To compare liver proteolysis and proteasome activation in steatotic liver grafts conserved in University of Wisconsin(UW)and Institut Georges Lopez-1(IGL-1)solutions.METHODSFatty liver grafts from male obese Zück...To compare liver proteolysis and proteasome activation in steatotic liver grafts conserved in University of Wisconsin(UW)and Institut Georges Lopez-1(IGL-1)solutions.METHODSFatty liver grafts from male obese Zücker rats were conserved in UW and IGL-1 solutions for 24 h at 4°Cand subjected to“ex vivo”normo-thermic perfusion(2 h;37°C).Liver proteolysis in tissue specimens and perfusate was measured by reverse-phase high performance liquid chromatography.Total free amino acid release was correlated with the activation of the ubiquitin proteasome system(UPS:measured as chymotryptic-like activity and 20S and 19S proteasome),the prevention of liver injury(transaminases),mitochondrial injury(confocal microscopy)and inflammation markers(TNF 1 alpha,high mobility group box-1(HGMB-1)and PPAR gamma),and liver apoptosis(TUNEL assay,cytochrome c and caspase 3).RESULTSProfiles of free AA(alanine,proline,leucine,isoleucine,methionine,lysine,ornithine,and threonine,among others)were similar for tissue and reperfusion effluent.In all cases,the IGL-1 solution showed a significantly higher prevention of proteolysis than UW(P<0.05)after cold ischemia reperfusion.Livers conserved in IGL-1 presented more effective prevention of ATP-breakdown and more inhibition of UPS activity(measured as chymotryptic-like activity).In addition,the prevention of liver proteolysis and UPS activation correlated with the prevention of liver injury(AST/ALT)and mitochondrial damage(revealed by confocal microscopy findings)as well as with the prevention of inflammatory markers(TNF1alpha and HMGB)after reperfusion.In addition,the liver grafts preserved in IGL-1 showed a significant decrease in liver apoptosis,as shown by TUNEL assay and the reduction of cytochrome c,caspase 3 and P62 levels.CONCLUSIONOur comparison of these two preservation solutions suggests that IGL-1 helps to prevent ATP breakdown more effectively than UW and subsequently achieves a higher UPS inhibition and reduced liver proteolysis.展开更多
Neurological diseases such as stroke,Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease are among the intractable diseases for which appropriate drugs and treatments are lacking.Proteolysis targeting ...Neurological diseases such as stroke,Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease are among the intractable diseases for which appropriate drugs and treatments are lacking.Proteolysis targeting chimera(PROTAC)technology is a novel strategy to solve this problem.PROTAC technology uses the ubiquitin-protease system to eliminate mutated,denatured,and harmful proteins in cells.It can be reused,and utilizes the protein destruction mechanism of the cells,thus making up for the deficiencies of traditional protein degradation methods.It can effectively target and degrade proteins,including proteins that are difficult to identify and bind.Therefore,it has extremely important implications for drug development and the treatment of neurological diseases.At present,the targeted degradation of mutant BTK,mHTT,Tau,EGFR,and other proteins using PROTAC technology is gaining attention.It is expected that corresponding treatment of nervous system diseases can be achieved.This review first focuses on the recent developments in PROTAC technology in terms of protein degradation,drug production,and treatment of central nervous system diseases,and then discusses its limitations.This review will provide a brief overview of the recent application of PROTAC technology in the treatment of central nervous system diseases.展开更多
The ASK1 （ARABIDOPSIS SKP1-LIKE） protein is a critical component of the SCF （Skpl-Cullin-F box protein） ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome. To investigate...The ASK1 （ARABIDOPSIS SKP1-LIKE） protein is a critical component of the SCF （Skpl-Cullin-F box protein） ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome. To investigate proteins that are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers, we compared the proteomes of the Arabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis （2-DE）. Ten protein spots with higher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to further mass spectrometry （MS） analysis. The results showed that they were proteins involved in photomorphogenesis, circadian oscillation, post-translation process, stress-responses and cell expansion or elongation, suggesting that those processes were affected in the ask1 mutant. The transcript levels of these genes were also compared based on the Affymetrix gene chip microarray data. No significant difference was observed for most of the genes, suggesting that the proteins with elevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASK 1-mediated proteolysis pathway. These results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes and also demonstrate the importance and necessity of studying protein levels with respect to gene functions.展开更多
The amino acid biosynthesis and proteolytic system of Lactobacillus bulgaricus (L.Bulgaricus ) is important for its growth in niche-specific environments, as well as for flavour formation in the food industry. Compara...The amino acid biosynthesis and proteolytic system of Lactobacillus bulgaricus (L.Bulgaricus ) is important for its growth in niche-specific environments, as well as for flavour formation in the food industry. Comparative analyses of 4 completed sequences of the L.Bulgaricus strain genome on a genomic scale revealed that genes involved in amino acids synthesis were undergoing reductive evolution. However, the selected industrial strains, namely, L.Bulgaricus 2038 and L.Bulgaricus ND02, retained more complete genes in the amino acid synthesis and proteolytic system category than the laboratory strains, and have some unique genes and pathways for obtaining amino acids that enable these bacteria to adapt to their various environmental niches.展开更多
We screened 95 kinase inhibitors whether they affect cAMP-dependent proteolysis of GATA-6 or not. Among them 7 inhibitors inhibited the proteolysis at the concentration range of μM around their IC50. They are inhibit...We screened 95 kinase inhibitors whether they affect cAMP-dependent proteolysis of GATA-6 or not. Among them 7 inhibitors inhibited the proteolysis at the concentration range of μM around their IC50. They are inhibitors for protein kinase A (H-89 and 4- cyano-3-methylisoquinoline), c-Jun N-terminal kinase (SP600125), phosphatidylinositol 3-kinase (Wort- mannin and LY-294002), casein kinase II (TBB) and cyclin dependent kinase (Cdk1/2 inhibitor III). It is of interest how these kinases play roles in the degradation process of GATA-6 since this transcription factor is essential for development and tissue-specific gene expression of mammals. Inhibitors identified in this study would be helpful to study molecular mechanisms of phenomena in which GATA-6 participates.展开更多
Dimethyl curcumin,a synthetic derivative of curcumin,has good anti-cancer,anti-arthritis and anti-inflammatory properties,but underline mechanism was unclear.Activation,differentiation,proliferation and apoptosis of l...Dimethyl curcumin,a synthetic derivative of curcumin,has good anti-cancer,anti-arthritis and anti-inflammatory properties,but underline mechanism was unclear.Activation,differentiation,proliferation and apoptosis of lymphocytes have important functions during arthritis development and immune homeostasis.Over-activation of lymphocyte causes inflammatory cytokines storm,leading to an immune imbalance and tissue damages.The aim of this study was to investigate the anti-inflammation mechanism of liposome encapsulated dimethyl curcumin(Lipo-DiMC)on over-activated rat spleen lymphocytes.In this study,Lipo-DiMC was produced to increase the solubility;the primary rat spleen lymphocytes were extracted and the Concanavalin A induced cell activation model was established to study the cellular responses to Lipo-DiMC treatment in-vitro.The results showed that Lipo-DiMC suppressed Concanavalin A-induced cell proliferation,inhibited cells entering G2/M phase,and reduced the ratio of necrosis/apoptosis by manipulating intracellular p53/p21/PCNA/JNK-1 pathways.Furthermore,Lipo-DiMC increased the accumulations of intracellular matrix metalloproteinase-9 and reduced matrix metalloproteinase-9 secretion of rat spleen lymphocytes.Also,Lipo-DiMC increased intracellular caspase-3 expression and reduced Cat-C activity in activated rat spleen lymphocytes,involving in intracellular proteolysis.Our findings suggest that dimethyl curcumin effectively alleviates Concanavalin A induced over-activation of rat spleen lymphocytes,thereby inhibiting inflammatory cytokines storm and restoring cell homeostasis.展开更多
Dipeptidyl peptidase-IV(DPP-4)enzyme inhibitors are a promising category of diabetes medications.Bioactive peptides,particularly those derived from bovine milk proteins,play crucial roles in inhibiting the DPP-4 enzym...Dipeptidyl peptidase-IV(DPP-4)enzyme inhibitors are a promising category of diabetes medications.Bioactive peptides,particularly those derived from bovine milk proteins,play crucial roles in inhibiting the DPP-4 enzyme.This study describes a comprehensive strategy for DPP-4 inhibitory peptide discovery and validation that combines machine learning and virtual proteolysis techniques.Five machine learning models,including GBDT,XGBoost,LightGBM,CatBoost,and RF,were trained.Notably,LightGBM demonstrated superior performance with an AUC value of 0.92±0.01.Subsequently,LightGBM was employed to forecast the DPP-4 inhibitory potential of peptides generated through virtual proteolysis of milk proteins.Through a series of in silico screening process and in vitro experiments,GPVRGPF and HPHPHL were found to exhibit good DPP-4 inhibitory activity.Molecular docking and molecular dynamics simulations further confirmed the inhibitory mechanisms of these peptides.Through retracing the virtual proteolysis steps,it was found that GPVRGPF can be obtained fromβ-casein through enzymatic hydrolysis by chymotrypsin,while HPHPHL can be obtained fromκ-casein through enzymatic hydrolysis by stem bromelain or papain.In summary,the integration of machine learning and virtual proteolysis techniques can aid in the preliminary determination of key hydrolysis parameters and facilitate the efficient screening of bioactive peptides.展开更多
Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems.In plants,the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largel...Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems.In plants,the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown.The O-fucosyltransferase SPINDLY(SPY)and the O-GIcNAc transferase SECRET AGENT(SEC)are two prominent O-glycosylation enzymes in higher plants,with both overlapped and unique functions in plant growth and development.Unlike the critical role of O-GIcNAc in regulating the animal circadian clock,here we report that nuclear-localized SPY,but not SEC,specifically modulates the pace of the Arabidopsis circadian clock.By identifying the interactome of SPY,we identified PSEUDORESPONSE REGULATOR 5(PRR5),one of the core circadian clock components,as a new SPY-interacting protein.PRR5 can be O-fucosylated by SPY in pianta,while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5.The protein abundance of PRR5 is strongly increased in spy mutants,while the degradation rate of PRR5 is much reduced,suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation.Moreover,multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock.Collectively,our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system,via O-GIcNAcylation in mammals,but via O-fuco-sylation in higher plants.展开更多
This review gives a broad glance on the progress of recent advances on proteolysis and peptide/protein separation by chroma-tographic strategies in the past ten years, covering the main research in these areas especia...This review gives a broad glance on the progress of recent advances on proteolysis and peptide/protein separation by chroma-tographic strategies in the past ten years, covering the main research in these areas especially in China. The reviewed research focused on enzymatic micro-reactors and peptide separation in bottom-up approaches, and protein and peptide separation in top-down approaches. The new enzymatic micro-reactor is able to accelerate proteolytic reaction rate from conventionally a couple of hours to a few seconds, and the multiple dimensional chromatographic-separation with various models or arrays could sufficiently separate the proteomic mixture. These advances have significantly promoted the research of protein/peptide separation and identification in proteomics.展开更多
The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on ...The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.展开更多
Liquid secondary ion mass spectrometry(LSIMS) was used to probe the proteolysis of melittin by trypsin and pepsin. The results showed that LSIMS is good for monitoring proteolytic reactions. It can not only identify...Liquid secondary ion mass spectrometry(LSIMS) was used to probe the proteolysis of melittin by trypsin and pepsin. The results showed that LSIMS is good for monitoring proteolytic reactions. It can not only identify the proteolytic products of proteins, but also be used to research the dynamics of proteolytic reactions. The proteolysis of melittin by trypsin gave seven main peptide fragments: 1, AA 1 AA 7 ; 2, AA 1 AA 21 ;3, AA 1 AA 22 ; 4, AA 1 AA 23 ; 5, AA 1 AA 24 ; 6, AA 8 AA 21 ; 7, AA 8 AA 22 . The proteolysis of melittin with pepsin gave thirteen peptide fragments: 1, AA 1 AA 3; 2, AA 4 AA 6;3, AA 1 AA 4;4, AA 1 AA 5 ;5, AA 1 AA 6 ;6, AA 1 AA 8;7, AA 7 AA 13 ;8, AA 10 AA 13 ;9, AA 14 AA 16 ;10, AA 14 AA 26 ;11, AA 15 AA 26 ;12, AA 17 AA 26 ;13, AA 20 AA 26 .展开更多
Over the past 20 years,great efforts have been invested in developing site-specific approaches to protein modification to dissect protein functions directly and accurately.Here,we report a proximitytriggered group tra...Over the past 20 years,great efforts have been invested in developing site-specific approaches to protein modification to dissect protein functions directly and accurately.Here,we report a proximitytriggered group transfer strategy from a sulfonium warhead to a Cysteine(Cys)residue of the target protein.With a guiding ligand,cargoes could be transferred selectively from a sulfonium center onto the Cys residue in the vicinity of their binding interface.The successful thalidomide transfer of sulfonium 1-X could be applied intracellularly for epidermal growth factor receptor degradation,highlighting the potential of group transfer strategy as a suite of chemical biology studies,including cell imaging,protein profiling,and protein degradation by simply employing different transferrable groups.展开更多
γ-Secretase,called“the proteasome of the membrane,”is a membrane-embedded protease complex that cleaves 150+peptide substrates with central roles in biology and medicine,including amyloid precursor protein and the ...γ-Secretase,called“the proteasome of the membrane,”is a membrane-embedded protease complex that cleaves 150+peptide substrates with central roles in biology and medicine,including amyloid precursor protein and the Notch family of cell-surface receptors.Mutations inγ-secretase and amyloid precursor protein lead to early-onset familial Alzheimer’s disease.γ-Secretase has thus served as a critical drug target for treating familial Alzheimer’s disease and the more common late-onset Alzheimer’s disease as well.However,critical gaps remain in understanding the mechanisms of processive proteolysis of substrates,the effects of familial Alzheimer’s disease mutations,and allosteric modulation of substrate cleavage byγ-secretase.In this review,we focus on recent studies of structural dynamic mechanisms ofγ-secretase.Different mechanisms,including the“Fit-Stay-Trim,”“Sliding-Unwinding,”and“Tilting-Unwinding,”have been proposed for substrate proteolysis of amyloid precursor protein byγ-secretase based on all-atom molecular dynamics simulations.While an incorrect registry of the Notch1 substrate was identified in the cryo-electron microscopy structure of Notch1-boundγ-secretase,molecular dynamics simulations on a resolved model of Notch1-boundγ-secretase that was reconstructed using the amyloid precursor protein-boundγ-secretase as a template successfully capturedγ-secretase activation for proper cleavages of both wildtype and mutant Notch,being consistent with biochemical experimental findings.The approach could be potentially applied to decipher the processing mechanisms of various substrates byγ-secretase.In addition,controversy over the effects of familial Alzheimer’s disease mutations,particularly the issue of whether they stabilize or destabilizeγ-secretase-substrate complexes,is discussed.Finally,an outlook is provided for future studies ofγ-secretase,including pathways of substrate binding and product release,effects of modulators on familial Alzheimer’s disease mutations of theγ-secretase-substrate complexes.Comprehensive understanding of the functional mechanisms ofγ-secretase will greatly facilitate the rational design of effective drug molecules for treating familial Alzheimer’s disease and perhaps Alzheimer’s disease in general.展开更多
F-box protein is an expanding family member of eukaryotic protein characterized by an F-box motif which has specificity of substrate recognition in the ubiquitin-mediated proteolysis.These proteins have been proved to...F-box protein is an expanding family member of eukaryotic protein characterized by an F-box motif which has specificity of substrate recognition in the ubiquitin-mediated proteolysis.These proteins have been proved to be critical for many physiological processes,such as cell-cycle transition,signal transduction,gene transcription,male sterility,programmed cell death （PCD） and so on.This paper mainly introduces the biological functions of the known F-box proteins and the analysis of F-box gene phylogeny.展开更多
Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light...Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for antophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.展开更多
Nitrogenous emissions from ruminant livestock production are of increasing public concern and, together with methane, contribute to environmental pollution. The main cause of nitrogen-(N)-containing emissions is the i...Nitrogenous emissions from ruminant livestock production are of increasing public concern and, together with methane, contribute to environmental pollution. The main cause of nitrogen-(N)-containing emissions is the inadequate provision of N to ruminants, leading to an excess of ammonia in the rumen, which is subsequently excreted. Depending on the size and molecular structure, various bacterial, protozoal and fungal species are involved in the ruminal breakdown of nitrogenous compounds(NC). Decelerating ruminal NC degradation by controlling the abundance and activity of proteolytic and deaminating microorganisms, but without reducing cellulolytic processes, is a promising strategy to decrease N emissions along with increasing N utilization by ruminants. Different dietary options, including among others the treatment of feedstuffs with heat or the application of diverse feed additives, as well as vaccination against rumen microorganisms or their enzymes have been evaluated. Thereby, reduced productions of microbial metabolites, e.g. ammonia, and increased microbial N flows give evidence for an improved N retention. However, linkage between these findings and alterations in the rumen microbiota composition, particularly NC-degrading microbes, remains sparse and contradictory findings confound the exact evaluation of these manipulating strategies, thus emphasizing the need for comprehensive research. The demand for increased sustainability in ruminant livestock production requests to apply attention to microbial N utilization efficiency and this will require a better understanding of underlying metabolic processes as well as composition and interactions of ruminal NC-degrading microorganisms.展开更多
Post-translational degradation of protein plays an important role in cell life. We employed chimeric molecules （dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]） to facilitate androgen ...Post-translational degradation of protein plays an important role in cell life. We employed chimeric molecules （dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]） to facilitate androgen receptor （AR） degradation via the ubiquitin-proteasome pathway （UPP） and to investigate the role of AR in cell proliferation and viability in androgen-sensitive prostate cancer cells. Western blot analysis and immunohistochemistry were applied to analyse AR levels in LNCaP cells after DHT-PROTAC treatment. Cell counting and the 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyl tetrazolium bromide （MTT） cell viability assay were used to evaluate cell proliferation and viability after AR elimination in both LNCaP and PC-3 cells. AR was tagged for elimination via the UPP by DHT-PROTAC, and this could be blocked by proteasome inhibitors. Degradation of AR depended on DHT-PROTAC concentration, and either DHT or an ALAPYIP-（arg）8 peptide could compete with DHT-PROTAC. Inhibition of cell proliferation and decreased viability were observed in LNCaP cells, but not in PC-3 or 786-0 cells after DHT-PROTAC treatment. These data indicate that AR elimination is facilitated via the UPP by DHT-PROTAC, and that the growth of LNCaP cells is repressed after AR degradation.展开更多
Aging is a popular method used by meat industry for improving the sensory attributes of meat.Despite the advent of many novel technologies,aging has not lost its charm and is still widely used commercially as a post-m...Aging is a popular method used by meat industry for improving the sensory attributes of meat.Despite the advent of many novel technologies,aging has not lost its charm and is still widely used commercially as a post-mortem intervention for tenderization.Aging improves the tenderness of meat through disruption of the muscle structure by intracellular proteolytic systems.Muscles undergo various molecular changes that cause proteolysis of key myofibrillar and cytoskeletal proteins,disrupting the overall integrity of muscle cells.Although several endogenous proteolytic systems are capable of post-mortem proteolysis,a great body of scientific evidence supports a major role for the calpain system.Calpains are intracellular calcium-dependent cysteine proteases found in most eukaryotes.At least three calpains(μ-and m-calpains and calpain 3)and calpastatin,their specific endogenous inhibitor,are found in muscle.They are known to be involved in the proteolysis of functionally relevant structural proteins such as the myofibrillar proteins and cytoskeletal anchorage complexes.These ubiquitous proteases are also present in mitochondria and play important roles in a variety of pathophysiological conditions including apoptotic and necrotic cell death phenomena.This review discusses the role and contribution of the calpain system and the factors that influence calpain activity during aging.展开更多
Objective.To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF)on Ca 2+ -CaM dependent phosphorylation of myosin light chains(CDPM)of smooth muscle.Method...Objective.To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF)on Ca 2+ -CaM dependent phosphorylation of myosin light chains(CDPM)of smooth muscle.Methods.Proteolysis of myosin light chain kinase(MLCK)by trypsin was used to obtain the con-stitutively active fragment of MLCK;Western blot was applied to demonstrate the homogeneity of typsin-digested MLCK fragments and intact MLCK.The phosphorylation of myosin light chains was detected us-ing SDS-PAGE and Scoin Image Software,and myosin Mg 2+ -ATPase activities were measured using spec-trophotometry to observe the characteristics of MLCKF in phosphorylating myosin light chains and stimu-lating myosin Mg 2+ -ATPase activities compared with intact MLCK.Results.Our assay demonstrated that MLCKF had a specific activity on CDPM.We also found that the extent of CDPM by MLCKF was greater than that by intact MLCK at different concentrations,differ-ent incubation time and different incubation temperatures.CDPM by MLCKF was also less influenced by the change of ionic strength of KCl than CDPM by intact MLCK.Mg 2+ -ATPase activities of phosphorylat-ed myosin light chains by MLCKF were higher than those by MLCK at different concentrations and dif-ferent reaction time.These differences were statistically significant ( P<0.01, P<0.05).Conclusion.MLCKF not only possessed a specific activity on CDPM but also was more efficient than MLCK in phosphorylating myosin light chains and stimulating myosin Mg 2+ -ATPase activities.Our results suggested that MLCKF possibly plays a certain role in smooth muscle contraction-relaxation cycle.展开更多
We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV inserti...We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In addition,the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes.Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish.We propose that prtV plays an important role in the pathogenesis of V.anguillarum.展开更多
基金Supported by Instituto de Salud Carlos III(ISCIII)through the FIS project PI12/0056,co-funded by FEDER from Regional Development European Funds(European Union)
文摘To compare liver proteolysis and proteasome activation in steatotic liver grafts conserved in University of Wisconsin(UW)and Institut Georges Lopez-1(IGL-1)solutions.METHODSFatty liver grafts from male obese Zücker rats were conserved in UW and IGL-1 solutions for 24 h at 4°Cand subjected to“ex vivo”normo-thermic perfusion(2 h;37°C).Liver proteolysis in tissue specimens and perfusate was measured by reverse-phase high performance liquid chromatography.Total free amino acid release was correlated with the activation of the ubiquitin proteasome system(UPS:measured as chymotryptic-like activity and 20S and 19S proteasome),the prevention of liver injury(transaminases),mitochondrial injury(confocal microscopy)and inflammation markers(TNF 1 alpha,high mobility group box-1(HGMB-1)and PPAR gamma),and liver apoptosis(TUNEL assay,cytochrome c and caspase 3).RESULTSProfiles of free AA(alanine,proline,leucine,isoleucine,methionine,lysine,ornithine,and threonine,among others)were similar for tissue and reperfusion effluent.In all cases,the IGL-1 solution showed a significantly higher prevention of proteolysis than UW(P<0.05)after cold ischemia reperfusion.Livers conserved in IGL-1 presented more effective prevention of ATP-breakdown and more inhibition of UPS activity(measured as chymotryptic-like activity).In addition,the prevention of liver proteolysis and UPS activation correlated with the prevention of liver injury(AST/ALT)and mitochondrial damage(revealed by confocal microscopy findings)as well as with the prevention of inflammatory markers(TNF1alpha and HMGB)after reperfusion.In addition,the liver grafts preserved in IGL-1 showed a significant decrease in liver apoptosis,as shown by TUNEL assay and the reduction of cytochrome c,caspase 3 and P62 levels.CONCLUSIONOur comparison of these two preservation solutions suggests that IGL-1 helps to prevent ATP breakdown more effectively than UW and subsequently achieves a higher UPS inhibition and reduced liver proteolysis.
基金This work was supported by the National Natural Science Foundation of China,No.81870975(to SLZ)the Nantong Science and Technology Innovation Program,China,No.JC2019028(to XMY).
文摘Neurological diseases such as stroke,Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease are among the intractable diseases for which appropriate drugs and treatments are lacking.Proteolysis targeting chimera(PROTAC)technology is a novel strategy to solve this problem.PROTAC technology uses the ubiquitin-protease system to eliminate mutated,denatured,and harmful proteins in cells.It can be reused,and utilizes the protein destruction mechanism of the cells,thus making up for the deficiencies of traditional protein degradation methods.It can effectively target and degrade proteins,including proteins that are difficult to identify and bind.Therefore,it has extremely important implications for drug development and the treatment of neurological diseases.At present,the targeted degradation of mutant BTK,mHTT,Tau,EGFR,and other proteins using PROTAC technology is gaining attention.It is expected that corresponding treatment of nervous system diseases can be achieved.This review first focuses on the recent developments in PROTAC technology in terms of protein degradation,drug production,and treatment of central nervous system diseases,and then discusses its limitations.This review will provide a brief overview of the recent application of PROTAC technology in the treatment of central nervous system diseases.
基金We thank Dr Yue Jun from Institute of Genetics at Fudan University for kind help and advice on 2-DE technique,Hasan Koc from the proteomic center at the Pennsylvania State University for help with protein identification with MS and Qing Zhang for assistance with the normalization of the microarray data.This work was supported by the Youth Exploration Funding of School of Life Sciences at Fudan Universityin part by a grant to H.M.from the US National Science Foundation(MCB-0092075).
文摘The ASK1 （ARABIDOPSIS SKP1-LIKE） protein is a critical component of the SCF （Skpl-Cullin-F box protein） ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome. To investigate proteins that are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers, we compared the proteomes of the Arabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis （2-DE）. Ten protein spots with higher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to further mass spectrometry （MS） analysis. The results showed that they were proteins involved in photomorphogenesis, circadian oscillation, post-translation process, stress-responses and cell expansion or elongation, suggesting that those processes were affected in the ask1 mutant. The transcript levels of these genes were also compared based on the Affymetrix gene chip microarray data. No significant difference was observed for most of the genes, suggesting that the proteins with elevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASK 1-mediated proteolysis pathway. These results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes and also demonstrate the importance and necessity of studying protein levels with respect to gene functions.
文摘The amino acid biosynthesis and proteolytic system of Lactobacillus bulgaricus (L.Bulgaricus ) is important for its growth in niche-specific environments, as well as for flavour formation in the food industry. Comparative analyses of 4 completed sequences of the L.Bulgaricus strain genome on a genomic scale revealed that genes involved in amino acids synthesis were undergoing reductive evolution. However, the selected industrial strains, namely, L.Bulgaricus 2038 and L.Bulgaricus ND02, retained more complete genes in the amino acid synthesis and proteolytic system category than the laboratory strains, and have some unique genes and pathways for obtaining amino acids that enable these bacteria to adapt to their various environmental niches.
文摘We screened 95 kinase inhibitors whether they affect cAMP-dependent proteolysis of GATA-6 or not. Among them 7 inhibitors inhibited the proteolysis at the concentration range of μM around their IC50. They are inhibitors for protein kinase A (H-89 and 4- cyano-3-methylisoquinoline), c-Jun N-terminal kinase (SP600125), phosphatidylinositol 3-kinase (Wort- mannin and LY-294002), casein kinase II (TBB) and cyclin dependent kinase (Cdk1/2 inhibitor III). It is of interest how these kinases play roles in the degradation process of GATA-6 since this transcription factor is essential for development and tissue-specific gene expression of mammals. Inhibitors identified in this study would be helpful to study molecular mechanisms of phenomena in which GATA-6 participates.
基金This work is supported by the Start-up Research Laboratory for Over-sea Talent Fund,China(No.Z391405)to ProfXiaoying Zhou and the Changzhou University Undergraduate Innovation and Entrepreneurship Fund,China(No.2019-02-C-96)to Ya-Ming Guo.
文摘Dimethyl curcumin,a synthetic derivative of curcumin,has good anti-cancer,anti-arthritis and anti-inflammatory properties,but underline mechanism was unclear.Activation,differentiation,proliferation and apoptosis of lymphocytes have important functions during arthritis development and immune homeostasis.Over-activation of lymphocyte causes inflammatory cytokines storm,leading to an immune imbalance and tissue damages.The aim of this study was to investigate the anti-inflammation mechanism of liposome encapsulated dimethyl curcumin(Lipo-DiMC)on over-activated rat spleen lymphocytes.In this study,Lipo-DiMC was produced to increase the solubility;the primary rat spleen lymphocytes were extracted and the Concanavalin A induced cell activation model was established to study the cellular responses to Lipo-DiMC treatment in-vitro.The results showed that Lipo-DiMC suppressed Concanavalin A-induced cell proliferation,inhibited cells entering G2/M phase,and reduced the ratio of necrosis/apoptosis by manipulating intracellular p53/p21/PCNA/JNK-1 pathways.Furthermore,Lipo-DiMC increased the accumulations of intracellular matrix metalloproteinase-9 and reduced matrix metalloproteinase-9 secretion of rat spleen lymphocytes.Also,Lipo-DiMC increased intracellular caspase-3 expression and reduced Cat-C activity in activated rat spleen lymphocytes,involving in intracellular proteolysis.Our findings suggest that dimethyl curcumin effectively alleviates Concanavalin A induced over-activation of rat spleen lymphocytes,thereby inhibiting inflammatory cytokines storm and restoring cell homeostasis.
基金supported by the National Key Research and Development Project(2018YFE0206300-02).
文摘Dipeptidyl peptidase-IV(DPP-4)enzyme inhibitors are a promising category of diabetes medications.Bioactive peptides,particularly those derived from bovine milk proteins,play crucial roles in inhibiting the DPP-4 enzyme.This study describes a comprehensive strategy for DPP-4 inhibitory peptide discovery and validation that combines machine learning and virtual proteolysis techniques.Five machine learning models,including GBDT,XGBoost,LightGBM,CatBoost,and RF,were trained.Notably,LightGBM demonstrated superior performance with an AUC value of 0.92±0.01.Subsequently,LightGBM was employed to forecast the DPP-4 inhibitory potential of peptides generated through virtual proteolysis of milk proteins.Through a series of in silico screening process and in vitro experiments,GPVRGPF and HPHPHL were found to exhibit good DPP-4 inhibitory activity.Molecular docking and molecular dynamics simulations further confirmed the inhibitory mechanisms of these peptides.Through retracing the virtual proteolysis steps,it was found that GPVRGPF can be obtained fromβ-casein through enzymatic hydrolysis by chymotrypsin,while HPHPHL can be obtained fromκ-casein through enzymatic hydrolysis by stem bromelain or papain.In summary,the integration of machine learning and virtual proteolysis techniques can aid in the preliminary determination of key hydrolysis parameters and facilitate the efficient screening of bioactive peptides.
基金suppoded by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB27030206)National Natural Science Foundation of China(No.31770287)+1 种基金National Key Research and Development Program of China(2016YFD0100600)to L.W.the National Institutes of Health(R01 GM100051)to T.P.S.
文摘Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems.In plants,the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown.The O-fucosyltransferase SPINDLY(SPY)and the O-GIcNAc transferase SECRET AGENT(SEC)are two prominent O-glycosylation enzymes in higher plants,with both overlapped and unique functions in plant growth and development.Unlike the critical role of O-GIcNAc in regulating the animal circadian clock,here we report that nuclear-localized SPY,but not SEC,specifically modulates the pace of the Arabidopsis circadian clock.By identifying the interactome of SPY,we identified PSEUDORESPONSE REGULATOR 5(PRR5),one of the core circadian clock components,as a new SPY-interacting protein.PRR5 can be O-fucosylated by SPY in pianta,while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5.The protein abundance of PRR5 is strongly increased in spy mutants,while the degradation rate of PRR5 is much reduced,suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation.Moreover,multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock.Collectively,our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system,via O-GIcNAcylation in mammals,but via O-fuco-sylation in higher plants.
基金support from the Major State Basie Research Development Program (Grant No. 2007CB914100)the National Natural Science Foundation of China (Grant Nos. 20875016 & 20735005)Shanghai Projects (Grant Nos. 08DZ2293601 & B109)
文摘This review gives a broad glance on the progress of recent advances on proteolysis and peptide/protein separation by chroma-tographic strategies in the past ten years, covering the main research in these areas especially in China. The reviewed research focused on enzymatic micro-reactors and peptide separation in bottom-up approaches, and protein and peptide separation in top-down approaches. The new enzymatic micro-reactor is able to accelerate proteolytic reaction rate from conventionally a couple of hours to a few seconds, and the multiple dimensional chromatographic-separation with various models or arrays could sufficiently separate the proteomic mixture. These advances have significantly promoted the research of protein/peptide separation and identification in proteomics.
文摘The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.
文摘Liquid secondary ion mass spectrometry(LSIMS) was used to probe the proteolysis of melittin by trypsin and pepsin. The results showed that LSIMS is good for monitoring proteolytic reactions. It can not only identify the proteolytic products of proteins, but also be used to research the dynamics of proteolytic reactions. The proteolysis of melittin by trypsin gave seven main peptide fragments: 1, AA 1 AA 7 ; 2, AA 1 AA 21 ;3, AA 1 AA 22 ; 4, AA 1 AA 23 ; 5, AA 1 AA 24 ; 6, AA 8 AA 21 ; 7, AA 8 AA 22 . The proteolysis of melittin with pepsin gave thirteen peptide fragments: 1, AA 1 AA 3; 2, AA 4 AA 6;3, AA 1 AA 4;4, AA 1 AA 5 ;5, AA 1 AA 6 ;6, AA 1 AA 8;7, AA 7 AA 13 ;8, AA 10 AA 13 ;9, AA 14 AA 16 ;10, AA 14 AA 26 ;11, AA 15 AA 26 ;12, AA 17 AA 26 ;13, AA 20 AA 26 .
基金Weare grateful for the financial support from the Natural Science Foundation of China(grant nos.21778009,21977010,and 22007033)National Key Research and Development Program“Synthetic Biology”Key Special Project of China(grant no.2018YFA0902504)+3 种基金China Postdoctoral Science Foundation(grant no.2021M690220)the Natural Science Foundation of Guangdong Province(grant nos.2020A1515010522,2020A1515010766,2019A1515110487,and 2019A151-5111184)the Foundation for Basic and Applied Research of Guangdong Province(grant no.2019A1515110489)and the Shenzhen Science and Technology Innovation Committee(grant nos.JCYJ20180507181527112,JCYJ-201805081522131455,and JCYJ20170817172023838).
文摘Over the past 20 years,great efforts have been invested in developing site-specific approaches to protein modification to dissect protein functions directly and accurately.Here,we report a proximitytriggered group transfer strategy from a sulfonium warhead to a Cysteine(Cys)residue of the target protein.With a guiding ligand,cargoes could be transferred selectively from a sulfonium center onto the Cys residue in the vicinity of their binding interface.The successful thalidomide transfer of sulfonium 1-X could be applied intracellularly for epidermal growth factor receptor degradation,highlighting the potential of group transfer strategy as a suite of chemical biology studies,including cell imaging,protein profiling,and protein degradation by simply employing different transferrable groups.
基金supported in part by Award 2121063 from National Science Foundation(to YM)AG66986 from the National Institutes of Health(to MSW).
文摘γ-Secretase,called“the proteasome of the membrane,”is a membrane-embedded protease complex that cleaves 150+peptide substrates with central roles in biology and medicine,including amyloid precursor protein and the Notch family of cell-surface receptors.Mutations inγ-secretase and amyloid precursor protein lead to early-onset familial Alzheimer’s disease.γ-Secretase has thus served as a critical drug target for treating familial Alzheimer’s disease and the more common late-onset Alzheimer’s disease as well.However,critical gaps remain in understanding the mechanisms of processive proteolysis of substrates,the effects of familial Alzheimer’s disease mutations,and allosteric modulation of substrate cleavage byγ-secretase.In this review,we focus on recent studies of structural dynamic mechanisms ofγ-secretase.Different mechanisms,including the“Fit-Stay-Trim,”“Sliding-Unwinding,”and“Tilting-Unwinding,”have been proposed for substrate proteolysis of amyloid precursor protein byγ-secretase based on all-atom molecular dynamics simulations.While an incorrect registry of the Notch1 substrate was identified in the cryo-electron microscopy structure of Notch1-boundγ-secretase,molecular dynamics simulations on a resolved model of Notch1-boundγ-secretase that was reconstructed using the amyloid precursor protein-boundγ-secretase as a template successfully capturedγ-secretase activation for proper cleavages of both wildtype and mutant Notch,being consistent with biochemical experimental findings.The approach could be potentially applied to decipher the processing mechanisms of various substrates byγ-secretase.In addition,controversy over the effects of familial Alzheimer’s disease mutations,particularly the issue of whether they stabilize or destabilizeγ-secretase-substrate complexes,is discussed.Finally,an outlook is provided for future studies ofγ-secretase,including pathways of substrate binding and product release,effects of modulators on familial Alzheimer’s disease mutations of theγ-secretase-substrate complexes.Comprehensive understanding of the functional mechanisms ofγ-secretase will greatly facilitate the rational design of effective drug molecules for treating familial Alzheimer’s disease and perhaps Alzheimer’s disease in general.
基金Supported by the Cloning and Genetics Analysis of Cold-Induced Genes in PA64S, Natural Science Foundation of Hunan Province(09JJ3044)
文摘F-box protein is an expanding family member of eukaryotic protein characterized by an F-box motif which has specificity of substrate recognition in the ubiquitin-mediated proteolysis.These proteins have been proved to be critical for many physiological processes,such as cell-cycle transition,signal transduction,gene transcription,male sterility,programmed cell death （PCD） and so on.This paper mainly introduces the biological functions of the known F-box proteins and the analysis of F-box gene phylogeny.
文摘Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for antophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.
基金supported by the"Deutsche Forschungsgemeinschaft"(DFG,German Research Foundation,SU124/33–1)
文摘Nitrogenous emissions from ruminant livestock production are of increasing public concern and, together with methane, contribute to environmental pollution. The main cause of nitrogen-(N)-containing emissions is the inadequate provision of N to ruminants, leading to an excess of ammonia in the rumen, which is subsequently excreted. Depending on the size and molecular structure, various bacterial, protozoal and fungal species are involved in the ruminal breakdown of nitrogenous compounds(NC). Decelerating ruminal NC degradation by controlling the abundance and activity of proteolytic and deaminating microorganisms, but without reducing cellulolytic processes, is a promising strategy to decrease N emissions along with increasing N utilization by ruminants. Different dietary options, including among others the treatment of feedstuffs with heat or the application of diverse feed additives, as well as vaccination against rumen microorganisms or their enzymes have been evaluated. Thereby, reduced productions of microbial metabolites, e.g. ammonia, and increased microbial N flows give evidence for an improved N retention. However, linkage between these findings and alterations in the rumen microbiota composition, particularly NC-degrading microbes, remains sparse and contradictory findings confound the exact evaluation of these manipulating strategies, thus emphasizing the need for comprehensive research. The demand for increased sustainability in ruminant livestock production requests to apply attention to microbial N utilization efficiency and this will require a better understanding of underlying metabolic processes as well as composition and interactions of ruminal NC-degrading microorganisms.
基金We are grateful to the National Natural Science Foundation of China （Grant No. 30600618） and the Science and Technology Commission of Shanghai Municipality （Grant No. 07QA14037） for their financial support. We thank Professor Qi-Xiang Guo and Liang Ding for providing the DHT-PROTAC.
文摘Post-translational degradation of protein plays an important role in cell life. We employed chimeric molecules （dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]） to facilitate androgen receptor （AR） degradation via the ubiquitin-proteasome pathway （UPP） and to investigate the role of AR in cell proliferation and viability in androgen-sensitive prostate cancer cells. Western blot analysis and immunohistochemistry were applied to analyse AR levels in LNCaP cells after DHT-PROTAC treatment. Cell counting and the 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyl tetrazolium bromide （MTT） cell viability assay were used to evaluate cell proliferation and viability after AR elimination in both LNCaP and PC-3 cells. AR was tagged for elimination via the UPP by DHT-PROTAC, and this could be blocked by proteasome inhibitors. Degradation of AR depended on DHT-PROTAC concentration, and either DHT or an ALAPYIP-（arg）8 peptide could compete with DHT-PROTAC. Inhibition of cell proliferation and decreased viability were observed in LNCaP cells, but not in PC-3 or 786-0 cells after DHT-PROTAC treatment. These data indicate that AR elimination is facilitated via the UPP by DHT-PROTAC, and that the growth of LNCaP cells is repressed after AR degradation.
文摘Aging is a popular method used by meat industry for improving the sensory attributes of meat.Despite the advent of many novel technologies,aging has not lost its charm and is still widely used commercially as a post-mortem intervention for tenderization.Aging improves the tenderness of meat through disruption of the muscle structure by intracellular proteolytic systems.Muscles undergo various molecular changes that cause proteolysis of key myofibrillar and cytoskeletal proteins,disrupting the overall integrity of muscle cells.Although several endogenous proteolytic systems are capable of post-mortem proteolysis,a great body of scientific evidence supports a major role for the calpain system.Calpains are intracellular calcium-dependent cysteine proteases found in most eukaryotes.At least three calpains(μ-and m-calpains and calpain 3)and calpastatin,their specific endogenous inhibitor,are found in muscle.They are known to be involved in the proteolysis of functionally relevant structural proteins such as the myofibrillar proteins and cytoskeletal anchorage complexes.These ubiquitous proteases are also present in mitochondria and play important roles in a variety of pathophysiological conditions including apoptotic and necrotic cell death phenomena.This review discusses the role and contribution of the calpain system and the factors that influence calpain activity during aging.
文摘Objective.To study the exact effects and characteristics of a constitutively active myosin light chain kinase fragment (MLCKF)on Ca 2+ -CaM dependent phosphorylation of myosin light chains(CDPM)of smooth muscle.Methods.Proteolysis of myosin light chain kinase(MLCK)by trypsin was used to obtain the con-stitutively active fragment of MLCK;Western blot was applied to demonstrate the homogeneity of typsin-digested MLCK fragments and intact MLCK.The phosphorylation of myosin light chains was detected us-ing SDS-PAGE and Scoin Image Software,and myosin Mg 2+ -ATPase activities were measured using spec-trophotometry to observe the characteristics of MLCKF in phosphorylating myosin light chains and stimu-lating myosin Mg 2+ -ATPase activities compared with intact MLCK.Results.Our assay demonstrated that MLCKF had a specific activity on CDPM.We also found that the extent of CDPM by MLCKF was greater than that by intact MLCK at different concentrations,differ-ent incubation time and different incubation temperatures.CDPM by MLCKF was also less influenced by the change of ionic strength of KCl than CDPM by intact MLCK.Mg 2+ -ATPase activities of phosphorylat-ed myosin light chains by MLCKF were higher than those by MLCK at different concentrations and dif-ferent reaction time.These differences were statistically significant ( P<0.01, P<0.05).Conclusion.MLCKF not only possessed a specific activity on CDPM but also was more efficient than MLCK in phosphorylating myosin light chains and stimulating myosin Mg 2+ -ATPase activities.Our results suggested that MLCKF possibly plays a certain role in smooth muscle contraction-relaxation cycle.
基金Supported by the National Basic Research Program of China (973 Program) (No. 2006CB101803)the National Natural Science Foundation of China (No. 30871935)the National High Technology Research and Development Program (863 Program) (No. 2003AA622070)
文摘We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In addition,the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes.Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish.We propose that prtV plays an important role in the pathogenesis of V.anguillarum.
