Faculty Bibliography

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.

Studies in continuously cultured cells have established that familial Alzheimer's disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for beta- and gamma-cleavage. This chimeric substrate generated excess Abeta42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a beta-/gamma-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Abeta42

PURPOSE OF REVIEW: Decades of literature have unambiguously demonstrated regression and remodeling of atherosclerotic lesions, including advanced plaques. Recent insights into underlying mechanisms are reviewed. RECENT FINDINGS: Factors promoting regression include decreased apolipoprotein B-lipoprotein retention within the arterial wall, efflux of cholesterol and other harmful lipids from plaques, and emigration of lesional foam cells followed by entry of healthy phagocytes that remove necrotic debris and other plaque components. Cellular lipid efflux and foam cell emigration can occur surprisingly rapidly once the plaque milieu is improved. Lipid efflux and foam cell emigration each involve specific molecular mediators, many of which have been identified. Necrotic debris removal can be surprisingly comprehensive, with essentially full disappearance documented in animal models. SUMMARY: The essential prerequisite for regression is robust improvement in plaque milieu, meaning large plasma reductions in atherogenic apolipoprotein B-lipoproteins or brisk enhancements in 'reverse' lipid transport from plaque into liver. Importantly, the processes of regression are consistent with rapid correction of features characteristic of the rupture-prone, vulnerable plaques responsible for acute coronary syndromes. New interventions to lower apolipoprotein B-lipoprotein levels and enhance reverse lipid transport may allow regression to become a widespread clinical goal. Strategies based on recent mechanistic insights may facilitate further therapeutic progress

Epidemiological studies support an association between vascular risk factors, including hypercholesterolemia, and Alzheimer's disease (AD). Recently, there has been much interest in the possibility that hypercholesterolemia might directly promote beta-amyloid (Abeta) production. Indeed, in vitro studies have shown that increasing cellular cholesterol levels enhances Abeta production. However, studies in AD transgenic mouse models have not consistently found that elevated plasma cholesterol leads to increased Abeta production or deposition in vivo. In this study, we determined whether elevated peripheral cholesterol influences Abeta production in mice with a null mutation of the low-density lipoprotein receptor (LDLR). We show that dramatically elevated plasma cholesterol levels, whether induced by high cholesterol, high fat, or high fat/high cholesterol diets, did not affect either levels of brain Abeta40, Abeta42, or APP, or the Abeta42/40 or APP-CTF/APP ratios, nor substantially alter brain cholesterol levels. ApoE protein levels in brain were, however, elevated, in LDLR-/- mice by post-transcriptional mechanisms. Collectively, these studies argue that plasma cholesterol levels do not normally regulate production of brain Abeta

The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

The thyroid is an endocrine gland in all vertebrates that develops from the ventral floor of the anterior pharyngeal endoderm. Unravelling the molecular mechanisms of thyroid development helps to understand congenital hypothyroidism caused by the absence or reduction of this gland in newborn humans. Severely reduced or absent thyroid-specific developmental genes concomitant with the complete loss of the functional gland in the zebrafish hands off (han, hand2) mutant reveals the han gene as playing a novel, crucial role in thyroid development. han-expressing tissues surround the thyroid primordium throughout development. Fate mapping reveals that, even before the onset of thyroid-specific developmental gene expression, thyroid precursor cells are in close contact with han-expressing cardiac lateral plate mesoderm. Grafting experiments show that han is required in surrounding tissue, and not in a cell-autonomous manner, for thyroid development. Loss of han expression in the branchial arches and arch-associated cells after morpholino knock-down of upstream regulator genes does not impair thyroid development, indicating that other han-expressing structures, most probably cardiac mesoderm, are responsible for the thyroid defects in han mutants. The zebrafish ace (fgf8) mutant has similar thyroid defects as han mutants, and chemical suppression of fibroblast growth factor (FGF) signalling confirms that this pathway is required for thyroid development. FGF-soaked beads can restore thyroid development in han mutants, showing that FGFs act downstream of or in parallel to han. These data suggest that loss of FGF-expressing tissue in han mutants is responsible for the thyroid defects.

BACKGROUND: Surgical resection of isolated hepatic or pulmonary colorectal metastases prolongs survival in selected patients. But the benefits of resection and appropriate selection criteria in patients who develop both hepatic and pulmonary metastases are ill defined. STUDY DESIGN: Data were prospectively collected from 131 patients with colorectal cancer who underwent resection of both hepatic and pulmonary metastases over a 20-year period. Median followup was 6.6 years from the time of resection of the primary tumor. Patient, treatment, and outcomes variables were analyzed using log-rank, Cox regression, and Kaplan-Meier methods. RESULTS: The site of first metastasis was the liver in 65% of patients, the lung in 11%, and both simultaneously in 24%. Multiple hepatic metastases were present in 51% of patients, and multiple pulmonary metastases were found in 48%. Hepatic lobectomy or trisegmentectomy was required in 61% of patients; most lung metastases (80%) were treated with wedge excisions. Median survival rates from resection of the primary disease, first site of metastasis, and second site of metastasis were 6.9, 5.0, and 3.3 years, respectively. After resection of disease at the second site of metastasis, the 1-, 3-, 5-, and 10-year disease-specific survival rates were 91%, 55%, 31%, and 19%, respectively. An analysis of prognostic factors revealed that survival was significantly longer when the disease-free interval between the development of the first and second sites of metastases exceeded 1 year, in patients with a single liver metastasis, and in patients younger than 55 years old. CONCLUSIONS: Surgical resection of both hepatic and pulmonary colorectal metastases is associated with prolonged survival in selected patients. Patients with a longer disease-free interval between metastases and those with single liver lesions had the best outcomes

Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity and chromosome segregation. In Drosophila the persistent presence of double strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGF+/--like molecule Gurken, which controls normal eggshell patterning. We used this easy scorable eggshell phenotype in a germ line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation and Gurken signaling. 118 new ventralizing mutants on the second chromosome fell into 17 complementation groups. Here we describe the analysis of eight complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba, the cohesin-related gene dPds5/cohiba and the Tudor domain gene montecristo. Our findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveals a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation and Gurken protein synthesis.