ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle.

In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules or two ATPγS plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.

Hepatic fat quantification in dual-layer computed tomography using a three-material decomposition algorithm.

OBJECTIVES: The purpose of this study was to evaluate a three-material decomposition algorithm for hepatic fat quantification using a dual-layer computed tomography (DL-CT) and MRI as reference standard on a large patient cohort. METHOD: A total of 104 patients were retrospectively included in our study, i.e., each patient had an MRI exam and a DL-CT exam in our institution within a maximum of 31 days. Four regions of interest (ROIs) were positioned blindly and similarly in the liver, by two independent readers on DL-CT and MRI images. For DL-CT exams, all imaging phases were included. Fat fraction agreement between CT and MRI was performed using intraclass correlation coefficients (ICC), determination coefficients R2, and Bland-Altman plots. Diagnostic performance was determined using sensitivity, specificity, and positive and negative predictive values. The cutoff for steatosis was 5%. RESULTS: Correlation between MRI and CT data was excellent for all perfusion phases with ICC calculated at 0.99 for each phase. Determination coefficients R2 were also good for all perfusion phases (about 0.95 for all phases). Performance of DL-CT in the diagnosis of hepatic steatosis was good with sensitivity between 89 and 91% and specificity ranging from 75 to 80%, depending on the perfusion phase. The positive predictive value was ranging from 78 to 93% and the negative predictive value from 82 to 86%. CONCLUSION: Multi-material decomposition in DL-CT allows quantification of hepatic fat fraction with a good correlation to MRI data. CLINICAL RELEVANCE STATEMENT: The use of DL-CT allows for detection of hepatic steatosis. This is especially interesting as an opportunistic finding CT performed for other reasons, as early detection can help prevent or slowdown the development of liver metabolic disease. KEY POINTS: • Hepatic fat fractions provided by the dual-layer CT algorithm is strongly correlated with that measured on MRI. • Dual-layer CT is accurate to detect hepatic steatosis ≥ 5%. • Dual-layer CT allows opportunistic detection of steatosis, on CT scan performed for various indications.

Bone marrow edema in traumatic vertebral compression fractures: Diagnostic accuracy of dual-layer detector CT using calcium suppressed images.

PURPOSE: To evaluate calcium suppressed images (CaSupp) in dual-layer detector computed tomography (DLCT) for the detection of bone marrow edema (BME) in vertebral fractures. MATERIALS AND METHODS: The retrospective study was approved by the institutional review board. 34 patients with synchronous DLCT and MRI, who were diagnosed with one or more acute vertebral fractures, were included. MRI were systematically analyzed as reference standard. Two blinded and independent readers evaluated CaSupp for vertebral BME. Additionally, both readers determined the optimal calcium suppression indices (CaSupp-I) for visualization of BME in consensus and correlated the CaSupp-I with parallel measurement of trabecular density as surrogate parameter for bone mineral density. ROI-based measurements of the contrast-to-noise ratios (CNR) were also conducted. Interrater agreement was determined by kappa-statistics. CNR were analyzed using Wilcoxon signed rank test. RESULTS: Fifty-seven acute fractured vertebrae out of 383 vertebrae (14.9%) were found. CaSupp yielded an average sensitivity of 87% and specificity of 99%, a positive predictive value of 95%, a negative predictive value of 98% and an accuracy of 97% for the detection of fracture-associated edema. Interrater agreement was excellent (kappa 0.91). Increase in CNR of BME correlated with increasing CaSupp-I. Edema adjacent to the cortical endplates was better visualized using CaSupp-I of 70 and 80, while extensive edema was better visualized using a CaSupp-I of 90 and 100 (chi2 < 0.0001). No correlation between optimal CaSupp-I and trabecular density was found (p > 0.2). CONCLUSION: CaSupp reconstructed from DLCT enable visualization and detection of BME in traumatic fractured vertebrae with high diagnostic accuracy using CaSupp-I of 70-100.

Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1.

The chaperonin CCT (chaperonin containing t-complex polypeptide 1 (TCP-1)) from bovine testis was mixed rapidly with different concentrations of ATP and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Two kinetic phases were observed and assigned by (i) analyzing the dependence of the corresponding observed rate constants on ATP concentration; and (ii) by carrying out mixing experiments also with ADP, ATPgammaS and ATP without K(+). The values of the observed rate constants corresponding to both phases are found to be dependent on ATP concentration. The observed rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are similar to those determined in steady-state ATPase experiments. This phase most likely reflects ATP binding-induced conformational changes. The rate constant of the conformational change in the presence of excess ATP is about 17s(-1) (at 25 degrees C) and is tenfold slower than the corresponding rate constant of GroEL. The observed rate constant corresponding to the second slower phase displays a hyperbolic dependence on ATP concentration. This phase is not observed in mixing experiments of CCT with ADP, ATPgammaS or ATP without K(+) and it, therefore, reflects a conformational change associated with ATP hydrolysis. Taken together, our results indicate that the kinetic mechanism of the allosteric transitions of CCT differs considerably from that of GroEL.

Virtual nonenhanced abdominal dual-energy MDCT: Analysis of image characteristics.

AIM: To evaluate abdominal and pelvic image characteristics and artifacts on virtual nonenhanced (VNE) images generated from contrast-enhanced dual-energy multidetector computed tomography (MDCT) studies. METHODS: Hadassah-Hebrew University Medical Institutional Review Board approval was obtained; 22 patients underwent clinically-indicated abdominal and pelvic single-source dual-energy MDCT (Philips Healthcare, Cleveland, OH, USA), pre- and post-IV administration of Omnipaque 300 contrast (100 cc). Various solid and vascular structures were evaluated. VNE images were generated from the portal contrast-enhanced phase using probabilistic separation. Contrast-enhanced-, regular nonenhanced (RNE)-, and VNE images were evaluated with a total of 1494 density measurements. The ratio of iodine contrast deletion was calculated. Visualization of calcifications, urinary tract stones, and image artifacts in VNE images were assessed. RESULTS: VNE images were successfully generated in all patients. Significant portal-phase iodine contrast deletion was seen in the kidney (61.7%), adrenal gland (55.3%), iliac artery (55.0%), aorta (51.6%), and spleen (34.5%). Contrast deletion was also significant in the right atrium (RA) (51.5%) and portal vein (39.3%), but insignificant in the iliac vein and inferior vena cava (IVC). Average post contrast-to-VNE HU differences were significant (P < 0.05) in the: RA -135.3 (SD 121.8), aorta -114.1 (SD 48.5), iliac artery -104.6 (SD 53.7), kidney -30.3 (SD 34.9), spleen -9.2 (SD 8.8), and portal vein -7.7 (SD 13.2). Average VNE-to-RNE HU differences were significant in all organs but the prostate and subcutaneous fat: aorta 38.0 (SD 9.3), RA 37.8 (SD 16.1), portal vein 21.8 (SD 12.0), IVC 12.2 (SD 11.6), muscle 3.3 (SD 4.9), liver 5.7 (SD 6.4), spleen 22.3 (SD 9.8), kidney 40.5 (SD 6.8), and adrenal 20.7 (SD 13.5). On VNE images, 196/213 calcifications (92%) and 5/6 renal stones (84%) were visualized. Lytic-like artifacts in the vertebral bodies were seen in all studies. CONCLUSION: Iodine deletion in VNE images is most significant in arteries, and less significant in solid organs and veins. Most vascular and intra-abdominal organ calcifications are preserved.

ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins.

The archaeal ATPase complex PAN, the homolog of the eukaryotic 26S proteasome-regulatory ATPases, was shown to associate transiently with the 20S proteasome upon binding of ATP or ATPgammaS, but not ADP. By electron microscopy (EM), PAN appears as a two-ring structure, capping the 20S, and resembles two densities in the 19S complex. The N termini of the archaeal 20S alpha subunits were found to function as a gate that prevents entry of seven-residue peptides but allows entry of tetrapeptides. Upon association with the 20S particle, PAN stimulates gate opening. Although degradation of globular proteins requires ATP hydrolysis, the PAN-20S complex with ATPgammaS translocates and degrades unfolded and denatured proteins. Rabbit 26S proteasomes also degrade these unfolded proteins upon ATP binding, without hydrolysis. Thus, although unfolding requires energy from ATP hydrolysis, ATP binding alone supports ATPase-20S association, gate opening, and translocation of unfolded substrates into the proteasome, which can occur by facilitated diffusion through the ATPase.

Phi value analysis of heterogeneity in pathways of allosteric transitions: Evidence for parallel pathways of ATP-induced conformational changes in a GroEL ring.

What are the mechanisms of ligand-induced allosteric transitions in proteins? A powerful method to characterize pathways and transition states of reactions is phi value analysis. A phi value is the ratio between the changes on a perturbation (e.g., mutation) in the activation and equilibrium free energies of a reaction. Here, phi value analysis is used to characterize the ATP-induced allosteric transitions of GroEL by using changes in ATP concentration as perturbations. GroEL consists of two stacked back-to-back heptameric rings that bind ATP with positive cooperativity within rings and negative cooperativity between rings. Evidence is presented for the existence of parallel pathways for the allosteric transition of each ring. In both allosteric transitions, there is an abrupt ATP-dependent switch from a pathway with ATP-binding sites in the transition state that are very similar to those of the initial T state (phi = 0) to a pathway with a phi value of approximately 0.3. The phi value procedure outlined here should be useful in mapping the energy landscape of allosteric transitions of other proteins.

Dissociation of the GroEL-GroES asymmetric complex is accelerated by increased cooperativity in ATP binding to the GroEL ring distal to GroES.

A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance. Excellent fits of the data were obtained using a double-exponential equation with a linear drift. Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP. The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES. Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring. It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex.