biochemical and pharmacological parameter determination,such as K_(D),K_(cat),K_(M),K_(i),IC_(50),and product inhibition kinetics parameters.The qFRET-based biochemical and pharmacological parameter assays and qFRET-based screenings are conducted in 384-well plates in a high-throughput assay mode.Therefore,the qFRET assay platform can provide a universal high-throughput assay platform for future large-scale protein characterizations and therapeutics development.
