RESUMEN
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.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas Hierro-Azufre/metabolismo , Poli U/metabolismo , Precursores del ARN/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Cobalto/química , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Mutación , Unión Proteica , Precursores del ARN/genética , Zinc/química , Dedos de Zinc , alfa-Sinucleína/genéticaRESUMEN
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as "zinc-finger" domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe-2S cluster and uses one of the Cys3His domains; 2Fe-2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Hierro/química , Señales de Poliadenilación de ARN 3'/genética , ARN Mensajero/química , Azufre/química , Factores de Escisión y Poliadenilación de ARNm/química , Sitios de Unión , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Simulación por Computador , Humanos , Modelos Químicos , Poliadenilación/genética , Unión Proteica , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Copper is an essential nutrient that is toxic to cells when present in excess. The fungal pathogen Candida albicans employs several mechanisms to survive in the presence of excess copper, but the molecular pathways that govern these responses are not completely understood. We report that deletion of GPA2, which specifies a G-protein α subunit, confers increased resistance to excess copper and propose that the increased resistance is due to a combination of decreased copper uptake and an increase in copper chelation by metallothioneins. This is supported by our observations that a gpa2Δ/Δ mutant has reduced expression of the copper uptake genes, CTR1 and FRE7, and a marked decrease in copper accumulation following exposure to high copper levels. Furthermore, deletion of GPA2 results in an increased expression of the copper metallothionein gene, CRD2. Gpa2p functions upstream in the cyclic AMP (cAMP)-protein kinase A (PKA) pathway to govern hyphal morphogenesis. The copper resistance phenotype of the gpa2Δ/Δ mutant can be reversed by artificially increasing the intracellular concentration of cAMP. These results provide evidence for a novel role of the PKA pathway in regulation of copper homeostasis. Furthermore, the connection between the PKA pathway and copper homeostasis appears to be conserved in the pathogen Cryptococcus neoformans but not in the nonpathogenic Saccharomyces cerevisiae.
Asunto(s)
Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Cobre/toxicidad , Proteínas Fúngicas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Cadmio/toxicidad , Candida albicans/citología , Candida albicans/genética , Cisplatino/farmacología , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Fúngicas/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos/genética , Homeostasis/efectos de los fármacos , Homeostasis/genética , Hierro/toxicidad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Plata/toxicidadRESUMEN
ZIF268, a member of the classical zinc finger protein family, contains three Cys(2)His(2) zinc binding domains that together recognize the DNA sequence 5'-AGCGTGGGCGT-3'. These domains can be fused to an endonuclease to make a chimeric protein to target and cleave specific DNA sequences. A peptide corresponding to these domains, named ZIF268-3D, has been prepared to determine if the zinc finger domain itself can promote DNA cleavage when a redox active metal ion, Fe(II), is coordinated. The UV-vis absorption spectrum of Fe(II)-ZIF268-3D is indicative of Fe(II) coordination. Using fluorescence anisotropy, we demonstrate that Fe(II)-ZIF268-3D binds selectively to its target DNA in the same manner as Zn(II)-ZIF268-3D. In the presence of added oxidant, H(2)O(2) or O(2), DNA cleavage is not observed by Fe(II)-ZIF268-3D. Instead, the peptide itself is rapidly oxidized. Similarly, Zn(II)-ZIF268-3D and apo-ZIF268-3D are rapidly oxidized by H(2)O(2) or O(2), and we propose that ZIF268-3D is highly susceptible to oxidation.
Asunto(s)
Cisteína/química , Histidina/química , Peróxido de Hidrógeno/química , Oxígeno/química , Péptidos/química , Dedos de Zinc , ADN/química , Oxidación-ReducciónRESUMEN
Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.
Asunto(s)
Oligopéptidos/química , Péptidos/química , Dedos de Zinc , Secuencia de Aminoácidos , Secuencia Conservada , Cobre/química , Hidrólisis , Iones/química , Metales/química , Posición Específica de Matrices de Puntuación , Zinc/químicaRESUMEN
Tristetraprolin [(TTP), also known as NUP475 and TIS11] is a non-classical zinc finger protein that regulates inflammatory response via a protein-RNA interaction. Specifically, TTP recognizes AU-rich RNA sequences located on the 3' untranslated region of messenger RNA associated with cytokines. Recently, TTP was shown to be upregulated when cells were exposed to cadmium. Other types of zinc finger proteins have been shown to bind Cd(II), thus the Cd(II) binding properties of TTP were pursued. Metal binding titrations using Co(II) as a spectroscopic probe for Cd(II) were performed. Cd(II) was found to coordinate to the two Cys(3)His structural zinc sites of TTP (TTP-2D) with an upper-limit dissociation constant, K(d), of 3.5±0.1 nM. Upon reconstitution of TTP-2D with Cd(II), the protein recognized target RNA, UUUAUUUAUUU, with a dissociation constant, K(d), of 2.4±0.2 nM. The Cd(II)TTP-2D/RNA binding event was more sensitive to base mutations than the Zn(II)TTP-2D/RNA binding event. A single base mutation within the UUUAUUUAUUU oligomer decreased the Cd(II)TTP-2D binding affinity 50-fold and a double mutation decreased the affinity 1000-2000 fold. In comparison, only 2-fold and 15-25 fold changes for Zn(II)TTP-2D binding to the identical RNA sequences are measured.
Asunto(s)
Cadmio/química , ARN Mensajero/química , Tristetraprolina/química , Dedos de Zinc , Zinc/química , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Cobalto/química , Ratones , Mutación , Estructura Terciaria de Proteína , ARN Mensajero/genética , Especificidad por Sustrato , Tristetraprolina/genéticaRESUMEN
Zinc finger proteins utilize zinc for structural purposes: zinc binds to a combination of cysteine and histidine ligands in a tetrahedral coordination geometry facilitating protein folding and function. While much is known about the classical zinc finger proteins, which utilize a Cys(2)His(2) ligand set to coordinate zinc and fold into an anti-parallel beta sheet/alpha helical fold, there are thirteen other families of 'non-classical' zinc finger proteins for which relationships between metal coordination and protein structure/function are less defined. This 'Perspective' article focuses on two classes of these non-classical zinc finger proteins: Cys(3)His type zinc finger proteins and Cys(2)His(2)Cys type zinc finger proteins. These proteins bind zinc in a tetrahedral geometry, like the classical zinc finger proteins, yet they adopt completely different folds and target different oligonucleotides. Our current understanding of the relationships between ligand set, metal ion, fold and function for these non-classical zinc fingers is discussed.