Functional importance of calcium binding sites in outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca(2+) as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca(2+) transfer to the interfacial calcium site.     

The chronic proliferative dermatitis mouse mutation (cpdm): mapping of the mutant gene locus

The chronic proliferative dermatitis (cpdm) mouse mutation was mapped to mouse Chromosome 15 using an intraspecific cross between C57BL/KaLawRij cpdm/cpdm and BALB/cJ mice. A second autosomal recessive mutation that resulted in a phenotype similar to that of cpdm arose spontaneously in a colony of OcB-3/Dem recombinant congenic mice. This new mutation was found to be allelic with cpdm. Therefore, the gene symbol for the allelic mutation is cpdm^Dem. The phenotype of these mutant mice consists of eosinophil-driven severe and progressive inflammatory changes in multiple organs including the skin and lungs.     

Organisational Factors Induce Prolonged Emergency Department Length of Stay in Elderly Patients – A Retrospective Cohort Study

Study objective

To assess the association of patient and organisational factors with emergency department length of stay (ED-LOS) in elderly ED patients (226565 years old) and in younger patients (<65 years old).

Methods

A retrospective cohort study of internal medicine patients visiting the emergency department between September 1^st 2010 and August 31^st 2011 was performed. All emergency department visits by internal medicine patients 226565 years old and a random sample of internal medicine patients <65 years old were included. Organisational factors were defined as non-medical factors. ED-LOS is defined as the time between ED arrival and ED discharge or admission. Prolonged ED-LOS is defined as ≥75^th percentile of ED-LOS in the study population, which was 208 minutes.

Results

Data on 1782 emergency department visits by elderly patients and 597 emergency department visits by younger patients were analysed. Prolonged ED-LOS in elderly patients was associated with three organisational factors: >1 consultation during the emergency department visit (odds ratio (OR) 3.2, 95% confidence interval (CI) 2.3–4.3), a higher number of diagnostic tests (OR 1.2, 95% CI 1.16–1.33) and evaluation by a medical student or non-trainee resident compared with a medical specialist (OR 4.2, 95% CI 2.0–8.8 and OR 2.3, 95% CI 1.4–3.9). In younger patients, prolonged ED-LOS was associated with >1 consultation (OR 2.6, 95% CI 1.4–4.6). Factors associated with shorter ED-LOS were arrival during nights or weekends as well as a high urgency level in elderly patients and self-referral in younger patients.

Conclusion

Organisational factors, such as a higher number of consultations and tests in the emergency department and a lower seniority of the physician, were the main aspects associated with prolonged ED-LOS in elderly patients. Optimisation of the organisation and coordination of emergency care is important to accommodate the needs of the continuously growing number of elderly patients in a better way.     

Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development,Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes—fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a—had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.     

BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: Implications for a proteasome-to-autophagy switch

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS).     

The Human Leukocyte Antigen–presented Ligandome of B Lymphocytes

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome.Peptides presented by human leukocyte antigen (HLA)¹ molecules on the cell surface play a crucial role in immunology and mediate the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action, the design of T-cell-mediated therapies such as tumor immunotherapy (1), and the treatment of hematological malignancies through a combination of hematopoietic stem cell transplantation and donor lymphocyte infusion (2). In addition, T cells can play an important role in organ rejection following transplantation.The presented HLA class I ligands are the products of the intracellular processing machinery, with its continuous cycle of protein synthesis and degradation (3). Much is known about the proteins involved in antigen processing, but high fidelity ligand/epitope predictions are at present not possible. The discovery of additional involved enzymes (3, 4) and the exciting discovery of peptide splicing (5) have shown that antigen processing is even more complex than was previously thought. Moreover, gene expression studies have shown many nonstandard, unexpected protein products, including the production of antigens derived from aberrant protein fragments as a result of expression in alternative reading frames (6). Several studies report the identification of HLA ligands (7–10). Many results have been collected and discussed in a recent review on the large-scale analysis of HLA class I ligands (11). Collectively, these reports illustrate the need for in-depth elucidation of the HLA ligandome.Elucidation of T cell epitopes has traditionally been achieved with the use of a forward immunological approach, as pioneered by Hunt and coworkers (12, 13). In this approach, the cognate peptide of T cells with the appropriate activity profile is elucidated via repeated rounds of chromatographic separation in combination with T cell recognition assays. Because T cells are not always available from the start, reverse immunological approaches (14–17) have been developed to predict T cell epitopes through a combination of bioinformatics and in vitro proteasome digests. Predicted epitopes are synthesized and tested for their capability to activate T cells. The main disadvantage of this approach is that less than 0.1% of the peptides that survive intracellular processing are presented on HLA class I molecules (3).Therefore, we developed a large-scale peptidomics approach that is a reverse immunology approach based not on algorithms but on the bona fide eluted ligandome, which means that the identified peptides are known to have survived processing and are bona fide HLA ligands. Once the ligandome has been identified as comprehensively as possible, T cells can subsequently be selected on the basis of the immunological question at hand, as will be illustrated in a separate paper.² The development of MHC exchange tetramers for finding relevant T cell epitopes is instrumental to this approach (18, 19).To improve ligandome coverage, we applied and compared three off-line first dimension separation techniques, followed by on-line nano-HPLC-tandem MS.The tandem mass spectra were interrogated by being matched against the International Protein Index (IPI) human database (20). In a second step, post-translation modifications (phosphorylation, cysteinylation) were allowed in the database search. In a third step, the tandem mass spectra were matched against a newly in-house developed database for the optimal identification of polymorphic ligands to find potential minor histocompatibility antigens (21). This led to the identification of ∼14,000 HLA class I ligands, the majority of which also were relatively quantitated. Next, we analyzed the peptides constituting our ligandome in as much detail as possible to confirm the correct identification of the vast majority of the ligands. We achieved this through a combination of several physicochemical and biological checks and comparison with existing ligand and epitope databases.Finally, as an additional quality check, we illustrated the functional relevance of the ligandome through the identification of both previously known and new minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands (phosphorylated ligands and cysteinylated ligands) (22–24). This is the largest ligandome reported to date, and it allows general insight into the presented peptide repertoire. This study supports the building of the “immunopeptidome” as has recently been suggested (25). A proteomics approach can be used as a starting point for contributions to immunology by providing a peptidome landscape in many immunological studies, both fundamental and applied.     

BAG3 directly interacts with mutated alphaB-crystallin to suppress its aggregation and toxicity

A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein αB-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type αB-crystallin and the αB-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by αB-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.     

The Circadian Clock Gates the Intestinal Stem Cell Regenerative State

1. Download : Download full-size image