Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Metal-induced oxidative stress and human plasma protein oxidation after SARS-CoV-2 infection.

Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis.

Pb(II) coordination to the nonclassical zinc finger tristetraprolin: retained function with an altered fold.

Tristetraprolin (TTP) is a nonclassical CCCH zinc finger (ZF) that plays a crucial role in regulating inflammation. TTP regulates cytokine mRNAs by specific binding of its two conserved ZF domains (CysX8CysX5CysX3His) to adenylate-uridylate-rich sequences (AREs) at the 3'-untranslated region, leading to degradation of the RNA. Dysregulation of TTP in animal models has demonstrated several cytokine-related syndromes, including chronic inflammation and autoimmune disorders. Exposure to Pb(II), a prevalent environmental toxin, is known to contribute to similar pathologies, in part by disruption of and/or competition with cysteine-rich metalloproteins. TTP's role during stress as a ubiquitous translational regulator of cell signaling (and dysfunction), which may underpin various phenotypes of Pb(II) toxicity, highlights the importance of understanding the interaction between TTP and Pb(II). The impact of Pb(II) binding on TTP's fold and RNA-binding function was analyzed via UV-Vis spectroscopy, circular dichroism, X-ray absorption spectroscopy, nuclear magnetic resonance spectroscopy, and fluorescence anisotropy. A construct containing the two ZF domains of TTP (TTP-2D) bound to Pb(II) with nanomolar affinity and exhibited a different geometry and fold in comparison to Zn2-TTP-2D. Despite the altered secondary structure, Pb(II)-substituted TTP-2D bound a canonical ARE sequence more selectively than Zn2-TTP-2D. Taken together, these data suggest that Pb(II) may interfere with proper TTP regulation and hinder the cell's ability to respond to inflammation.

Fe-S clusters masquerading as zinc finger proteins.

Metal ions are commonly found as protein co-factors in biology, and it is estimated that over a quarter of all proteins require a metal cofactor. The distribution and utilization of metals in biology has changed over time. As the earth evolved, the atmosphere became increasingly oxygen rich which affected the bioavailability of certain metals such as iron, which in the oxidized ferric form is significantly less soluble than its reduced ferrous counterpart. Additionally, proteins that utilize metal cofactors for structural purposes grew in abundance, necessitating the use of metal co-factors that are not redox active, such as zinc. One common class of Zn co-factored proteins are zinc finger proteins (ZFs). ZFs bind zinc utilizing cysteine and histidine ligands to promote structure and function. Bioinformatics has annotated 5% of the human genome as ZFs; however, many of these proteins have not been studied empirically. In recent years, examples of annotated ZFs that instead harbor Fe-S clusters have been reported. In this review we highlight four examples of mis-annotated ZFs: mitoNEET, CPSF30, nsp12, and Fep1 and describe methods that can be utilized to differentiate the metal-cofactor.

Radiographic presentation of artifactual dyed hair on lateral cephalograms, chemical processing, and forensic application: Novel case report.

Forensic assessment employs an array of methods to identify human remains. Radiologic examinations with panoramic radiographs, computed tomography scans, Waters view, and nuclear magnetic resonance imaging may offer evidentiary clues in challenging cases, such as mass disasters. In these cases, alternative forensic tools are used to narrow lists of target victims using their biological features. This study aims to I) report on the unusual radiographic aspect of chemicals used for hair dyeing, and II) discuss the potential forensic application of this finding for human identification. The case depicts an asymptomatic 14-years-old female who presented for orthodontic therapy. During radiographic examination on a lateral cephalogram, numerous thin radiopaque streaks were visible, extending to the posterior neck between the occipital region of the skull base and vertebra C6. Clinical investigations revealed that these were artifactual hair images (possibly documented for the first time in the scientific literature). Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the patient's scalp hair was performed for 10 heavy metals, including zinc, copper, iron, chromium, nickel, cadmium, tin, lead, antimony, and bismuth. Eight of these metals were detected at normal levels, ranging from 160 parts per million (ppm) for zinc to less than 1 ppm for nickel, cadmium, tin, lead, and antimony. Conversely, slightly elevated levels of chromium at 0.41 ppm and bismuth at 0.025 ppm were found in the hair sample. The distinctive radiographic presentation of artifactual hair images combined with the chemical properties of hair exposed to dye products may provide useful traces for human identification, especially in mass disasters.

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

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.

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

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.

Evaluation of the Physicochemical Properties of the Iron Nanoparticle Drug Products: Brand and Generic Sodium Ferric Gluconate.

Complex iron nanoparticle-based drugs are one of the oldest and most frequently administered classes of nanomedicines. In the US, there are seven FDA-approved iron nanoparticle reference drug products, of which one also has an approved generic drug product (i.e., sodium ferric gluconate (SFG)). These products are indicated for the treatment of iron deficiency anemia and are administered intravenously. On the molecular level, iron nanomedicines are colloids composed of an iron oxide core with a carbohydrate coating. This formulation makes nanomedicines more complex than conventional small molecule drugs. As such, these products are often referred to as nonbiological complex drugs (e.g., by the nonbiological complex drugs (NBCD) working group) or complex drug products (e.g., by the FDA). Herein, we report a comprehensive study of the physiochemical properties of the iron nanoparticle product SFG. SFG is the single drug for which both an innovator (Ferrlecit) and generic product are available in the US, allowing for comparative studies to be performed. Measurements focused on the iron core of SFG included optical spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), X-ray powder diffraction (XRPD), 57Fe Mössbauer spectroscopy, and X-ray absorbance spectroscopy (XAS). The analysis revealed similar ferric-iron-oxide structures. Measurements focused on the carbohydrate shell comprised of the gluconate ligands included forced acid degradation, dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and gel permeation chromatography (GPC). Such analysis revealed differences in composition for the innovator versus the generic SFG. These studies have the potential to contribute to future quality assessment of iron complex products and will inform on a pharmacokinetic study of two therapeutically equivalent iron gluconate products.

Understanding RNA Binding by the Nonclassical Zinc Finger Protein CPSF30, a Key Factor in Polyadenylation during Pre-mRNA Processing.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.

Cigalike electronic nicotine delivery systems e-liquids contain variable levels of metals.

Electronic nicotine delivery systems (ENDS) are prefilled, battery-operated products intended to deliver nicotine to the user via an inhaled complex aerosol formed by heating a liquid composed of propylene glycol and glycerol, also referred to as vegetable glycerin and collectively called e-liquid, that contains nicotine and various flavor ingredients. Since their introduction in 2006, the number of ENDS on the market has increased exponentially. Despite their growing ubiquity, the possible health risks associated with ENDS use remain poorly understood. One potential concern is the presence of toxic metals in the e-liquid and aerosol. Herein, we report the evaluation of the metal content in the e-liquids from a series of commercially available cigalike ENDS brands (various flavors) determined using inductively coupled plasma mass spectrometry (ICP-MS) following e-liquid extraction. Each brand of cigalike ENDS was purchased at least three times at retail outlets in the Baltimore, Maryland metropolitan region over a period of six months (September 2017 to February 2018). This allowed for comparison of batch-to-batch variability. Several potentially toxic metals, including lead, chromium, copper, and nickel were detected in the e-liquids. In addition, high variability in metal concentrations within and between brands and flavors was observed . The internal assembled parts of each cartridge were analyzed by X-ray imaging, before dissembling so that the materials used to manufacture each cartridge could be evaluated to determine the metals they contained. Following washing to remove traces of e-liquid, lead, chromium, copper and nickel were all detected in the cigalike ENDS prefilled cartridges, suggesting one potential source for the metals found in the e-liquids. Collectively, these findings can inform further evaluation of product design and manufacturing processes, including quantification of metal concentrations in e-liquids over foreseeable storage times, safeguards against high concentrations of metals in the e-liquid before and after aerosolization (by contact with a metal heating coil), and control over batch-to-batch variability.

Characterization of Acinetobacter baumannii Copper Resistance Reveals a Role in Virulence.

Acinetobacter baumannii is often highly drug-resistant and causes severe infections in compromised patients. These infections can be life threatening due to limited treatment options. Copper is inherently antimicrobial and increasing evidence indicates that copper containing formulations may serve as non-traditional therapeutics against multidrug-resistant bacteria. We previously reported that A. baumannii is sensitive to high concentrations of copper. To understand A. baumannii copper resistance at the molecular level, herein we identified putative copper resistance components and characterized 21 strains bearing mutations in these genes. Eight of the strains displayed a copper sensitive phenotype (pcoA, pcoB, copB, copA/cueO, copR/cusR, copS/cusS, copC, copD); the putative functions of these proteins include copper transport, oxidation, sequestration, and regulation. Importantly, many of these mutant strains still showed increased sensitivity to copper while in a biofilm. Inductively coupled plasma mass spectrometry revealed that many of these strains had defects in copper mobilization, as the mutant strains accumulated more intracellular copper than the wild-type strain. Given the crucial antimicrobial role of copper-mediated killing employed by the immune system, virulence of these mutant strains was investigated in Galleria mellonella; many of the mutant strains were attenuated. Finally, the cusR and copD strains were also investigated in the murine pneumonia model; both were found to be important for full virulence. Thus, copper possesses antimicrobial activity against multidrug-resistant A. baumannii, and copper sensitivity is further increased when copper homeostasis mechanisms are interrupted. Importantly, these proteins are crucial for full virulence of A. baumannii and may represent novel drug targets.

Unraveling the RNA Binding Properties of the Iron-Sulfur Zinc Finger Protein CPSF30.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a "zinc finger" protein that plays a crucial role in the transition of pre-mRNA to RNA. CPSF30 contains five conserved CCCH domains and a CCHC "zinc knuckle" domain. CPSF30 activity is critical for pre-mRNA processing. A truncated form of the protein, in which only the CCCH domains are present, has been shown to specifically bind AU-rich pre-mRNA targets; however, the RNA binding and recognition properties of full-length CPSF30 are not known. Herein, we report the isolation and biochemical characterization of full-length CPSF30. We report that CPSF30 contains one 2Fe-2S cluster in addition to five zinc ions, as measured by inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, and X-ray absorption spectroscopy. Utilizing fluorescence anisotropy RNA binding assays, we show that full-length CPSF30 has high binding affinity for two types of pre-mRNA targets, AAUAAA and polyU, both of which are conserved sequence motifs present in the majority of pre-mRNAs. Binding to the AAUAAA motif requires that the five CCCH domains of CPSF30 be present, whereas binding to polyU sequences requires the entire, full-length CPSF30. These findings implicate the CCHC "zinc knuckle" present in the full-length protein as being critical for mediating polyU binding. We also report that truncated forms of the protein, containing either just two CCCH domains (ZF2 and ZF3) or the CCHC "zinc knuckle" domain, do not exhibit any RNA binding, indicating that CPSF30/RNA binding requires several ZF (and/or Fe-S cluster) domains working in concert to mediate RNA recognition.

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Role of Gold in Inflammation and Tristetraprolin Activity.

The zinc finger protein tristetraprolin (TTP) regulates inflammation by downregulating cytokine mRNAs. Misregulation results in arthritis, sepsis and cancer, and there is an interest in modulating TTP activity with exogenous agents. Gold has anti-inflammatory properties and has recently been shown to modulate the signaling pathway that produces TTP, suggesting that TTP may be a target of gold. The reactivity of [AuIII (terpy)Cl]Cl2 with TTP was investigated by UV/Vis spectroscopy, spin-filter inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy and native electrospray ionization mass spectrometry. AuIII was found to replace zinc in the protein active site in the reduced AuI form, with the AuI ion coordinated to two cysteine residues in a linear geometry. The replacement of ZnII with AuI results in loss of both secondary structure and RNA binding function. In contrast, when ZnII TTP is bound to its RNA target, no replacement of ZnII with AuI is observed, even in the presence of excess AuIII terpy. This discovery of differential reactivity of gold with TTP versus TTP/RNA offers a potential strategy for selective targeting with gold complexes to control inflammation.

Direct Zinc Finger Protein Persulfidation by H2 S Is Facilitated by Zn2.

H2 S is a gaseous signaling molecule that modifies cysteine residues in proteins to form persulfides (P-SSH). One family of proteins modified by H2 S are zinc finger (ZF) proteins, which contain multiple zinc-coordinating cysteine residues. Herein, we report the reactivity of H2 S with a ZF protein called tristetraprolin (TTP). Rapid persulfidation leading to complete thiol oxidation of TTP mediated by H2 S was observed by low-temperature ESI-MS and fluorescence spectroscopy. Persulfidation of TTP required O2 , which reacts with H2 S to form superoxide, as detected by ESI-MS, a hydroethidine fluorescence assay, and EPR spin trapping. H2 S was observed to inhibit TTP function (binding to TNFα mRNA) by an in vitro fluorescence anisotropy assay and to modulate TNFα in vivo. H2 S was unreactive towards TTP when the protein was bound to RNA, thus suggesting a protective effect of RNA.

Measuring Intracellular Metal Concentration via ICP-MS Following Copper Exposure.

This chapter describes protocols for measuring fluctuation in intracellular metal concentration of A. baumannii isolates in response to copper exposure using inductively coupled plasma mass spectrometry (ICP-MS).

Snapshots of Iron Speciation: Tracking the Fate of Iron Nanoparticle Drugs via a Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometric Approach.

Nanomedicines are nanoparticle-based therapeutic or diagnostic agents designed for targeted delivery or enhanced stability. Nanotechnology has been successfully employed to develop various drug formulations with improved pharmacokinetic characteristics, and current research efforts are focused on the development of new innovator and generic nanomedicines. Nanomedicines, which are often denoted as complex or nonbiological complex drugs, have inherently different physicochemical and pharmacokinetic properties than conventional small molecule drugs. The tools necessary to fully evaluate nanomedicines in clinical settings are limited, which can hamper their development. One of the most successful families of nanomedicines are iron-carbohydrate nanoparticles, which are administered intravenously (IV) to treat iron-deficiency anemia. In the U.S., the FDA has approved six distinct iron-carbohydrate nanoparticles but only one generic version (sodium ferric gluconate for Ferrlecit). There is significant interest in approving additional generic iron-carbohydrate drugs; however, the lack of a direct method to monitor the fate of the iron nanoparticles in clinical samples has impeded this approval. Herein we report a novel liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) method that allows for the direct quantification of the iron-carbohydrate drugs in clinical samples, while simultaneously measuring the speciation of the iron released from the nanoparticles in biological samples. To our knowledge, this is the first time that iron nanoparticles have been observed in clinical samples, opening the door for direct pharmacokinetic studies of this family of drugs. This method has potential applications not only for iron-nanoparticle drugs but also for any nanomedicine with an inorganic component.

Iron-Sulfur Clusters in Zinc Finger Proteins.

Zinc finger (ZF) proteins are proteins that use zinc as a structural cofactor. The common feature among all ZFs is that they contain repeats of four cysteine and/or histidine residues within their primary amino acid sequence. With the explosion of genome sequencing in the early 2000s, a large number of proteins were annotated as ZFs based solely upon amino acid sequence. As these proteins began to be characterized experimentally, it was discovered that some of these proteins contain iron-sulfur sites either in place of or in addition to zinc. Here, we describe methods to isolate and characterize one such ZF protein, cleavage and polyadenylation specificity factor 30 (CPSF3O) with respect to its metal-loading and RNA-binding activity.

Equivalence and regulatory approaches of nonbiological complex drug products across the United States, the European Union, and Turkey.

Regulatory agencies around the world may have different standards and approaches to evaluate and approve drug products and biological products. We describe the U.S. Food and Drug Administration's (FDA) Generic Drug User Fee Act program, as well as their approach to complex products. We discuss regulatory approaches for the development of nonbiological complex drug follow-ons and approval pathways in the United States. We compare FDA policies with other regulatory agencies (i.e., the European Medicines Agency and the Turkish Medicines and Medical Devices Agency). In particular, we describe the policies/pathways across these three agencies to assess equivalence of glatiramer acetate, enoxaparin sodium, and sodium ferric gluconate complex products. We also examine the Turkish market for these selected nonbiological complex drugs.

Cu(I) Disrupts the Structure and Function of the Nonclassical Zinc Finger Protein Tristetraprolin (TTP).

Tristetraprolin (TTP) is a nonclassical zinc finger (ZF) protein that plays a key role in regulating inflammatory response. TTP regulates cytokines at the mRNA level by binding to AU-rich sequences present at the 3'-untranslated region, forming a complex that is then degraded. TTP contains two conserved CCCH domains with the sequence CysX8CysX5CysX3His that are activated to bind RNA when zinc is coordinated. During inflammation, copper levels are elevated, which is associated with increased inflammatory response. A potential target for Cu(I) during inflammation is TTP. To determine whether Cu(I) binds to TTP and how Cu(I) can affect TTP/RNA binding, two TTP constructs were prepared. One construct contained just the first CCCH domain (TTP-1D) and serves as a peptide model for a CCCH domain; the second construct contains both CCCH domains (TTP-2D) and is functional (binds RNA) when Zn(II) is coordinated. Cu(I) binding to TTP-1D was assessed via electronic absorption spectroscopy titrations, and Cu(I) binding to TTP-2D was assessed via both absorption spectroscopy and a spin filter/inductively coupled plasma mass spectrometry (ICP-MS) assay. Cu(I) binds to TTP-1D with a 1:1 stoichiometry and to TTP-2D with a 3:1 stoichiometry. The CD spectrum of Cu(I)-TTP-2D did not exhibit any secondary structure, matching that of apo-TTP-2D, while Zn(II)-TTP-2D exhibited a secondary structure. Measurement of RNA binding via fluorescence anisotropy revealed that Cu(I)-TTP-2D does not bind to the TTP-2D RNA target sequence UUUAUUUAUUU with any measurable affinity, while Zn(II)-TTP-2D binds to this site with nanomolar affinity. Similarly, addition of Cu(I) to the Zn(II)-TTP-2D/RNA complex resulted in inhibition of RNA binding. Together, these data indicate that, while Cu(I) binds to TTP-2D, it does not result in a folded or functional protein and that Cu(I) inhibits Zn(II)-TTP-2D/RNA binding.