Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites.

The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1-3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography-tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.

Evaluation of the Abbott BinaxNOW COVID-19 Test Ag Card for rapid detection of SARS-CoV-2 infection by a local public health district with a rural population.

SARS-CoV-2 RT-PCR, the gold standard for diagnostic testing, may not be readily available or logistically applicable for routine COVID-19 testing in many rural communities in the United States. In this validation study, we compared the BinaxNOW™ COVID-19 Test Ag Card with SARS-CoV-2 RT-PCR in 214 participants who sought COVID-19 testing from a local public health district in Idaho, USA. The median age of participants was 35 and 82.7% were symptomatic. Thirty-seven participants (17.3%) had positive RT-PCR results. Results between the two tests were 94.4% concordant. The sensitivity of the BinaxNOW™ COVID-19 Test Ag Card was 67.6% (95% CI: 50.2-81.9%), and the specificity was 100.0% (95% CI: 97.9-100.0%). The positive predictive value (PPV) for the BinaxNOW™ COVID-19 Test Ag Card was 100.0% (95% CI: 86.2-100.0%), and the negative predictive value (NPV) was 93.6% (95% CI: 89.1-96.6%). Although the sensitivity of BinaxNOW™ COVID-19 Test Ag Card was lower than RT-PCR, rapid results and high specificity support its use for early detection of COVID-19, especially in settings where SARS-CoV-2 RT-PCR testing is not readily available. Rapid antigen tests, such as the BinaxNOW™ COVID-19 Test Ag Card, may be a more convenient tool in quickly identifying and preventing COVID-19 transmission, especially in rural settings.

Optimized procedures for producing biologically active chemokines.

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.

Biochemical characterization of human prolyl hydroxylase domain protein 2 variants associated with erythrocytosis.

Prolyl hydroxylase domain proteins (PHD isozymes 1-3) regulate levels of the alpha-subunit of the hypoxia inducible factor (HIF) through proline hydroxylation, earmarking HIFalpha for proteosome-mediated degradation. Under hypoxic conditions, HIF stabilization leads to enhanced transcription and regulation of a multitude of processes, including erythropoiesis. Herein, we examine the biochemical characterization of PHD2 variants, Arg371His and Pro317Arg, identified from patients with familial erythrocytosis. The variants display differential effects on catalytic rate and substrate binding, implying that partial inhibition or selective inhibition with regard to HIFalpha isoforms of PHD2 could result in the phenotype displayed by patients with familial erythrocytosis.