2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana.

The role of the RNA degradation product 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) is poorly understood. Recent studies have identified 2',3'-cAMP in plant material and determined its role in stress signaling. The level of 2',3'-cAMP increases upon wounding, in the dark, and under heat, and 2',3'-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2',3'-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2',3'-cAMP treatment. We demonstrated that 2',3'-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2',3'-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2',3'-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2',3'-cAMP treatment influences the movement of processing bodies, confirming the role of 2',3'-cAMP in the formation and motility of membraneless organelles.

Zn(II) to Ag(I) Swap in Rad50 Zinc Hook Domain Leads to Interprotein Complex Disruption through the Formation of Highly Stable Agx(Cys)y Cores.

The widespread application of silver nanoparticles in medicinal and daily life products increases the exposure to Ag(I) of thiol-rich biological environments, which help control the cellular metallome. A displacement of native metal cofactors from their cognate protein sites is a known phenomenon for carcinogenic and otherwise toxic metal ions. Here, we examined the interaction of Ag(I) with the peptide model of the interprotein zinc hook (Hk) domain of Rad50 protein from Pyrococcus furiosus, a key player in DNA double-strand break (DSB) repair. The binding of Ag(I) to 14 and 45 amino acid long peptide models of apo- and Zn(Hk)2 was experimentally investigated by UV-vis spectroscopy, circular dichroism, isothermal titration calorimetry, and mass spectrometry. The Ag(I) binding to the Hk domain was found to disrupt its structure via the replacement of the structural Zn(II) ion by multinuclear Agx(Cys)y complexes. The ITC analysis indicated that the formed Ag(I)-Hk species are at least 5 orders of magnitude stronger than the otherwise extremely stable native Zn(Hk)2 domain. These results show that Ag(I) ions may easily disrupt the interprotein zinc binding sites as an element of silver toxicity at the cellular level.

An Extremely Stable Interprotein Tetrahedral Hg(Cys)4 Core Forms in the Zinc Hook Domain of Rad50 Protein at Physiological pH.

In nature, thiolate-based systems are the primary targets of divalent mercury (HgII ) toxicity. The formation of Hg(Cys)x cores in catalytic and structural protein centers mediates mercury's toxic effects and ultimately leads to cellular damage. Multiple studies have revealed distinct HgII -thiolate coordination preferences, among which linear HgII complexes are the most commonly observed in solution at physiological pH. Trigonal or tetrahedral geometries are formed at basic pH or in tight intraprotein Cys-rich metal sites. So far, no interprotein tetrahedral HgII complex formed at neutral pH has been reported. Rad50 protein is a part of the multiprotein MRN complex, a major player in DNA damage-repair processes. Its central region consists of a conserved CXXC motif that enables dimerization of two Rad50 molecules by coordinating ZnII . Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is critical for the biological activity of the MRN. Using a series of length-differentiated peptide models of the Pyrococcus furiosus zinc hook domain, we investigated its interaction with HgII . Using UV-Vis, CD, PAC, and 199 Hg NMR spectroscopies as well as anisotropy decay, we discovered that all Rad50 fragments preferentially form homodimeric Hg(Hk)2 species with a distorted tetrahedral HgS4 coordination environment at physiological pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in solution. At higher HgII content, monomeric HgHk complexes with linear geometry are formed. The Hg(Cys)4 core of Rad50 is extremely stable and does not compete with cyanides, NAC, or DTT. Applying ITC, we found that the stability constant of the Rad50 Hg(Hk)2 complex is approximately three orders of magnitude higher than those of the strongest HgII complexes known to date.

An Extremely Stable Interprotein Tetrahedral Hg(Cys)4 Core Forms in the Zinc Hook Domain of Rad50 Protein at Physiological pH.

Invited for the cover of this issue is the group of Artur Krezel at the University of Wroclaw in collaboration with Lars Hemmingsen at The University of Copenhagen and Eva Freisinger at the University of Zürich. The image depicts the outcomes of HgII interactions with Rad50 protein. Read the full text of the article at 10.1002/chem.202202738.

Metal Exchange in the Interprotein ZnII -Binding Site of the Rad50 Hook Domain: Structural Insights into CdII -Induced DNA-Repair Inhibition.

CdII is a major genotoxic agent that readily displaces ZnII in a multitude of zinc proteins, abrogates redox homeostasis, and deregulates cellular metalloproteome. To date, this displacement has been described mostly for cysteine(Cys)-rich intraprotein binding sites in certain zinc finger domains and metallothioneins. To visualize how a ZnII -to-CdII swap can affect the target protein's status and thus understand the molecular basis of CdII -induced genotoxicity an intermolecular ZnII -binding site from the crucial DNA repair protein Rad50 and its zinc hook domain were examined. By using a length-varied peptide base, ZnII -to-CdII displacement in Rad50's hook domain is demonstrated to alter it in a bimodal fashion: 1) CdII induces around a two-orders-of-magnitude stabilization effect (log K 12 Zn II =20.8 vs. log K 12 Cd II =22.7), which defines an extremely high affinity of a peptide towards a metal ion, and 2) the displacement disrupts the overall assembly of the domain, as shown by NMR spectroscopic and anisotropy decay data. Based on the results, a new model explaining the molecular mechanism of CdII genotoxicity that underlines CdII 's impact on Rad50's dimer stability and quaternary structure that could potentially result in abrogation of the major DNA damage response pathway is proposed.

Interaction of 2',3'-cAMP with Rbp47b Plays a Role in Stress Granule Formation.

2',3'-cAMP is an intriguing small molecule that is conserved among different kingdoms. 2',3'-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 2',3'-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 2',3'-cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 2',3'-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 2',3'-cAMP. Furthermore, SG formation was promoted in 2',3'-cAMP-treated Arabidopsis seedlings, and interactions between 2',3'-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 2',3'-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.

Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains.

Metal ions are essential elements present in biological systems able to facilitate many cellular processes including proliferation, signaling, DNA synthesis and repair. Zinc ion (Zn(II)) is an important cofactor for numerous biochemical reactions. Commonly, structural zinc sites demonstrate high Zn(II) affinity and compact architecture required for sequence-specific macromolecule binding. However, how Zn(II)-dependent proteins fold, how their dissociation occurs, and which factors modulate zinc protein affinity as well as stability remains not fully understood. The molecular rules governing precise regulation of zinc proteins function are hidden in the relationship between sequence and structure, and hence require deep understanding of their folding mechanism under metal load, reactivity and metal-to-protein affinity. Even though, this sequence-structure relationship has an impact on zinc proteins function, it has been shown that other biological factors including cellular localization and Zn(II) availability influence overall protein behavior. Taking into account all of the mentioned factors, in this review, we aim to describe the relationship between structure-function-stability of zinc structural sites, found in a zinc finger, zinc hook and zinc clasps, and reach far beyond a structural point of view in order to appreciate the balance between chemistry and biology that govern the protein world.