Differential global structural changes in the core particle of yeast and mouse proteasome induced by ligand binding

Two clusters of configurations of the main proteolytic subunit β5 were identified by principal component analysis of crystal structures of the yeast proteasome core particle (yCP). The apo-cluster encompasses unliganded species and complexes with nonpeptidic ligands, and the pep-cluster comprises complexes with peptidic ligands. The murine constitutive CP structures conform to the yeast system, with the apo-form settled in the apo-cluster and the PR-957 (a peptidic ligand) complex in the pep-cluster. In striking contrast, the murine immune CP classifies into the pep-cluster in both the apo and the PR-957–liganded species. The two clusters differ essentially by multiple small structural changes and a domain motion enabling enclosure of the peptidic ligand and formation of specific hydrogen bonds in the pep-cluster. The immune CP species is in optimal peptide binding configuration also in its apo form. This favors productive ligand binding and may help to explain the generally increased functional activity of the immunoproteasome. Molecular dynamics simulations of the representative murine species are consistent with the experimentally observed configurations. A comparison of all 28 subunits of the unliganded species with the peptidic liganded forms demonstrates a greatly enhanced plasticity of β5 and suggests specific signaling pathways to other subunits.Among the many factors involved in protein degradation through the ubiquitin-proteasome pathway, the core particle (CP) 20S proteasome plays the key role of the protease component. With the regulatory particle (RP), it forms a complex that selectively degrades ubiquitin-protein conjugates (1, 2). The CP in eukaryotes is a multisubunit complex composed of four stacked heptameric rings: two identical outer rings formed by seven different α subunits and two identical inner rings formed by seven different β subunits. The α_1–7β_1–7β_1–7α_1–7 organization defines a cylindrical structure (3). The α-rings control substrate entry into the lumen of the particle, where it is processed at the peptidolytic active centers, which are located at the inner walls of the β rings, specifically at subunits β1, β2, and β5. These active subunits are characterized by an N-terminal Thr residue. The other four β subunits have unprocessed N-terminal propeptides and are enzymatically inactive.All three active subunits share a common peptide hydrolyzing mechanism with two main steps (4): (i) the positioning of the substrate peptide in the active site by antiparallel alignment in between segments 47–49 and 21 of the active β subunits and (ii) peptide bond cleavage initiated by a nucleophilic attack of the hydroxyl group of the N-terminal Thr1 on the carbonyl carbon atom of the scissile peptide. Sequence diversity among β subunits endows them with distinctive structural features and different specificity pockets (S1, S2, S3, etc.) where the substrate side chains (P1, P2, P3, etc.) are bound (5). Consequently, the correlation of structural features of the S1 pockets with the distinctive cleavage products has led to the association of β1, β2, and β5 with caspase-like, trypsin-like, and chymotrypsin-like activities, respectively (6).The catalytically active subunits are substituted in immune cells of vertebrate organisms by the immune β-subunits β1i, β2i, and β5i as part of an adaptive immune response. These substitutions cause substantial functional differences between the constitutive (cCP) and immuno (iCP) species, reflected in higher yield of peptides that are recognized by the major histocompatibility complex (MHC) class I generated by iCP (7). Additionally, it has been observed that iCP achieves higher degradation rates than cCP, in both in vitro and cellular assays (8–13).Some sequence variations between the constitutive and immune subunits provide explanations to the observed catalytic differences. Most conspicuously, and first seen in the eukaryotic proteasome crystal structure from yeast (yCP) (3) and confirmed by the murine constitutive and immune CP structures (mcCP and miCP) (14), Arg45 of the β1 subunit, located at the base of the S1 pocket, is replaced by leucine in β1i, thereby causing a specific change of the electrostatic milieu, in line with the observed low postacidic activity of the iCP (15).Despite the high sequence similarity between β5 subunits of mcCP and miCP including identical active sites, a peptidic α-β-epoxyketone inhibitor, PR-957, showed higher affinity to iCP by one order of magnitude. The structural comparison of cCP and iCP in their apo and PR-957 liganded states suggested an explanation. On binding of PR-957, the cCP β5 backbone displays significant deformations, whereas the iCP β5 backbone remains unchanged. This observation, together with our experience in constructing β5 models for virtual screening purposes, prompted us to reinvestigate the vast amount of structural data for yCP by a procedure that facilitates discovery of global changes: principal component analysis (PCA).We focus our study on the β5 subunit, because β5 inactivation in yeast renders a lethal phenotype (16) and therefore β5 harbors an essential enzymatic activity, and because almost all crystallographically defined complexes are liganded at their β5 active site.Here we present a detailed investigation of the wealth of yeast and mouse proteasome ligand complex structures that led us to embark on structural comparisons beyond the immediate vicinity of the ligands to obtain a view of the global response of the core particle of yeast and mouse proteasome to complex formation. This study (i) is evidence of the structural plasticity of the β, specifically β5, subunits; (ii) offers perspectives for the analysis of the structure-function relationship of the CP; and (iii) provides an aid for the design and development of ligands as drugs for this intensively studied target for cancer and autoimmune diseases.     

Evaluation of an anorganic bovine-derived mineral with P-15 hydrogel bone graft: preliminary study in a rabbit cranial bone model

Objectives: The present investigation aimed to assess the bone‐regenerative potential of two formulations of anorganic bovine‐derived mineral bound to a P‐15 (ABM/P‐15) bone graft – the particulate and the hydrogel forms – in a delayed healing rabbit cranial defect model. Material and methods: Ten adult male New Zealand White rabbits were used to create two 8 mm transcortical cranial defects per rabbit and each one received randomly the test material (ABM/P‐15 carboxymethyl cellulose (CMC)‐hydrogel graft), the standard control material (ABM/P‐15 particulate graft) or remained empty as a negative control. The defects were allowed to heal for 2 and 4 weeks. Qualitative and quantitative histological outcomes were assessed on undecalcified sections. Results: In the defects grafted with the test material, at both time points, there was a marked random migration of the bone substitute particles. As a consequence, the space maintenance provision was lost and new bone formation was reduced compared with the control particulate graft material. The histomorphometric analysis showed that the control material attained better results, with an average of 13.8 ± 1.9% and 18.2 ± 4.4% of new bone at 2 and 4 weeks, compared with 8.5 ± 2.4% and 13 ± 2.9% for the test material. These differences were significant at 2 weeks (P≤0.05), but not at 4 weeks (P>0.05). Additionally, there was a significant difference in the total area of mineralized tissue (new bone plus particles), favoring the standard control over the test material: 43.2 ± 14.4% vs. 14.2 ± 5.3% at 2 weeks and 56.9 ± 4.2% vs. 24.2 ± 9.6% at 4 weeks, respectively. Conclusions: The test ABM/P‐15 CMC‐hydrogel graft material behaved in this animal model by migration of the graft particles, what determined an unpredictable osseoconduction and, consequently, a decreased quality and quantity of bone regeneration as compared with the osseopromotive behavior exhibited by the standard particulate form of the ABM/P‐15 control graft. It is therefore suggested to restrain the application of the hydrogel graft form in non‐contained anatomical bone defects.     

Sleep-disordered breathing and oxidative stress in preclinical chronic mountain sickness (excessive erythrocytosis)

Chronic mountain sickness (CMS) is considered to be a loss of ventilatory acclimatization to high altitude (>2500 m) resulting in marked arterial hypoxemia and polycythemia. This case–control study explores the possibility that sleep-disordered breathing (SDB) and associated oxidative stress contribute to the etiology of CMS. Nocturnal respiratory and Sa_O2${\text{Sa}}_{{\text{O}}_2}$ patterns were measured using standard polysomnography techniques and compared between male high-altitude residents (aged 18–25) with preclinical CMS (excessive erythrocytosis (EE), n = 20) and controls (n = 19). Measures of oxidative stress and antioxidant status included isoprostanes (8-iso-PGF2_alpha), superoxide dismutase and ascorbic acid. EE cases had a greater apnea–hypopnea index, a higher frequency of apneas (central and obstructive) and hypopneas during REM sleep, and lower nocturnal Sa_O2${\text{Sa}}_{{\text{O}}_2}$ compared to controls. 8-iso-PGF2_alpha was greater in EE than controls, negatively associated with nocturnal Sa_O2${\text{Sa}}_{{\text{O}}_2}$, and positively associated with hemoglobin concentration. Mild sleep-disordered breathing and oxidative stress are evident in preclinical CMS, suggesting that the resolution of nocturnal hypoxemia or antioxidant treatment may prevent disease progression.     

Diagnosis, Clinical Presentation, and In-Hospital Mortality of Severe Malaria in HIV-Coinfected Children and Adults in Mozambique

Background.?Severe falciparum malaria with human immunodeficiency virus (HIV) coinfection is common in settings with a high prevalence of both diseases, but there is little information on whether HIV affects the clinical presentation and outcome of severe malaria. Methods.?HIV status was assessed prospectively in hospitalized parasitemic adults and children with severe malaria in Beira, Mozambique, as part of a clinical trial comparing parenteral artesunate versus quinine (ISRCTN50258054). Clinical signs, comorbidity, complications, and disease outcome were compared according to HIV status. Results.?HIV-1 seroprevalence was 11% (74/655) in children under 15 years and 72% (49/68) in adults with severe malaria. Children with HIV coinfection presented with more severe acidosis, anemia, and respiratory distress, and higher peripheral blood parasitemia and plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP2). During hospitalization, deterioration in coma score, convulsions, respiratory distress, and pneumonia were more common in HIV-coinfected children, and mortality was 26% (19/74) versus 9% (53/581) in uninfected children (P?相似文献   

Genetic Expression Profile of Human Liver Grafts in Ischemia-Reperfusion Injury: Comparison of Familial Amyloidotic Polyneuropathy and Deceased-Donor Liver Grafts

This study aimed to compare the histologic and molecular gene expression at several surgical times (beginning of harvesting, T0; end of cold ischemia period, T1; and after reperfusion, T2) to characterize the ischemia-reperfusion injury (IRI) in deceased-donor liver grafts harvested from patients with familial amyloidotic polyneuropathy (FAP). For this purpose, 54 patients undergoing liver transplantation were studied and divided into 3 groups: deceased donor to cirrhotic recipient (group 1; n = 27), deceased donor to FAP recipient (group 2; n = 15), and FAP donor to cirrhotic recipient (group 3; n = 12). The main comparison was performed between a histologic score (Suzuki score, adding steatosis and neutrophil infiltration), and molecular gene expression of the following genes: interleukin (IL) 1β, IL-6, E-selectin, Fas-ligand, granzyme B, heme oxygenase 1 (HO1), and nitric oxide synthetase (iNOS2A). We observed less neutrophil infiltration levels in group 3 in sample T0 (P = .0082), which was associated with gene expression of HO1 in the biopsies at T2 (P = .022). In group 3, the molecular expression of genes related to attenuated proinflammatory reaction during IRI, iNOS2A at T0 and HO1 at T2, was detected. We conclude that FAP liver grafts express differently the genes associated with an attenuated proinflammatory reaction, presenting less neutrophil infiltration at harvesting. These findings add more knowledge about the better short-term outcomes in patients receiving this type of liver graft.     

Is There a Relationship Between Intraoperative Hemodynamic Instability and Calcineurin Inhibitor-Related Toxicity,Early After Liver Transplantation? A Single-Center Observational Study

This study evaluated the relationship between intraoperative hemodynamic instability (IOHI) and the development of calcineurin inhibitor (CNI) toxicity in the early postoperative period after liver transplantation (LT). Eighty-two patients were enrolled during a 1-year period and a 3-month follow-up. IOHI, requiring continuous infusion of vasopressors, was observed in 31 patients (38%, group 1; control group 2, n = 51). Acute kidney injury (AKI) developed in 28 patients (52% in group 1 vs 24% in group 2, P = .02), and CNI-related neurotoxicity (CNI-NT) in 26 (48% in group 1 vs 22% in group 2, P = .03). Group 1 patients received mainly deceased donor grafts (87% vs 57% in group 2, P < .001). An independent association between IOHI and CNI-NT (P = .029) and AKI (P = .016) was observed. The receiver-operator characteristic curve revealed an area under the curve of 0.63 for IHI (sensitivity 56%; specificity 75%) and 0.65 for AKI (sensitivity 56%; specificity 70.2%). In conclusion, patients undergoing LT with IOHI may be more prone to developing CNI-NT and AKI in the early postoperative period.     

Detection of Epstein-Barr virus in children and adolescents with Burkitt's lymphoma by in situ hybridization using tissue microarrays

In this retrospective study, we evaluated 53 patients diagnosed with Burkitt's lymphoma (BL). Patients were admitted to the Pediatric Department of Santa Casa de S?o Paulo, Brazil from August 1988 to September 2006. The male gender was found more frequently in the proportion of three males to one female. At the time of the diagnosis, the mean age was 6 years. The most frequent clinical presentation was abdominal (extra nodal) in 42 (79.2%) cases. The patients were treated with chemotherapy using two different treatment methods, 30 cases until September 1994 with BGLTI -87 and the other 23 until September of 2006 with BGLTI -96. According to Murphy's classification, 90.2% of the patients were diagnosed as stages III and VI showing an advanced disease at time of diagnosis. Using Tissue Microarrays (TMA); we searched for the expression of the Epstein-Barr virus (EBV) RNA in 50 patients. The presence of EBV using in situ hybridization was found in 33/50 (66%) and there was no association between the presence of the virus in the tumor cells or patient age, as well as the survival rate. There was 74.5% global survival rate until September, 2005 of 12 years and 8 months. The disease free survival rate was 86.5%. The frequency of the EBV association with this neoplasia in Brazil is intermediate between the endemic and sporadic forms of disease.     

Diarrhoea-related hospitalizations in children before and after implementation of monovalent rotavirus vaccination in Mexico

Objective

To assess, by socioeconomic setting, the effect of nationwide vaccination against species A rotavirus (RVA) on childhood diarrhoea-related hospitalizations in Mexico.

Methods

Data on children younger than 5 years who were hospitalized for diarrhoea in health ministry hospitals between 1 January 2003 and 31 December 2011 were collected from monthly discharge reports. Human development indexes were used to categorize the states where hospitals were located as having generally high, intermediate or low socioeconomic status. Annual rates of hospitalization for diarrhoea – per 10 000 hospitalizations for any cause – were calculated. Administrative data were used to estimate vaccine coverage.

Findings

In the states with high, intermediate and low socioeconomic status, coverage with a two-dose monovalent RVA vaccine – among children younger than 5 years – had reached 93%, 86% and 71%, respectively, by 2010. The corresponding median annual rates of hospitalization for diarrhoea – per 10 000 admissions – fell from 1001, 834 and 1033 in the “prevaccine” period of 2003–2006, to 597, 497 and 705 in the “postvaccine” period from 2008 to 2011, respectively. These decreases correspond to rate reductions of 40% (95% confidence interval, CI: 38–43), 41% (95% CI: 38–43) and 32% (95% CI: 29–34), respectively. Nationwide, RVA vaccination appeared to have averted approximately 16 500 hospitalizations for childhood diarrhoea in each year of the postvaccine period.

Conclusion

Monovalent RVA vaccination has substantially reduced childhood diarrhoea-related hospitalizations for four continuous years in discretely different socioeconomic populations across Mexico.