A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

Intracellular hypoxia of tumor tissue estimated by noninvasive electron paramagnetic resonance oximetry technique using paramagnetic probes.

Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.

Continuous wave EPR oximetric imaging at 300 MHz using radiofrequency power saturation effects.

A novel continuous wave (CW), radiofrequency (RF), electron paramagnetic resonance (EPR) oximetric imaging technique is proposed, based on the influence of oxygen concentration on the RF power saturation of the EPR resonance. A linear relationship is demonstrated between the partial oxygen pressure (pO(2)) and the normalized signal intensity (I(N)), defined as, I(N) = (I(HP) - I(LP))/I(LP), where I(LP) and I(HP) refer to signal intensities at low (P(L)) and high (P(H)) RF power levels, respectively. A formula for the determination of pO(2), derived on the basis of the experimental results, reliably estimated various oxygen concentrations in a five-tube phantom. This new technique was time-efficient and also avoided the missing angle problem associated with conventional spectral-spatial CW EPR oximetric imaging. In vivo power saturation oximetric imaging in a tumor bearing mouse clearly depicted the hypoxic foci within the tumor.

A new strategy for fast radiofrequency CW EPR imaging: direct detection with rapid scan and rotating gradients.

Rapid field scan on the order of T/s using high frequency sinusoidal or triangular sweep fields superimposed on the main Zeeman field, was used for direct detection of signals without low-frequency field modulation. Simultaneous application of space-encoding rotating field gradients have been employed to perform fast CW EPR imaging using direct detection that could, in principle, approach the speed of pulsed FT EPR imaging. The method takes advantage of the well-known rapid-scan strategy in CW NMR and EPR that allows arbitrarily fast field sweep and the simultaneous application of spinning gradients that allows fast spatial encoding. This leads to fast functional EPR imaging and, depending on the spin concentration, spectrometer sensitivity and detection band width, can provide improved temporal resolution that is important to interrogate dynamics of spin perfusion, pharmacokinetics, spectral spatial imaging, dynamic oxymetry, etc.

Absolute oxygen tension (pO(2)) in murine fatty and muscle tissue as determined by EPR.

The absolute partial pressure of oxygen (pO(2)) in the mammary gland pad and femoral muscle of female mice was measured using EPR oximetry at 700 MHz. A small quantity of lithium phthalocyanine (LiPc) crystals was implanted in both mammary and femoral muscle tissue of female C3H mice. Subsequent EPR measurements were carried out 1-30 days after implantation with or without control of core body temperature. The pO(2) values in the tissue became stable 2 weeks after implantation of LiPc crystals. The pO(2) level was found to be higher in the femoral muscle than in the mammary tissue. However, the pO(2) values showed a strong dependence on the core body temperature of the mice. The pO(2) values were responsive to carbogen (95% O(2), 5% CO(2)) breathing even 44-58 days after the implantation of LiPc. The LiPc linewidth was also sensitive to changes in the blood supply even 60 days after implantation of the crystals. This study further validates the use of LiPc crystals and EPR oximetry for long-term non-invasive assessment of pO(2) levels in tissues, underscores the importance of maintaining normal body core temperature during the measurements, and demonstrates that mammary tissue functions at a lower pO(2) level than muscle in female C3H mice.