Bioavailable affinity label for collagen prolyl 4-hydroxylase.

Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the nonheme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases.

Site-specific folate conjugation to a cytotoxic protein.

Conjugation to folic acid is known to enhance the uptake of molecules by human cells that over-produce folate receptors. Variants of bovine pancreatic ribonuclease (RNase A) that have attenuated affinity for the endogenous ribonuclease inhibitor protein (RI) are toxic to mammalian cells. Here, the random acylation of amino groups in wild-type RNase A with folic acid is shown to decrease its catalytic activity dramatically, presumably because of the alteration to a key active-site residue, Lys41. To effect site-specific coupling, N(δ)-bromoacetyl-N(α)-pteroyl-l-ornithine, which is a folate analogue with an electrophilic bromoacetamido group, was synthesized and used to S-alkylate Cys88 of the G88C variant of RNase A. The pendant folate moiety does not decrease enzymatic activity, enables RI-evasion, and endows toxicity for cancer cells that over-produce the folate receptor. These data reveal a propitious means for targeting proteins and other molecules to cancer cells.

Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p.

Pumilio/FBF (PUF) family proteins are found in eukaryotic organisms and regulate gene expression post-transcriptionally by binding to sequences in the 3' untranslated region of target transcripts. PUF proteins contain an RNA binding domain that typically comprises eight alpha-helical repeats, each of which recognizes one RNA base. Some PUF proteins, including yeast Puf4p, have altered RNA binding specificity and use their eight repeats to bind to RNA sequences with nine or ten bases. Here we report the crystal structures of Puf4p alone and in complex with a 9-nucleotide (nt) target RNA sequence, revealing that Puf4p accommodates an 'extra' nucleotide by modest adaptations allowing one base to be turned away from the RNA binding surface. Using structural information and sequence comparisons, we created a mutant Puf4p protein that preferentially binds to an 8-nt target RNA sequence over a 9-nt sequence and restores binding of each protein repeat to one RNA base.

Production of human prolyl 4-hydroxylase in Escherichia coli.

Prolyl 4-hydroxylase (P4H) catalyzes the post-translational hydroxylation of proline residues in collagen strands. The enzyme is an alpha2beta2 tetramer in which the alpha subunits contain the catalytic active sites and the beta subunits (protein disulfide isomerase) maintain the alpha subunits in a soluble and active conformation. Heterologous production of the native alpha2beta2 tetramer is challenging and had not been reported previously in a prokaryotic system. Here, we describe the production of active human P4H tetramer in Escherichia coli from a single bicistronic vector. P4H production requires the relatively oxidizing cytosol of Origami B(DE3) cells. Induction of the wild-type alpha(I) cDNA in these cells leads to the production of a truncated alpha subunit (residues 235-534), which assembles with the beta subunit. This truncated P4H is an active enzyme, but has a high Km value for long substrates. Replacing the Met235 codon with one for leucine removes an alternative start codon and enables production of full-length alpha subunit and assembly of the native alpha2beta2 tetramer in E. coli cells to yield 2 mg of purified P4H per liter of culture (0.2 mg/g of cell paste). We also report a direct, automated assay of proline hydroxylation using high-performance liquid chromatography. We anticipate that these advances will facilitate structure-function analyses of P4H.

Activation of the prolyl hydroxylase oxygen-sensor results in induction of GLUT1, heme oxygenase-1, and nitric-oxide synthase proteins and confers protection from metabolic inhibition to cardiomyocytes.

Recently an oxygen-sensing/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase domain-containing enzymes (PHD). In normoxia, PHD hydroxylates a specific proline residue that directs the degradation of constitutively synthesized hypoxia-inducible factor-1alpha. During hypoxia, the cessation of hydroxylation of this proline results in less degradation and thus increases hypoxia-inducible factor-1alpha protein levels. In this study we have examined the consequences of activating the PHD oxygen-sensing pathway in cultured neonatal myocytes using ethyl-3,4 dihydroxybenzoate and dimethyloxalylglycine, inhibitors that, similar to hypoxia, inhibit this family of O2-dependent PHD enzymes. Increased glucose uptake and enhanced glycolytic metabolism are classical cellular responses to hypoxia. Ethyl-3,4 dihydroxybenzoate treatment of cardiomyocyte cultures for 24 h increased [3H]deoxy-4-glucose uptake concurrent with an induction of GLUT1 protein. In addition, ethyl-3,4 dihydroxybenzoate, dimethyloxalylglycine, and hypoxia treatments were found to induce protein levels of nitricoxide synthase-2 and heme oxygenase-1, two important cardioregulatory proteins whose expression in response to hypoxic conditions is poorly understood. In conjunction with these changes in gene expression, activation of the PHD oxygen-sensing mechanism was found to preserve myocyte viability in the face of metabolic inhibition with cyanide and 2-deoxyglucose. These results point to a key role for the PHD pathway in the phenotypic changes that are observed in a hypoxic myocyte and may suggest a strategy to pharmacologically induce protection in heart.

Zinc(II)-mediated inhibition of a ribonuclease by an N-hydroxyurea nucleotide.

The inhibition of ribonuclease Bi by 3'-N-hydroxyurea-3'-deoxythymidine 5'-phosphate is enhanced by 30-fold in the presence of Zn(2+). Thus, an N-hydroxyurea nucleotide can recruit Zn(2+) to inhibit the enzymatic activity of a ribonuclease. This result engenders a general strategy for the inhibition of non-metalloenzymes by metal complexes.

Zinc(II)-mediated inhibition of ribonuclease Sa by an N-hydroxyurea nucleotide and its basis.

Ribonuclease Sa (RNase Sa) is a secretory ribonuclease from Streptomyces aureofaciens. Herein, 3'-N-hydroxyurea-3'-deoxythymidine 5'-phosphate is shown to be a competitive inhibitor of catalysis by RNase Sa. Inhibition is enhanced by nearly 10-fold in the presence of Zn(2+), which could coordinate to the N-hydroxyurea group along with enzymic residues. The carboxylate of Glu54 is the putative base that abstracts a proton from the 2' hydroxyl group during catalysis of RNA cleavage by RNase Sa. Replacing Glu54 with a glutamine residue has no effect on the affinity of N-hydroxyurea 1 for the enzyme, but eliminates the zinc(II)-dependence of that affinity. These data indicate that an N-hydroxyurea nucleotide can recruit Zn(2+) to inhibit the enzymatic activity of RNase Sa, and suggest that the carboxylate of Glu54 is a ligand for that Zn(2+). These findings further the development of a new class of ribonuclease inhibitors based on the complex of an N-hydroxyurea nucleotide and zinc(II).