Protein complexes associated with β-catenin differentially influence the differentiation profile of neonatal and adult CD8+ T cells

The canonical Wnt signaling pathway is a master cell regulator involved in CD8⁺ T cell proliferation and differentiation. In human CD8⁺ T cells, this pathway induces differentiation into memory cells or a “stem cell memory like” population, which is preferentially present in cord blood. To better understand the role of canonical Wnt signals in neonatal or adult blood, we compared the proteins associated with β-catenin, in nonstimulated and Wnt3a-stimulated human neonatal and adult naive CD8⁺ T cells. Differentially recruited proteins established different complexes in adult and neonatal cells. In the former, β-catenin-associated proteins were linked to cell signaling and immunological functions, whereas those of neonates were linked to proliferation and metabolism. Wnt3a stimulation led to the recruitment and overexpression of Wnt11 in adult cells and Wnt5a in neonatal cells, suggesting a differential connexion with planar polarity and Wnt/Ca²⁺ noncanonical pathways, respectively. The chromatin immunoprecipitation polymerase chain reaction β-catenin was recruited to a higher level on the promoters of cell renewal genes in neonatal cells and of differentiation genes in those of adults. We found a preferential association of β-catenin with CBP in neonatal cells and with p300 in the adult samples, which could be involved in a higher self-renewal capacity of the neonatal cells and memory commitment in those of adults. Altogether, our results show that different proteins associated with β-catenin during Wnt3a activation mediate a differential response of neonatal and adult human CD8⁺ T cells.     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.     

Epistasis between ADIPOQ rs1501299 and PON1 rs662 polymorphisms is potentially associated with the development of knee osteoarthritis

Molecular Biology Reports - Overweight produces oxidative stress (OS) on the articular cartilage, with the subsequent risk of developing knee osteoarthritis (OA). Associations between genetic...     

First proposal of quality indicators and standards and recommendations to improve the healthcare in the Spanish National Registry of Hip Fracture

BackgroundThe Spanish National Hip Fracture Registry (or Registro Nacional de Fractura de Cadera, RNFC) is a database of hip fracture patients admitted to Spanish hospitals. Its goals include assessment and continuous improvement of the care process.ObjectivesTo (1) establish a series of indicators, (2) evaluate their initial fulfillment, (3) propose quality standards, (4) suggest recommendations to facilitate standards compliance, and (5) monitor the indicators.MethodThe indicators fulfilled the criteria of (1) evaluating the process or outcome, (2) being clinically relevant for patients, (3) being modifiable through changes in healthcare practice, and (4) being considered important by the RNFC participants. The first quartile obtained by the group of hospitals in each of the respective variables was proposed as the standard. The Indicators Advisory Committee (IAC) elaborated a list of recommendations for each indicator, based on the available evidence.ResultsSeven indicators were chosen. These indicators (its baseline compliance vs. the standard to be reached, respectively) were: the proportion of patients receiving surgery within 48 h (44% vs. 63%), mobilized the first postoperative day (56% vs. 86%), with antiosteoporotic medication at discharge (32% vs. 61%), with calcium supplements at discharge (46% vs. 77%), with vitamin D supplements at discharge (67% vs. 92%), who developed pressure ulcers during hospitalization (7.2% vs. 2.1%) and with independent mobility at 30 days (58% vs. 70%). The IAC has established 25 recommendations for improving care.ConclusionThe indicators and standards chosen are presented, as well as the list of recommendations. This process completes the first step to improve quality of care. The results will be evaluated 6 months after implementing the recommendations.     

Oxethazaine inhibits esophageal squamous cell carcinoma proliferation and metastasis by targeting aurora kinase A

Esophageal squamous cell carcinoma (ESCC), a malignant neoplasm with high incidence, is a severe global public health threat. The current modalities used for treating ESCC include surgery, chemotherapy, and radiotherapy. Although ESCC management and treatment strategies have improved over the last decade, the overall 5-year survival rate remains <20%. Therefore, the identification of novel therapeutic strategies that can increase ESCC patient survival rates is urgently needed. Oxethazaine, an amino-amide anesthetic agent, is mainly prescribed in combination with antacids to relieve esophagitis, dyspepsia, and other gastric disorders. In the present study, we found that oxethazaine inhibited the proliferation and migration of esophageal cancer cells. According to the results of in vitro screening and binding assays, oxethazaine binds directly to AURKA, suppresses AURKA activity, and inhibits the downstream effectors of AURKA. Notably, we found that oxethazaine suppressed tumor growth in three patient-derived esophageal xenograft mouse models and tumor metastasis in vivo. Our findings suggest that oxethazaine can inhibit ESCC proliferation and metastasis in vitro and in vivo by targeting AURKA.Subject terms: Cancer prevention, Cell growth     

CHMP2B regulates TDP-43 phosphorylation and cytotoxicity independent of autophagy via CK1

The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy–endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2B^Intron5 mutation. In this study, we identify CHMP2B as a modifier of TDP-43–mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2B^Intron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, we find that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.     

Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.     

Identification of the motif in versican G3 domain that plays a dominant-negative effect on astrocytoma cell proliferation through inhibiting versican secretion and binding

This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanDeltaEGF) and a G3 mutant (G3DeltaEGF) exerted a dominant-negative effect on cell proliferation. G3DeltaEGF-transfected cells formed smaller colonies, arrested cell cycle at G(1) phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3DeltaEGF products exhibited decreased binding activity, but higher levels of G3DeltaEGF products were able to inhibit the binding of G3 to the cell surface. G3DeltaEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3DeltaEGF constructs. The effect seems to depend on the expression efficiency of G3DeltaEGF, and it occurred via the carbohydrate recognition domain.     

Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and bacteriophage T4's Gp31

Bacteriophage T4-encoded Gp31 is a functional ortholog of the Escherichia coli GroES cochaperonin protein. Both of these proteins form transient, productive complexes with the GroEL chaperonin, required for protein folding and other related functions in the cell. However, Gp31 is specifically required, in conjunction with GroEL, for the correct folding of Gp23, the major capsid protein of T4. To better understand the interaction between GroEL and its cochaperonin cognates, we determined whether the so-called "pseudo-T-even bacteriophages" are dependent on host GroEL function and whether they also encode their own cochaperonin. Here, we report the isolation of an allele-specific mutation of bacteriophage RB49, called epsilon22, which permits growth on the E. coli groEL44 mutant but not on the isogenic wild type host. RB49 epsilon22 was used in marker rescue experiments to identify the corresponding wild type gene, which we have named cocO (cochaperonin cognate). CocO has extremely limited identity to GroES but is 34% identical and 55% similar at the protein sequence level to T4 Gp31, sharing all of the structural features of Gp31 that distinguish it from GroES. CocO can substitute for Gp31 in T4 growth and also suppresses the temperature-sensitive phenotype of the E. coli groES42 mutant. CocO's predicted mobile loop is one residue longer than that of Gp31, with the epsilon22 mutation resulting in a Q36R substitution in this extra residue. Both the CocO wild type and epsilon22 proteins have been purified and shown in vitro to assist GroEL in the refolding of denatured citrate synthase.     

Structural basis of electron transfer modulation in the purple CuA center

The X-ray structure of an engineered purple CuA center in azurin from Pseudomonas aeruginosa has been determined and refined at 1.65 A resolution. Two independent purple CuA azurin molecules are in the asymmetric unit of a new P21 crystal, and they have nearly identical conformations (rmsd of 0.27 A for backbone atoms). The purple CuA azurin was produced by the loop-engineering strategy, and the resulting overall structure is unperturbed. The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other. The high-resolution structure reveals the detailed environment of the delocalized mixed-valence [Cu(1.5).Cu(1.5)] binuclear purple CuA center, which serves as a useful reference model for other native proteins, and provides a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology. The two independent Cu-Cu distances of 2.42 and 2.35 A (with respective concomitant adjustments of ligand-Cu distances) are consistent with that (2.39 A) obtained from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complexes. A comparison of the purple CuA azurin structure with those of other CuA centers reveals an important relationship between the angular position of the two His imidazole rings with respect to the Cu2S2(Cys) core plane and the distance between the Cu and the axial ligand. This relationship strongly suggests that the fine structural variation of different CuA centers can be correlated with the angular positions of the two histidine rings because, from these positions, one can predict the relative axial ligand interactions, which are responsible for modulating the Cu-Cu distance and the electron transfer properties of the CuA centers.