Mechanistic Studies of Protein Lipidation: Yeast Palmitoyltransferase Akr1p and Protein Farnesyltransferase.

Protein palmitoylation is a widespread lipid modification in which cysteine thiols on a substrate protein are modified with a palmitoyl group. Mutations in palmitoyltransferases responsible for this modification are associated with a number of neurological diseases and cancer progression. Defining the active site and catalytic mechanism of palmitoyltransferases represents a key step towards understanding its biological significance. Akr1p, one of the first identified protein palmitoyltransferases, is an 86 kDa yeast integral membrane protein. Mutagenesis studies of Akr1p suggest that a conserved DHYC motif serves as a potential active site; the hypothesized mechanism is a two-step mechanism where the palmitoyl group is transferred from palmitoyl-CoA to Akr1p, and finally from Akr1p to the substrate protein. A covalent intermediate has been detected using radioactive assays. In this study, we elucidated the role of each amino acid in the DHYC motif using mutagenesis. In addition, mutagenesis of all of the cysteine residues in Akr1p along with mass spectrometric analysis demonstrated that the DHYC cysteine is the site in Akr1p where a covalent thioester intermediate forms. Based on these data, we propose a detailed mechanism for palmitoylation catalyzed by Akr1p, which may shed light on the mechanism of other palmitoyltransferases within the DHHC protein family. Protein farnesylation is another important lipid modification in which the cysteine of a substrate protein is modified by attachment of a 15-carbon farnesyl group which results in membrane localization of the protein. Protein farnesyltransferase (FTase) catalyzes farnesylation of a specific C-terminal “Ca1a2X” sequence of substrate proteins. Here we analyze the determinants of recognition of the a2 residue by FTase, demonstrating that completely conserved tryptophan residues in FTase, although not essential for maintaining the farnesylation activity, play an important role in modulating the substrate selectivity of FTase. Mutagenesis studies demonstrate that the conserved W102β and W106β residues modulate both the reactivity of FTase and substrate selectivity based on the size of the binding pocket. The complete conservation of these two amino acids suggests that maintenance of the exact substrate selectivity of FTase is crucial for the in vivo activity.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89718/1/xmguan_1.pd

E-cadherin Can Replace N-cadherin During Secretory-Stage Enamel Development

Orginal data for qPCR, microhardness measurement and microCT scannin

E-cadherin can replace N-cadherin during secretory-stage enamel development.

N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development.The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ.The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue

Simultaneous Indoor Tracking and Activity Recognition Using Pyroelectric Infrared Sensors

Indoor human tracking and activity recognition are fundamental yet coherent problems for ambient assistive living. In this paper, we propose a method to address these two critical issues simultaneously. We construct a wireless sensor network (WSN), and the sensor nodes within WSN consist of pyroelectric infrared (PIR) sensor arrays. To capture the tempo-spatial information of the human target, the field of view (FOV) of each PIR sensor is modulated by masks. A modified partial filter algorithm is utilized to decode the location of the human target. To exploit the synergy between the location and activity, we design a two-layer random forest (RF) classifier. The initial activity recognition result of the first layer is refined by the second layer RF by incorporating various effective features. We conducted experiments in a mock apartment. The mean localization error of our system is about 0.85 m. For five kinds of daily activities, the mean accuracy for 10-fold cross-validation is above 92%. The encouraging results indicate the effectiveness of our system

Abnormal Activity Detection Using Pyroelectric Infrared Sensors

Healthy aging is one of the most important social issues. In this paper, we propose a method for abnormal activity detection without any manual labeling of the training samples. By leveraging the Field of View (FOV) modulation, the spatio-temporal characteristic of human activity is encoded into low-dimension data stream generated by the ceiling-mounted Pyroelectric Infrared (PIR) sensors. The similarity between normal training samples are measured based on Kullback-Leibler (KL) divergence of each pair of them. The natural clustering of normal activities is discovered through a self-tuning spectral clustering algorithm with unsupervised model selection on the eigenvectors of a modified similarity matrix. Hidden Markov Models (HMMs) are employed to model each cluster of normal activities and form feature vectors. One-Class Support Vector Machines (OSVMs) are used to profile the normal activities and detect abnormal activities. To validate the efficacy of our method, we conducted experiments in real indoor environments. The encouraging results show that our method is able to detect abnormal activities given only the normal training samples, which aims to avoid the laborious and inconsistent data labeling process

E-Cadherin Can Replace N-Cadherin during Secretory-Stage Enamel Development - Figure 1

<p>(<b>a</b>) qPCR analysis of N-cadherin gene expression in wild-type (WT) and N-cadherin conditional knockout (cKO) mouse enamel organs. mRNA was extracted from postnatal day-5 enamel organs from 3 mice per genotype for qPCR analysis. Results are presented as expression ratios relative to the WT levels. The ablated N-cadherin cKO enamel organs (K14-<i>Cre</i>-N-cadherin-<i>LoxP</i>+/+) had 0.19 fold of the WT N-cadherin expression level (***, p<0.001) and the heterozygous ablated mice (K14-<i>Cre</i>-N-cadherin-<i>LoxP</i>+/−) had 0.75 fold of the WT expression level (*, p<0.05). (<b>b</b>) N-cadherin protein expression was ablated in the ameloblast layer of K14-<i>Cre</i>-N-cadherin-<i>LoxP</i>+/+ mice. Immunohistochemical staining of N-cadherin was performed on paraffin-imbedded incisor sections from both WT and N-cadherin cKO mice. In WT mice N-cadherin was not expressed highly in pre-secretory stage ameloblasts, but was strongly up-regulated during the secretory stage and was later down-regulated when the ameloblasts progressed into the maturation stage. In contrast, regardless of developmental stage, N-cadherin expression was not observed in the N-cadherin cKO ameloblasts demonstrating that N-cadherin expression was successfully deleted in these mice. A, ameloblast layer; O, odontoblast layer.</p

Gene expression analysis in WT and N-cadherin cKO mouse enamel organs.

<p>qPCR was performed on WT and N-cadherin cKO postnatal day-5 enamel organs with 7 mice assessed per genotype. No significant difference was observed in expression levels of p120 and β-catenin between WT and N-cadherin ablated enamel organs. Various cadherins were assessed for expression in secretory stage enamel organ and for those that were expressed, expression levels were assessed. A comparison of expression levels between WT and N-cadherin cKO enamel organs revealed that VE-cadherin expression was slightly but significantly reduced compared to WT and that E-cadherin expression was significantly increased by approximately 1.5 fold compared to WT. No significant differences by genotype were observed for P-cadherin or cadherin-11 (*, p<0.05).</p

Primers used for quantitative real-time PCR (qPCR).

<p>Primers used for quantitative real-time PCR (qPCR).</p

No difference in Enamel hardness between WT and N-cadherin cKO mice.

<p>Adult mouse incisors were harvested and indented for Vickers microhardness measurements. Enamel hardness from 4 mice per genotype was measured and results were averaged. WT samples have an averaged Vickers hardness number of 219.6±24.5, while N-cadherin cKO samples possess a slightly higher value of 235.1±23.8.</p

BMP2 signaling may be responsible for maintaining E-cadherin expression during the secretory stage in N-cadherin cKO enamel organs.

<p>qPCR was performed on WT and N-cadherin cKO postnatal day-5 enamel organs. <i>Bmp2</i> expression increased approximately 1.7 fold over WT levels in the N-cadherin ablated enamel organs (**, p<0.005). Conversely, the BMP signaling antagonist <i>Nog</i> decreased by approximately 80% of the WT expression level (*, p<0.05). Results were obtained from 4 mice per genotype.</p