RESUMO
The cellular prion protein (PrPC) is a membrane-anchored copper binding protein that undergoes proteolytic processing. ß-cleavage of PrPC is associated with a pathogenic condition and it yields two fragments: N2 with residues 23-89, and C2 including residues 90-231. The membrane-bound C2 fragment retains the Cu binding sites at His96 and His111, but it also has a free N-terminal NH2 group. In this study, the impact of ß-cleavage of PrPC in its Cu(II) binding properties was evaluated, using the peptide of the human prion protein hPrP(90-115) as a model for the C2 fragment. The Cu(II) coordination properties of hPrP(90-115) were studied using circular dichroism (CD) and electron paramagnetic resonance (EPR); while the H96A and H111A substitutions and its acetylated variants were also studied. Cu binding to hPrP(90-115) is dependent on metal ion concentration: At low copper concentrations the participation of His96 and free NH2-terminus is evident, while at high copper concentrations the His111 site is populated without participation of the N-terminal NH2 group. The presence of a free NH2-terminal group in the C2 fragment significantly impacts the Cu(II) coordination properties of the His96 site, where the NH2 group also anchors the metal ion. This study provides further insights into the impact of proteolytic processing of PrPC in the Cu binding properties of this important neuronal protein.
Assuntos
Cobre/química , Doenças Priônicas/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Sítios de Ligação , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Histidina/química , Humanos , Peptídeos/química , Príons/química , Príons/metabolismo , Ligação ProteicaRESUMO
In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Transporte/química , Cloroplastos/enzimologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroplastos/genética , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leucina/química , Leucina/metabolismo , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways.
Assuntos
Técnicas Biossensoriais , Humanos , Metionina , Metionina Sulfóxido Redutases/metabolismo , Oxirredução , Especificidade por SubstratoRESUMO
The N-terminus of the prion protein is a large intrinsically disordered region encompassing approximately 125 amino acids. In this paper, we review its structural and functional properties, with a particular emphasis on its binding to copper ions. The latter is exploited by the region's conformational flexibility to yield a variety of biological functions. Disease-linked mutations and proteolytic processing of the protein can impact its copper-binding properties, with important structural and functional implications, both in health and disease progression.
Assuntos
Cobre/química , Cobre/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Humanos , Modelos Moleculares , Proteínas Priônicas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Relação Estrutura-AtividadeRESUMO
The cellular prion protein (PrPC) is a copper binding protein that undergoes post-translational modifications, such as endoproteolytic alpha cleavage, which occurs in the vicinity of the His111 Cu binding site. Alpha cleavage processing of PrPC is considered to be neuroprotective since the cleavage site is located in a region that is key to the conversion of PrPC into the infectious scrapie isoform (PrPSc), yielding a membrane bound C1 fragment of PrPC that still contains His111. In this work, we use hPrP(111-115) fragment as a model peptide to evaluate the impact of alpha cleavage processing of PrPC in its ability to coordinate Cu(ii) ions at His111. By using different spectroscopic techniques such as electronic absorption, circular dichroism, nuclear magnetic resonance, and electron paramagnetic resonance, this study demonstrates that Cu(ii) binding to the cleaved His111 site is highly dependent on Cu and proton concentrations. The imidazole group of His111 and its free NH2 terminus emerge as the main anchoring sites for Cu(ii) coordination, yielding very different complexes from those characterized for the intact His111 site in the full protein. Different Cu(ii) coordination modes that could form with the alpha cleaved PrPC under physiological conditions are identified and characterized. Overall, this study contributes to understand how alpha cleavage processing of PrPC impacts its Cu(ii) binding properties at His111. While the functional implications of Cu binding to the cleaved PrPC remain to be discovered, proteolytic processing of PrPC and its Cu binding features appear to be molecular events that might be strongly linked to its cellular function.
RESUMO
Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits found in pancreatic ß-cells of patients with type 2 diabetes (T2D). Copper ions have an inhibitory effect on the amyloid aggregation of hIAPP, and they may play a role in the etiology of T2D. However, deeper knowledge of the structural details of the copper-hIAPP interaction is required to understand the molecular mechanisms involved. Here, we performed a spectroscopic study of Cu(II) binding to hIAPP and several variants, using electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), electronic absorption, and circular dichroism (CD) in the UV-vis region in combination with Born-Oppenheimer molecular dynamics (BOMD) and density functional theory geometry optimizations. We find that Cu(II) binds to the imidazole N1 of His18, the deprotonated amides of Ser19 and Ser20, and an oxygen-based ligand provided by Ser20, either via its hydroxyl group or its backbone carbonyl, while Asn22 might also play a role as an axial ligand. Ser20 plays a crucial role in stabilizing Cu(II) coordination toward the C-terminal, providing a potential link between the S20G mutation associated with early onset of T2D, its impact in Cu binding properties, and hIAPP amyloid aggregation. Our study defines the nature of the coordination environment in the Cu(II)-hIAPP complex, revealing that the amino acid residues involved in metal ion binding are also key residues for the formation of ß-sheet structures and amyloid fibrils. Cu(II) binding to hIAPP may lead to the coexistence of more than one coordination mode, which in turn could favor different sets of Cu-induced conformational ensembles. Cu-induced hIAPP conformers would display a higher energetic barrier to form amyloid fibrils, hence explaining the inhibitory effect of Cu ions in hIAPP aggregation. Overall, this study provides further structural insights into the bioinorganic chemistry of T2D.
RESUMO
Conversion of prion protein (PrP) to an altered conformer, the scrapie PrP (PrP(Sc)), is a critical step in the development of transmissible spongiform encephalopathies. Both Cu(II) and nucleic acid molecules have been implicated in this conversion. Full-length PrP can bind up to six copper ions; four Cu(II) binding sites are located in the octarepeat domain (residues 60-91), and His-96 and His-111 coordinate two additional copper ions. Experimental evidence shows that PrP binds different molecules, resulting in diverse cellular signaling events. However, there is little information about the interaction of macromolecular ligands with Cu(II)-bound PrP. Both RNA and DNA sequences can bind PrP, and this interaction results in reciprocal conformational changes. Here, we investigated the interaction of Cu(II) and nucleic acids with amyloidogenic non-octarepeat PrP peptide models (comprising human PrP residues 106-126 and hamster PrP residues 109-149) that retain His-111 as the copper-anchoring residue. The effect of Cu(II) and DNA or RNA sequences in the aggregation, conformation, and toxicity of PrP domains was investigated at low and neutral pH. Circular dichroism and EPR spectroscopy data indicate that interaction of the PrP peptides with Cu(II) and DNA occurs at pH 7. This dual interaction induces conformational changes in the peptides, modulating their aggregation, and affecting the morphology of the aggregated species, resulting in different cytotoxic effects. These results provide new insights into the role of Cu(II) and nucleic acid sequences in the structural conversion and aggregation of PrP, which are both critical events related to prion pathogenesis.