Msh2 Acts in Medium-Spiny Striatal Neurons as an Enhancer of CAG Instability and Mutant Huntingtin Phenotypes in Huntington's Disease Knock-In Mice

The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.     

The Sortase A Enzyme That Attaches Proteins to the Cell Wall of Bacillus anthracis Contains an Unusual Active Site Architecture

The pathogen Bacillus anthracis uses the Sortase A (SrtA) enzyme to anchor proteins to its cell wall envelope during vegetative growth. To gain insight into the mechanism of protein attachment to the cell wall in B. anthracis we investigated the structure, backbone dynamics, and function of SrtA. The NMR structure of SrtA has been determined with a backbone coordinate precision of 0.40 ± 0.07 Å. SrtA possesses several novel features not previously observed in sortase enzymes including the presence of a structurally ordered amino terminus positioned within the active site and in contact with catalytically essential histidine residue (His¹²⁶). We propose that this appendage, in combination with a unique flexible active site loop, mediates the recognition of lipid II, the second substrate to which proteins are attached during the anchoring reaction. pK_a measurements indicate that His¹²⁶ is uncharged at physiological pH compatible with the enzyme operating through a “reverse protonation” mechanism. Interestingly, NMR relaxation measurements and the results of a model building study suggest that SrtA recognizes the LPXTG sorting signal through a lock-in-key mechanism in contrast to the prototypical SrtA enzyme from Staphylococcus aureus.     

Evolutionary Gain of Function for the ER Membrane Protein Sec62 from Yeast to Humans

Because of similarity to their yeast orthologues, the two membrane proteins of the human endoplasmic reticulum (ER) Sec62 and Sec63 are expected to play a role in protein biogenesis in the ER. We characterized interactions between these two proteins as well as the putative interaction of Sec62 with ribosomes. These data provide further evidence for evolutionary conservation of Sec62/Sec63 interaction. In addition, they indicate that in the course of evolution Sec62 of vertebrates has gained an additional function, the ability to interact with the ribosomal tunnel exit and, therefore, to support cotranslational mechanisms such as protein transport into the ER. This view is supported by the observation that Sec62 is associated with ribosomes in human cells. Thus, the human Sec62/Sec63 complex and the human ER membrane protein ERj1 are similar in providing binding sites for BiP in the ER-lumen and binding sites for ribosomes in the cytosol. We propose that these two systems provide similar chaperone functions with respect to different precursor proteins.     

Ligand Depletion in vivo Modulates the Dynamic Range and Cooperativity of Signal Transduction

Biological signal transduction commonly involves cooperative interactions in the binding of ligands to their receptors. In many cases, ligand concentrations in vivo are close to the value of the dissociation constant of their receptors, resulting in the phenomenon of ligand depletion. Using examples based on rotational bias of bacterial flagellar motors and calcium binding to mammalian calmodulin, we show that ligand depletion diminishes cooperativity and broadens the dynamic range of sensitivity to the signaling ligand. As a result, the same signal transducer responds to different ranges of signal with various degrees of cooperativity according to its effective cellular concentration. Hence, results from in vitro dose-response analyses cannot be applied directly to understand signaling in vivo. Moreover, the receptor concentration is revealed to be a key element in controlling signal transduction and we propose that its modulation constitutes a new way of controlling sensitivity to signals. In addition, through an analysis of the allosteric enzyme aspartate transcarbamylase, we demonstrate that the classical Hill coefficient is not appropriate for characterizing the change in conformational state upon ligand binding to an oligomeric protein (equivalent to a dose-response curve), because it ignores the cooperativity of the conformational change for the corresponding equivalent monomers, which are generally characterized by a Hill coefficient . Therefore, we propose a new index of cooperativity based on the comparison of the properties of oligomers and their equivalent monomers.     

Time‐resolved quantitative proteome profiling of host–pathogen interactions: The response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells

Staphylococcus aureus is a versatile Gram‐positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse‐chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on‐membrane digestion, and high‐sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof‐of‐principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.     

A putative transporter is essential for integrating nutrient and hormone signaling with lateral root growth and nodule development in Medicago truncatula

Legume root architecture involves not only elaboration of the root system by the formation of lateral roots but also the formation of symbiotic root nodules in association with nitrogen‐fixing soil rhizobia. The Medicago truncatula LATD/NIP gene plays an essential role in the development of both primary and lateral roots as well as nodule development. We have cloned the LATD/NIP gene and show that it encodes a member of the NRT1(PTR) transporter family. LATD/NIP is expressed throughout the plant. pLATD/NIP‐GFP promoter–reporter fusions in transgenic roots establish the spatial expression of LATD/NIP in primary root, lateral root and nodule meristems and the surrounding cells. Expression of LATD/NIP is regulated by hormones, in particular by abscisic acid which has been previously shown to rescue the primary and lateral root meristem arrest of latd mutants. latd mutants respond normally to ammonium but have defects in responses of the root architecture to nitrate. Taken together, these results suggest that LATD/NIP may encode a nitrate transporter or transporter of another compound.