Premature ageing in mice expressing defective mitochondrial DNA polymerase

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.     

Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase

In higher organisms the formation of the steroid scaffold is catalysed exclusively by the membrane-bound oxidosqualene cyclase (OSC; lanosterol synthase). In a highly selective cyclization reaction OSC forms lanosterol with seven chiral centres starting from the linear substrate 2,3-oxidosqualene. Valuable data on the mechanism of the complex cyclization cascade have been collected during the past 50 years using suicide inhibitors, mutagenesis studies and homology modelling. Nevertheless it is still not fully understood how the enzyme catalyses the reaction. Because of the decisive role of OSC in cholesterol biosynthesis it represents a target for the discovery of novel anticholesteraemic drugs that could complement the widely used statins. Here we present two crystal structures of the human membrane protein OSC: the target protein with an inhibitor that showed cholesterol lowering in vivo opens the way for the structure-based design of new OSC inhibitors. The complex with the reaction product lanosterol gives a clear picture of the way in which the enzyme achieves product specificity in this highly exothermic cyclization reaction.     

Crystal symmetry and the reversibility of martensitic transformations

Martensitic transformations are diffusionless, solid-to-solid phase transitions, and have been observed in metals, alloys, ceramics and proteins. They are characterized by a rapid change of crystal structure, accompanied by the development of a rich microstructure. Martensitic transformations can be irreversible, as seen in steels upon quenching, or they can be reversible, such as those observed in shape-memory alloys. In the latter case, the microstructures formed on cooling are easily manipulated by loads and disappear upon reheating. Here, using mathematical theory and numerical simulation, we explain these sharp differences in behaviour on the basis of the change in crystal symmetry during the transition. We find that a necessary condition for reversibility is that the symmetry groups of the parent and product phases be included in a common finite symmetry group. In these cases, the energy barrier to lattice-invariant shear is generically higher than that pertaining to the phase change and, consequently, transformations of this type can occur with virtually no plasticity. Irreversibility is inevitable in all other martensitic transformations, where the energy barrier to plastic deformation (via lattice-invariant shears, as in twinning or slip) is no higher than the barrier to the phase change itself. Various experimental observations confirm the importance of the symmetry of the stable states in determining the macroscopic reversibility of martensitic transformations.     

Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome

We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both alleles of POLR1C in three individuals with TCS. These findings identify two additional genes involved in TCS, confirm the genetic heterogeneity of TCS and support the hypothesis that TCS is a ribosomopathy.     

A genome-wide association study of metabolic traits in human urine

We present a genome-wide association study of metabolic traits in human urine, designed to investigate the detoxification capacity of the human body. Using NMR spectroscopy, we tested for associations between 59 metabolites in urine from 862 male participants in the population-based SHIP study. We replicated the results using 1,039 additional samples of the same study, including a 5-year follow-up, and 992 samples from the independent KORA study. We report five loci with joint P values of association from 3.2 × 10(-19) to 2.1 × 10(-182). Variants at three of these loci have previously been linked with important clinical outcomes: SLC7A9 is a risk locus for chronic kidney disease, NAT2 for coronary artery disease and genotype-dependent response to drug toxicity, and SLC6A20 for iminoglycinuria. Moreover, we identify rs37369 in AGXT2 as the genetic basis of hyper-β-aminoisobutyric aciduria.     

Structure-based activity prediction for an enzyme of unknown function

With many genomes sequenced, a pressing challenge in biology is predicting the function of the proteins that the genes encode. When proteins are unrelated to others of known activity, bioinformatics inference for function becomes problematic. It would thus be useful to interrogate protein structures for function directly. Here, we predict the function of an enzyme of unknown activity, Tm0936 from Thermotoga maritima, by docking high-energy intermediate forms of thousands of candidate metabolites. The docking hit list was dominated by adenine analogues, which appeared to undergo C6-deamination. Four of these, including 5-methylthioadenosine and S-adenosylhomocysteine (SAH), were tested as substrates, and three had substantial catalytic rate constants (10(5) M(-1 )s(-1)). The X-ray crystal structure of the complex between Tm0936 and the product resulting from the deamination of SAH, S-inosylhomocysteine, was determined, and it corresponded closely to the predicted structure. The deaminated products can be further metabolized by T. maritima in a previously uncharacterized SAH degradation pathway. Structure-based docking with high-energy forms of potential substrates may be a useful tool to annotate enzymes for function.