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Cadmium Exchange with Zinc in the Non-Classical Zinc Finger Protein Tristetraprolin.

ABSTRACT: Tristetraprolin (TTP) is a nonclassical CCCH zinc finger protein that regulates inflammation. TTP targets AU-rich RNA sequences of cytokine mRNAs forming a TTP/mRNA complex. This complex is then degraded, switching off the inflammatory response. Cadmium, a known carcinogen, triggers proinflammatory effects, and there is evidence that Cd increases TTP expression in cells, suggesting that Zn-TTP may be a target for cadmium toxicity. We sought to determine whether Cd exchanges with Zn in the TTP active site and measure the effect of RNA binding on this exchange. A construct of TTP that contains the two CCCH domains (TTP-2D) was employed to investigate these interactions. A spin-filter ICP-MS experiment to quantify the metal that is bound to the ZF after metal exchange was performed, and it was determined that Cd exchanges with Zn in Zn₂-TTP-2D and that Zn exchanges with Cd in Cd₂-TTP-2D. A native ESI-MS experiment to identify the metal-ZF complexes formed after metal exchange was performed, and M-TTP-2D complexes with singular and double metal exchange were observed. Metal exchange was measured in both the absence and presence of TTP's partner RNA, with retention of RNA binding. These data show that Cd can exchange with Zn in TTP without affecting function.

SUBMITTER: Brandis JEP

PROVIDER: S-EPMC8501473 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Zinc finger protein tristetraprolin interacts with CCL3 mRNA and regulates tissue inflammation.

Project description:Zinc finger protein tristetraprolin (TTP) modulates macrophage inflammatory activity by destabilizing cytokine mRNAs. In this study, through a screen of TTP-bound mRNAs in activated human macrophages, we have identified CCL3 mRNA as the most abundantly bound TTP target mRNA and have characterized this interaction via conserved AU-rich elements. Compared to the wild-type cells, TTP(-/-) macrophages produced higher levels of LPS-induced CCL3. In addition, the plasma level of CCL3 in TTP(-/-) mice was markedly higher than that in wild-type mice. To determine the in vivo significance of TTP-regulated CCL3, we generated CCL3(-/-)TTP(-/-) double-knockout mice. Along with decreased proinflammatory cytokines in their paw joints, there were significant functional and histologic improvements in the inflammatory arthritis of TTP(-/-) mice when CCL3 was absent, although cachexia, reflecting systemic inflammation, was notably unaffected. Furthermore, the marked exacerbation of aortic plaque formation caused by TTP deficiency in the APOE(-/-) mouse model of atherosclerosis was also rescued by disrupting CCL3. Taken together, our data indicate that the interaction between TTP and CCL3 mRNA plays an important role in modulating localized inflammatory processes in tissues that are dissociated from the systemic manifestations of chronic inflammation.

| S-EPMC3159726 | biostudies-literature

The tandem zinc finger RNA binding domain of members of the tristetraprolin protein family.

Project description:Tristetraprolin (TTP), the prototype member of the protein family of the same name, was originally discovered as the product of a rapidly inducible gene in mouse cells. Development of a knockout (KO) mouse established that absence of the protein led to a severe inflammatory syndrome, due in part to elevated levels of tumor necrosis factor (TNF). TTP was found to bind directly and with high affinity to specific AU-rich sequences in the 3'-untranslated region of the TNF mRNA. This initial binding led to promotion of TNF mRNA decay and inhibition of its translation. Many additional TTP target mRNAs have since been identified, some of which are cytokines and chemokines involved in the inflammatory response. There are three other proteins in the mouse with similar activities and domain structures, but whose KO phenotypes are remarkably different. Moreover, proteins with similar domain structures and activities have been found throughout eukaryotes, demonstrating that this protein family arose from an ancient ancestor. The defining characteristic of this protein family is the tandem zinc finger (TZF) domain, a 64 amino acid sequence with many conserved residues that is responsible for the direct RNA binding. We discuss here many aspects of this protein domain that have been elucidated since the original discovery of TTP, including its sequence conservation throughout eukarya; its apparent continued evolution in some lineages; its functional dependence on many key conserved residues; its "interchangeability" among evolutionarily distant species; and the evidence that RNA binding is required for the physiological functions of the proteins. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

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Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining.

Project description:DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a 'Ku-adaptor' that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.

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Targeting Zinc Finger Proteins with Exogenous Metals and Molecules: Lessons learned from Tristetraprolin, a CCCH type Zinc Finger.

Project description:ZF proteins are ubiquitous eukaryotic proteins that play important roles in gene regulation. ZFs contain small domains made up of a combination of four cysteine and histidine residues, and are classified based up on the identity of these residues and their spacing. One emerging class of ZFs are the Cys3His (or CCCH) class of ZFs. These ZFs play key roles in regulating RNA. In this minireview, an overview of the CCCH class of ZFs, with a focus on tristetraprolin (TTP) is provided. TTP regulates inflammation by controlling cytokine mRNAs, and there is an interest in modulating TTP activity to control inflammation. Two methods to control TTP activity are to target with exogenous metals (a 'metals in medicine' approach) or to target with endogenous signaling molecules. Work that has been done to target TTP with Fe, Cu, Cd and Au as well as with H2S is reviewed. This includes attention to new methods that have been developed to monitor metal exchange with the spectroscopically silent ZnII including native electro-spray ionization mass spectrometry (ESI-MS), spin-filter inductively coupled plasma mass spectrometry (ICP-MS) and cryo-electro-spray mass spectrometry (CSI-MS); along with fluorescence anisotropy (FA) to follow RNA binding.

| S-EPMC8635303 | biostudies-literature

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

Project description: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.

| S-EPMC1064869 | biostudies-literature

Mutational and structural analysis of the tandem zinc finger domain of tristetraprolin.

Project description:Tristetraprolin (TTP), the best known member of a class of tandem (R/K)YKTELCX8CX5CX3H zinc finger proteins, can destabilize target mRNAs by first binding to AU-rich elements (AREs) in their 3'-untranslated regions (UTRs) and subsequently promoting deadenylation and ultimate destruction of those mRNAs. This study sought to determine the roles of selected amino acids in the RNA binding domain, known as the tandem zinc finger (TZF) domain, in the ability of the full-length protein to bind to AREs within the tumor necrosis factor ? (TNF) mRNA 3'-UTR. Within the CX8C region of the TZF domain, mutation of some of the residues specific to TTP, not found in other members of the TTP protein family, resulted in decreased binding to RNA as well as inhibited mRNA deadenylation and decay. Evaluation of simulation solution models revealed a distinct structure in the second zinc finger of TTP that was induced by the presence of these TTP-specific residues. In addition, mutations within the lead-in sequences preceding the first C of highly conserved residues within the CX5C or CX3H regions or within the linker region between the two fingers also perturbed both RNA binding and the simulation model of the TZF domain in complex with RNA. We conclude that, although the majority of conserved residues within the TZF domain of TTP are required for productive binding, not all residues at sequence-equivalent positions in the two zinc fingers of the TZF domain of TTP are functionally equivalent.

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The RNA-binding zinc-finger protein tristetraprolin regulates AU-rich mRNAs involved in breast cancer-related processes.

Project description:Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc-finger RNA-binding protein that regulates the stability of certain AU-rich element (ARE) mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared with normal cell types. In this study we found that TTP expression was lower in invasive breast cancer cells (MDAMB231) compared with normal breast cell lines MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc-finger TTP mutant that functions as dominant negative and increases target mRNA expression. In contrast to wild-type TTP, C124R TTP was able to increase certain ARE-mRNA expressions in serum-stimulated breast cancer cells. Using an ARE-gene microarray, novel targets of TTP regulation were identified, namely, urokinase plasminogen activator (uPA), uPA receptor and matrix metalloproteinase-1, all known to have prominent roles in breast cancer invasion and metastasis. Expression of these targets was upregulated in tumorigenic types, particularly in highly invasive MDAMB231. The mRNA half-lives of these TTP-regulated genes were increased in TTP-knockout embryonic mouse fibroblasts, as assessed using real-time polymerase chain reaction, whereas forced restoration of TTP by transfection led to a reduction in their mRNA levels. RNA immunoprecipitation confirmed an association of TTP, but not C124R, with these target transcripts. Moreover, TTP reduced, whereas the mutant C124R TTP increased, the activity of reporter constructs fused to target ARE. As a result of TTP regulation, invasiveness of MDAMB231 cells was reduced. The data suggest that TTP, in a 3' untranslated region-and ARE-dependent manner, regulates an important subset of cancer-related genes that are involved in cellular growth, invasion and metastasis.

| S-EPMC3422647 | biostudies-literature

Structural Basis of the Disorder in the Tandem Zinc Finger Domain of the RNA-Binding Protein Tristetraprolin.

Project description:Tristetraprolin (TTP) and TIS11d are two human RNA-binding proteins that belong to the CCCH-type tandem zinc finger family. In the RNA-free state, TIS11d coordinates a zinc ion in each of its two fingers, while TTP coordinates a single zinc ion with the N-terminal zinc finger. We have previously identified three residues, located in the C-terminal half of a short α-helix in the second zinc finger, that control how structured the RNA-binding domain is in these two proteins: Y151, L152, and Q153 in TTP and H201, T202, and I203 in TIS11d. Here, we have used molecular dynamics, NMR spectroscopy, and other biochemical methods to investigate the role of these three residues in the stability of the RNA-binding domain. We found that the intrahelical hydrogen bond formed by the T202 hydroxyl group in the C-terminal zinc finger of TIS11d is necessary to allow for π-π stacking between the side chains of a conserved phenylalanine and the zinc-coordinating histidine. We demonstrated that the lack of this hydrogen bond in TTP is responsible for the reduced zinc affinity of the C-terminal zinc finger.

| S-EPMC5137377 | biostudies-literature

Zinc finger protein ZNF384 is an adaptor of KU to DNA during classical non-homologous end-joining

Project description:DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damages as they can lead to mutations and chromosomal rearrangements, which underlie cancer development and progression. Non-homologous end-joining (NHEJ) is the dominant pathway for the repair of DSBs in human cells, which involves the DNA binding proteins Ku70/Ku80. Other DNA binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair. However, their role in NHEJ has remained elusive. Here we show that ZNF384, which is a member of the C2H2 family of ZnF proteins that binds DNA in vitro, is recruited to DSBs in vivo. ZNF384 recruitment requires the PARP/PAR-dependent expansion of damaged chromatin followed by binding of ZNF384 to the exposed DNA via its unique C2H2 motifs. Mass-spectrometry-based experiments revealed that ZNF384 interacts with the Ku70/Ku80 NHEJ complex via its N-terminus. This interaction promotes the assembly of Ku70/Ku80 at DBSs as revealed by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. By regulating Ku70/80 dynamics, ZNF384 facilitates the assembly of several downstream NHEJ factors (e.g. APLF and XRCC4) and repair by cNHEJ at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds to DSBs and Ku70/80 to facilitate their binding and the subsequent build-up of a functional cNHEJ repairosome at these lesions, high-lighting a role for ZNF384 in DSB repair and genome maintenance.

2021-11-15 | PXD020417 | Pride

A role for hydrogen bonding in DNA recognition by the non-classical CCHHC type zinc finger, NZF-1.

Project description:The non-classical zinc finger protein, Neural Zinc Finger Factor-1, contains six Cys2His2Cys domains. All three cysteines and the second histidine directly bind Zn(II). Using a combination of mutagenesis, metal coordination and DNA binding studies, we report that the first histidine is involved in a functionally important hydrogen bonding interaction.

| S-EPMC4066409 | biostudies-literature

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