Faculty Bibliography

Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (P(i)) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate:phosphate antiporters

BACKGROUND: Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time. RESULTS: The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10-15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias. CONCLUSION: Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease

Wnt target gene transcription is mediated by nuclear translocation of stabilized beta-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of beta-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13

Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle-blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division

Melanoma inhibitor of apoptosis (ML-IAP) is a potent inhibitor of apoptosis, which is highly expressed in melanomas and likely contributes to their resistance to chemotherapeutic treatments. Herein, we show that the lineage survival oncogene microphthalmia-associated transcription factor (MITF) is a critical regulator of ML-IAP transcription in melanoma cells. The ML-IAP promoter contains two MITF consensus sites, and analysis of MITF and ML-IAP mRNA levels revealed a high correlation in melanoma tumor samples and cell lines. In reporter assays, MITF promoted a strong stimulation of transcriptional activity from the ML-IAP promoter, and MITF bound the endogenous ML-IAP promoter in melanoma cells by chromatin immunoprecipitation and electrophoretic mobility shift assay. Strikingly, small interfering RNA (siRNA)-mediated knockdown of MITF in melanoma cells led to a dramatic decrease in ML-IAP mRNA and protein levels, establishing that ML-IAP expression in melanoma cells is MITF dependent. Additionally, cyclic AMP-mediated induction of MITF expression in melanocytes resulted in increased ML-IAP expression, suggesting that melanocytes can express ML-IAP when MITF levels are heightened. Disruption of MITF by siRNA led to a decrease in melanoma cell viability, which could be rescued by ectopic expression of ML-IAP. Collectively, these findings implicate MITF as a major transcriptional regulator of ML-IAP expression in melanomas, and suggest that ML-IAP contributes to the prosurvival activity of MITF in melanoma progression.

Small heat shock proteins (sHsps) are one of the most ubiquitous molecular chaperones. They are grouped together based on a conserved domain, the alpha-crystallin domain. Generally, sHsps exist as oligomers of 9-40 subunits, and the oligomers undergo reversible temperature-dependent dissociation into smaller species as dimers, which interact with denaturing substrate proteins. Previous studies have shown that the C-terminal region, especially the consensus IXI/V motif, is responsible for oligomer assembly. In this study, we examined deletions or mutations in the C-terminal region on the oligomer assembly and function of StHsp14.0, an sHsp from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Mutated StHsp14.0 with C-terminal deletion or replacement of IIe residues in the IXI/V motif to Ala, Ser, or Phe residues could not form large oligomers and lost chaperone activity. StHsp14.0WKW, whose Ile residues in the IXI/V motif are changed to Trp, existed as an oligomer like that of the wild type. However, it dissociates to small oligomers and exhibits chaperone activity at relatively lowered temperature. Replacement of two Ile residues in the motif to relatively small residues, Ala or Ser, also resulted in the change of beta-sheet rich secondary structure and decrease of hydrophobicity. Interestingly, StHsp14.0 mutant with amino acid replacements to Phe kept almost the same secondary structure and relatively high hydrophobicity despite that it could not form an oligomeric structure. The results show that hydrophobicity and size of the amino acids in the IXI/V motif in the C-terminal region are responsible not only for assembly of the oligomer but also for the maintenance of beta-sheet rich secondary structure and hydrophobicity, which are important for the function of sHsp.

PURPOSE: Previous study has shown that the absence of uroplakin II can cause urinary tract dysfunction, including vesicoureteral reflux and renal abnormalities, as well as micturition pattern changes. We developed a simple surrogate measure of bladder function using ultraviolet visualization of urinary voiding patterns in a uroplakin II knockout mouse animal model. MATERIALS AND METHODS: Three male and 3 female WT mice, and 3 male and 3 female uroplakin II knockout mice were evaluated by cystometric analysis and voiding pattern markings. Voiding pattern markings were graded by independent observers on a scale of 1 to 5 according to the degree of dispersion of voided urine. Statistical analysis was then used to correlate voiding dispersion grades with cystometric parameters in the same mice. RESULTS: The degree of dispersion of voiding pattern markings correlated with several measures of bladder function. Specifically the Pearson correlation coefficients for the observed voiding patterns highly correlated with baseline pressure, threshold pressure and intermicturition pressure measurements made during conscious cystometry in these mice (p <0.05). CONCLUSIONS: Ultraviolet visualization of urinary voiding patterns of mice correlated well with certain measures of standard cystometric evaluations. As such, this method provides a simple, noninvasive method of evaluating mouse bladder function. Implementation of this methodology, which can potentially be automated for high throughput analysis, can accelerate the development of novel therapy for certain important aspects of bladder disease/dysfunction

Id proteins play important roles in osteogenic differentiation; however, the molecular mechanism remains unknown. In this study, we established that inhibitor of differentiation (Id) proteins, including Id1, Id2, and Id3, associate with core binding factor alpha-1 (Cbfa1) to cause diminished transcription of the alkaline phosphatase (ALP) and osteocalcin (OCL) gene, leading to less ALP activity and osteocalcin (OCL) production. Id acts by inhibiting the sequence-specific binding of Cbfa1 to DNA and by decreasing the expression of Cbfa1 in cells undergoing osteogenic differentiation. p204, an interferon-inducible protein that interacts with both Cbfa1 and Id2, overcame the Id2-mediated inhibition of Cbfa1-induced ALP activity and OCL production. We show that 1) p204 disturbed the binding of Id2 to Cbfa1 and enabled Cbfa1 to bind to the promoters of its target genes and 2) that p204 promoted the translocation from nucleus to the cytoplasm and accelerated the degradation of Id2 by ubiquitin-proteasome pathway during osteogenesis. Nucleus export signal (NES) of p204 is required for the p204-enhanced cytoplasmic translocation and degradation of Id2, because a p204 mutant lacking NES lost these activities. Together, Cbfa1, p204, and Id proteins form a regulatory circuit and act in concert to regulate osteoblast differentiation

In both normal development and in a variety of pathological conditions, epithelial cells can acquire migratory and invasive properties. Border cells in the Drosophila ovary provide a genetically tractable model for elucidating the mechanisms controlling such behaviors. Here we report the identification of a mutant, apontic (apt), in which the migratory population expanded and separation from the epithelium was impeded. This phenotype resembled gain-of-function of JAK/STAT activity. Gain-of-function of APT also mimicked loss of function of STAT and its key downstream target, SLBO. APT expression was induced by STAT, which bound directly to sites in the apt gene. The data suggest that a regulatory circuit between STAT, APT, and SLBO functions to convert an initially graded signal into an all-or-nothing activation of JAK/STAT and thus to proper cell specification and migration. These findings are supported by a mathematical model, which accurately simulates wild-type and mutant phenotypes.

PURPOSE: Beta-adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting RESULTS: With the use of an integrated pharmacologic and genetic approach, the authors investigated how the beta-AR impacts corneal epithelial healing. METHODS: Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in beta2-AR(+/+) and beta2-AR(-/-) mice. Signaling pathways were evaluated by immunoblotting. results. The beta-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the beta-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P < 0.05). The same treatment protocols had no effect on AMCE cells derived from beta2-AR(-/-) mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In beta2-AR(+/+) animals, the beta-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas beta-AR antagonist (timolol) treatment increased the rate of healing by 16% (P < 0.05) compared with saline-treated controls. In contrast, in the beta2-AR(-/-) mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein). CONCLUSIONS: The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact beta2-AR for the modulation of corneal epithelial wound repair