Faculty Bibliography

The molecular architecture of the yeast plasma membrane H(+)-ATPase phosphorylation region was explored by Fe(2+)-catalyzed cleavage. An ATP-Mg(2+).Fe(2+) complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H(2)O(2). Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg(2+). The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT(558)GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA(340)V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D(646)TGIAVE and DMPGS(595)ELADF, respectively. The latter are spatially close to the highly conserved motif GD(634)GVND(638)APSL and conserved residues Thr(558) and Lys(615), which have been implicated in coordinating Mg(2+) and ATP. Overall, these results demonstrate that Fe(2+) associated with ATP and Mg(2+) acts as an affinity cleavage agent of the H(+)-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H(+)-ATPase that is consistent with, but not identical to, typical P-type enzymes.

Mammalian eggs are surrounded by a thick extracellular coat, the zona pellucida, that is composed of three glycoproteins, called ZP1-3. Sperm recognize and bind to O-linked oligosaccharides attached to Ser-332 and Ser-334 at the sperm combining-site of mouse ZP3 (mZP3). Mutation of either of these Ser residues to a small aliphatic amino acid results in the loss of sperm binding to mZP3 in vitro. Here, we converted both Ser-332 and Ser-334 to Thr residues by site-directed mutagenesis. Recombinant mutant glycoprotein made by stably transfected EC cells was purified and then assayed for its ability to inhibit binding of sperm to ovulated eggs in vitro. Results of these experiments suggest that Thr residues can replace the two evolutionarily conserved Ser residues as acceptors for essential O-linked oligosaccharides at the sperm combining-site of mZP3 without affecting the glycoprotein's sperm receptor activity.

Transcriptional activation is associated commonly with recruitment of histone acetylases, while repression involves histone deacetylases (HDACs). Here, we provide evidence to suggest that STAT5 activates gene expression by recruiting HDAC. The interleukin-3 (IL-3)-dependent expression of the Id-1 gene, encoding a helix-loop-helix (HLH) transcriptional inhibitor, is activated by both C/EBPbeta and STAT5 transcription factors bound to its pro-B-cell enhancer (PBE), but is inhibited by HDAC inhibitors in Ba/F3 cells. STAT5 interacts with HDAC1 in the PBE region, resulting in deacetylation of histones, as well as C/EBPbeta, whose acetylation diminishes its DNA-binding activity. Consistently, expression of an acetylation-resistant mutant of C/EBPbeta results in IL-3-independent expression of the Id-1 gene. Thus, we propose a novel mechanism by which STAT5 mediates the deacetylation of C/EBPbeta, allowing transcriptional activation.

Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years

During neuroinflammation, activated microglial cells migrate toward dying neurons, where they exacerbate local cell damage. The signaling molecules that trigger microglial cell migration are poorly understood. In this paper, we show that pathological overstimulation of neurons by glutamate plus carbachol dramatically increases the production of the endocannabinoid 2-arachidonylglycerol (2-AG) but only slightly increases the production of anandamide and does not affect the production of two putative endocannabinoids, homo-gamma-linolenylethanolamide and docosatetraenylethanolamide. We further show that pathological stimulation of microglial cells with ATP also increases the production of 2-AG without affecting the amount of other endocannabinoids. Using a Boyden chamber assay, we provide evidence that 2-AG triggers microglial cell migration. This effect of 2-AG occurs through CB2 and abnormal-cannabidiol-sensitive receptors, with subsequent activation of the extracellular signal-regulated kinase 1/2 signal transduction pathway. It is important to note that cannabinol and cannabidiol, two nonpsychotropic ingredients present in the marijuana plant, prevent the 2-AG-induced cell migration by antagonizing the CB2 and abnormal-cannabidiol-sensitive receptors, respectively. Finally, we show that microglial cells express CB2 receptors at the leading edge of lamellipodia, which is consistent with the involvement of microglial cells in cell migration. Our study identifies a cannabinoid signaling system regulating microglial cell migration. Because this signaling system is likely to be involved in recruiting microglial cells toward dying neurons, we propose that cannabinol and cannabidiol are promising nonpsychotropic therapeutics to prevent the recruitment of these cells at neuroinflammatory lesion sites.

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes

Subthreshold-activating somatodendritic A-type potassium channels have fundamental roles in neuronal signaling and plasticity which depend on their unique cellular localization, voltage dependence, and kinetic properties. Some of the components of A-type K(+) channels have been identified; however, these do not reproduce the properties of the native channels, indicating that key molecular factors have yet to be unveiled. We purified A-type K(+) channel complexes from rat brain membranes and found that DPPX, a protein of unknown function that is structurally related to the dipeptidyl aminopeptidase and cell adhesion protein CD26, is a novel component of A-type K(+) channels. DPPX associates with the channels' pore-forming subunits, facilitates their trafficking and membrane targeting, reconstitutes the properties of the native channels in heterologous expression systems, and is coexpressed with the pore-forming subunits in the somatodendritic compartment of CNS neurons

In vivo, cardiac-gated, black-blood, and ex vivo magnetic resonance microscopy (MRM) images of the aortic root, and histopathology data were obtained from 12 transgenic and wild-type (WT) mice. MRM was performed using a black-blood imaging spin-echo sequence with upstream and downstream in-flow saturation pulses to obtain aortic root images in three contrast techniques: proton density-weighted (PDW), T(1)- (T(1)W), and T(2)-weighted (T(2)W). Aortic wall thickness and area were measured and correlated with histopathology data (R > 0.90). Ex vivo lesion components (lipid core, fibrous tissue, and cell tissue) were identified and characterized by differing image contrast in PDW, T(1)W, and T(2)W MRM, and by histopathology. The differences between WT and transgenic mice for maximal wall thickness and area were statistically significant (P < 0.05). This study demonstrates the feasibility of in vivo murine aortic root lesion assessment and ex vivo plaque characterization by MRM

In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA(i) (Met-tRNA(i)) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2. GTP. Met-tRNA(i) complex is thought to bind to the 40S ribosomal subunit before the eIF4G. eIF4E. mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the alpha-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes

The biological functions of vitamin A in the epidermis are mediated by all-trans retinoic acid, which is biosynthesized from retinol in two oxidative reactions. The first step involves enzymatic conversion of retinol to retinaldehyde. The physiological significance and relative contributions of the various retinol dehydrogenases to the oxidation of retinol in epidermal cells remain unclear. We report the characterization of a retinol dehydrogenase/reductase of the SDR superfamily, hRoDH-E2, which is abundantly expressed in the epidermis, epidermal appendages and in cultured epidermal keratinocytes. Both in live keratinocytes and in isolated keratinocyte microsomes, where the enzyme normally localizes, hRoDH-E2 functions as a bona fide retinol dehydrogenase. In the prevailing oxidative reaction it recognizes both free- and CRBP-bound retinol, and shows preference toward NADP as a co-substrate. In comparison, hRoDH-E2 retinol dehydrogenase activity in the simple epithelial HEK 293 cells is much lower and in CHO cells is non-existent. hRoDH-E2 transcripts are distributed throughout the epidermal layers but are more abundant in the basal cells. In contrast, the protein is detected predominantly in the basal and the most differentiated living layers. Its synthesis is negatively regulated by retinoic acid. The biochemical properties and the differential expression of hRoDH-E2 in the strata where retinoic acid signaling is critical for epidermal homeostasis support a conclusion that hRoDH-E2 bears the characteristics of the major microsomal retinol dehydrogenase activity in the epidermal keratinocytes in physiological circumstances.