采用PCR-SSCP方法对58头草原红牛钙激活中性蛋白酶3基因(CAPN3)的单核苷酸多态性进行研究分析,发现其外显子1存在三种基因型AA、AB和BB,测序发现,在外显子1的124位存在碱基突变,由原来的A突变为G,氨基酸水平由天冬氨酸Asp变为天冬酰胺A...采用PCR-SSCP方法对58头草原红牛钙激活中性蛋白酶3基因(CAPN3)的单核苷酸多态性进行研究分析,发现其外显子1存在三种基因型AA、AB和BB,测序发现,在外显子1的124位存在碱基突变,由原来的A突变为G,氨基酸水平由天冬氨酸Asp变为天冬酰胺Asn,此SNP位点在此前的研究中未见报道。经遗传多样性分析表明,该位点等位基因A占优势地位,且三种基因型分布符合Hardy-W e inberg平衡。通过显著性差异检验发现,CAPN3基因此位点的多态性对该牛群的屠宰率、净肉率和大理石花纹这三个性状有显著影响(P<0.05),这将为CAPN3作为牛肉肉质性状候选基因提供进一步依据。展开更多
为了揭示脂肪型的藏猪和瘦肉型的杜洛克猪肌肉生长及嫩度相关基因的表达差异,采用QRT-PCR方法检测了两猪种背最长肌中钙蛋白酶3(CAPN3)基因在180日龄的表达差异,分析其表达与肌纤维面积(CSA)、肌肉剪切力的相关性。结果表明,180日龄藏猪...为了揭示脂肪型的藏猪和瘦肉型的杜洛克猪肌肉生长及嫩度相关基因的表达差异,采用QRT-PCR方法检测了两猪种背最长肌中钙蛋白酶3(CAPN3)基因在180日龄的表达差异,分析其表达与肌纤维面积(CSA)、肌肉剪切力的相关性。结果表明,180日龄藏猪CAPN3 m RNA表达量显著高于杜洛克猪(P<0.05)。相关性分析结果显示,CAPN3 m RNA表达量与肌纤维面积、剪切力均呈显著负相关(P<0.05)。以上结果初步揭示了两猪种在180日龄CAPN3基因表达的品种差异,为深入研究猪肌肉生长及剪切力的调控机制提供了基础数据。展开更多
目的对一个2A型肢带型肌营养不良(limb-girdle muscular dystrophy type 2A)家系进行CAPN3基因的致病突变分析。方法收集先证者及家系成员的外周血,提取DNA,应用全外显子测序技术对先证者进行致病基因检测,然后用Sanger测序技术对先证...目的对一个2A型肢带型肌营养不良(limb-girdle muscular dystrophy type 2A)家系进行CAPN3基因的致病突变分析。方法收集先证者及家系成员的外周血,提取DNA,应用全外显子测序技术对先证者进行致病基因检测,然后用Sanger测序技术对先证者家系成员进行突变位点的验证。结果全外显子测序发现先证者携带CAPN3基因c.1194-9A>G和c.1437C>T(p.ser479=)的复合杂合突变。Sanger测序验证先证者母亲为CAPN3基因c.1194-9A>G变异携带者。家系中其他患者均存在相同的复合杂合突变,其未发病的姐姐和女儿为CAPN3基因c.1437C>T(p.ser479=)变异携带者,先证者的女婿未检测到上述位点变异。结论CAPN3基因c.1194-9A>G和c.1437C>T(p.ser479=)的复合杂合突变为该家系的致病原因。展开更多
The nucleolus,as the‘nucleus of the nucleus’,is a prominent subcellular organelle in a eukaryocyte.The nucleolus serves as the centre for ribosome biogenesis,as well as an important site for cell-cycle regulation,ce...The nucleolus,as the‘nucleus of the nucleus’,is a prominent subcellular organelle in a eukaryocyte.The nucleolus serves as the centre for ribosome biogenesis,as well as an important site for cell-cycle regulation,cellular senescence,and stress response.The protein composition of the nucleolus changes dynamically through protein turnover to meet the needs of cellular activities or stress responses.Recent studies have identified a nucleolus-localized protein degradation pathway in zebrafish and humans,namely the Def-CAPN3 pathway,which is essential to ribosome production and cell-cycle progression,by controlling the turnover of multiple substrates(e.g.,ribosomal small-subunit[SSU]processome component Mpp10,transcription factor p53,check-point proteins Chk1 and Wee1).This pathway relies on the Ca2þ-dependent cysteine proteinase CAPN3 and is independent of the ubiquitin-mediated proteasome pathway.CAPN3 is recruited by nucleolar protein Def from cytoplasm to nucleolus,where it proteolyzes its substrates which harbor a CAPN3 recognition-motif.Def depletion leads to the exclusion of CAPN3 and accumulation of p53,Wee1,Chk1,and Mpp10 in the nucleolus that result in cell-cycle arrest and rRNA processing abnormality.Here,we summarize the discovery of the Def-CAPN3 pathway and propose its biological role in cell-cycle control and ribosome biogenesis.展开更多
Mutations in calcium-dependent papain-like protease CALPAIN3(CAPN3)cause Limb-Girdle Muscular Dystrophy Recessive Type 1(LGMDR1),the most common limb-girdle muscular dystrophy in humans.In addition to progressive musc...Mutations in calcium-dependent papain-like protease CALPAIN3(CAPN3)cause Limb-Girdle Muscular Dystrophy Recessive Type 1(LGMDR1),the most common limb-girdle muscular dystrophy in humans.In addition to progressive muscle weakness,persistent inflammatory infiltration is also a feature of LGMDR1.Despite the underlying mechanism remaining poorly understood,we consider that it may relate to the newly defined role of CAPN3/Capn3b in the nucleolus.Here,we report that the loss of function of zebrafish capn3b,the counterpart of human CAPN3,induces an autoimmune response akin to that in LGMDR1 patients.capn3b mutant larvae are more susceptible to Listeria monocytogenes injection,characterized by recruiting more macrophages.Under germ-free conditions,transcriptome analysis of the capn3b mutant muscle reveals a significant upregulation of the chemokine-production-related genes.Coincidently,more neutrophils are recruited to the injury site imposed by either muscle stabbing or tail fin amputation.Nucleolar proteomic analysis and enzymatic assays reveal NKAP,an activating factor of the NF-k B pathway,to be a target of CAPN3.We conclude that the accumulation of Nkap and other factors in the capn3b mutant may be involved in the over-activation of innate immunity.Our studies indicate that the zebrafish capn3b mutant is a powerful model for studying the immunity-related progression of human LGMDR1.展开更多
Recovery of liver mass to a healthy liver donor by compensatory regeneration after partial hepatectomy(PH)is a prerequisite for liver transplantation.Synchronized cell cycle reentry of the existing hepatocytes after P...Recovery of liver mass to a healthy liver donor by compensatory regeneration after partial hepatectomy(PH)is a prerequisite for liver transplantation.Synchronized cell cycle reentry of the existing hepatocytes after PH is seemingly a hallmark of liver compensatory regeneration.Although the molecular control of the PH-triggered cell cycle reentry has been extensively studied,little is known about how the synchronization is achieved after PH.The nucleolus-localized protein cleavage complex formed by the nucleolar protein Digestive-organ expansion factor(Def)and cysteine proteinase Calpain 3(Capn3)has been implicated to control wounding healing during liver regeneration through selectively cleaving the tumor suppressor p53 in the nucleolus.However,whether the Def-Capn3 complex participates in regulating the synchronization of cell cycle reentry after PH is unknown.In this report,we generated a zebrafish capn3b null mutant(capn3b^(Δ19Δ14)).The homozygous mutant was viable and fertile,but suffered from a delayed liver regeneration after PH.Delayed liver regeneration in capn3b^(Δ19Δ14)was due to disruption of synchronized cell proliferation after PH.Mass spectrometry(MS)analysis of nuclear proteins revealed that a number of negative regulators of cell cycle are accumulated in the capn3b^(Δ19Δ14)liver after PH.Moreover,we demonstrated that Check-point kinase 1(Chk1)and Wee1,two key negative regulators of G2 to M transition,are substrates of Capn3.We also demonstrated that Chk1 and Wee1 were abnormally accumulated in the nucleoli of amputated capn3bΔ19Δ14 liver.In conclusion,our findings suggest that the nucleolar-localized Def-Capn3 complex acts as a novel regulatory pathway for the synchronization of cell cycle reentry,at least partially,through inactivating Chk1 and Wee1 during liver regeneration after PH.展开更多
Gene knockdown approaches using antisense oligo nucleotides or analogs such as siRNAs and morpholinos have been widely adopted to study gene functions although the off-target issue has been always a concern in these s...Gene knockdown approaches using antisense oligo nucleotides or analogs such as siRNAs and morpholinos have been widely adopted to study gene functions although the off-target issue has been always a concern in these studies.On the other hand,classic genetic analysis relies on the availability of loss-offunction or gain-of-function mutants.The fast development of genome editing technologies such as TALEN and CRISPR/Cas9 has greatly facilitated the generation of null mutants for the functional studies of target genes in a variety of organisms such as zebrafish.Surprisingly,an unexpected discrepancy was observed between morphant phenotype and mutant phenotype for many genes in zebrafish,i.e.,while the morphant often displays an obvious phenotype,the corresponding null mutant appears relatively normal or only exhibits a mild phenotype due to gene compensation.Two recent reports have partially answered this intriguing question by showing that a pre-mature termination codon and homologous sequence are required to elicit the gene compensation and the histone modifying complex COMPASS is involved in activating the expression of the compensatory genes.Here,I summarize these exciting new progress and try to redefine the concept of genetic compensation and gene compensation.展开更多
文摘采用PCR-SSCP方法对58头草原红牛钙激活中性蛋白酶3基因(CAPN3)的单核苷酸多态性进行研究分析,发现其外显子1存在三种基因型AA、AB和BB,测序发现,在外显子1的124位存在碱基突变,由原来的A突变为G,氨基酸水平由天冬氨酸Asp变为天冬酰胺Asn,此SNP位点在此前的研究中未见报道。经遗传多样性分析表明,该位点等位基因A占优势地位,且三种基因型分布符合Hardy-W e inberg平衡。通过显著性差异检验发现,CAPN3基因此位点的多态性对该牛群的屠宰率、净肉率和大理石花纹这三个性状有显著影响(P<0.05),这将为CAPN3作为牛肉肉质性状候选基因提供进一步依据。
文摘为了揭示脂肪型的藏猪和瘦肉型的杜洛克猪肌肉生长及嫩度相关基因的表达差异,采用QRT-PCR方法检测了两猪种背最长肌中钙蛋白酶3(CAPN3)基因在180日龄的表达差异,分析其表达与肌纤维面积(CSA)、肌肉剪切力的相关性。结果表明,180日龄藏猪CAPN3 m RNA表达量显著高于杜洛克猪(P<0.05)。相关性分析结果显示,CAPN3 m RNA表达量与肌纤维面积、剪切力均呈显著负相关(P<0.05)。以上结果初步揭示了两猪种在180日龄CAPN3基因表达的品种差异,为深入研究猪肌肉生长及剪切力的调控机制提供了基础数据。
文摘目的对一个2A型肢带型肌营养不良(limb-girdle muscular dystrophy type 2A)家系进行CAPN3基因的致病突变分析。方法收集先证者及家系成员的外周血,提取DNA,应用全外显子测序技术对先证者进行致病基因检测,然后用Sanger测序技术对先证者家系成员进行突变位点的验证。结果全外显子测序发现先证者携带CAPN3基因c.1194-9A>G和c.1437C>T(p.ser479=)的复合杂合突变。Sanger测序验证先证者母亲为CAPN3基因c.1194-9A>G变异携带者。家系中其他患者均存在相同的复合杂合突变,其未发病的姐姐和女儿为CAPN3基因c.1437C>T(p.ser479=)变异携带者,先证者的女婿未检测到上述位点变异。结论CAPN3基因c.1194-9A>G和c.1437C>T(p.ser479=)的复合杂合突变为该家系的致病原因。
基金the National Key R&D Program of China and the Natural Science Foundation of China in the order of 2018YFA0800502,2017YFA0504501,31330050.
文摘The nucleolus,as the‘nucleus of the nucleus’,is a prominent subcellular organelle in a eukaryocyte.The nucleolus serves as the centre for ribosome biogenesis,as well as an important site for cell-cycle regulation,cellular senescence,and stress response.The protein composition of the nucleolus changes dynamically through protein turnover to meet the needs of cellular activities or stress responses.Recent studies have identified a nucleolus-localized protein degradation pathway in zebrafish and humans,namely the Def-CAPN3 pathway,which is essential to ribosome production and cell-cycle progression,by controlling the turnover of multiple substrates(e.g.,ribosomal small-subunit[SSU]processome component Mpp10,transcription factor p53,check-point proteins Chk1 and Wee1).This pathway relies on the Ca2þ-dependent cysteine proteinase CAPN3 and is independent of the ubiquitin-mediated proteasome pathway.CAPN3 is recruited by nucleolar protein Def from cytoplasm to nucleolus,where it proteolyzes its substrates which harbor a CAPN3 recognition-motif.Def depletion leads to the exclusion of CAPN3 and accumulation of p53,Wee1,Chk1,and Mpp10 in the nucleolus that result in cell-cycle arrest and rRNA processing abnormality.Here,we summarize the discovery of the Def-CAPN3 pathway and propose its biological role in cell-cycle control and ribosome biogenesis.
基金financially supported by National Natural Science Foundation of China(U21A20198 and 32100658)the National Key R&D Program of China(2018YFA0800502)。
文摘Mutations in calcium-dependent papain-like protease CALPAIN3(CAPN3)cause Limb-Girdle Muscular Dystrophy Recessive Type 1(LGMDR1),the most common limb-girdle muscular dystrophy in humans.In addition to progressive muscle weakness,persistent inflammatory infiltration is also a feature of LGMDR1.Despite the underlying mechanism remaining poorly understood,we consider that it may relate to the newly defined role of CAPN3/Capn3b in the nucleolus.Here,we report that the loss of function of zebrafish capn3b,the counterpart of human CAPN3,induces an autoimmune response akin to that in LGMDR1 patients.capn3b mutant larvae are more susceptible to Listeria monocytogenes injection,characterized by recruiting more macrophages.Under germ-free conditions,transcriptome analysis of the capn3b mutant muscle reveals a significant upregulation of the chemokine-production-related genes.Coincidently,more neutrophils are recruited to the injury site imposed by either muscle stabbing or tail fin amputation.Nucleolar proteomic analysis and enzymatic assays reveal NKAP,an activating factor of the NF-k B pathway,to be a target of CAPN3.We conclude that the accumulation of Nkap and other factors in the capn3b mutant may be involved in the over-activation of innate immunity.Our studies indicate that the zebrafish capn3b mutant is a powerful model for studying the immunity-related progression of human LGMDR1.
基金This study was supported by the National Key R&D Program of China and the Natural Science Foundation of China in the order of 2018YFA0800502,31830113 and 2017YFA0504501.
文摘Recovery of liver mass to a healthy liver donor by compensatory regeneration after partial hepatectomy(PH)is a prerequisite for liver transplantation.Synchronized cell cycle reentry of the existing hepatocytes after PH is seemingly a hallmark of liver compensatory regeneration.Although the molecular control of the PH-triggered cell cycle reentry has been extensively studied,little is known about how the synchronization is achieved after PH.The nucleolus-localized protein cleavage complex formed by the nucleolar protein Digestive-organ expansion factor(Def)and cysteine proteinase Calpain 3(Capn3)has been implicated to control wounding healing during liver regeneration through selectively cleaving the tumor suppressor p53 in the nucleolus.However,whether the Def-Capn3 complex participates in regulating the synchronization of cell cycle reentry after PH is unknown.In this report,we generated a zebrafish capn3b null mutant(capn3b^(Δ19Δ14)).The homozygous mutant was viable and fertile,but suffered from a delayed liver regeneration after PH.Delayed liver regeneration in capn3b^(Δ19Δ14)was due to disruption of synchronized cell proliferation after PH.Mass spectrometry(MS)analysis of nuclear proteins revealed that a number of negative regulators of cell cycle are accumulated in the capn3b^(Δ19Δ14)liver after PH.Moreover,we demonstrated that Check-point kinase 1(Chk1)and Wee1,two key negative regulators of G2 to M transition,are substrates of Capn3.We also demonstrated that Chk1 and Wee1 were abnormally accumulated in the nucleoli of amputated capn3bΔ19Δ14 liver.In conclusion,our findings suggest that the nucleolar-localized Def-Capn3 complex acts as a novel regulatory pathway for the synchronization of cell cycle reentry,at least partially,through inactivating Chk1 and Wee1 during liver regeneration after PH.
基金supported by National Key R&D Program of China (2018YFA0800501)National Natural Science Foundation of China (Nos.31571495, 31830113 and 31330050)
文摘Gene knockdown approaches using antisense oligo nucleotides or analogs such as siRNAs and morpholinos have been widely adopted to study gene functions although the off-target issue has been always a concern in these studies.On the other hand,classic genetic analysis relies on the availability of loss-offunction or gain-of-function mutants.The fast development of genome editing technologies such as TALEN and CRISPR/Cas9 has greatly facilitated the generation of null mutants for the functional studies of target genes in a variety of organisms such as zebrafish.Surprisingly,an unexpected discrepancy was observed between morphant phenotype and mutant phenotype for many genes in zebrafish,i.e.,while the morphant often displays an obvious phenotype,the corresponding null mutant appears relatively normal or only exhibits a mild phenotype due to gene compensation.Two recent reports have partially answered this intriguing question by showing that a pre-mature termination codon and homologous sequence are required to elicit the gene compensation and the histone modifying complex COMPASS is involved in activating the expression of the compensatory genes.Here,I summarize these exciting new progress and try to redefine the concept of genetic compensation and gene compensation.
