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Formation and Electronic Structure of an Atypical CuA Site.

ABSTRACT: PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper CuA center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD CuA bear similarities to those of canonical CuA sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric CuA site to two mononuclear Cu2+ sites, as well as its electronic and geometric structure, using stopped-flow optical and advanced paramagnetic resonance spectroscopies. PmoD CuA formation occurs rapidly and involves a short-lived intermediate with a ?max of 360 nm. Unlike other CuA sites, the PmoD CuA is unstable, decaying to two type 2 Cu2+ centers. Surprisingly, NMR data indicate that the PmoD CuA has a pure ?u* ground state rather than the typical equilibrium between ?u* and ?u of all other CuA proteins. EPR, ENDOR, ESEEM, and HYSCORE data indicate the presence of two histidine and two cysteine ligands coordinating the CuA core in a highly symmetrical fashion. This report significantly expands the diversity and understanding of known CuA sites.

SUBMITTER: Ross MO

PROVIDER: S-EPMC6953997 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Formation and Electronic Structure of an Atypical CuA Site.

Ross Matthew O MO Fisher Oriana S OS Morgada Marcos N MN Krzyaniak Matthew D MD Wasielewski Michael R MR Vila Alejandro J AJ Hoffman Brian M BM Rosenzweig Amy C AC

Journal of the American Chemical Society 20190307 11

PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper CuA center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD CuA bear similarities to those of canonical CuA sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric CuA site to two monon ...[more]

PMID: 30807125

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Project description:Introduction and Objectives: Optimizing targeted therapy in patients with metastatic renal cell carcinoma (RCC) would improve clinical outcomes but patient derived xenograft (PDX) models are lacking. We present a novel pre-clinical model that is superior to nude mice for accommodating RCC PDXs. This preclinical model implants RC PDXs into the chorioallantoic membrane (CAM) of avian embryos and is a patient-specific platform that could be advantageous for physicians in the future when deciding what treatment options are best for their patients. This drug panelling platform is rapid, cost-effective, and relies on the highly angiogenic CAM to support RCC PDXs. Methods: Commercial and patient derived RCC cell lines, were grown to full confluence and transduced to generate fluorescently labeled versions of each cell line. Cells were implanted into the CAM. Tumors were treated every two days by applying 10 μL (10 μM) of indicated drug onto the tumor onplant. The drugs that were paneled include Sunitinib, Sorafenib, Pazopanib, Axitinib and a vehicle treatment. After 7–8 days of incubation post-implantation, tumor take rate was determined by the presence of tumor growth in the CAM using a fluorescent stereoscope. Results: The highest tumor take rates were observed in the vehicle treatments of the embryos, ranging from 50–86%. Both commercial and primary cell lines saw a reduction in tumor take rate with the application of various anti-angiogenic drugs. Specifically, XP121 tumors were resistant to Sorafenib; 786-0, XP121; XP206 were resistant to Pazopanib; T258, XP121 were resistant to Sunitinib; and lastly, T258 tumors were resistant to Axitinib (Table 1). Conclusions: RCC PDXs onplanted in the CAM of avian embryos offer a robust and cost-effective platform to predict sensitivity/resistance to targeted therapies. When evaluating several patient-derived RCC cell lines, drug paneling revealed other alternative treatment options for these PDXs. More importantly, RCC PDXs that were shown to be Sunitinib-resistant in both the patient and in mouse-based PDXs, also produced the same resistance phenotype in the CAM.

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Formation and Electronic Structure of an Atypical CuA Site.

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Formation and Electronic Structure of an Atypical Cu<sub>A</sub> Site.

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