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Ming-daw Tsai - One of the best experts on this subject based on the ideXlab platform.
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Tumor suppressor Ink4: refinement of p16Ink4A structure and determination of p15Ink4B structure by comparative modeling and NMR data
Protein Science, 2000Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:Within the tumor suppressor protein Ink4 ~inhibitor of cyclin-dependent kinase 4! family, p15 Ink4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein’s conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16 Ink4A as the template, followed by restrained energy minimization with NMR constraints ~NOE and H-bonds!. For this purpose, the solution structure of p16 Ink4A , whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-meansquare deviation ~RMSD! improved from 1.23 to 0.68 A for backbone heavy atoms. The completion of p15 Ink4B coupled with refinement of p16 Ink4A made it possible to compare the structures of the four Ink4 members in depth, and to compare the structures of p16 Ink4A in the free form and in the p16 Ink4A -CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each Ink4 member.
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Tumor suppressor Ink4: quantitative structure-function analyses of p18Ink4C as an inhibitor of cyclin-dependent kinase 4.
Biochemistry, 2000Co-Authors: Ming Poi, Thomas L. Selby, In-ja L. Byeon, Dongyan Qin, Ming-daw TsaiAbstract:We report the first detailed structure -function analyses of p18 Ink4C (p18), which is a homologue of the important tumor suppressor p16 Ink4A (p16). Twenty-four mutants were designed rationally. The global conformations of the mutants were characterized by NMR, while the function was assayed by inhibition of cyclin-dependent kinase 4 (CDK4). Most of these mutants have unperturbed global structures, thus the changes in their inhibitory abilities can be attributed to the mutated residues. The important results are summarized as follows: (a) some residues at loops 1 and 2, but not 3, are important for the inhibitory function of p18, similar to the results for p16; (b) two residues at the first helix-turn-helix motif and two at the third are important for inhibition; (c) while the results generally agree with the prediction based on the crystal structures of p16-CDK6 and p19-CDK6 binary complexes, there are significant differences in a few residues, suggesting that the interactions in the binary complexes may not accurately represent the interactions in the ternary complexes (in the presence of cyclin D2); (d) most importantly, the extra loop of p18 appears to contribute to the function of p18, even though the crystal structure of the p19 Ink4D -CDK6 complex indicates no interactions involving this loop; (e) detailed analyses of the crystal structures and the functional results suggest that there are notable differences in the interactions between different members of the Ink4 family and CDKs.
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tumor suppressor Ink4 comparisons of conformational properties between p16 Ink4a and p18 Ink4c
Journal of Molecular Biology, 1999Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:The Ink4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D. While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear 1H-15N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/2H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18
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Tumor suppressor Ink4: comparisons of conformational properties between p16(Ink4A) and p18(Ink4C).
Journal of Molecular Biology, 1999Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:The Ink4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D. While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear 1H-15N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/2H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18
Manuel Serrano - One of the best experts on this subject based on the ideXlab platform.
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the Ink4 arf locus is a barrier for ips cell reprogramming
Nature, 2009Co-Authors: Manuel Collado, Marta Cañamero, Aranzazu Villasante, Katerina Strati, Sagrario Ortega, Maria A. Blasco, Manuel SerranoAbstract:The Ink4/Arf tumour suppressor locus encodes three potent tumour suppressors, namely p16Ink4a, p15Ink4b and p19Arf. Here Li et al. show that the locus is rate limiting for reprogramming, and its transient inhibition significantly improves the generation of iPS cells. The also show ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms. The Ink4/Arf tumour suppressor locus encodes the three potent tumour suppressors p16Ink4a, p15Ink4b and p19Arf. Here the locus is shown to be rate-limiting for reprogramming, and its transient inhibition significantly improves the generation of induced pluripotent stem cells. Furthermore, ageing is shown to upregulate the Ink4/Arf locus, with less efficient reprogramming seen in cells from old organisms. The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood1. The Ink4/Arf locus comprises the Cdkn2a–Cdkn2b genes encoding three potent tumour suppressors, namely p16Ink4a, p19Arf and p15Ink4b, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals2,3,4. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of ‘stemness’. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, Ink4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.
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The Ink4/Arf locus is a barrier for iPS cell reprogramming
Nature, 2009Co-Authors: Manuel Collado, Marta Cañamero, Aranzazu Villasante, Katerina Strati, Sagrario Ortega, Maria A. Blasco, Manuel SerranoAbstract:The Ink4/Arf tumour suppressor locus encodes three potent tumour suppressors, namely p16Ink4a, p15Ink4b and p19Arf. Here Li et al. show that the locus is rate limiting for reprogramming, and its transient inhibition significantly improves the generation of iPS cells. The also show ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms. The Ink4/Arf tumour suppressor locus encodes the three potent tumour suppressors p16Ink4a, p15Ink4b and p19Arf. Here the locus is shown to be rate-limiting for reprogramming, and its transient inhibition significantly improves the generation of induced pluripotent stem cells. Furthermore, ageing is shown to upregulate the Ink4/Arf locus, with less efficient reprogramming seen in cells from old organisms. The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood1. The Ink4/Arf locus comprises the Cdkn2a–Cdkn2b genes encoding three potent tumour suppressors, namely p16Ink4a, p19Arf and p15Ink4b, which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals2,3,4. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of ‘stemness’. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, Ink4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus2,5 and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.
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Polycomb mediated epigenetic silencing and replication timing at the Ink4a/ARF locus during senescence.
PLoS ONE, 2009Co-Authors: Hanane Agherbi, Anne Gaussmann-wenger, Christophe Verthuy, Lionel Chasson, Manuel Serrano, Malek DjabaliAbstract:BACKGROUND: The Ink4/ARF locus encodes three tumor suppressor genes (p15(Ink4b), Arf and p16(Ink4a)) and is frequently inactivated in a large number of human cancers. Mechanisms regulating Ink4/ARF expression are not fully characterized. PRINCIPAL FINDINGS: Here we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the Ink4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the Ink4a/ARF locus during senescence. CONCLUSIONS: We identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the Ink4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the Ink4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the Ink4/ARF locus.
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A new mechanism of inactivation of the Ink4/ARF locus.
Cell Cycle, 2006Co-Authors: Susana González, Manuel SerranoAbstract:The Ink4/ARF locus encodes three tumor suppressors, p15(Ink4b), p16(Ink4a) and ARF, which together constitute one of the main anti-oncogenic defenses of mammalian organisms. The activity of these tumor suppressors depends mostly on the transcriptional status of the locus. Recently, we have identified a conserved DNA element with the capacity to regulate the locus in a global manner. Inactivation of this element, which we have named RD(Ink4/ARF), results in the silencing of the entire Ink4/ARF locus. Interestingly, RD(Ink4/ARF) is both a transcriptional regulatory element and a replication origin. The replication protein Cdc6 binds to RD(Ink4/ARF) and is able to recruit histone deacetylases that, in turn, result in the heterochromatinization and repression of the Ink4/ARF locus. This model has striking parallelisms with the silencing of the yeast mating-type loci, and it is a novel oncogenic mechanism that connects the replication machinery with the inactivation of tumor suppressors.
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a new mechanism of inactivation of the Ink4 arf locus
Cell Cycle, 2006Co-Authors: Susana González, Manuel SerranoAbstract:The Ink4/ARF locus encodes three tumor suppressors, p15(Ink4b), p16(Ink4a) and ARF, which together constitute one of the main anti-oncogenic defenses of mammalian organisms. The activity of these tumor suppressors depends mostly on the transcriptional status of the locus. Recently, we have identified a conserved DNA element with the capacity to regulate the locus in a global manner. Inactivation of this element, which we have named RD(Ink4/ARF), results in the silencing of the entire Ink4/ARF locus. Interestingly, RD(Ink4/ARF) is both a transcriptional regulatory element and a replication origin. The replication protein Cdc6 binds to RD(Ink4/ARF) and is able to recruit histone deacetylases that, in turn, result in the heterochromatinization and repression of the Ink4/ARF locus. This model has striking parallelisms with the silencing of the yeast mating-type loci, and it is a novel oncogenic mechanism that connects the replication machinery with the inactivation of tumor suppressors.
Mike Fried - One of the best experts on this subject based on the ideXlab platform.
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One Ink4 gene and no ARF at the Fugu equivalent of the human Ink4A/ARF/Ink4B tumour suppressor locus
Oncogene, 2001Co-Authors: Jonathan Gilley, Mike FriedAbstract:The Ink4A/ARF/Ink4B locus, conserved in mammals, encodes three polypeptides that regulate cell proliferation via the pRb and p53 tumour suppressor pathways. The locus is mutated in many cancers. The related, tandemly-linked Ink4A and Ink4B genes encode the p16(Ink4A) and p15(Ink4B) members of the Ink4 family of cyclin-dependent kinase inhibitors which block phosphorylation of pRb, whereas the third product, ARF, derived from an alternative reading frame of Ink4A, regulates p53 activity. We assessed the status of this unusual locus in the puffer fish, Fugu rubripes, and identified two Ink4 genes using degenerate PCR and hybridization analyses. Sequence conservation and conservation of synteny between human and Fugu predict one gene to be an Ink4A or Ink4B homologue and the other an Ink4D homologue. Analysis of the Fugu Ink4A/B gene and the surrounding 40-kb of genomic DNA did not reveal the presence of any ARF-encoding potential or another related Ink4 gene. We conclude that the gene duplication event that generated adjacent Ink4A and Ink4B genes and the association of ARF with the ancestral Ink4A gene occurred after the divergence of the lineage leading to mammals from fish. Thus, unlike mammals, the fish p53 and pRb tumour suppressor pathways are not regulated by a single locus.
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one Ink4 gene and no arf at the fugu equivalent of the human Ink4a arf Ink4b tumour suppressor locus
Oncogene, 2001Co-Authors: Jonathan Gilley, Mike FriedAbstract:The Ink4A/ARF/Ink4B locus, conserved in mammals, encodes three polypeptides that regulate cell proliferation via the pRb and p53 tumour suppressor pathways. The locus is mutated in many cancers. The related, tandemly-linked Ink4A and Ink4B genes encode the p16(Ink4A) and p15(Ink4B) members of the Ink4 family of cyclin-dependent kinase inhibitors which block phosphorylation of pRb, whereas the third product, ARF, derived from an alternative reading frame of Ink4A, regulates p53 activity. We assessed the status of this unusual locus in the puffer fish, Fugu rubripes, and identified two Ink4 genes using degenerate PCR and hybridization analyses. Sequence conservation and conservation of synteny between human and Fugu predict one gene to be an Ink4A or Ink4B homologue and the other an Ink4D homologue. Analysis of the Fugu Ink4A/B gene and the surrounding 40-kb of genomic DNA did not reveal the presence of any ARF-encoding potential or another related Ink4 gene. We conclude that the gene duplication event that generated adjacent Ink4A and Ink4B genes and the association of ARF with the ancestral Ink4A gene occurred after the divergence of the lineage leading to mammals from fish. Thus, unlike mammals, the fish p53 and pRb tumour suppressor pathways are not regulated by a single locus.
Chunhua Yuan - One of the best experts on this subject based on the ideXlab platform.
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Tumor suppressor Ink4: refinement of p16Ink4A structure and determination of p15Ink4B structure by comparative modeling and NMR data
Protein Science, 2000Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:Within the tumor suppressor protein Ink4 ~inhibitor of cyclin-dependent kinase 4! family, p15 Ink4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein’s conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16 Ink4A as the template, followed by restrained energy minimization with NMR constraints ~NOE and H-bonds!. For this purpose, the solution structure of p16 Ink4A , whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-meansquare deviation ~RMSD! improved from 1.23 to 0.68 A for backbone heavy atoms. The completion of p15 Ink4B coupled with refinement of p16 Ink4A made it possible to compare the structures of the four Ink4 members in depth, and to compare the structures of p16 Ink4A in the free form and in the p16 Ink4A -CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each Ink4 member.
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tumor suppressor Ink4 comparisons of conformational properties between p16 Ink4a and p18 Ink4c
Journal of Molecular Biology, 1999Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:The Ink4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D. While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear 1H-15N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/2H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18
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Tumor suppressor Ink4: comparisons of conformational properties between p16(Ink4A) and p18(Ink4C).
Journal of Molecular Biology, 1999Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:The Ink4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D. While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear 1H-15N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/2H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18
In-ja L. Byeon - One of the best experts on this subject based on the ideXlab platform.
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Tumor suppressor Ink4: refinement of p16Ink4A structure and determination of p15Ink4B structure by comparative modeling and NMR data
Protein Science, 2000Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:Within the tumor suppressor protein Ink4 ~inhibitor of cyclin-dependent kinase 4! family, p15 Ink4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein’s conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16 Ink4A as the template, followed by restrained energy minimization with NMR constraints ~NOE and H-bonds!. For this purpose, the solution structure of p16 Ink4A , whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-meansquare deviation ~RMSD! improved from 1.23 to 0.68 A for backbone heavy atoms. The completion of p15 Ink4B coupled with refinement of p16 Ink4A made it possible to compare the structures of the four Ink4 members in depth, and to compare the structures of p16 Ink4A in the free form and in the p16 Ink4A -CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each Ink4 member.
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Tumor suppressor Ink4: quantitative structure-function analyses of p18Ink4C as an inhibitor of cyclin-dependent kinase 4.
Biochemistry, 2000Co-Authors: Ming Poi, Thomas L. Selby, In-ja L. Byeon, Dongyan Qin, Ming-daw TsaiAbstract:We report the first detailed structure -function analyses of p18 Ink4C (p18), which is a homologue of the important tumor suppressor p16 Ink4A (p16). Twenty-four mutants were designed rationally. The global conformations of the mutants were characterized by NMR, while the function was assayed by inhibition of cyclin-dependent kinase 4 (CDK4). Most of these mutants have unperturbed global structures, thus the changes in their inhibitory abilities can be attributed to the mutated residues. The important results are summarized as follows: (a) some residues at loops 1 and 2, but not 3, are important for the inhibitory function of p18, similar to the results for p16; (b) two residues at the first helix-turn-helix motif and two at the third are important for inhibition; (c) while the results generally agree with the prediction based on the crystal structures of p16-CDK6 and p19-CDK6 binary complexes, there are significant differences in a few residues, suggesting that the interactions in the binary complexes may not accurately represent the interactions in the ternary complexes (in the presence of cyclin D2); (d) most importantly, the extra loop of p18 appears to contribute to the function of p18, even though the crystal structure of the p19 Ink4D -CDK6 complex indicates no interactions involving this loop; (e) detailed analyses of the crystal structures and the functional results suggest that there are notable differences in the interactions between different members of the Ink4 family and CDKs.
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tumor suppressor Ink4 comparisons of conformational properties between p16 Ink4a and p18 Ink4c
Journal of Molecular Biology, 1999Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:The Ink4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D. While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear 1H-15N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/2H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18
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Tumor suppressor Ink4: comparisons of conformational properties between p16(Ink4A) and p18(Ink4C).
Journal of Molecular Biology, 1999Co-Authors: Chunhua Yuan, Thomas L. Selby, In-ja L. Byeon, Ming-daw TsaiAbstract:The Ink4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D. While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear 1H-15N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/2H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18