Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.     

An empirical assessment of individual-based population genetic statistical techniques: application to British pig breeds

Recently developed Bayesian genotypic clustering methods for analysing genetic data offer a powerful tool to evaluate the genetic structure of domestic farm animal breeds. The unit of study with these approaches is the individual instead of the population. We aimed to empirically evaluate various individual-based population genetic statistical methods for characterization of genetic diversity and structure of livestock breeds. Eighteen British pig populations, comprising 819 individuals, were genotyped at 46 microsatellite markers. Three Bayesian genotypic clustering approaches, principle component analysis (PCA) and phylogenetic reconstruction were applied to individual multilocus genotypes to infer the genetic structure and diversity of the British pig breeds. Comparisons of the three Bayesian genotypic clustering methods (, and ) revealed some broad similarities but also some notable differences. Overall, the methods agreed that majority of the British pig breeds are independent genetic units with little evidence of admixture. The three Bayesian genotypic clustering methods provided complementary, biologically credible clustering solutions but at different levels of resolution. detected finer genetic differentiation and in some cases, populations within breeds. Consequently, it estimated a greater number of underlying genetic populations (K, in the notation of Bayesian clustering methods). Two of the Bayesian methods ( and ) and phylogenetic reconstruction provided similar success in assignment of individuals, supporting the use of these methods for breed assignment.     

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

O₂ sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1–F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1–cullin-1–F-box protein–Rbx1 subcomplex of E3^SCFUb ligases. E3^SCFUb ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O₂ availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA–Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O₂-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3^SCFUb ligases that regulate O₂-dependent developmental progression.Timely protein degradation is a cornerstone of cell cycling and the regulation of numerous physiological and developmental processes. Eukaryotes have evolved an extensive array of polyubiquitination enzymes to tag proteins on a protein-by-protein basis as a recognition marker for degradation in the 26S proteasome. The cullin-RING ubiquitin ligases (CRLs)¹ are a prominent subgroup of these enzymes (1) and consist of an E3 architecture that includes a substrate receptor, an adaptor (in most cases), the cullin scaffold, the RING protein, and an exchangeable E2 ubiquitin donor that has been charged with ubiquitin (Ub) by an E1 enzyme. The first discovered and still prototypic example is the CRL1 class (2), also referred to as SCF on account of the names of its founding subunits, Skp1, cullin-1, and F-box proteins (FBPs). The CRL1 (or SCF) complexes utilize FBPs as substrate receptors, Skp1 as the adaptor linking the FBP to the N-terminal region of cullin-1 (Cul1), and Rbx1 as the RING protein that tethers the E2 Ub donor to the Cul1 C-terminal region (see Fig. 2B). CRL1s can be activated by neddylation of Cul1 by a Nedd8-specific E2, which mobilizes Rbx1 to afford rotational flexibility of the E2 and displaces the inhibitor Cand1, permitting docking of the Skp1–FBP heterodimer (3–5). Deneddylation mediated by the eight-subunit COP9 signalosome is required for in vivo activity, suggesting that Cand1 serves as a substrate exchange factor to allow for re-equilibration of SCF complexes from preexisting subunits. Each reaction cycle requires the exchange of a new E2-Ub and typically assembles a K48-linked polyUb chain that is recognized by the proteasome. Substrate specificity is conferred by FBPs, a gene family that numbers 69 in humans, 20 in budding yeast, 300 in Caenorhabditis elegans, and ∼800 in Arabidopsis. Some characterized FBPs can recognize perhaps a dozen or more substrates, and the coding of recognition and the meaning of their control by the same FBP is under intense investigation (6). Recognition is often activated by posttranslational modification of the substrate (often phosphorylation). Regulation of SCF Ub ligases has centered on the neddylation cycle, which potentially influences all seven known CRLs. Regulation of Skp1, investigated in this paper, would be specific to CRLs possessing Skp1, which include CRL1 and possibly the minor class CRL7 (7).Open in a separate windowFig. 2.Skp1 modification pathway and global analysis of Skp1 interactions. A, Skp1 is sequentially modified by the indicated enzymes (in blue), resulting in the formation of a pentasaccharide at Pro143. B, model of the SCF complex in the context of the overall E3 Ub ligase, from studies in yeast, plants, and animals. Catalysis involves transfer of Ub from an exchangeable Ub-E2 conjugate to the substrate. Removal of Nedd8 by the COP9 signalosome facilitates binding of Cand1 to Cul1, which inhibits binding of Skp1 to Cul1. C, D, vegetative (growth stage) cells were filter-lysed, and a cytosolic fraction prepared via ultracentrifugation was chromatographed on a Superose 12 gel filtration column. Fractions were analyzed via Western blotting (representative examples are shown in C) followed by densitometry (D). The elution position of free Skp1 from a separate trial is indicated.The basic SCF model is thought to be widespread among eukaryotes but has been extensively studied only in fungi/yeasts, plants, and animals. The broad phylogeny represented by protists includes many benign and pathogenic unicellular organisms of great economic, health, and environmental impact. Emerging evidence reveals that Skp1 in some of these groups is subject to a novel form of prolyl 4(trans)-hydroxylation and complex glycosylation (8). The roles of these Skp1 modifications have been most studied in the social amoeba Dictyostelium, which undergoes a starvation-induced developmental program during which individual amoebae chemotactically aggregate into an initial mound that then elongates into a migratory slug. Under appropriate conditions, the slug reorganizes to form a fruiting body consisting of a ball of spores supported by a vertical cellular stalk. The slug-to-fruit switch, referred to as culmination, and sporulation are regulated by checkpoints that are sensitive to multiple factors, including O₂ (9–11). Functional studies of Dictyostelium Skp1 hydroxylation and glycosylation reveal roles in regulating the O₂ dependence of culmination and sporulation (12–14). For example, wild-type (wt) cells require 7% to 10% O₂ and phyA^– requires 18% to 21% O₂ in order to achieve 50% spore formation (a quantitative measure of fruiting body formation), whereas glycosylation mutants exhibit a complex pattern of intermediate requirements (13). In addition, at 21% O₂, phyA^– cells require an additional 3 to 4 h to complete development relative to their wt counterparts (14). In the apicomplexan Toxoplasma gondii, PhyA is also required for Skp1 glycosylation, and phyA^– parasites are deficient in proliferation, especially at low O₂ (15).The idea that O₂ availability is rate limiting for Skp1 modification was originally based on the observation that the Dictyostelium phyA^– phenotype mimics that of wt cells in low O₂ (9). However, the majority of Skp1 is hydroxylated and glycosylated in wt cells even at low O₂ levels where culmination is blocked or delayed. Further analysis of a submerged development model, in which terminal development depended on an atmosphere of 70% to 100% O₂ in order to overcome the diffusion barrier posed by the water layer, showed that at atmospheric O₂ levels of 5% to 21% where sporulation was blocked, unmodified Skp1 accumulated to a higher level than at permissive O₂ levels (10). As Skp1 modifications are thought to be irreversible, this likely resulted from slow hydroxylation of newly synthesized Skp1. To address this in a more physiological setting, we investigated nascent Skp1 directly using metabolic labeling with [³⁵S]Met/Cys and verified that the rate of hydroxylation of newly synthesized Skp1 polypeptide was indeed inversely proportional to O₂ levels, which makes PhyA-mediated hydroxylation of Skp1 an excellent candidate for the primary O₂ sensor for culmination.These modifications of Skp1 are of interest as a novel mechanism regulating the SCF ligase. Previously, we showed that hydroxylation and glycosylation of Dictyostelium Skp1 affect its conformation and promote binding to a soluble FBP, guinea pig Fbs1, in studies of purified proteins (16). Here we show that Dictyostelium Skp1 is indeed a subunit of a canonical SCF complex, as expected. The significance of undermodified Skp1 was examined via interactome analysis of Skp1 isoforms that accumulate in modification pathway mutants. Our findings revealed a lower abundance of SCF complexes than in wt cells, suggesting that Skp1 modification may promote SCF assembly and E3^SCFUb ligase activities that control timely turnover of select proteins involved in developmental progression.     

Is Female Visual Signaling to Male Song Socially Regulated in Brown‐headed Cowbirds?

This research focused on how adult female brown‐headed cowbirds, Molothrus ater, regulate social feedback on a group level to shape the development of male song. Specifically, females produce rapid wing movements in response to male song, termed ‘wing strokes,’ which have been shown to shape male song and predict song quality. These effects have been documented in captive dyads and triads, but not in more naturalistic flocks, where song development actually occurs. Here, we studied wing stroking in small seminatural flocks of differing female‐to‐male ratios. Despite differences in the number of females and their social selectivity, the same pattern of female feedback emerged in seven of eight flocks: One female produced the majority of wing strokes to male song, making her the primary wing stroker in her flock. Previous studies on large flocks have demonstrated females to facilitate male song improvisation and development if they exhibited higher social selectivity by approaching immature males less. Here, we found that primary wing strokers were indeed more socially selective than non‐primary wing strokers. This research is the first to document social stimulation being facilitated at the group level to ensure that more highly selective females deliver the most feedback.     

Point-of-care versus central testing of hemoglobin during large volume blood transfusion

Hypoxic preconditioning (HPC) may protect multiple organs from various injuries. We hypothesized that HPC would reduce lung injury in patients undergoing thoracoscopic lobectomy. In a prospective randomized controlled trial, 70 patients undergoing elective thoracoscopic lobectomy were randomly allocated to the HPC group or the control group. Three cycles of 5-min hypoxia and 3-min ventilation applied to the nondependent lung served as the HPC intervention. The primary outcome was the PaO2/FiO2 ratio. Secondary outcomes included postoperative pulmonary complications, pulmonary function, and duration of hospital stay. HPC significantly increased the PaO2/FiO2 ratio compared with the control at 30 min after one-lung ventilation and 7 days after operation. Compared with the control, it also significantly improved postoperative pulmonary function and markedly reduced the postoperative hospital stay duration. No significant differences between groups were observed in the incidence of pulmonary complications or overall postoperative morbidity. HPC improves postoperative oxygenation, enhances the recovery of pulmonary function, and reduces the duration of hospital stay in patients undergoing thoracoscopic lobectomy. This study was registered in the Chinese Clinical Trial Registry (ChiCTR-IPR-17011249) on April 27, 2017.     

Preclinical pharmacokinetics,pharmacodynamics, tissue distribution,and tumor penetration of anti-PD-L1 monoclonal antibody,an immune checkpoint inhibitor

MPDL3280A is a human monoclonal antibody that targets programmed cell death-1 ligand 1 (PD-L1), and exerts anti-tumor activity mainly by blocking PD-L1 interaction with programmed cell death-1 (PD-1) and B7.1. It is being investigated as a potential therapy for locally advanced or metastatic malignancies. The purpose of the study reported here was to characterize the pharmacokinetics, pharmacodynamics, tissue distribution and tumor penetration of MPDL3280A and/or a chimeric anti-PD-L1 antibody PRO304397 to help further clinical development.

The pharmacokinetics of MPDL3280A in monkeys at 0.5, 5 and 20 mg·kg^?1 and the pharmacokinetics / pharmacodynamics of PRO304397 in mice at 1, 3 10 mg·kg^?1 were determined after a single intravenous dose. Tissue distribution and tumor penetration for radiolabeled PRO304397 in tumor-bearing mouse models were determined.

The pharmacokinetics of MPDL3280A and PRO304397 were nonlinear in monkeys and mice, respectively. Complete saturation of PD-L1 in blood in mice was achieved at serum concentrations of PRO304397 above ～0.5 µg·mL^?1. Tissue distribution and tumor penetration studies of PRO304397 in tumor-bearing mice indicated that the minimum tumor interstitial to plasma radioactivity ratio was ～0.3; saturation of target-mediated uptake in non–tumor tissues and desirable exposure in tumors were achieved at higher serum concentrations, and the distribution into tumors was dose-and time-dependent.

The biodistribution data indicated that the efficacious dose is mostly likely higher than that estimated based on simple pharmacokinetics/pharmacodynamics in blood. These data also allowed for estimation of the target clinical dose for further development of MPDL3280A.     

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.