Faculty Bibliography

The mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. Here, we report that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. We identified cdk7 through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppressed apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid failed to localize to the mitochondria and failed to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid.

BACKGROUND: The high affinity tyrosine kinase receptor, TrkB, is the primary receptor for brain derived neurotrophic factor (BDNF) and plays an important role in development, maintenance and plasticity of the striatal output medium size spiny neuron. The striatal BDNF/TrkB system is thereby implicated in many physiologic and pathophysiologic processes, the latter including mood disorders, addiction, and Huntington's disease. We crossed a mouse harboring a transgene directing cre-recombinase expression primarily to postnatal, dorsal striatal medium spiny neurons, to a mouse containing a floxed TrkB allele (fB) mouse designed for deletion of TrkB to determine its role in the adult striatum. RESULTS: We found that there were sexually dimorphic alterations in behaviors in response to stressful situations and drugs of abuse. Significant sex and/or genotype differences were found in the forced swim test of depression-like behaviors, anxiety-like behaviors on the elevated plus maze, and cocaine conditioned reward. Microarray analysis of dorsal striatum revealed significant dysregulation in individual and groups of genes that may contribute to the observed behavioral responses and in some cases, represent previously unidentified downstream targets of TrkB. CONCLUSIONS: The data point to a set of behaviors and changes in gene expression following postnatal deletion of TrkB in the dorsal striatum distinct from those in other brain regions.

Integrin alphaIIbbeta3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified alphaIIbbeta3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away from the membrane. Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the alphaIIb lower leg is bent between the calf-1 and calf-2 domains and the beta3 Integrin-Epidermal Growth Factor (I-EGF) 2 to 4 domains are freely coiled rather than in a cleft between the beta3 headpiece and the alphaIIb lower leg. Our data indicate an important role for the region that links the distal calf-2 and beta-tail domains to their respective transmembrane (TM) domains in transmitting the conformational changes in the TM domains associated with inside-out activation.

K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho-K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL-dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP3 regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors.

The adult mouse prostate has a seemingly endless capacity for regeneration, and sonic hedgehog (SHH) signaling has been implicated in this stem cell-driven process. However, it is not clear whether SHH acts on the epithelium or stromal cells that secrete factors required for epithelial expansion. Because little is known about stromal stem cells compared with their epithelial counterparts, we used in vivo mouse genetics tools to characterize four prostate stromal subtypes and their stem cells. Using knockin reporter alleles, we uncovered that SHH signals from prostate basal epithelial cells to adjacent stromal cells. Furthermore, the SHH target gene Gli1 is preferentially expressed in subepithelial fibroblast-like cells, one of four prostate stromal subtypes and the subtype closest to the epithelial source of SHH. Using Genetic Inducible Fate Mapping to mark adult Gli1- or Smooth muscle actin-expressing cells and follow their fate during regeneration, we uncovered that Gli1-expressing cells exhibit long-term self-renewal capacity during multiple rounds of androgen-mediated regeneration after castration-induced involution, and depleted smooth muscle cells are mainly replenished by preexisting smooth muscle cells. Based on our Genetic Inducible Fate Mapping studies, we propose a model where SHH signals to multiple stromal stem cells, which are largely unipotent in vivo.

Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options.

The concentration of UV filters (UVFs) and UV light stabilizers (UVLSs) were measured in seawater and river water collected from sites at four beaches, two reefs, and one river on Okinawa Island, Japan. UVFs and/or UVLSs of 8-10 types were detected in beaches samples and 6-9 types were detected in reef samples. The total UVF concentrations at the beach sites were highest either in July or August with a maximum of 1.4 mug L(-1). The concentrations at the reef sites did not show peaks in summer and the maximum values were close to 10 ng L(-1). The detected UVF profiles reflected the ingredients of sunscreens used in each region. The highest UVLS concentrations at the reefs were observed not only in summer but also in June and September. The UVLS concentrations at the reefs were similar to or even higher than that at the beaches or in the river.

The INrf2 (Keap1)-Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3-Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCepsilon is essential for INrf2-Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCepsilon, knockdown of PKCepsilon and the INrf2S602A mutant all failed to phosphorylate INrf2, leading to loss of the INrf2-Nrf2 interaction, Nrf2 degradation and enhanced cytoprotection and drug resistance. Molecular modeling analyses revealed that phosphorylation of S599 exposes the deeply buried S602 for phosphorylation and enhanced INrf2-Nrf2 interaction. Analysis of human lung and liver tumor protein arrays showed lower PKCepsilon and higher Nrf2 levels, which presumably promoted cancer cell survival and drug resistance. In conclusion, phosphorylation of INrf2 by PKCepsilon leads to regulation of Nrf2, with significant implications for the survival of cancer cells, which often express lower levels of PKCepsilon.

BACKGROUND: Although transposable element (TE) derived DNA accounts for more than half of mammalian genomes and initiates a significant proportion of RNA transcripts, high throughput methods are rarely leveraged specifically to detect expression from interspersed repeats. RESULTS: To characterize the contribution of transposons to mammalian transcriptomes, we developed a custom microarray platform with probes covering known human and mouse transposons in both sense and antisense orientations. We termed this platform the "TE-array" and profiled TE repeat expression in a panel of normal mouse tissues. Validation with nanoString(R) and RNAseq technologies demonstrated that TE-array is an effective method. Our data show that TE transcription occurs preferentially from the sense strand and is regulated in highly tissue-specific patterns. CONCLUSIONS: Our results are consistent with the hypothesis that transposon RNAs frequently originate within genomic TE units and do not primarily accumulate as a consequence of random 'read-through' from gene promoters. Moreover, we find TE expression is highly dependent on the tissue context. This suggests that TE expression may be related to tissue-specific chromatin states or cellular phenotypes. We anticipate that TE-array will provide a scalable method to characterize transposable element RNAs.

BACKGROUND: Although it is well established that the extracellular matrix affects tumour progression, not much is known about the various components and their effect on head and neck squamous cell carcinoma (HNSCC) progression. Levels of collagen type XI alpha1 (colXIalpha1), a minor fibrillar collagen, have been shown to be increased in tumour compared with normal tissue in several cancers, including colorectal, breast, and non-small cell lung cancer. Currently, the functional significance of colXIalpha1 is not understood. METHODS: We examined the expression levels of colXIalpha1 mRNA and elucidated the functional role of colXIalpha1 in HNSCC. Cell proliferation, invasion, and migration were examined with and without colXIalpha1 knockdown with siRNA in HNSCC cells. RESULTS: Our data demonstrate that colXIalpha1 expression is increased in tumour samples compared with levels in normal adjacent tissue in 16/23 HNSCC patients. In addition, colalpha11 is increased in HNSCC cell lines compared with normal immortalised epithelial cells and is increased in tumour-derived fibroblasts compared with normal fibroblasts. Using an siRNA approach, we demonstrate that colXIalpha1 contributes to proliferation, migration, and invasion of HNSCC. CONCLUSION: Our cumulative findings suggest that colXIalpha1 contributes to HNSCC tumorigenesis and may serve as a potential therapeutic target.