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The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis.

Carrick DM, Lai WS, Blackshear PJ - Arthritis Res. Ther. (2004)

Bottom Line: The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine.Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex.The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Clinical Research, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.

ABSTRACT
Tristetraprolin (TTP) is the best-studied member of a small family of three proteins in humans that is characterized by a tandem CCCH zinc finger (TZF) domain with highly conserved sequences and spacing. Although initially discovered as a gene that could be induced rapidly and transiently by the stimulation of fibroblasts with growth factors and mitogens, it is now known that TTP can bind to AU-rich elements in mRNA, leading to the removal of the poly(A) tail from that mRNA and increased rates of mRNA turnover. This activity was discovered after TTP-deficient mice were created and found to have a systemic inflammatory syndrome with severe polyarticular arthritis and autoimmunity, as well as medullary and extramedullary myeloid hyperplasia. The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine. The myeloid hyperplasia might be due in part to increased stability of granulocyte-macrophage colony-stimulating factor (GM-CSF). This review highlights briefly the characteristics of the TTP-deficiency syndrome in mice and its possible genetic modifiers, as well as recent data on the characteristics of the TTP-binding site in the TNF-alpha and GM-CSF mRNAs. Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex. We review the current knowledge of TTP sequence variants in humans and discuss the possible contributions of the TTP-related proteins in mouse physiology and in human monocytes. The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

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Effects of tristetraprolin (TTP)-related tandem CCCH zinc finger (TZF) proteins to bind AU-rich element (ARE)-containing probes and to promote their deadenylation. HEK-293 cells were maintained, and transient transfection of 1.2 × 106 cells with expression plasmid constructs in calcium phosphate precipitates was performed, as described [22]. To each plate of HEK-293 cells was added 0.2 μg of the TZF protein expression constructs CMV.hTTP.tag (hTTP), a human TTP (hTTP) zinc finger mutant (C124R), CMV.cMG1.tag (cMG1), CMV.mTis11D.tag (mTis11D), 0.1 μg of human poly(A) exonuclease (hPARN) expression plasmid CMV.hPARN.flag (hPARN), or plasmid DNA alone (BS+). The zinc finger protein expression constructs were transfected either with vector alone or together with CMV.hPARN.flag; vector DNA (BS+) was added to each transfection to make the total amount of co-transfected DNA 5 μg per plate. Cytosolic extracts were prepared and used in deadenylation assays as described [23]. (a) Extracts (10 μg of protein per sample) were incubated with probes ARE or ARE-A50 at 37°C for 60 min in the presence (+) or absence (-) of 20 mM EDTA, as indicated. The samples were processed as described previously [23]. The arrow indicates the migration position of the ARE probe (lanes 1–6) and the deadenylated product of probe ARE-A50 (lanes 9, 11, 12, 14, 16 and 17). (b) The extracts used in lanes 7–13 of (a) were incubated with the ARE-A50 probe and used in a gel-shift assay. Lane 7 (P') was loaded with probe alone (digested with RNase T1). The migration positions of the zinc finger protein-RNA complexes are indicated by the bracket to the right of the gel, and the position of the free probe (FP) is also indicated. The bands present in the gel in lane 1 represent endogenous HEK-293 cell proteins shifting the probe; note that this pattern is identical in lane 3, representing a zinc finger mutant of TTP, and in lane 6, representing hPARN alone.
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Figure 7: Effects of tristetraprolin (TTP)-related tandem CCCH zinc finger (TZF) proteins to bind AU-rich element (ARE)-containing probes and to promote their deadenylation. HEK-293 cells were maintained, and transient transfection of 1.2 × 106 cells with expression plasmid constructs in calcium phosphate precipitates was performed, as described [22]. To each plate of HEK-293 cells was added 0.2 μg of the TZF protein expression constructs CMV.hTTP.tag (hTTP), a human TTP (hTTP) zinc finger mutant (C124R), CMV.cMG1.tag (cMG1), CMV.mTis11D.tag (mTis11D), 0.1 μg of human poly(A) exonuclease (hPARN) expression plasmid CMV.hPARN.flag (hPARN), or plasmid DNA alone (BS+). The zinc finger protein expression constructs were transfected either with vector alone or together with CMV.hPARN.flag; vector DNA (BS+) was added to each transfection to make the total amount of co-transfected DNA 5 μg per plate. Cytosolic extracts were prepared and used in deadenylation assays as described [23]. (a) Extracts (10 μg of protein per sample) were incubated with probes ARE or ARE-A50 at 37°C for 60 min in the presence (+) or absence (-) of 20 mM EDTA, as indicated. The samples were processed as described previously [23]. The arrow indicates the migration position of the ARE probe (lanes 1–6) and the deadenylated product of probe ARE-A50 (lanes 9, 11, 12, 14, 16 and 17). (b) The extracts used in lanes 7–13 of (a) were incubated with the ARE-A50 probe and used in a gel-shift assay. Lane 7 (P') was loaded with probe alone (digested with RNase T1). The migration positions of the zinc finger protein-RNA complexes are indicated by the bracket to the right of the gel, and the position of the free probe (FP) is also indicated. The bands present in the gel in lane 1 represent endogenous HEK-293 cell proteins shifting the probe; note that this pattern is identical in lane 3, representing a zinc finger mutant of TTP, and in lane 6, representing hPARN alone.

Mentions: As noted above, there are now known to be three members of the TZF protein family in humans, and extensive blasting of the human genome and EST collections has not yielded any further members, despite the presence of a group of sequences of closely related fourth members in fish and frogs [53]. Much less is known about the physiological roles of these proteins in mammalian systems. As shown in Fig. 7a, all three members of the family can bind readily to a TNF-α ARE probe, as demonstrated by RNA gel-shift analysis. In addition, all three family members can promote the deadenylation of ARE-containing polyadenylated RNA probes, both in intact cell transfection systems and in cell-free deadenylation assays (Fig. 7b) [23,54]. This occurs whether or not the proteins are used to 'effectively activate' endogenous deadenylating activities in HEK-293 cell extracts, or co-transfected PARN in the same cells (Fig. 7b). This and other types of evidence suggest that all three proteins have similar roles to TTP in the physiology of some cell types; that is, they are capable of binding to specific ARE sequences in certain transcripts and promoting their deadenylation and degradation. Many questions remain, including the following. First, in what cell types does each protein function as an mRNA destabilizing factor, and in what physiological or pathological situations? Second, how are these interactions regulated, by biosynthetic and post-translational events, as well as interactions with other cellular proteins? Third, what are the mRNA targets for each family member in normal physiology?


The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis.

Carrick DM, Lai WS, Blackshear PJ - Arthritis Res. Ther. (2004)

Effects of tristetraprolin (TTP)-related tandem CCCH zinc finger (TZF) proteins to bind AU-rich element (ARE)-containing probes and to promote their deadenylation. HEK-293 cells were maintained, and transient transfection of 1.2 × 106 cells with expression plasmid constructs in calcium phosphate precipitates was performed, as described [22]. To each plate of HEK-293 cells was added 0.2 μg of the TZF protein expression constructs CMV.hTTP.tag (hTTP), a human TTP (hTTP) zinc finger mutant (C124R), CMV.cMG1.tag (cMG1), CMV.mTis11D.tag (mTis11D), 0.1 μg of human poly(A) exonuclease (hPARN) expression plasmid CMV.hPARN.flag (hPARN), or plasmid DNA alone (BS+). The zinc finger protein expression constructs were transfected either with vector alone or together with CMV.hPARN.flag; vector DNA (BS+) was added to each transfection to make the total amount of co-transfected DNA 5 μg per plate. Cytosolic extracts were prepared and used in deadenylation assays as described [23]. (a) Extracts (10 μg of protein per sample) were incubated with probes ARE or ARE-A50 at 37°C for 60 min in the presence (+) or absence (-) of 20 mM EDTA, as indicated. The samples were processed as described previously [23]. The arrow indicates the migration position of the ARE probe (lanes 1–6) and the deadenylated product of probe ARE-A50 (lanes 9, 11, 12, 14, 16 and 17). (b) The extracts used in lanes 7–13 of (a) were incubated with the ARE-A50 probe and used in a gel-shift assay. Lane 7 (P') was loaded with probe alone (digested with RNase T1). The migration positions of the zinc finger protein-RNA complexes are indicated by the bracket to the right of the gel, and the position of the free probe (FP) is also indicated. The bands present in the gel in lane 1 represent endogenous HEK-293 cell proteins shifting the probe; note that this pattern is identical in lane 3, representing a zinc finger mutant of TTP, and in lane 6, representing hPARN alone.
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Related In: Results  -  Collection

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Figure 7: Effects of tristetraprolin (TTP)-related tandem CCCH zinc finger (TZF) proteins to bind AU-rich element (ARE)-containing probes and to promote their deadenylation. HEK-293 cells were maintained, and transient transfection of 1.2 × 106 cells with expression plasmid constructs in calcium phosphate precipitates was performed, as described [22]. To each plate of HEK-293 cells was added 0.2 μg of the TZF protein expression constructs CMV.hTTP.tag (hTTP), a human TTP (hTTP) zinc finger mutant (C124R), CMV.cMG1.tag (cMG1), CMV.mTis11D.tag (mTis11D), 0.1 μg of human poly(A) exonuclease (hPARN) expression plasmid CMV.hPARN.flag (hPARN), or plasmid DNA alone (BS+). The zinc finger protein expression constructs were transfected either with vector alone or together with CMV.hPARN.flag; vector DNA (BS+) was added to each transfection to make the total amount of co-transfected DNA 5 μg per plate. Cytosolic extracts were prepared and used in deadenylation assays as described [23]. (a) Extracts (10 μg of protein per sample) were incubated with probes ARE or ARE-A50 at 37°C for 60 min in the presence (+) or absence (-) of 20 mM EDTA, as indicated. The samples were processed as described previously [23]. The arrow indicates the migration position of the ARE probe (lanes 1–6) and the deadenylated product of probe ARE-A50 (lanes 9, 11, 12, 14, 16 and 17). (b) The extracts used in lanes 7–13 of (a) were incubated with the ARE-A50 probe and used in a gel-shift assay. Lane 7 (P') was loaded with probe alone (digested with RNase T1). The migration positions of the zinc finger protein-RNA complexes are indicated by the bracket to the right of the gel, and the position of the free probe (FP) is also indicated. The bands present in the gel in lane 1 represent endogenous HEK-293 cell proteins shifting the probe; note that this pattern is identical in lane 3, representing a zinc finger mutant of TTP, and in lane 6, representing hPARN alone.
Mentions: As noted above, there are now known to be three members of the TZF protein family in humans, and extensive blasting of the human genome and EST collections has not yielded any further members, despite the presence of a group of sequences of closely related fourth members in fish and frogs [53]. Much less is known about the physiological roles of these proteins in mammalian systems. As shown in Fig. 7a, all three members of the family can bind readily to a TNF-α ARE probe, as demonstrated by RNA gel-shift analysis. In addition, all three family members can promote the deadenylation of ARE-containing polyadenylated RNA probes, both in intact cell transfection systems and in cell-free deadenylation assays (Fig. 7b) [23,54]. This occurs whether or not the proteins are used to 'effectively activate' endogenous deadenylating activities in HEK-293 cell extracts, or co-transfected PARN in the same cells (Fig. 7b). This and other types of evidence suggest that all three proteins have similar roles to TTP in the physiology of some cell types; that is, they are capable of binding to specific ARE sequences in certain transcripts and promoting their deadenylation and degradation. Many questions remain, including the following. First, in what cell types does each protein function as an mRNA destabilizing factor, and in what physiological or pathological situations? Second, how are these interactions regulated, by biosynthetic and post-translational events, as well as interactions with other cellular proteins? Third, what are the mRNA targets for each family member in normal physiology?

Bottom Line: The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine.Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex.The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Clinical Research, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.

ABSTRACT
Tristetraprolin (TTP) is the best-studied member of a small family of three proteins in humans that is characterized by a tandem CCCH zinc finger (TZF) domain with highly conserved sequences and spacing. Although initially discovered as a gene that could be induced rapidly and transiently by the stimulation of fibroblasts with growth factors and mitogens, it is now known that TTP can bind to AU-rich elements in mRNA, leading to the removal of the poly(A) tail from that mRNA and increased rates of mRNA turnover. This activity was discovered after TTP-deficient mice were created and found to have a systemic inflammatory syndrome with severe polyarticular arthritis and autoimmunity, as well as medullary and extramedullary myeloid hyperplasia. The syndrome seemed to be due predominantly to excess circulating tumor necrosis factor-alpha (TNF-alpha), resulting from the increased stability of the TNF-alpha mRNA and subsequent higher rates of secretion of the cytokine. The myeloid hyperplasia might be due in part to increased stability of granulocyte-macrophage colony-stimulating factor (GM-CSF). This review highlights briefly the characteristics of the TTP-deficiency syndrome in mice and its possible genetic modifiers, as well as recent data on the characteristics of the TTP-binding site in the TNF-alpha and GM-CSF mRNAs. Recent structural data on the characteristics of the complex between RNA and one of the TTP-related proteins are reviewed, and used to model the TTP-RNA binding complex. We review the current knowledge of TTP sequence variants in humans and discuss the possible contributions of the TTP-related proteins in mouse physiology and in human monocytes. The TTP pathway of TNF-alpha and GM-CSF mRNA degradation is a possible novel target for anti-TNF-alpha therapies for rheumatoid arthritis, and also for other conditions proven to respond to anti-TNF-alpha therapy.

Show MeSH
Related in: MedlinePlus