The S-layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C-terminal hydrophobic domain

PS2 is the S-layer protein of Corynebacterium glutamicum . The S-layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70°C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S-layer, which remains organized. Because we show that trypsin cleaves the C-terminal part of the protein, we conclude that this domain is involved in the association of the S-layer to the cell but is not essential in the interaction between individual PS2 proteins within the S-layer. A modified form of PS2, deleted of its C-terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S-layer and is mainly released into the medium. We suggest that PS2 is anchored via its C-terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane.     

Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) have opposing but complementary functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore, regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal truncation products all bind to TGF-β1 and TGF-β2 with a similar but somewhat less affinity than they bind BMP-2; that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-β with greater affinity than do the truncation products; that FL-Spp24 inhibits TGF-β2 induced bone formation in vivo, but Spp14.5 does not; and that co-administration of FL-Spp24 or Spp14.5 with TGF-β2 in vivo is associated with a reduction in the amount of cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-dose TGF-β administration in vivo is associated with greater bone formation than high-dose TGF-β administration, and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-β activity or availability during bone growth and development. The similarities and differences of the interactions between Spp24 proteins and TGF-β compared to the interaction of the Spp24 proteins and BMPs have significant implications with respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal disorders.     

Animal digestive strategies versus anaerobic digestion bioprocesses for biogas production from lignocellulosic biomass

Herbivorous mammals and wood-eating insects are fairly effective in the digestion of plant polymers, such as lignocellulosics. In order to improve methane production from the lignocellulosic biomass, several kinds of anaerobic digestion processes derived from animal models have been devised. However, the rates of biodegradation occurring in the anaerobic bioreactors still remain lower than in animal guts. The effectiveness of the digestive systems of those animals results from the concerted action of the various enzymes (e.g. cellulases, xylanases, esterases, ligninases) produced in their guts as well as their integration with mechanical and chemical actions. Powerful pretreatment (prefermentation) operations are integrated to and support efficiently the microbial fermentation system, e.g. the rumination (i.e. mechanical) in ruminants and the secretion of endogenous cellulases (i.e. enzymatic) or the alkaline treatment (chemical) at mid-way in xylophagous insects. The oxygen gradients along the gastrointestinal tract may also stimulate the hydrolytic activities of some microbial populations. In addition, the solid retention time, the digesta flow and the removal of the end-products are well ordered to enable animals to thrive on a complex polymer such as lignocellulose. At the same time, technologies were developed to degrade lignocellulosic biomass, such as the rumen derived anaerobic digestion (RUDAD) process and the rumen simulating technique (RUSITEC), more elaborated and using rumen microbial consortia. Overall, they showed that the fermentation taking place in the rumen fermentation and even in the hindgut are biological processes that go beyond the limited environmental conditions generally found in anaerobic digesters. Hence, knowledge on herbivores' digestion mechanisms might be better exploited in the design and operation of anaerobic digesters. This literature review is a cross-analysis of the relevant information about the digestive strategies of herbivorous and wood-eating animals and the bioengineering techniques in lignocelluloses degradation. The aim is to highlight strategies of animals' digestion simulation for more effective anaerobic digestion of lignocellulosic compounds and other solid residues.     

Mitochondrial hsp60 chaperonopathy causes an autosomal-recessive neurodegenerative disorder linked to brain hypomyelination and leukodystrophy

Hypomyelinating leukodystrophies (HMLs) are disorders involving aberrant myelin formation. The prototype of primary HMLs is the X-linked Pelizaeus-Merzbacher disease (PMD) caused by mutations in PLP1. Recently, homozygous mutations in GJA12 encoding connexin 47 were found in patients with autosomal-recessive Pelizaeus-Merzbacher-like disease (PMLD). However, many patients of both genders with PMLD carry neither PLP1 nor GJA12 mutations. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD, in which linkage to PLP1 and GJA12 was excluded. Using homozygosity mapping and mutation analysis, we have identified a homozygous missense mutation (D29G) not previously described in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60) in all affected individuals. The D29G mutation completely segregates with the disease-associated phenotype. The pathogenic effect of D29G on Hsp60-chaperonin activity was verified by an in vivo E. coli complementation assay, which demonstrated compromised ability of the D29G-Hsp60 mutant protein to support E. coli survival, especially at high temperatures. The disorder, which we have termed MitCHAP-60 disease, can be distinguished from spastic paraplegia 13 (SPG13), another Hsp60-associated autosomal-dominant neurodegenerative disorder, by its autosomal-recessive inheritance pattern, as well as by its early-onset, profound cerebral involvement and lethality. Our findings suggest that Hsp60 defects can cause neurodegenerative pathologies of varying severity, not previously suspected on the basis of the SPG13 phenotype. These findings should help to clarify the important role of Hsp60 in myelinogenesis and neurodegeneration.     

Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL MscL does not need the insertase YidC for insertion in vitro

The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.     

Alternative chemical modifications reverse the binding orientation of a pharmacophore scaffold in the active site of macrophage migration inhibitory factor

Pharmacophores are chemical scaffolds upon which changes in chemical moieties (R-groups) at specific sites are made to identify a combination of R-groups that increases the therapeutic potency of a small molecule inhibitor while minimizing adverse effects. We developed a pharmacophore based on a carbonyloxime (OXIM) scaffold for macrophage migration inhibitory factor (MIF), a protein involved in the pathology of sepsis, to validate that inhibition of a catalytic site could produce therapeutic benefits. We studied the crystal structures of MIF.OXIM-based inhibitors and found two opposite orientations for binding to the active site that were dependent on the chemical structures of an R-group. One orientation was completely unexpected based on previous studies with hydroxyphenylpyruvate and (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1). We further confirmed that the unexpected binding mode targets MIF in cellular studies by showing that one compound, OXIM-11, abolished the counter-regulatory activity of MIF on anti-inflammatory glucocorticoid action. OXIM-11 treatment of mice, initiated 24 h after the onset of cecal ligation and puncture-induced sepsis, significantly improved survival when compared with vehicle-treated controls, confirming that inhibition of the MIF catalytic site could produce therapeutic effects. The crystal structures of the MIF inhibitor complexes provide insight for further structure-based drug design efforts.     

Correction to: Solid-Phase Synthesis and Antibacterial Activity of an Artificial Cyclic Peptide Containing Two Disulfide Bridges

International Journal of Peptide Research and Therapeutics - The original version of this article unfortunately contained a typo in the author name. The co-author name should be Khalil K.Y. Saleh...     

The cultural contagion of conflict

Anecdotal evidence abounds that conflicts between two individuals can spread across networks to involve a multitude of others. We advance a cultural transmission model of intergroup conflict where conflict contagion is seen as a consequence of universal human traits (ingroup preference, outgroup hostility; i.e. parochial altruism) which give their strongest expression in particular cultural contexts. Qualitative interviews conducted in the Middle East, USA and Canada suggest that parochial altruism processes vary across cultural groups and are most likely to occur in collectivistic cultural contexts that have high ingroup loyalty. Implications for future neuroscience and computational research needed to understand the emergence of intergroup conflict are discussed.     

S-layer protein transport across the cell wall of Corynebacterium glutamicum: in vivo kinetics and energy requirements

Corynebacteria are Gram-positive bacteria with a very peculiar cell envelope structure as it is constituted of an inner membrane and an outer membrane-like structure. Protein secretion in Corynebacterium glutamicum was studied in vivo, using the S-layer protein PS2 as a model. We show that different variants of PS2 protein are exported through the whole cell envelope with a half-life ranging between 2 and 4 min, by a two-step mechanism. The first step, which is over after about 1.5 min, is ATP- and proton motive force-dependent and may correspond to translocation across the inner membrane via the 'Sec' machinery. The second step, across the cell wall and the outer mycolate layer, is rapid but independent of energy sources. This very efficient secretion process across the mycolate layer raises the question of the existence in this layer of a specific machinery.