Faculty Bibliography

Transporting epithelia posed formidable conundrums right from the moment that Du Bois Raymond discovered their asymmetric behavior, a century and a half ago. It took a century and a half to start unraveling the mechanisms of occluding junctions and polarity, but we now face another puzzle: lest its cells died in minutes, the first high metazoa (i.e., higher than a sponge) needed a transporting epithelium, but a transporting epithelium is an incredibly improbable combination of occluding junctions and cell polarity. How could these coincide in the same individual organism and within minutes? We review occluding junctions (tight and septate) as well as the polarized distribution of Na(+)-K(+)-ATPase both at the molecular and the cell level. Junctions and polarity depend on hosts of molecular species and cellular processes, which are briefly reviewed whenever they are suspected to have played a role in the dawn of epithelia and metazoan. We come to the conclusion that most of the molecules needed were already present in early protozoan and discuss a few plausible alternatives to solve the riddle described above.

The albino mouse was already known in ancient times and was apparently selectively bred in Egypt, China, and Japan. Thus, it is not surprising that the c or albino locus (now the Tyr locus) was among the first used to demonstrate Mendelian inheritance in mammals at the dawn of the past century. This locus is now known to encode tyrosinase, the rate-limiting enzyme in the production of melanin pigment, and the molecular basis of the albino ( Tyr(c)) mutation is known. Here we describe the congenic series of Tyr-locus alleles, from wild type to null ( albino). We compare eye and skin pigmentation phenotypes and the genetic lesions that cause each. We suggest that this panel of congenic mutants contains rich, untapped resources for the study of many questions of basic cell biological interest

E2A transcription factors, E12 and E47, are important regulators of lymphocyte development. Notch signaling pathways have been shown to regulate E2A function by accelerating the degradation of E2A proteins through a mitogen-activated protein kinase-dependent and ubiquitin-mediated pathway. To further understand the mechanism underlying E2A ubiquitination and degradation, we conducted a yeast two-hybrid screen and identified the carboxyl terminus of Hsc70-interacting protein (CHIP) as an E47 binding protein. Here, we show that CHIP associates with E2A proteins in vivo and that overexpression of CHIP induces E47 degradation in a phosphorylation-dependent manner. Conversely, knocking down CHIP with small interfering RNA alleviates Notch-induced E47 degradation. CHIP binds E47 through the E protein homology domains 2 and 3 (EHD2 and EHD3). This interaction between CHIP and E47 is independent of the U-box domain with E3 ubiquitin ligase activity but requires the chaperone binding tetratricopeptide repeats domain. The ability of CHIP to induce E47 ubiquitination and degradation correlates with its ability to bind E47. We propose that CHIP, together with its partner Hsc70, forms a preubiquitination complex (PUC) with E47 and Skp2, thus facilitating the interaction between E47 and Skp2. CHIP also associates with Cul1, which introduces PUC to the SCF E3 ligase complex, responsible for E47 ubiquitination. Therefore, CHIP plays a crucial role in the ubiquitination and degradation of E2A proteins.

The uptake of the echinocandin drug caspofungin acetate in Candida albicans was evaluated at drug levels at or near the MIC for the organism. Maximal uptake was achieved in 10 min and was energy independent. A saturable transport system, consistent with a facilitated-diffusion carrier, was observed with the unlabeled drug competing with the labeled drug for uptake and efflux. More than 90% of the transported drug was observed in a single kinetic compartment that was available for efflux, indicating that the drug was free in the cytoplasm following uptake. Efflux was also energy independent but was sensitive to the presence of a fully loaded carrier on both faces of the bilayer. Overall, the data presented are consistent with the presence of a high-affinity facilitated-diffusion transporter that mediates caspofungin uptake and could be a potential source of transport-related reduced susceptibility.

RACK1 serves as a scaffold protein for a wide range of kinases and membrane-bound receptors. It is a WD-repeat family protein and is predicted to have a beta-propeller architecture with seven blades like a Gbeta protein. Mass spectrometry studies have identified its association with the small subunit of eukaryotic ribosomes and, most recently, it has been shown to regulate initiation by recruiting protein kinase C to the 40S subunit. Here we present the results of a cryo-EM study of the 80S ribosome that positively locate RACK1 on the head region of the 40S subunit, in the immediate vicinity of the mRNA exit channel. One face of RACK1 exposes the WD-repeats as a platform for interactions with kinases and receptors. Using this platform, RACK1 can recruit other proteins to the ribosome

Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics

The potential of amyloid-beta (Abeta) immunization as a disease-modifying therapy for Alzheimer's disease is limited by the occurrence of encephalitic side effects in a subset of treated patients. The encephalitis was not predicted from immunization studies in transgenic, Abeta-depositing mice. More recently, studies in these same mice indicate that passive immunization with certain anti-Abeta antibodies can induce microhemorrhage. Cerebral amyloid angiopathy (CAA) may play a key role in determining the risk for these complications. Because aged nonhuman primates (NHPs) have a more human-like immune system than rodents, and because NHPs naturally develop senile plaques and CAA with age, NHPs appear to be important, adjunctive models for assessing the efficacy and safety of immunotherapeutics for Alzheimer's disease. Conversely, the ability to model the complications of Abeta immunotherapy will be important for elucidating the bases of these complications, and for developing protocols that minimize or eliminate the risks of these serious adverse effects

To understand the actions of morphogens, it is crucial to determine how they elicit different transcriptional responses in different cell types. Here, we identify a BMP-responsive enhancer of Msx2, an immediate early target of bone morphogenetic protein (BMP) signaling. We show that the BMP-responsive region of Msx2 consists of a core element, required generally for BMP-dependent expression, and ancillary elements that mediate signaling in diverse developmental settings. Analysis of the core element identified two classes of functional sites: GCCG sequences related to the consensus binding site of Mad/Smad-related BMP signal transducers; and a single TTAATT sequence, matching the consensus site for Antennapedia superclass homeodomain proteins. Chromatin immunoprecipitation and mutagenesis experiments indicate that the GCCG sites are direct targets of BMP restricted Smads. Intriguingly, however, these sites are not sufficient for BMP responsiveness in mouse embryos; the TTAATT sequence is also required. DNA sequence comparisons reveal this element is highly conserved in Msx2 promoters from mammalian orders but is not detectable in other vertebrates or non-vertebrates. Despite this lack of conservation outside mammals, the Msx2 BMP-responsive element serves as an accurate readout of Dpp signaling in a distantly related bilaterian - Drosophila. Strikingly, in Drosophila embryos, as in mice, both TTAATT and GCCG sequences are required for Dpp responsiveness, showing that a common cis-regulatory apparatus can mediate the transcriptional activation of BMP-regulated genes in widely divergent bilaterians