RESUMEN

Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal ß-hexosaminidase A (HexA). We report that (1) recombinant HEXA alone increased HexA activity and decreased GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein composed of HEXA linked to two blood-brain barrier (BBB) entry elements, a transferrin receptor binding sequence and granulocyte-colony stimulating factor, associates with HEXB in vitro; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, in vivo; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by approximately 40% within 6 weeks, when injected intravenously (IV) to adult TS-mutant mice mimicking the slow course of late-onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, decrease the accumulation of GM2, and improve muscle strength, thereby providing potential treatment for late-onset TS.

RESUMEN

The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtype-selective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the Nav1.7 channel based on tarantula ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to the Nav1.7 channel. The resulting microproteins are highly potent (IC50 to Nav1.7 of 2.5 nm) and selective. We achieved 80- and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000 nm The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

Asunto(s)

Canal de Sodio Activado por Voltaje NAV1.7/química , Manejo del Dolor/métodos , Ingeniería de Proteínas , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Células HEK293 , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Técnicas de Placa-Clamp , Filogenia , Venenos de Araña/química

RESUMEN

Increasing the in vivo residence times of protein therapeutics could decrease their dosing frequencies. We show that genetic fusion of an unstructured recombinant polypeptide of 864 amino acids, called XTEN, to a peptide or protein provides an apparently generic approach to extend plasma half-life. Allometric scaling suggests that a fusion of XTEN to the exenatide peptide should increase exenatide half-life in humans from 2.4 h to a projected time of 139 h. We confirmed the biological activity of the exenatide-XTEN fusion in mice. As extended stability might exacerbate undesirable side effects in some cases, we show that truncating the XTEN sequence can regulate plasma half-life. XTEN lacks hydrophobic amino acid residues that often contribute to immunogenicity and complicate manufacture. Based on data on XTEN fusions to exenatide, glucagon, GFP and human growth hormone, we expect that XTEN will enable dosing of otherwise rapidly cleared protein drugs at up to monthly intervals in humans.

Asunto(s)

Péptidos/química , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Animales , Ratones , Proteínas Recombinantes de Fusión/sangre

RESUMEN

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP(+)/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450-458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal-ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal-ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.

Asunto(s)

Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/metabolismo , Bacterias Anaerobias/enzimología , Dominio Catalítico , Cobalto/metabolismo , Zinc/metabolismo , Catálisis , Cristalografía por Rayos X , Ligandos , Agua/metabolismo

RESUMEN

Collagen VIII is a major component of Descemet's membrane and is also found in vascular subendothelial matrices. The C-terminal non-collagenous domain (NC1) domain of collagen VIII, which is a member of the C1q-like protein family, forms a stable trimer and is thought to direct the assembly of the collagen triple helix, as well as polygonal supramolecular structures. We have solved the crystal structure of the mouse alpha1(VIII)(3) NC1 domain trimer at 1.9 A resolution. Each subunit of the intimate NC1 trimer consists of a ten-stranded beta-sandwich. The surface of the collagen VIII NC1 trimer presents three strips of partially exposed aromatic residues shown to interact with the non-ionic detergent CHAPS, which are likely to be involved in supramolecular assemblies. Equivalent strips exist in the NC1 domain of the closely related collagen X, suggesting a conserved assembly mechanism. Surprisingly, the collagen VIII NC1 trimer lacks the buried calcium cluster of the collagen X NC1 trimer. The mouse alpha1(VIII) and alpha2(VIII) NC1 domains are 71.5% identical in sequence, with the differences being concentrated on the NC1 trimer surface. A few non-conservative substitutions map to the subunit interfaces near the surface, but it is not obvious from the structure to what extent they determine the preferred assembly of collagen VIII alpha1 and alpha2 chains into homotrimers.

Asunto(s)

Colágeno Tipo VIII/química , Secuencia de Aminoácidos/genética , Animales , Colágeno Tipo VIII/genética , Colágeno Tipo X/química , Cristalografía , Ratones , Datos de Secuencia Molecular , Estructura Molecular , Estructura Terciaria de Proteína

RESUMEN

gp210 is a major constituent of the nuclear pore complex (NPC) with possible structural and regulatory roles. It interacts with components of the NPC via its C-terminal domain (CTD), which follows a transmembrane domain and a massive ( approximately 200 kDa) N-terminal region that resides in the lumen of the perinuclear space. Here, we report the solution structure of the human gp210 CTD as determined by various spectroscopic techniques. In water, the CTD adopts an extended, largely unordered conformation, which contains a significant amount of left-handed polyproline type II (PII) helical structure. The conformation of the CTD is altered by high pH, charged detergents, and the hydrogen bond-promoting reagent trifluoroethanol (TFE), which decrease the PII fraction of the fragment. TFE also induces a conformational change in a region containing an SPXX motif whose serine becomes specifically phosphorylated during mitosis. We propose that PII elements in the CTD may play a role in its interaction with the NPC and may serve as recognition sites for regulatory proteins bearing WW or other, unknown PII-binding motifs.

Asunto(s)

Glicoproteínas de Membrana/química , Poro Nuclear/química , Proteínas Nucleares/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Secuencia Conservada , Drosophila/genética , Proteínas de Drosophila/genética , Humanos , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/genética , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Soluciones , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , Xenopus/genética , Proteínas de Xenopus

RESUMEN

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.

Asunto(s)

Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/genética , Clostridium/enzimología , Alcohol Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Clostridium/genética , Cristalografía por Rayos X , Estabilidad de Enzimas , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Sales (Química)/química , Alineación de Secuencia , Temperatura

RESUMEN

Collagen X is expressed specifically in the growth plate of long bones. Its C1q-like C-terminal NC1 domain forms a stable homotrimer and is crucial for collagen X assembly. Mutations in the NC1 domain cause Schmid metaphyseal chondrodysplasia (SMCD). The crystal structure at 2.0 A resolution of the human collagen X NC1 domain reveals an intimate trimeric assembly strengthened by a buried cluster of calcium ions. Three strips of exposed aromatic residues on the surface of NC1 trimer are likely to be involved in the supramolecular assembly of collagen X. Most internal SMCD mutations probably prevent protein folding, whereas mutations of surface residues may affect the collagen X suprastructure in a dominant-negative manner.

Asunto(s)

Colágeno Tipo X/química , Exostosis Múltiple Hereditaria/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Colágeno Tipo X/genética , Cristalografía por Rayos X , Exostosis Múltiple Hereditaria/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Homología de Secuencia de Aminoácido , Solventes , Relación Estructura-Actividad , Propiedades de Superficie