秀丽线虫蛋白质酪氨酸磷酸酶组的生物信息学及ceMTM3生物学功能的研究
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摘要
蛋白质酪氨酸磷酸酶在细胞信号传导过程中发挥着非常关键的作用,并与多种人类疾病的发生、发展密切相关。对于PTPs生物学功能的研究已经越来越受到人们的重视,而秀丽线虫已经被证明是一个非常优秀的模式生物,可以用来研究PTPs的生物学功能以及相关的信号传导通路。首先,我们应用丰富的数据库资源,从秀丽线虫基因组中搜索得到了所有编码蛋白质酪氨酸磷酸酶的基因,并对这些基因及其编码的蛋白质进行了整理和分析。我们发现,秀丽线虫相对人类及其它物种来说,具有一个非常大的PTP组,包含127个成员,而且经过序列比对发现,秀丽线虫PTPs具有非常高的多样性,其中27个以丝氨酸为活性中心位点的PTPs在其它物种中并未发现。另外,我们对秀丽线虫中具有FYVE结构域的MTM家族酶ceMTM3进行了克隆、表达、抗体制备及生物化学性质的表征,发现了一个新的ceMTM3基因亚型,而且证明ceMTM3是作用于底物磷酸肌醇的D3位点上。将ceMTM3基因进行RNA干扰后,秀丽线虫的运动能力在成虫期以后受到了严重的损害,说明ceMTM3在秀丽线虫肌肉保持中发挥着重要的作用,这为研究人类骨骼肌减少症提供了一个优秀的模型并打下了良好的理论基础。
Reversible tyrosine phosphorylation governed by balanced actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) regulates important signaling pathways that control various cell activities including proliferation, differentiation, transformation, adhesion, and migration. PTPs constitute a large, structurally diverse family of highly specific enzymes. They have both inhibitory and stimulatory effects on signaling processes, and deregulation of PTP function causes various human diseases.
As an excellent model organism, Caenorhabditis elegans has been widely used from its initial use in nervous system development to diverse fields such as development, signal transduction, cell death, and aging. C. elegans possesses particularly favorable characteristics for laboratory studies. It is generally believed that similar mechanisms direct equivalent biological processes in all animal species including humans. It is thus conceivable that C.elegans should serve as an excellent model for studies of phosphatase/kinase involved in signal transduction pathways. The C. elegans genome project was successfully completed in 1998, making C. elegans the first multicellular organism for which the genome has been sequenced. However, C.elegans protein phosphatases family had not been analyzed systematically so far. Therefore, a detailed analysis of the C.elegans phosphatome is highly desirable.
By searching of the NCBI and Wormbase databases using the BLAST program with the cDNA sequences of 37 human classical PTPs, we identified a large number of genes in C. elegans which encode protein tyrosine phosphatases. Further detailed sequence alignment analyses by using various online resources revealed unique as well common features of the C. elegans phosphatatome in comparison with those of many other species.
The results demonstrated that C. elegans contains a larger PTP family with more structural diversities. Excluding several PTPs fragments which correspond to incomplete PTP catalytic domain, 118 tyrosine phosphatase-conding genes are identified in the C.elegans genome. Among them are 25 VH1-like phosphatases, 4 CDC25 subfamily members,1 low molecular weight phosphatase, and 1 eye-absent (EYA) protein. These 31 enzymes all have human homologues, suggesting high structural conservativities and perhaps similar basic functions. The remaining 87 genes encode tyrosine-specific PTP1B-like phosphatase, far more than 37, 32, 47, 30, and 16 members found in human, chicken, zebrafish, sea urchin, and fruit fly, respectively.
In addition, the protein tyrosine phosphatases in C.elegans display much more diversity than vertebrates and other species. Based on the results of complete multiple sequences alignment and the phylogenetic tree containing 87 cePTPs and 37 hPTPs, 10 of the PTP1B-like PTPs in C.elegans are homologous to human classical PTPs and thus form the conventional PTP subfmaily. There are additional 4 subfamilies which do not have human homologues. Importantly, 27 of these enzymes contain a serine residue at the position expected for the conserved active site cysteine and are predicted inactive, suggesting new structural features, catalytic mechanisms, and biological functions. Another characteristics of the worm PTP family is the presence of the so-called worm specific N-terminal (WSN) domain in some members. WSN has unknown function and is usually found at the N-terminal end of nematode proteins. It is defined as a 70 aa conserved sequence in our study and can be detected in 17 worm transmembrane PTPs.
Unlike the protein tyrosine kinases which are thought metazoan-specific family, protein tyrosine phosphatases widely exist in almost all the eukaryotic organisms including plant rice (2 PTPs) and Arabidopsis (1 PTP), fungi yeast (3 PTPs), the unicellular Monosiga brevicollis (27 PTPs), some protist Dictyostelium discoideum (3 PTPs), and Leishmania major (2 PTPs).
The above analysis provides valuable information for the further investigation of structures, functions, regulation of C. elegans PTPs in C.elegans in comparison with those found in humans.
Myotubularin phosphatases (MTMs) are members of PTP superfamily and are reported to dephosphorylate PI3P or PI3’5P2. Previous studies indicated that MTMs were associated with several human diseases such as X-link myotubular myopathy and Charcot-Marie-Tooth disease 4B1 etc. Binding of FYVE domain with PI3P presumably targets FYVE domain-containing MTMs to PI3P-rich cell compartments, thereby dephosphorylating PI3P and PI3’5P2 more efficiently. PI3P is known to play an important role in membrane trafficking.
To verify the cDNA sequence of the C.elegans MTM3, we employed the rapid amplification cDNA ends (RACE) by using Smart-RACE kit from Clontech. This resulted in the isolation of two cDNA sequences. One of them represents a new isoform and was named ceMTM3b and another is re-designated as ceMTM3a. ceMTM3a has 9 exons while ceMTM3b contains a 12 bp-shorter exon 2 and a distinct exon 8 compared with ceMTM3a exon 8 and 9. The two cDNA sequences resulted from alternative splicing in gene transcription.
To analyze the expression of ceMTM3 in C.elegans, we first generated a rabbit polyclonal antibody against GST-ceMTM3CT, a GST fusion molecule containing the C-terminal region (aa 701-961) of ceMTM3b expressed in E.coli. As expected, ceMTM3 preferably dephosphorylated PI3P. It also displayed a significant activity toward PI3’5P2 but essentially no activity to PI4P and PI5P. To determine the binding specificity of the FYVE domains to phosphoinositides, we employed the commonly used lipid membrane overlay assays. GST-ceMTM3CT binds specifically to PI3P, whereas no binding was found with GST control. The data indicate that ceMTM contains a typical FYVE domain.
We employed immunofluorescent cell staining to detect the tissue expression of the enzyme in C.elegans. Staining of whole-mount gravid worms showed expression of the enzyme in body wall muscle and in eggs. This was further verified by staining paraffin-embedded cross-sections of adult worms. Note that the staining also revealed partial expression of the enzyme in the intestine. On western blot analysis, ceMTM3 expressed at the egg and young larva (L1 and L2) stages. It decreases to near absence in the L3 to young adult stages, and then regains high expression in the egg-laying period and post-reproductive age.
To study the biological function of ceMTM3, feeding-based RNA interference was employed to knockdown the expression of enzyme in C.elegans. A clear phenotype was the impaired locomotion of the worms when they reached late and post-reproductive stages (day 5 and thereafter). At the larva and young adult stages, their locomotion appeared normal. As we know, human MTM1 is responsible for X-linked recessive myotubular myopathy. Mutation of hMTM1 appears to affect organization of muscle cells during myogenesis since the disease was believed to result from an arrest in the normal development of muscle fibers at the myotubular stage. Interestingly, MTM1-deficient mice develop a progressive centronuclear myopathy during postnatal life that severely reduces their life expectancy. The data indicate that MTM1 plays a role in muscle maintenance in mice rather than in myogenesis seen in human. Apparently, ceMTM3 may be similar to mouse MTM1 in muscle maintenance. We believe that studying the MTM enzymes may have clinical implications in the prevention and treatment of human sarcopenia.
Reversible tyrosine phosphorylation governed by balanced actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) regulates important signaling pathways that control various cell activities including proliferation, differentiation, transformation, adhesion, and migration. PTPs constitute a large, structurally diverse family of highly specific enzymes. They have both inhibitory and stimulatory effects on signaling processes, and deregulation of PTP function causes various human diseases.
As an excellent model organism, Caenorhabditis elegans has been widely used from its initial use in nervous system development to diverse fields such as development, signal transduction, cell death, and aging. C. elegans possesses particularly favorable characteristics for laboratory studies. It is generally believed that similar mechanisms direct equivalent biological processes in all animal species including humans. It is thus conceivable that C.elegans should serve as an excellent model for studies of phosphatase/kinase involved in signal transduction pathways. The C. elegans genome project was successfully completed in 1998, making C. elegans the first multicellular organism for which the genome has been sequenced. However, C.elegans protein phosphatases family had not been analyzed systematically so far. Therefore, a detailed analysis of the C.elegans phosphatome is highly desirable.
By searching of the NCBI and Wormbase databases using the BLAST program with the cDNA sequences of 37 human classical PTPs, we identified a large number of genes in C. elegans which encode protein tyrosine phosphatases. Further detailed sequence alignment analyses by using various online resources revealed unique as well common features of the C. elegans phosphatatome in comparison with those of many other species.
The results demonstrated that C. elegans contains a larger PTP family with more structural diversities. Excluding several PTPs fragments which correspond to incomplete PTP catalytic domain, 118 tyrosine phosphatase-conding genes are identified in the C.elegans genome. Among them are 25 VH1-like phosphatases, 4 CDC25 subfamily members,1 low molecular weight phosphatase, and 1 eye-absent (EYA) protein. These 31 enzymes all have human homologues, suggesting high structural conservativities and perhaps similar basic functions. The remaining 87 genes encode tyrosine-specific PTP1B-like phosphatase, far more than 37, 32, 47, 30, and 16 members found in human, chicken, zebrafish, sea urchin, and fruit fly, respectively.
In addition, the protein tyrosine phosphatases in C.elegans display much more diversity than vertebrates and other species. Based on the results of complete multiple sequences alignment and the phylogenetic tree containing 87 cePTPs and 37 hPTPs, 10 of the PTP1B-like PTPs in C.elegans are homologous to human classical PTPs and thus form the conventional PTP subfmaily. There are additional 4 subfamilies which do not have human homologues. Importantly, 27 of these enzymes contain a serine residue at the position expected for the conserved active site cysteine and are predicted inactive, suggesting new structural features, catalytic mechanisms, and biological functions. Another characteristics of the worm PTP family is the presence of the so-called worm specific N-terminal (WSN) domain in some members. WSN has unknown function and is usually found at the N-terminal end of nematode proteins. It is defined as a 70 aa conserved sequence in our study and can be detected in 17 worm transmembrane PTPs.
Unlike the protein tyrosine kinases which are thought metazoan-specific family, protein tyrosine phosphatases widely exist in almost all the eukaryotic organisms including plant rice (2 PTPs) and Arabidopsis (1 PTP), fungi yeast (3 PTPs), the unicellular Monosiga brevicollis (27 PTPs), some protist Dictyostelium discoideum (3 PTPs), and Leishmania major (2 PTPs).
The above analysis provides valuable information for the further investigation of structures, functions, regulation of C. elegans PTPs in C.elegans in comparison with those found in humans.
Myotubularin phosphatases (MTMs) are members of PTP superfamily and are reported to dephosphorylate PI3P or PI3’5P2. Previous studies indicated that MTMs were associated with several human diseases such as X-link myotubular myopathy and Charcot-Marie-Tooth disease 4B1 etc. Binding of FYVE domain with PI3P presumably targets FYVE domain-containing MTMs to PI3P-rich cell compartments, thereby dephosphorylating PI3P and PI3’5P2 more efficiently. PI3P is known to play an important role in membrane trafficking.
To verify the cDNA sequence of the C.elegans MTM3, we employed the rapid amplification cDNA ends (RACE) by using Smart-RACE kit from Clontech. This resulted in the isolation of two cDNA sequences. One of them represents a new isoform and was named ceMTM3b and another is re-designated as ceMTM3a. ceMTM3a has 9 exons while ceMTM3b contains a 12 bp-shorter exon 2 and a distinct exon 8 compared with ceMTM3a exon 8 and 9. The two cDNA sequences resulted from alternative splicing in gene transcription.
To analyze the expression of ceMTM3 in C.elegans, we first generated a rabbit polyclonal antibody against GST-ceMTM3CT, a GST fusion molecule containing the C-terminal region (aa 701-961) of ceMTM3b expressed in E.coli. As expected, ceMTM3 preferably dephosphorylated PI3P. It also displayed a significant activity toward PI3’5P2 but essentially no activity to PI4P and PI5P. To determine the binding specificity of the FYVE domains to phosphoinositides, we employed the commonly used lipid membrane overlay assays. GST-ceMTM3CT binds specifically to PI3P, whereas no binding was found with GST control. The data indicate that ceMTM contains a typical FYVE domain.
We employed immunofluorescent cell staining to detect the tissue expression of the enzyme in C.elegans. Staining of whole-mount gravid worms showed expression of the enzyme in body wall muscle and in eggs. This was further verified by staining paraffin-embedded cross-sections of adult worms. Note that the staining also revealed partial expression of the enzyme in the intestine. On western blot analysis, ceMTM3 expressed at the egg and young larva (L1 and L2) stages. It decreases to near absence in the L3 to young adult stages, and then regains high expression in the egg-laying period and post-reproductive age.
To study the biological function of ceMTM3, feeding-based RNA interference was employed to knockdown the expression of enzyme in C.elegans. A clear phenotype was the impaired locomotion of the worms when they reached late and post-reproductive stages (day 5 and thereafter). At the larva and young adult stages, their locomotion appeared normal. As we know, human MTM1 is responsible for X-linked recessive myotubular myopathy. Mutation of hMTM1 appears to affect organization of muscle cells during myogenesis since the disease was believed to result from an arrest in the normal development of muscle fibers at the myotubular stage. Interestingly, MTM1-deficient mice develop a progressive centronuclear myopathy during postnatal life that severely reduces their life expectancy. The data indicate that MTM1 plays a role in muscle maintenance in mice rather than in myogenesis seen in human. Apparently, ceMTM3 may be similar to mouse MTM1 in muscle maintenance. We believe that studying the MTM enzymes may have clinical implications in the prevention and treatment of human sarcopenia.
引文
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