The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1

Crystal structures of a membrane protein transporter in three different conformational states provide insights into the transport mechanism

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1

Identification of novel genes associated with longevity in Drosophila melanogaster - a computational approach

Despite a growing number of studies on longevity in Drosophila, genetic factors influencing lifespan are still poorly understood. In this paper we propose a conceptually new approach for the identification of novel longevity-associated genes and potential target genes for SNPs in non-coding regions by utilizing the knowledge of co-location of various loci, governed by the three-dimensional architecture of the Drosophila genome. Firstly, we created networks between genes/genomic regions harboring SNPs deemed to be significant in two longevity GWAS summary statistics datasets using intra- and inter-chromosomal interaction frequencies (Hi-C data) as a measure of co-location. These networks were further extended to include regions strongly interacting with previously selected regions. Using various network measures, literature search and additional bioinformatics resources, we investigated the plausibility of genes found to have genuine association with longevity. Several of the newly identified genes were common between the two GWAS datasets and these possessed human orthologs. We also found that the proportion of non-coding SNPs in borders between topologically associated domains is significantly higher than expected by chance. Assuming co-location, we investigated potential target genes for non-coding SNPs. This approach therefore offers a stepping stone to identification of novel genes and SNP targets linked to human longevity

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Reinigung, Kristallisation und Röntgenstrukturanalyse der Proteine des Lysinbiosyntheseweges von Mycobacterium Tuberculosis und Strukturanalyse von Membranproteinen von Deinococcus radiodurans R1 und Escherichia coli K12

Since the lysine biosynthetic pathway is present in bacteria, fungi and plants but absent from mammals its 9 annotated enzymes have drawn considerable attention as potential candidates for new drugs against tuberculosis. Within this work the 3 proteins Rv0858c, Rv1201c and Rv1293 have been selected for structural analysis. Rv0858c protein has been purified, crystallised and the structure determined to a resolution of 2.0 Å. In addition, pure protein of Rv1201c has been crystallised successfully and first attempts towards structure determination were carried out. For the third target, Rv1293, an existing dataset was used to re-build the model and to refine the structure. In addition this structure was compared in detail to the same target solved earlier in a different space group.A detailed bioinformatical analysis was carried out for all 9 annotated proteins of the lysine biosynthetic pathway. The entire analysis was based on the questions: are too many or too few proteins annotated within the pathway?, is the order of proteins correctly annotated and is there any hint towards complex formation?A second project dealt with the analysis of structures of several outer and inner membrane proteins. A new bacterial strain was therefore introduced to the lab and basic membrane protein laboratory procedures were established. Different well established protocols were applied in order to extract all the present membrane proteins from the outer membranes. Different candidate proteins were analysed by Maldi-tof mass spectroscopy and sequence comparisons.Additionally the inner membrane protein Nramp of Deinococcus radiodurans R1 was selected for structural analysis. The target sequence of this protein was amplified by PCR, cloned into different vectors and the gene product overexpressed in different host organisms. The overexpression procedure was optimised and a purification protocol established. The protein was crystallised in lipidic cubic phases as well as by counter diffusion. These crystals were optimised and diffraction was obtained to a resolution of approximately 30 Å.In order to set up a SAXS approach for membrane proteins, the model protein OmpF of E.coli was selected. The experimental set up was optimised and the shape of the obtained model reflected the high resolution structure which was clearly reproduced. Also, the Nramp transporter of the inner membrane was measured using this technique, but could not structurally characterised so far

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapC (Rv0858c) from Mycobacterium tuberculosis

M. tuberculosis N-succinyldiaminopimelate aminotransferase, the enzyme which catalyzes the sixth reaction in the lysine-biosynthesis pathway, has been cloned, expressed, purified and crystallized

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of tetrahydrodipicolinate-N-succinyltransferase (Rv1201c) from Mycobacterium tuberculosis

M. tuberculosis tetrahydrodipicolinate-N-succinyltransferase, the enzyme that catalyses the fifth reaction step of the lysine-biosynthesis pathway, has been cloned, expressed, purified and crystallized

Mesothelial/monocytic incidental cardiac excrescences (cardiac MICE) associated with acute aortic dissection: a study of two cases

Acute aortic dissection is a life-threatening condition mainly caused by hypertension, atherosclerotic disease and other degenerative diseases of the connective tissue of the aortic wall. Mesothelial/monocytic incidental cardiac excrescences (cardiac MICE) is a rare benign reactive tumor-like lesion composed of admixture of histiocytes, mesothelial cells, and inflammatory cells set within a fibrinous meshwork without a vascular network or supporting stroma. Cardiac MICE occurring in association with aortic dissection is exceptionally rare (only one such case reported to date). We herein report on the surgical repair of two Stanford type A aortic dissections caused by idiopathic giant cell aortitis in a 66-year-old-woman and by atherosclerotic disease in a 58-year-old-man, respectively. In both cases, the dissections could be visualized via computed tomography. Histopathology showed cardiac incidental MICE within the external aortic wall near the pericardial surface which was confirmed by immunohistochemistry