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1.
J Mol Biol ; 432(14): 3989-4009, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32304700

RESUMO

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.

2.
Nat Struct Mol Biol ; 25(3): 270-278, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29483648

RESUMO

Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. However, their exploitation has been hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression, screening and large-scale production of these peptides: 46 were successfully produced in multimilligram quantities, and >600 more were deemed expressible through stringent screening criteria. Many showed extreme resistance to temperature, proteolysis and/or reduction, and all displayed inhibitory activity against at least 1 of 20 ion channels tested, thus confirming their biological functionality. Crystal structures of 12 confirmed proper cystine topology and the utility of crystallography to study these molecules but also highlighted the need for rational classification. Previous categorization attempts have focused on limited subsets featuring distinct motifs. Here we present a global definition, classification and analysis of >700 structures of cystine-dense peptides, providing a unifying framework for these molecules.


Assuntos
Cistina/química , Peptídeos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Células HEK293 , Humanos , Canais Iônicos/antagonistas & inibidores , Modelos Moleculares , Biossíntese Peptídica , Peptídeos/classificação , Peptídeos/farmacologia
3.
Nat Chem ; 9(9): 843-849, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28837177

RESUMO

Berkelium (Bk) has been predicted to be the only transplutonium element able to exhibit both +III and +IV oxidation states in solution, but evidence of a stable oxidized Bk chelate has so far remained elusive. Here we describe the stabilization of the heaviest 4+ ion of the periodic table, under mild aqueous conditions, using a siderophore derivative. The resulting Bk(IV) complex exhibits luminescence via sensitization through an intramolecular antenna effect. This neutral Bk(IV) coordination compound is not sequestered by the protein siderocalin-a mammalian metal transporter-in contrast to the negatively charged species obtained with neighbouring trivalent actinides americium, curium and californium (Cf). The corresponding Cf(III)-ligand-protein ternary adduct was characterized by X-ray diffraction analysis. Combined with theoretical predictions, these data add significant insight to the field of transplutonium chemistry, and may lead to innovative Bk separation and purification processes.

4.
Nat Commun ; 7: 12973, 2016 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-27796299

RESUMO

Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe-/-) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway.


Assuntos
Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , Lipocalina-2/genética , Lipocalina-2/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Inflamação , Quelantes de Ferro , Sobrecarga de Ferro/genética , Rim/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , Mutação , Oxirredução , Ligação Proteica , Sideróforos , Transferrina/metabolismo
5.
Inorg Chem ; 55(22): 11930-11936, 2016 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-27802058

RESUMO

Targeted α therapy holds tremendous potential as a cancer treatment: it offers the possibility of delivering a highly cytotoxic dose to targeted cells while minimizing damage to surrounding healthy tissue. The metallic α-generating radioisotopes 225Ac and 227Th are promising radionuclides for therapeutic use, provided adequate chelation and targeting. Here we demonstrate a new chelating platform composed of a multidentate high-affinity oxygen-donating ligand 3,4,3-LI(CAM) bound to the mammalian protein siderocalin. Respective stability constants log ß110 = 29.65 ± 0.65, 57.26 ± 0.20, and 47.71 ± 0.08, determined for the EuIII (a lanthanide surrogate for AcIII), ZrIV, and ThIV complexes of 3,4,3-LI(CAM) through spectrophotometric titrations, reveal this ligand to be one of the most powerful chelators for both trivalent and tetravalent metal ions at physiological pH. The resulting metal-ligand complexes are also recognized with extremely high affinity by the siderophore-binding protein siderocalin, with dissociation constants below 40 nM and tight electrostatic interactions, as evidenced by X-ray structures of the protein:ligand:metal adducts with ZrIV and ThIV. Finally, differences in biodistribution profiles between free and siderocalin-bound 238PuIV-3,4,3-LI(CAM) complexes confirm in vivo stability of the protein construct. The siderocalin:3,4,3-LI(CAM) assembly can therefore serve as a "lock" to consolidate binding to the therapeutic 225Ac and 227Th isotopes or to the positron emission tomography emitter 89Zr, independent of metal valence state.


Assuntos
Quelantes/química , Complexos de Coordenação/química , Proteínas/química , Radioterapia/métodos , Tório/química , Zircônio/química , Animais , Complexos de Coordenação/farmacocinética , Feminino , Ligantes , Camundongos , Modelos Químicos , Distribuição Tecidual
6.
Proc Natl Acad Sci U S A ; 112(33): 10342-7, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26240330

RESUMO

Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin-transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein-ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications.


Assuntos
Elementos da Série Actinoide/química , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas/química , Elementos da Série Actinoide/farmacocinética , Quelantes/química , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Íons , Cinética , Elementos da Série dos Lantanídeos , Ligantes , Luminescência , Metais/química , Conformação Molecular , Centrais Nucleares , Fotoquímica , Ligação Proteica , Liberação Nociva de Radioativos , Espectrofotometria , Eletricidade Estática , Difração de Raios X
7.
PLoS Pathog ; 9(9): e1003639, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086134

RESUMO

The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies.


Assuntos
Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Receptores de Inositol 1,4,5-Trifosfato/imunologia , Animais , Anticorpos Monoclonais/genética , Autoanticorpos/genética , Autoantígenos/genética , Anticorpos Amplamente Neutralizantes , Cardiolipinas/genética , Cardiolipinas/imunologia , Regiões Determinantes de Complementaridade/genética , Epitopos/genética , Anticorpos Anti-HIV/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Camundongos , Camundongos Transgênicos , Proteoma/genética , Proteoma/imunologia
8.
Methods Mol Biol ; 252: 303-11, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15017059

RESUMO

Conditions and techniques that result in successful crystallization differ from RNA to RNA. However, there are some general principles that facilitate crystallization of most RNAs. Three procedures that were instrumental in obtaining well-ordered crystals of the hairpin ribozyme are described in this chapter. These are: i) the design of a series of candidate crystallization constructs; ii) the evaluation of conditions to obtain monodisperse RNA; and iii) the use of seeding techniques to separate nucleation and growth events during crystallization. These procedures can be usefully adapted for the crystallization of other RNAs.


Assuntos
RNA Catalítico/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X/métodos , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , RNA Catalítico/biossíntese , Taq Polimerase
9.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 8): 1521-4, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12876372

RESUMO

The human U1A RNA-binding domain (RBD1) adopts one of the most common protein folds, the RNA-recognition motif, and is a paradigm for understanding RNA-protein interactions. A 2.8 A resolution structure of the unbound RBD1 has previously been determined [Nagai et al. (1990). Nature (London), 348, 515-520] and revealed a well defined alpha/beta core with disordered termini. Using a longer construct, a 1.8 A resolution structure of the unbound domain was determined that reveals an ordered C-terminal helix. The presence of this helix is consistent with a solution structure of the free domain [Avis et al. (1996). J. Mol. Biol. 257, 398-411]; however, in the solution structure the helix occludes the RNA-binding surface. In the present structure, the helix occupies a position similar to that seen in a 1.9 A resolution RNA-RBD1 complex structure [Oubridge et al. (1994). Nature (London), 372, 432-438]. The crystals in this study were grown from 2.2 M sodium malonate. It is possible that the high salt concentration helps to orient the C-terminal helix in the RNA-bound conformation by strengthening hydrophobic interactions between the buried face of the helix and the alpha/beta core of the protein. Alternatively, the malonate (several molecules of which are bound in the vicinity of the RNA-binding surface) may mimic RNA.


Assuntos
Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U1/química , Motivos de Aminoácidos , Cristalização , Cristalografia por Raios X , Escherichia coli/enzimologia , Humanos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA/química , Proteínas Recombinantes/química
10.
Science ; 298(5597): 1421-4, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12376595

RESUMO

The hairpin ribozyme catalyzes sequence-specific cleavage of RNA through transesterification of the scissile phosphate. Vanadate has previously been used as a transition state mimic of protein enzymes that catalyze the same reaction. Comparison of the 2.2 angstrom resolution structure of a vanadate-hairpin ribozyme complex with structures of precursor and product complexes reveals a rigid active site that makes more hydrogen bonds to the transition state than to the precursor or product. Because of the paucity of RNA functional groups capable of general acid-base or electrostatic catalysis, transition state stabilization is likely to be an important catalytic strategy for ribozymes.


Assuntos
RNA Catalítico/química , RNA Catalítico/metabolismo , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Conformação de Ácido Nucleico , Oxigênio/química , Oxigênio/metabolismo , Vanadatos/química , Vanadatos/metabolismo
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