Design principles for robust biochemical reaction networks: what works, what cannot work, and what might almost work

We bring together recent results that connect the structure of a mass-action reaction network to its capacity for concentration robustness — that is, its capacity to keep the concentration of a critical bio-active species within narrow limits, even against large fluctuations in the overall supply of the network’s constituents.     

Human mutation within Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) causes basal insulin hypersecretion

PAS kinase (PASK) is a glucose-regulated protein kinase involved in the control of pancreatic islet hormone release and insulin sensitivity. We aimed here to identify mutations in the PASK gene that may be associated with young-onset diabetes in humans. We screened 18 diabetic probands with unelucidated maturity-onset diabetes of the young (MODY). We identified two rare nonsynonymous mutations in the PASK gene (p.L1051V and p.G1117E), each of which was found in a single MODY family. Wild type or mutant PASKs were expressed in HEK 293 cells. Kinase activity of the affinity-purified proteins was assayed as autophosphorylation at amino acid Thr307 or against an Ugp1p-derived peptide. Whereas the PASK p.G1117E mutant displayed a ～25% increase with respect to wild type PASK in the extent of autophosphorylation, and a ～2-fold increase in kinase activity toward exogenous substrates, the activity of the p.L1051V mutant was unchanged. Amino acid Gly1117 is located in an α helical region opposing the active site of PASK and may elicit either: (a) a conformational change that increases catalytic efficiency or (b) a diminished inhibitory interaction with the PAS domain. Mouse islets were therefore infected with adenoviruses expressing wild type or mutant PASK and the regulation of insulin secretion was examined. PASK p.G1117E-infected islets displayed a 4-fold decrease in glucose-stimulated (16.7 versus 3 mM) insulin secretion, chiefly reflecting a 4.5-fold increase in insulin release at low glucose. In summary, we have characterized a rare mutation (p.G1117E) in the PASK gene from a young-onset diabetes family, which modulates glucose-stimulated insulin secretion.     

Transit defect of potassium-chloride Co-transporter 3 is a major pathogenic mechanism in hereditary motor and sensory neuropathy with agenesis of the corpus callosum

Missense and protein-truncating mutations of the human potassium-chloride co-transporter 3 gene (KCC3) cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe neurodegenerative disease characterized by axonal dysfunction and neurodevelopmental defects. We previously reported that KCC3-truncating mutations disrupt brain-type creatine kinase-dependent activation of the co-transporter through the loss of its last 140 amino acids. Here, we report a novel and more distal HMSN/ACC-truncating mutation (3402C → T; R1134X) that eliminates only the last 17 residues of the protein. This small truncation disrupts the interaction with brain-type creatine kinase in mammalian cells but also affects plasma membrane localization of the mutant transporter. Although it is not truncated, the previously reported HMSN/ACC-causing 619C → T (R207C) missense mutation also leads to KCC3 loss of function in Xenopus oocyte flux assay. Immunodetection in Xenopus oocytes and in mammalian cultured cells revealed a decreased amount of R207C at the plasma membrane, with significant retention of the mutant proteins in the endoplasmic reticulum. In mammalian cells, curcumin partially corrected these mutant protein mislocalizations, with more protein reaching the plasma membrane. These findings suggest that mis-trafficking of mutant protein is an important pathophysiological feature of HMSN/ACC causative KCC3 mutations.     

Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an α-helix. NMR analysis identifies two putative α-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.     

Benzo[f]naphtyridones: a new family of topical antibacterial agents active on multi-resistant Gram-positive pathogens

The synthesis and antibacterial activity of benzo[f][1,7]naphtyridone derivatives are reported. These compounds are potent antibacterial agents with a Gram-positive spectrum of activity. They are active against multi-resistant cocci, especially Staphylococcus aureus strains. Their physico-chemical and biological properties make them particularly suitable for topical antibacterial use.     

Modulation of TRPV4 gating by intra- and extracellular Ca2+

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4alphaPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+](e)) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+](e). It was also much slower in the presence of [Ca2+](e) in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797), were constitutively open. In conclusion, gating of the 4alphaPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.     

Crosstalk between nitric oxide and zinc pathways to neuronal cell death involving mitochondrial dysfunction and p38-activated K+ channels

Nitric oxide (NO) and zinc (Zn2+) are implicated in the pathogenesis of cerebral ischemia and neurodegenerative diseases. However, their relationship and the molecular mechanism of their neurotoxic effects remain unclear. Here we show that addition of exogenous NO or NMDA (to increase endogenous NO) leads to peroxynitrite (ONOO-) formation and consequent Zn2+ release from intracellular stores in cerebrocortical neurons. Free Zn2+ in turn induces respiratory block, mitochondrial permeability transition (mPT), cytochrome c release, generation of reactive oxygen species (ROS), and p38 MAP kinase activation. This pathway leads to caspase-independent K+ efflux with cell volume loss and apoptotic-like death. Moreover, Zn2+ chelators, ROS scavengers, Bcl-xL, dominant-interfering p38, or K+ channel blockers all attenuate NO-induced K+ efflux, cell volume loss, and neuronal apoptosis. Thus, these data establish a new form of crosstalk between NO and Zn2+ apoptotic signal transduction pathways that may contribute to neurodegeneration.     

Genetically Engineered Immunomodulatory Streptococcus thermophilus Strains Producing Antioxidant Enzymes Exhibit Enhanced Anti-Inflammatory Activities

The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.