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Induction of apoptosis by the retinoid inducible growth regulator RIG1 depends on the NC motif in HtTA cervical cancer cells.

Tsai FM, Shyu RY, Lin SC, Wu CC, Jiang SY - BMC Cell Biol. (2009)

Bottom Line: The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N(112)C(113) motif double- (NC-->FG) or triple- (NCR-->FGE) mutated RIG1 variants.Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1(111-123) or Leu121-mutated RIG1(111-123):L--> C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1(111-123):NC-->FG or RIG1(111-123):NCR-->FGE, were cytoplasmically localized and did not induce apoptosis.The RIG1(111-123) dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, Buddhist Tzu Chi General Hospital Taipei Branch, Taipei county 231, Taiwan, Republic of China. afu2215@gmail.com

ABSTRACT

Background: Retinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 or retinoic-acid receptor responder 3, is a growth regulator, which induces apoptosis and differentiation. RIG1 is classified into the NC protein family. This study investigated functional domains and critical amino acids associated with RIG1-mediated cell death and apoptosis.

Results: Using enhanced green fluorescence protein (EGFP)-tagged RIG1 variants, RIG1 proteins with deletion at the NC domain significantly decreased cell death induced by RIG1, and fusion variants containing only the NC domain significantly induced apoptosis of HtTA cervical cancer cells. The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N(112)C(113) motif double- (NC-->FG) or triple- (NCR-->FGE) mutated RIG1 variants. Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1(111-123) or Leu121-mutated RIG1(111-123):L--> C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1(111-123):NC-->FG or RIG1(111-123):NCR-->FGE, were cytoplasmically localized and did not induce apoptosis. The RIG1(111-123) also induced apoptosis of A2058 melanoma cells but not normal human fibroblasts.

Conclusion: The NC domain, especially the NC motif, plays the major role in RIG1-mediated pro-apoptotic activity. The RIG1(111-123) dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.

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Designation of mutated DP of HREV107 family protein. (A) Sequence alignment of the NC domains of human HREV107 family proteins and LRAT. Identical amino acids are indicated by black boxes and the DP is underlined. (B) Comparison of amino acid sequences of DP and DP variations (changed amino acids underlined) used in this study. Sequences of DPs derived from HREV107 and LRAT are also indicated.
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Figure 3: Designation of mutated DP of HREV107 family protein. (A) Sequence alignment of the NC domains of human HREV107 family proteins and LRAT. Identical amino acids are indicated by black boxes and the DP is underlined. (B) Comparison of amino acid sequences of DP and DP variations (changed amino acids underlined) used in this study. Sequences of DPs derived from HREV107 and LRAT are also indicated.

Mentions: A multiple alignment of amino acid sequences of the NC domain among the HREV107 family proteins and LRAT is shown in Fig 3A. Thirteen amino acids within the NC domain are identical among all 6 members, and the DP region exhibits the highest degree of homology. Fluorescein (FITC)-labeled wild type and mutated RIG1111–123 peptides (Fig 3B) were introduced into HtTA cells to analyze activities and critical amino acids of DP affecting cell death. The wild type FITC-RIG1111–123 was predominantly distributed within the nucleus of HtTA cells 48 h after electroporation (Fig 4A). The Leu120-mutated FITC-RIG1111–123:L → C was distributed homogenously between cytoplasm and nucleus. In contrast, DPs containing mutation at one (R), two (NC) or three (NCR) amino acids were all cytoplasmically localized. Similar nuclear and cytoplasm distribution of wild type and NCR-mutated RIG1111–123 respectively was observed in A2058 melanoma cells. However, both the wild type and NCR-mutated RIG1-DPs were distributed only within the cytoplasm of normal human fibroblasts (Fig 4B, C). In addition to nuclear targeting, RIG1111–123 also induced nuclear shrinkage, a characteristic of cellular apoptosis, of A2058 cells.


Induction of apoptosis by the retinoid inducible growth regulator RIG1 depends on the NC motif in HtTA cervical cancer cells.

Tsai FM, Shyu RY, Lin SC, Wu CC, Jiang SY - BMC Cell Biol. (2009)

Designation of mutated DP of HREV107 family protein. (A) Sequence alignment of the NC domains of human HREV107 family proteins and LRAT. Identical amino acids are indicated by black boxes and the DP is underlined. (B) Comparison of amino acid sequences of DP and DP variations (changed amino acids underlined) used in this study. Sequences of DPs derived from HREV107 and LRAT are also indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656461&req=5

Figure 3: Designation of mutated DP of HREV107 family protein. (A) Sequence alignment of the NC domains of human HREV107 family proteins and LRAT. Identical amino acids are indicated by black boxes and the DP is underlined. (B) Comparison of amino acid sequences of DP and DP variations (changed amino acids underlined) used in this study. Sequences of DPs derived from HREV107 and LRAT are also indicated.
Mentions: A multiple alignment of amino acid sequences of the NC domain among the HREV107 family proteins and LRAT is shown in Fig 3A. Thirteen amino acids within the NC domain are identical among all 6 members, and the DP region exhibits the highest degree of homology. Fluorescein (FITC)-labeled wild type and mutated RIG1111–123 peptides (Fig 3B) were introduced into HtTA cells to analyze activities and critical amino acids of DP affecting cell death. The wild type FITC-RIG1111–123 was predominantly distributed within the nucleus of HtTA cells 48 h after electroporation (Fig 4A). The Leu120-mutated FITC-RIG1111–123:L → C was distributed homogenously between cytoplasm and nucleus. In contrast, DPs containing mutation at one (R), two (NC) or three (NCR) amino acids were all cytoplasmically localized. Similar nuclear and cytoplasm distribution of wild type and NCR-mutated RIG1111–123 respectively was observed in A2058 melanoma cells. However, both the wild type and NCR-mutated RIG1-DPs were distributed only within the cytoplasm of normal human fibroblasts (Fig 4B, C). In addition to nuclear targeting, RIG1111–123 also induced nuclear shrinkage, a characteristic of cellular apoptosis, of A2058 cells.

Bottom Line: The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N(112)C(113) motif double- (NC-->FG) or triple- (NCR-->FGE) mutated RIG1 variants.Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1(111-123) or Leu121-mutated RIG1(111-123):L--> C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1(111-123):NC-->FG or RIG1(111-123):NCR-->FGE, were cytoplasmically localized and did not induce apoptosis.The RIG1(111-123) dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, Buddhist Tzu Chi General Hospital Taipei Branch, Taipei county 231, Taiwan, Republic of China. afu2215@gmail.com

ABSTRACT

Background: Retinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 or retinoic-acid receptor responder 3, is a growth regulator, which induces apoptosis and differentiation. RIG1 is classified into the NC protein family. This study investigated functional domains and critical amino acids associated with RIG1-mediated cell death and apoptosis.

Results: Using enhanced green fluorescence protein (EGFP)-tagged RIG1 variants, RIG1 proteins with deletion at the NC domain significantly decreased cell death induced by RIG1, and fusion variants containing only the NC domain significantly induced apoptosis of HtTA cervical cancer cells. The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N(112)C(113) motif double- (NC-->FG) or triple- (NCR-->FGE) mutated RIG1 variants. Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1(111-123) or Leu121-mutated RIG1(111-123):L--> C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1(111-123):NC-->FG or RIG1(111-123):NCR-->FGE, were cytoplasmically localized and did not induce apoptosis. The RIG1(111-123) also induced apoptosis of A2058 melanoma cells but not normal human fibroblasts.

Conclusion: The NC domain, especially the NC motif, plays the major role in RIG1-mediated pro-apoptotic activity. The RIG1(111-123) dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.

Show MeSH
Related in: MedlinePlus