Faculty Bibliography

In mammals, the prototypical endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1 (IRE1) has diverged into two paralogs. IRE1alpha is broadly expressed and mediates the unconventional splicing of X-box binding protein 1 (XBP1) mRNA during ER stress. By contrast, IRE1beta is expressed selectively in the digestive tract, and its function remains unclear. Here, we report that IRE1beta plays a distinctive role in mucin-secreting goblet cells. In IRE1beta(-/-) mice, aberrant mucin 2 (MUC2) accumulated in the ER of goblet cells, accompanied by ER distension and elevated ER stress signaling such as increased XBP1 mRNA splicing. In contrast, conditional IRE1alpha(-/-) mice showed no such ER distension but a marked decrease in spliced XBP1 mRNA. mRNA stability assay revealed that MUC2 mRNA was greatly stabilized in IRE1beta(-/-) mice. These findings suggest that in goblet cells, IRE1beta, but not IRE1alpha, promotes efficient protein folding and secretion in the ER by optimizing the level of mRNA encoding their major secretory product, MUC2.

Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrP(C)) into an amyloidogenic beta-sheet infectious form (PrP(Sc)). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnp(a), encoding the polymorphism Leu-108/Thr-189) or b (Prnp(b), Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrP(Sc). The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrP(C) into PrP(Sc) remain unclear. Here we show that recombinant PrP(a) and PrP(b) aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrP(b) exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrP(b) is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP.

Accumulation of double-stranded DNA breaks and inhibition of DNA repair contributes to reproductive aging by diminishing ovarian reserves in mice and women.

SNPs in the first intron of FTO (fat mass and obesity associated) are strongly associated with human obesity. While it is not yet formally established that this effect is mediated through the actions of the FTO protein itself, loss of function mutations in FTO or its murine homologue Fto result in severe growth retardation, and mice globally overexpressing FTO are obese. The mechanisms through which FTO influences growth and body composition are unknown. We describe a role for FTO in the coupling of amino acid levels to mammalian target of rapamycin complex 1 signaling. These findings suggest that FTO may influence body composition through playing a role in cellular nutrient sensing.

Energy-coupling factor (ECF) transporters are a recently discovered family of primary active transporters for micronutrients and vitamins, such as biotin, thiamine, and riboflavin. Found exclusively in archaea and bacteria, including the human pathogens Listeria, Streptococcus, and Staphylococcus, ECF transporters may be the only means of vitamin acquisition in these organisms. The subunit composition of ECF transporters is similar to that of ATP binding cassette (ABC) importers, whereby both systems share two homologous ATPase subunits (A and A'), a high affinity substrate-binding subunit (S), and a transmembrane coupling subunit (T). However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between the two transporter families. Moreover, the subunit stoichiometry of ECF transporters is controversial, and the detailed molecular interactions between subunits and the conformational changes during substrate translocation are unknown. We have characterized the ECF transporters from Thermotoga maritima and Streptococcus thermophilus. Our data suggests a subunit stoichiometry of 2S:2T:1A:1A' and that S subunits for different substrates can be incorporated into the same transporter complex simultaneously. In the first crystal structure of the A-A' heterodimer, each subunit contains a novel motif called the Q-helix that plays a key role in subunit coupling with the T subunits. Taken together, these findings suggest a mechanism for coupling ATP binding and hydrolysis to transmembrane transport by ECF transporters.

The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or nonhomologous end joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ, and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1; limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage; and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.

To discover interordinal relationships of living and fossil placental mammals and the time of origin of placentals relative to the Cretaceous-Paleogene (K-Pg) boundary, we scored 4541 phenomic characters de novo for 86 fossil and living species. Combining these data with molecular sequences, we obtained a phylogenetic tree that, when calibrated with fossils, shows that crown clade Placentalia and placental orders originated after the K-Pg boundary. Many nodes discovered using molecular data are upheld, but phenomic signals overturn molecular signals to show Sundatheria (Dermoptera + Scandentia) as the sister taxon of Primates, a close link between Proboscidea (elephants) and Sirenia (sea cows), and the monophyly of echolocating Chiroptera (bats). Our tree suggests that Placentalia first split into Xenarthra and Epitheria; extinct New World species are the oldest members of Afrotheria.

YiiP is a dimeric Zn(2+)/H(+) antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-A resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn(2+). Starting from the X-ray structure in the presence of Zn(2+), we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.

During animal development, SDF1 simultaneously guides various cell types to different targets. As many targets are in close proximity to one another, it is unclear how the system avoids mistargeting. Zebrafish trigeminal sensory neurons express the SDF1 receptor Cxcr4b and encounter multiple SDF1 sources during migration, but ignore all but the correct one. We show that miR-430 and Cxcr7b regulation of SDF1a are required for precise guidance. In the absence of miR-430 or Cxcr7b, neurons responded to ectopic SDF1a sources along their route and did not reach their target. This was due to a failure to clear SDF1a transcript and protein from sites of expression that the migrating neurons had already passed. Our findings suggest an "attractive path" model in which migrating cells closely follow a dynamic SDF1a source that is refined on a transcript and protein level by miR-430 and Cxcr7b, respectively.