Comorbid anxiety in chronic pain is clinically common,with a comorbidity rate of over 50%.The main treatments are based on pharmacological,interventional,and implantable approaches,which have limited efficacy and carr...Comorbid anxiety in chronic pain is clinically common,with a comorbidity rate of over 50%.The main treatments are based on pharmacological,interventional,and implantable approaches,which have limited efficacy and carry a risk of side effects.Here,we report a terahertz(THz,1012 Hz)wave stimulation(THS)technique,which exerts nonthermal,long-term modulatory effects on neuronal activity by reducing the binding between nano-sized glutamate molecules and GluA2,leading to the relief of pain and comorbid anxiety-like behaviors in mice.In mice with co-occurring anxiety and chronic pain induced by complete Freund’s adjuvant(CFA)injection,hyperactivity was observed in glutamatergic neurons in the anterior cingulate cortex(ACCGlu).Using whole-cell recording in ACC slices,we demonstrated that THS(34 THz)effectively inhibited the excitability of ACCGlu.Moreover,molecular dynamics simulations showed that THS reduced the number of hydrogen bonds bound between glutamate molecules and GluA2.Furthermore,THS target to the ACC in CFA-treatment mice suppressed ACCGlu hyperactivity and,as a result,alleviated pain and anxiety-like behaviors.Consistently,inhibition of ACCGlu hyperactivity by chemogenetics mimics THS-induced antinociceptive and antianxiety behavior.Together,our study provides evidence for THS as an intervention technique for modulating neuronal activity and a viable clinical treatment strategy for pain and comorbid anxiety.展开更多
Dear Editor,Pyruvate dehydrogenase complex(PDHc) is a large multienzyme assembly(Mr = 4–10 million Daltons) consisting of three essential components: pyruvate dehydrogenase(E1p), dihydrolipoyl transacetylase(E2p), an...Dear Editor,Pyruvate dehydrogenase complex(PDHc) is a large multienzyme assembly(Mr = 4–10 million Daltons) consisting of three essential components: pyruvate dehydrogenase(E1p), dihydrolipoyl transacetylase(E2p), and dihydrolipoyl dehydrogenase(E3). These three enzymes perform distinct functions sequentially to catalyze the oxidative decarboxylation of pyruvate with formation of nicotinamide adenine dinucleotide(NADH) and acetyl-coenzyme A(Patel and Roche, 1990).展开更多
Mammalian mitochondrial electron transport chain complexes are the most important and complicated protein machinery in mitochondria.Although this system has been studied for more than a century,its composition and mol...Mammalian mitochondrial electron transport chain complexes are the most important and complicated protein machinery in mitochondria.Although this system has been studied for more than a century,its composition and molecular mechanism are still largely unknown.Here we report the high-resolution cryo-electron microscopy(Cryo-EM)structures of porcine respiratory chain megacomplex-Ⅰ_(2)Ⅲ_(2)Ⅳ_(2)(MCⅠ_(2)Ⅲ_(2)Ⅳ_(2))in five different conformations,including State 1,State 2,Mid 1,Mid 2,and Mid 3.High-resolution Cryo-EM imaging,combined with super-resolution gated stimulated emission depletion microscopy(gSTED),strongly supports the formation of MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)in live cells.Each MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)structure contains 141 subunits(70 different kinds of peptides,2.9 MDa)in total with 240 transmembrane helices.The mutual influence among CⅠ,CⅢ,and CⅣshown in the MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)structure suggests this megacomplex could act as an integral unit in electron transfer and proton pumping.The conformational changes from different states suggest a plausible regulatory mechanism for the MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)activation/deactivation process.展开更多
Spinal cord injury(SCI)disrupts the structural and functional connectivity between the higher center and the spinal cord,resulting in severe motor,sensory,and autonomic dysfunction with a variety of complications.The ...Spinal cord injury(SCI)disrupts the structural and functional connectivity between the higher center and the spinal cord,resulting in severe motor,sensory,and autonomic dysfunction with a variety of complications.The pathophysiology of sci is complicated and multifaceted,and thus individual treatments acting on a specific aspect or process are inadequate to elicit neuronal regeneration and functional recovery after ScI.Combinatory strategies targeting multiple aspects of scI pathology have achieved greater beneficial effects than individuai therapy alone.Although many problems and challenges remain,the encouraging outcomes that have been achieved in preclinical models offer a promising foothold for the development of novel clinical strategies to treat scl.In this review,we characterize the mechanisms underlying axon regeneration of adult neurons and summarize recent advances in facilitating functional recovery following scI at both the acute and chronic stages.In addition,we analyze the current status,remaining problems,and realistic challenges towards clinical translation.Finally,we consider the future of scI treatment and provide insights into how to narrow the translational gap that currently exists between preclinical studies and clinical practice.Going forward,clinical trials should emphasize multidisciplinary conversation and cooperation to identify optimal combinatorial approaches to maximize therapeuticbenefitinhumanswithscl.展开更多
Respirasome, a huge molecular machine that carries out cellular respiration, has gained growing attention since its discovery, because respiration is the most indis- pensable biological process in almost all living cr...Respirasome, a huge molecular machine that carries out cellular respiration, has gained growing attention since its discovery, because respiration is the most indis- pensable biological process in almost all living crea- tures. The concept of respirasome has renewed our understanding of the respiratory chain organization, and most recently, the structure of respirasome solved by Yang＇s group from Tsinghua University （Gu et al. Nature 237（7622）：639-643, 2016） firstly presented the detailed interactions within this huge molecular machine, and provided important information for drug design and screening. However, the study of cellular respiration went through a long history. Here, we briefly showed the detoured history of respiratory chain investigation, and then described the amazing structure of respirasome.展开更多
The melanoma antigen(MAGE)family proteins are well known as tumor-specific antigens and comprise more than 60 genes,which share a conserved MAGE homology domain(MHD).Type I MAGEs are highly expressed cancer antigens,a...The melanoma antigen(MAGE)family proteins are well known as tumor-specific antigens and comprise more than 60 genes,which share a conserved MAGE homology domain(MHD).Type I MAGEs are highly expressed cancer antigens,and they play an important role in tumorigenesis and cancer cell survival.Recently,several MAGE proteins were identified to interact with RING domain proteins,including a sub-family of E3 ubiquitin ligases.The binding mode between MAGEs and RING proteins was investigated and one important structure of these MAGE-RING complexes was solved:the MAGE-G1-NSE1 complex.Structural and biochemical studies indicated that MAGE proteins could adjust the E3 ubiquitin ligase activity of its cognate RING partner both in vitro and in vivo.However,the underlying mechanism was not fully understood.Here,we review these exciting advances in the studies on MAGE family,suggest potential mechanisms by which MAGEs activate the E3 activity of their binding RING proteins and highlight the anticancer potential of this family proteins.展开更多
Staphylococcus aureus is the most important Gram-positive colonizer of human skin and nasal passage,causing high morbidity and mortality.SD-repeat containing protein D(SdrD),an MSCRAMM(Microbial Surface Components Rec...Staphylococcus aureus is the most important Gram-positive colonizer of human skin and nasal passage,causing high morbidity and mortality.SD-repeat containing protein D(SdrD),an MSCRAMM(Microbial Surface Components Recognizing Adhesive Matrix Molecules)family surface protein,plays an important role in S.aureus adhesion and pathogenesis,while its binding target and molecular mechanism remain largely unknown.Here we solved the crystal structures of SdrD N2-N3 domain and N2-N3-B1 domain.Through structural analysis and comparisons,we characterized the ligand binding site of SdrD,and proposed a featured sequence motif of its potential ligands.In addition,the structures revealed for the first time the interactions between B1 domain and N2-N3 domain among B domain-containing MSCRAMMs.Our results may help in understanding the roles SdrD plays in S.aureus adhesion and shed light on the development of novel antibiotics.展开更多
Respirasome,as a vital part of the oxidative phosphorylation system,undertakes the task of transferring electrons from the electron donors to oxygen and produces a proton concentration gradient across the inner mitoch...Respirasome,as a vital part of the oxidative phosphorylation system,undertakes the task of transferring electrons from the electron donors to oxygen and produces a proton concentration gradient across the inner mitochondrial membrane through the coupled translocation of protons.Copious research has been carried out on this lynchpin of respiration.From the discovery of individual respiratory complexes to the report of the high-resolution structure of mammalian respiratory supercomplex I1III2IV1,scientists have gradually uncovered the mysterious veil of the electron transport chain(ETC).With the discovery of the mammalian respiratory mega complex I2III2IV2,a new perspective emerges in the research field of the ETC.Behind these advances glitters the light of the revolution in both theory and technology.Here,we give a short review about how scientists‘see’the structure and the mechanism of respirasome from the macroscopic scale to the atomic scale during the past decades.展开更多
The transition metal cobalt,an essential cofactor for many enzymes in prokaryotes,is taken up by several specifi c transport systems.The CbiMNQO protein complex be-longs to type-1 energy-coupling factor(ECF)transporte...The transition metal cobalt,an essential cofactor for many enzymes in prokaryotes,is taken up by several specifi c transport systems.The CbiMNQO protein complex be-longs to type-1 energy-coupling factor(ECF)transporters and is a widespread group of microbial cobalt transport-ers.CbiO is the ATPase subunit(A-component)of the cobalt transporting system in the gram-negative thermo-philic bacterium Thermoanaerobacter tengcongensis.Here we report the crystal structure of a nucleotide-free CbiO at a resolution of 2.3Å.CbiO contains an N-terminal canonical nucleotide-binding domain(NBD)and C-termi-nal helical domain.Structural and biochemical data show that CbiO forms a homodimer mediated by the NBD and the C-terminal domain.Interactions mainly via conserved hydrophobic amino acids between the two C-terminal do-mains result in formation of a four-helix bundle.Structural comparison with other ECF transporters suggests that non-conserved residues outside the T-component bind-ing groove in the A component likely act as a specifi city determinant for T components.Together,our data provide information on understanding of the structural organiza-tion and interaction of the CbiMNQO system.展开更多
Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom st...Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom strand; indicates nicking on the top strand）. However, wild type Dralll shows significant star activity. In this study, it was found that the prominent star site is CATSGTT;GTG, consisting of a star 5＇ half （CAT） and a canonical 3＇ half （GTG）. Dralll nicks the 3＇ canonical half site at a faster rate than the 5＇ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the Dralll protein was solved. It indicated, as supported by mutagenesis, that Dralll possesses a ~13a- metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site- directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.展开更多
Dear Editor,Mitochondrial Ca2+homeostasis regulates energy production,cell division,and cell death.The basic properties of mitochondrial Ca2+uptake have been firmly established.The Ca2+influx is mediated by MCU,driven...Dear Editor,Mitochondrial Ca2+homeostasis regulates energy production,cell division,and cell death.The basic properties of mitochondrial Ca2+uptake have been firmly established.The Ca2+influx is mediated by MCU,driven by membrane potential and using a uniporter mechanism(Vasington and Murphy,1962).Patch-clamp analysis of MCU currents demonstrated that MCU is a channel with exceptionally high Ca2+selectivity(Kirichok et al.,2004).展开更多
Dear Editor, Mitochondrial respiratory chain consists of four multimeric protein complexes, Complex I-IV （CI, NADH dehydroge- nase; CII, succinate：ubiquinone oxidoreductase; Clll, cyto- chrome bcl complex; and CIV, ...Dear Editor, Mitochondrial respiratory chain consists of four multimeric protein complexes, Complex I-IV （CI, NADH dehydroge- nase; CII, succinate：ubiquinone oxidoreductase; Clll, cyto- chrome bcl complex; and CIV, cytochrome c oxidase）. These four complexes transfer electrons from NADH or FADH2 to oxygen and pump protons from mitochondrial matrix to intermembrane space, generating electrochemical gradient across the inner membrane which is harnessed by complex V to synthesize ATP, providing the majority of energy acquired by living organisms. Respiratory chain complexes were reported to interact with each other to form supercomplexes.展开更多
TRPML1 channel is a non-selective group-2 transient receptor potential （TRP） channel with Ca2＋ permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the ...TRPML1 channel is a non-selective group-2 transient receptor potential （TRP） channel with Ca2＋ permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV （MLIV）. In the present study, we determined the cryo-electron microscopy （cryo-EM） structures of Mus musculus TRPML1 （mTRPMLI） in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain （PMD） may sense the luminal/extracellular stimuli and undergo a ＂move upward＂ motion during endocytosis, thus triggering the overall conformational change in TRPMLI. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca2＋, and phosphoinosi- tides in a combined manner so as to accommodate the dynamic endocytosis process.展开更多
基金supported by the National Natural Science Foundation of China(grants T2241002 and 12225511)support from the Xplore Prize No.2020-1023by China Academy of Traditional Chinese Medicine Science and Technology Innovation Project(grant:CI2023C017YL).
文摘Comorbid anxiety in chronic pain is clinically common,with a comorbidity rate of over 50%.The main treatments are based on pharmacological,interventional,and implantable approaches,which have limited efficacy and carry a risk of side effects.Here,we report a terahertz(THz,1012 Hz)wave stimulation(THS)technique,which exerts nonthermal,long-term modulatory effects on neuronal activity by reducing the binding between nano-sized glutamate molecules and GluA2,leading to the relief of pain and comorbid anxiety-like behaviors in mice.In mice with co-occurring anxiety and chronic pain induced by complete Freund’s adjuvant(CFA)injection,hyperactivity was observed in glutamatergic neurons in the anterior cingulate cortex(ACCGlu).Using whole-cell recording in ACC slices,we demonstrated that THS(34 THz)effectively inhibited the excitability of ACCGlu.Moreover,molecular dynamics simulations showed that THS reduced the number of hydrogen bonds bound between glutamate molecules and GluA2.Furthermore,THS target to the ACC in CFA-treatment mice suppressed ACCGlu hyperactivity and,as a result,alleviated pain and anxiety-like behaviors.Consistently,inhibition of ACCGlu hyperactivity by chemogenetics mimics THS-induced antinociceptive and antianxiety behavior.Together,our study provides evidence for THS as an intervention technique for modulating neuronal activity and a viable clinical treatment strategy for pain and comorbid anxiety.
基金supported by the National Key R&D Program of China(2022YFA1302701)the National Natural Science Foundation of China(32030056 to M.Y.+4 种基金32241031 and 32171195 to S.L.)the scientific project of Beijing Life Science Academy(2023300CA0090)Tsinghua University Initiative Scientific Research Program(2023Z11DSZ001)the King Abdullah University of Science and Technology(KAUST)Office of Sponsored Research(OSR)under Award(OSR-2020-CRG9-4352)Office of Research Administration(ORA)under Award No.URF/1/4352-01-01,FCC/1/1976-44-01,FCC/1/1976-45-01,REI/1/5234-01-01,and REI/1/5414-01-01.
文摘Dear Editor,Pyruvate dehydrogenase complex(PDHc) is a large multienzyme assembly(Mr = 4–10 million Daltons) consisting of three essential components: pyruvate dehydrogenase(E1p), dihydrolipoyl transacetylase(E2p), and dihydrolipoyl dehydrogenase(E3). These three enzymes perform distinct functions sequentially to catalyze the oxidative decarboxylation of pyruvate with formation of nicotinamide adenine dinucleotide(NADH) and acetyl-coenzyme A(Patel and Roche, 1990).
基金supported by funds from the National Natural Science Foundation of China(32030056 and 32100962)the Tsinghua University Spring Breeze Fund(20201080572)+4 种基金the National Science Fund for Distinguished Young Scholars(3210110055)the China Postdoctoral Science Foundation(2020TQ0178,2020M680519,and 2020M680521)the Guangdong Basic and Applied Basic Research Foundation(2023B1515020039)the Shenzhen Science and Technology Program(RCYX20221008092904016)the Shenzhen University 2035 Program for Excellent Research(2022C012).
文摘Mammalian mitochondrial electron transport chain complexes are the most important and complicated protein machinery in mitochondria.Although this system has been studied for more than a century,its composition and molecular mechanism are still largely unknown.Here we report the high-resolution cryo-electron microscopy(Cryo-EM)structures of porcine respiratory chain megacomplex-Ⅰ_(2)Ⅲ_(2)Ⅳ_(2)(MCⅠ_(2)Ⅲ_(2)Ⅳ_(2))in five different conformations,including State 1,State 2,Mid 1,Mid 2,and Mid 3.High-resolution Cryo-EM imaging,combined with super-resolution gated stimulated emission depletion microscopy(gSTED),strongly supports the formation of MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)in live cells.Each MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)structure contains 141 subunits(70 different kinds of peptides,2.9 MDa)in total with 240 transmembrane helices.The mutual influence among CⅠ,CⅢ,and CⅣshown in the MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)structure suggests this megacomplex could act as an integral unit in electron transfer and proton pumping.The conformational changes from different states suggest a plausible regulatory mechanism for the MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)activation/deactivation process.
文摘Spinal cord injury(SCI)disrupts the structural and functional connectivity between the higher center and the spinal cord,resulting in severe motor,sensory,and autonomic dysfunction with a variety of complications.The pathophysiology of sci is complicated and multifaceted,and thus individual treatments acting on a specific aspect or process are inadequate to elicit neuronal regeneration and functional recovery after ScI.Combinatory strategies targeting multiple aspects of scI pathology have achieved greater beneficial effects than individuai therapy alone.Although many problems and challenges remain,the encouraging outcomes that have been achieved in preclinical models offer a promising foothold for the development of novel clinical strategies to treat scl.In this review,we characterize the mechanisms underlying axon regeneration of adult neurons and summarize recent advances in facilitating functional recovery following scI at both the acute and chronic stages.In addition,we analyze the current status,remaining problems,and realistic challenges towards clinical translation.Finally,we consider the future of scI treatment and provide insights into how to narrow the translational gap that currently exists between preclinical studies and clinical practice.Going forward,clinical trials should emphasize multidisciplinary conversation and cooperation to identify optimal combinatorial approaches to maximize therapeuticbenefitinhumanswithscl.
基金This work was supported by funds from the National Basic Research Program （973 Program） （Nos. 2016YFA0501101 and 2012CB911101）, and the National Science Fund for Distinguished Young Scholars （No. 3163000168） and the National Natural Science Foundation of China （Grant Nos. 31030020 and 31170679）.
文摘Respirasome, a huge molecular machine that carries out cellular respiration, has gained growing attention since its discovery, because respiration is the most indis- pensable biological process in almost all living crea- tures. The concept of respirasome has renewed our understanding of the respiratory chain organization, and most recently, the structure of respirasome solved by Yang＇s group from Tsinghua University （Gu et al. Nature 237（7622）：639-643, 2016） firstly presented the detailed interactions within this huge molecular machine, and provided important information for drug design and screening. However, the study of cellular respiration went through a long history. Here, we briefly showed the detoured history of respiratory chain investigation, and then described the amazing structure of respirasome.
基金This work was supported by NSFC 31030020 to Maojun Yang.
文摘The melanoma antigen(MAGE)family proteins are well known as tumor-specific antigens and comprise more than 60 genes,which share a conserved MAGE homology domain(MHD).Type I MAGEs are highly expressed cancer antigens,and they play an important role in tumorigenesis and cancer cell survival.Recently,several MAGE proteins were identified to interact with RING domain proteins,including a sub-family of E3 ubiquitin ligases.The binding mode between MAGEs and RING proteins was investigated and one important structure of these MAGE-RING complexes was solved:the MAGE-G1-NSE1 complex.Structural and biochemical studies indicated that MAGE proteins could adjust the E3 ubiquitin ligase activity of its cognate RING partner both in vitro and in vivo.However,the underlying mechanism was not fully understood.Here,we review these exciting advances in the studies on MAGE family,suggest potential mechanisms by which MAGEs activate the E3 activity of their binding RING proteins and highlight the anticancer potential of this family proteins.
基金supported by the National Key R&D Program of China (2019YFA0704903)the National Natural Science Foundation of China (11834012, 52172232, 52130203, and 91963122)Foshan Xianhu Laboratory of the Advanced Energy Science and Technology Guangdong Laboratory (XHT2020-004)。
基金supported by the National Basic Research Program(973 Program)(Nos.2011CB910502 and 2012CB911101)National Natural Science Foundation of China(Grant Nos.31030020 and 31170679).
文摘Staphylococcus aureus is the most important Gram-positive colonizer of human skin and nasal passage,causing high morbidity and mortality.SD-repeat containing protein D(SdrD),an MSCRAMM(Microbial Surface Components Recognizing Adhesive Matrix Molecules)family surface protein,plays an important role in S.aureus adhesion and pathogenesis,while its binding target and molecular mechanism remain largely unknown.Here we solved the crystal structures of SdrD N2-N3 domain and N2-N3-B1 domain.Through structural analysis and comparisons,we characterized the ligand binding site of SdrD,and proposed a featured sequence motif of its potential ligands.In addition,the structures revealed for the first time the interactions between B1 domain and N2-N3 domain among B domain-containing MSCRAMMs.Our results may help in understanding the roles SdrD plays in S.aureus adhesion and shed light on the development of novel antibiotics.
基金Tsinghua University Branch of China National Center for Protein Sciences(Beijing)for providing the facility support.
文摘Respirasome,as a vital part of the oxidative phosphorylation system,undertakes the task of transferring electrons from the electron donors to oxygen and produces a proton concentration gradient across the inner mitochondrial membrane through the coupled translocation of protons.Copious research has been carried out on this lynchpin of respiration.From the discovery of individual respiratory complexes to the report of the high-resolution structure of mammalian respiratory supercomplex I1III2IV1,scientists have gradually uncovered the mysterious veil of the electron transport chain(ETC).With the discovery of the mammalian respiratory mega complex I2III2IV2,a new perspective emerges in the research field of the ETC.Behind these advances glitters the light of the revolution in both theory and technology.Here,we give a short review about how scientists‘see’the structure and the mechanism of respirasome from the macroscopic scale to the atomic scale during the past decades.
基金the National Basic Research Program(973 Program)(Nos.2011CB910502 and 2012CB911101)the National Natural Science Foundation of China(Grant Nos.31030020 and 31170679).
文摘The transition metal cobalt,an essential cofactor for many enzymes in prokaryotes,is taken up by several specifi c transport systems.The CbiMNQO protein complex be-longs to type-1 energy-coupling factor(ECF)transporters and is a widespread group of microbial cobalt transport-ers.CbiO is the ATPase subunit(A-component)of the cobalt transporting system in the gram-negative thermo-philic bacterium Thermoanaerobacter tengcongensis.Here we report the crystal structure of a nucleotide-free CbiO at a resolution of 2.3Å.CbiO contains an N-terminal canonical nucleotide-binding domain(NBD)and C-termi-nal helical domain.Structural and biochemical data show that CbiO forms a homodimer mediated by the NBD and the C-terminal domain.Interactions mainly via conserved hydrophobic amino acids between the two C-terminal do-mains result in formation of a four-helix bundle.Structural comparison with other ECF transporters suggests that non-conserved residues outside the T-component bind-ing groove in the A component likely act as a specifi city determinant for T components.Together,our data provide information on understanding of the structural organiza-tion and interaction of the CbiMNQO system.
文摘Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom strand; indicates nicking on the top strand）. However, wild type Dralll shows significant star activity. In this study, it was found that the prominent star site is CATSGTT;GTG, consisting of a star 5＇ half （CAT） and a canonical 3＇ half （GTG）. Dralll nicks the 3＇ canonical half site at a faster rate than the 5＇ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the Dralll protein was solved. It indicated, as supported by mutagenesis, that Dralll possesses a ~13a- metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site- directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.
文摘Dear Editor,Mitochondrial Ca2+homeostasis regulates energy production,cell division,and cell death.The basic properties of mitochondrial Ca2+uptake have been firmly established.The Ca2+influx is mediated by MCU,driven by membrane potential and using a uniporter mechanism(Vasington and Murphy,1962).Patch-clamp analysis of MCU currents demonstrated that MCU is a channel with exceptionally high Ca2+selectivity(Kirichok et al.,2004).
基金We thank the Tsinghua University Branch of China National Center for Protein Sciences （Beijing） for providing the facility support. The computation was completed on the ＂Explorer 100＂ cluster system of Tsinghua National Laboratory for Information Science and Technol- ogy. This work was supported by funds from the National Key R＆D Program of China （Nos. 2017YFA0504600 and 2016YFA0501100）. The National Science Fund for Distinguished Young Scholars（31625008）, and the National Natural Science Foundation of China （Grant Nos. 21532004 and 31570733）.
文摘Dear Editor, Mitochondrial respiratory chain consists of four multimeric protein complexes, Complex I-IV （CI, NADH dehydroge- nase; CII, succinate：ubiquinone oxidoreductase; Clll, cyto- chrome bcl complex; and CIV, cytochrome c oxidase）. These four complexes transfer electrons from NADH or FADH2 to oxygen and pump protons from mitochondrial matrix to intermembrane space, generating electrochemical gradient across the inner membrane which is harnessed by complex V to synthesize ATP, providing the majority of energy acquired by living organisms. Respiratory chain complexes were reported to interact with each other to form supercomplexes.
文摘TRPML1 channel is a non-selective group-2 transient receptor potential （TRP） channel with Ca2＋ permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV （MLIV）. In the present study, we determined the cryo-electron microscopy （cryo-EM） structures of Mus musculus TRPML1 （mTRPMLI） in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain （PMD） may sense the luminal/extracellular stimuli and undergo a ＂move upward＂ motion during endocytosis, thus triggering the overall conformational change in TRPMLI. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca2＋, and phosphoinosi- tides in a combined manner so as to accommodate the dynamic endocytosis process.
