Regulating the human HECT E3 ligases

Ubiquitination, the covalent attachment of ubiquitin to proteins, by E3 ligases of the HECT (homologous to E6AP C terminus) family is critical in controlling diverse physiological pathways. Stringent control of HECT E3 ligase activity and substrate specificity is essential for cellular health, whereas deregulation of HECT E3s plays a prominent role in disease. The cell employs a wide variety of regulatory mechanisms to control HECT E3 activity and substrate specificity. Here, we summarize the current understanding of these regulatory mechanisms that control HECT E3 function. Substrate specificity is generally determined by interactions of adaptor proteins with domains in the N-terminal extensions of HECT E3 ligases. These N-terminal domains have also been found to interact with the HECT domain, resulting in the formation of inhibitory conformations. In addition, catalytic activity of the HECT domain is commonly regulated at the level of E2 recruitment and through HECT E3 oligomerization. The previously mentioned regulatory mechanisms can be controlled through protein–protein interactions, post-translational modifications, the binding of calcium ions, and more. Functional activity is determined not only by substrate recruitment and catalytic activity, but also by the type of ubiquitin polymers catalyzed to the substrate. While this is often determined by the specific HECT member, recent studies demonstrate that HECT E3s can be modulated to alter the type of ubiquitin polymers they catalyze. Insight into these diverse regulatory mechanisms that control HECT E3 activity may open up new avenues for therapeutic strategies aimed at inhibition or enhancement of HECT E3 function in disease-related pathways

Connectivity, Sprawl, and the Cul-de-sac: An Analysis of Cul-de-sacs and Dead-end Streets in Burlington and the Surrounding Suburbs

For over six decades, professionals within the real estate industry, homebuyers, and planners have favored the cul-de-sac, an iconic symbol of suburban America. The cul-de-sac is a dead-end street characterized by a circular end to facilitate the turnaround of an automobile in one seamless motion. However, recent debates have challenged this street design. Cul-de-sac development and its connection with concerns related to urban sprawl have sparked a great deal of controversy between planners, developers, and policy makers. Relationships between physical neighborhood factors such as security, street connectivity, green spaces, and communal integration are often highlighted as the key elements that cause conflict between the proponents of the cul-de-sac and those who favor a grid street layout. Supporters of Smart Growth, a movement to highlight the need to develop more sustainable communities, claim that neighborhoods with cul-de-sacs tend to be more car-dependent, less safe for pedestrians, and can encourage crime by reducing levels of social cohesion and connectivity, while grid layouts foster non-car transport, can improve walkability, and lessen crime because of their enhanced permeability. This study uses a mixed-methods approach to understand: what is the experience of, and specifically, what is attractive about cul-de-sacs to residents in Chittenden County, Vermont, and what opinions do planning professionals have of cul-de-sacs

Teredinibacter turnerae gen. nov., sp. nov., a dinitrogen-fixing, cellulolytic, endosymbiotic c-proteobacterium isolated from the gills of wood-boring molluscs (Bivalvia: Teredinidae)

Author Posting. © Society for General Mircobiology, 2002. This article is posted here by permission of Society for General Mircobiology for personal use, not for redistribution. The definitive version was published in International Journal of Systematic Bacteriology 52 (2002): 2261-2269, doi:10.1099/ijs.0.02184-0.A cellulolytic, dinitrogen-fixing bacterium isolated from the gill tissue of a wood-boring mollusc (shipworm) Lyrodus pedicellatus of the bivalve family Teredinidae and 58 additional strains with similar properties, isolated from gills of 24 bivalve species representing 9 of 14 genera of Teredinidae, are described. The cells are Gram-negative, rigid, rods (0<4–0<6x3–6 lm) that bear a single polar flagellum. All isolates are capable of chemoheterotrophic growth in a simple mineral medium supplemented with cellulose as a sole source of carbon and energy. Xylan, pectin, carboxymethylcellulose, cellobiose and a variety of sugars and organic acids also support growth. Growth requires addition of combined nitrogen when cultures are vigorously aerated, but all isolates fix dinitrogen under microaerobic conditions. The pH, temperature and salinity optima for growth were determined for six isolates and are approximately 8<5, 30–35 °C and 0<3 M NaCl respectively. The isolates are marine. In addition to NaCl, growth requires elevated concentrations of Ca2M and Mg2M that reflect the chemistry of seawater. The DNA GMC content ranged from 49 to 51 mol%. Four isolates were identical with respect to small-subunit rRNA sequence over 891 positions compared and fall within a unique clade in the c-subclass of the Proteobacteria. Based on morphological, physiological and phylogenetic characteristics and specific symbiotic association with teredinid bivalves, a new genus and species, Teredinibacter turnerae gen. nov., sp. nov., is proposed. The type strain is T7902T (vATCC 39867TvDSM 15152T).This work was supported by grants from the National Science Foundation no. NSF DEB-9420051 and IBN- 9982982, the Maine Science and Technology Foundation's Center for Innovation in Biotechnology, and the University of Maine's Faculty Research program

Conditioned Flavor Aversion: A Mechanism for Goats to Avoid Condensed Tannins in Blackbrush

It has been hypothesized that herbivores instinctively avoid tannin-containing plant parts in response to the adverse effects of tannins on forage digestion. However, we found that goats learned to avoid condensed tannins (CTs) from blackbrush current season\u27s growth by associating the flavor of foods containing CTs with aversive postingestive consequences. The aversive consequences experienced by goats apparently are not related to digestion inhibition and may depend on the structure of CTs and on how CTs are bound with other cell constituents. These observations suggest several areas of inquiry related to the interaction between CTs and herbivores. A better understanding of the physiological effects of CTs and how herbivores perceive these effects is essential to our knowledge of chemically mediated interactions between plants and mammalian herbivores. With few exceptions, the effects of food flavor have not been separated from those associated with postingestive consequences, even though our data show that postingestive consequences strongly influence palatability. We also need to know how herbivores learn which plant species to eat and which to avoid while foraging in areas that contain a variety of plant species and parts with different kinds and concentrations of CTs. Condensed tannins are pervasive in nature and can defend plants from herbivory, but since many important forages contain high levels of tannins, the presence or absence of tannins per se does not reliably indicate food quality. To predict the ability of a tannin-producing plant to deter herbivores requires a full understanding of how changes in CT structure and binding affect herbivores

Genetic interaction screen for severe neurodevelopmental disorders reveals a functional link between Ube3a and Mef2 in Drosophila melanogaster

Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene (Da, Mef2, Ube3a, Zfh1, XNP) and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities

Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis

The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis

A versatile plasmid system for reconstitution and analysis of mammalian ubiquitination cascades in yeast

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex posttranslational modifications in a cellular setting

Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure.

We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions. The selection is based on the lethality of hydrogen peroxide (H2O2) that is produced in wild type cells during the peroxisomal beta-oxidation of fatty acids. In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes. The mutants were shown to have monogenetic defects and to fall into 12 complementation groups. Representative members of each complementation group were morphologically examined by immunocytochemistry using EM. In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase. These mutants suggest the existence of multiple components involved in peroxisomal protein import. Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation. In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisome