A Modular BAM Complex in the Outer Membrane of the α-Proteobacterium Caulobacter crescentus

Mitochondria are organelles derived from an intracellular α-proteobacterium. The biogenesis of mitochondria relies on the assembly of β-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an α-proteobacterium, and the BAM (β-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A ∼150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane

Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM

The β-barrel assembly machinery (BAM) is a ~203 kDa complex of five proteins (BamA-E) which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating’ motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex, and interactions between BamA, B, D, and E and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM

Identification and Characterization of the Lamprey High-Mobility Group Box 1 Gene

High-mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. We identified a homolog of HMGB1 in the Japanese lamprey (Lampetra japonica). The Lampetra japonica HMGB1 gene (Lj-HMGB1) has over 70% sequence identity with its homologs in jawed vertebrates. Despite the reasonably high sequence identity with other HMGB1 proteins, Lj-HMGB1 did not group together with these proteins in a phylogenetic analysis. We examined Lj-HMGB1 expression in lymphocyte-like cells, and the kidneys, heart, gills, and intestines of lampreys before and after the animals were challenged with lipopolysaccharide (LPS) and concanavalin A (ConA). Lj-HMGB1 was initially expressed at a higher level in the heart, but after treatment with LPS and ConA only the gills demonstrated a significant up-regulation of expression. The recombinant Lj-HMGB1 (rLj-HMGB1) protein bound double-stranded DNA and induced the proliferation of human adenocarcinoma cells to a similar extent as human HMGB1. We further revealed that Lj-HMGB1 was able to induce the production of tumor necrosis factor-α (TNF-α), a pro-inflammatory mediator, in activated human acute monocytic leukemia cells. These results suggest that lampreys use HMGB1 to activate their innate immunity for the purpose of pathogen defense

Laforin, a Dual Specificity Phosphatase Involved in Lafora Disease, Is Present Mainly as Monomeric Form with Full Phosphatase Activity

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding

A unified model for BAM function that takes into account type Vc secretion and species differences in BAM composition

Transmembrane proteins in the outer membrane of Gram-negative bacteria are almost exclusively β-barrels. They are inserted into the outer membrane by a conserved and essential protein complex called the BAM (for β-barrel assembly machinery). In this commentary, we summarize current research into the mechanism of this protein complex and how it relates to type V secretion. Type V secretion systems are autotransporters that all contain a β-barrel transmembrane domain inserted by BAM. In type Vc systems, this domain is a homotrimer. We argue that none of the current models are sufficient to explain BAM function particularly regarding type Vc secretion. We also find that current models based on the well-studied model system Escherichia coli mostly ignore the pronounced differences in BAM composition between different bacterial species. We propose a more holistic view on how all OMPs, including autotransporters, are incorporated into the lipid bilayer

Book spreads in PG (7, 2)

An (n,q,r,s) book is a collection of r-subspaces in PG(n,q) called pages, which cover the whole projective space and intersect in a common s-subspace called the spine such that any point outside the spine is in exactly one page. An (n,q,r,s) book t-spread is a t-spread in PG(n,q) for which there exists an (n,q,r,s) book, such that the points of each page of this book and hence the points of the spine are partitioned by t-subspaces of the t-spread. We commence by showing that an (n,q,r,s)book t-spread exists if and only if the following three conditions hold: (i) (r-s)|(n-s),(ii) (t+1)|(s+1),(iii) (t+1)|(r+1). In general the number of different kinds of (n,q,r,s) book t-spreads is a tiny proportion of the number of different kinds of t-spreads in PG(n,q). In the rest of this paper we present computer-aided classification results for certain types of (7,2,5,3) book 1-spreads. © 2014 Published by Elsevier B.V

Investigation of Materials used by Edvard Munch

The pigments and paint binders used by Edvard Munch have been investigated in several studies. Munch used a mixture of media in his works of art. The two versions of The Scream studied here were found to include oil paints and oil paints thickened with beeswax and also oil crayons containing beeswax and Japan wax, as well as casein pastels, a paraffin wax crayon and at least one gum-bound paint. His sketches on canvas make use of oil paints and tempera paints including egg and casein, as well as casein pastels in at least one instance. His oil paintings on canvas seem to have been executed using a more conventional technique, with most having one or a few paint layers bound with linseed oil on a ground formed from lead white in oil on top of a ground made of chalk in glue. Munch's palette is not extensive, though he was reasonably willing to introduce new materials, such as his use of a petroleum-based wax crayon in 1893, oil pastel - possibly as early as 1893 and certainly by 1910, and his use of cadmium red by 1927-1929. The identification of materials has informed conservators who are planning and carrying out conservation treatment

Influence of p-isopropenylcalixarenestyrene copolymer buffer layer over Alq

The characteristics of organic light-emitting devices based on aluminum tris-(8-hydroxy-quinoline) (Alq3), N,N'-bis(3-methylphenyl)-N,N'-diphenylbenzidine (TPD) with novel p-isopropenyl-calix[8]arenestyrene copolymer buffer layer and Al cathode were investigated. The devices with TPD/Alq3/Al were also fabricated in the same way for comparison. The p-isopropenylcalix[8]arenestyrene copolymer used as buffer layer greatly improved the performance of the device and increased the device efficiency and stability