Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

The ankyrin and SOCS (suppressor of cytokine signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting 18 members in humans, the identity of the physiological targets of the Asb proteins remains largely unexplored. To increase our understanding of the function of ASB proteins, we conducted a family-wide SILAC (stable isotope labeling by amino acids in cell culture)-based protein/protein interaction analysis. This investigation led to the identification of novel as well as known ASB-associated proteins like Cullin 5 and Elongins B/C. We observed that several proteins can be bound by more than one Asb protein. The additional exploration of this phenomenon demonstrated that ASB-Cullin 5 complexes can oligomerize and provides evidence that Cullin 5 forms heterodimeric complexes with the Cullin 4a-DDB1 complex. We also demonstrated that ASB11 is a novel endoplasmic reticulum-associated ubiquitin ligase with the ability to interact and promote the ubiquitination of Ribophorin 1, an integral protein of the oligosaccharyltransferase (OST) glycosylation complex. Moreover, expression of ASB11 can increase Ribophorin 1 protein turnover in vivo. In summary, we provide a comprehensive protein/protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases.     

Interactions of the natural product kendomycin and the 20S proteasome

Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds, the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin (KM) was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of KM–proteasome interactions in vitro and in vivo, including complex structures of wild type and mutant yeast 20S proteasome with KM. Our results provide evidence for a polypharmacological mode of action for KM's cytotoxic effect on cancer cells.     

Assessing the Susceptibility of Transgenic Mice Overexpressing Deer Prion Protein to Bovine Spongiform Encephalopathy

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536^+/−, to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536^+/− mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.     

Trans-activation Response (TAR) RNA-binding Protein 2 Is a Novel Modulator of Transient Receptor Potential Canonical 4 (TRPC4) Protein

TRPC4 proteins function as Ca²⁺ conducting, non-selective cation channels in endothelial, smooth muscle, and neuronal cells. To further characterize the roles of TRPC4 in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. Therefore, a mouse brain cDNA library was searched for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified Trans-activation Response RNA-binding protein 2 (Tarpb2), a protein that recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Tarbp2 was found to bind to the C terminus of TRPC4 and TRPC5 and to modulate agonist-dependent TRPC4-induced Ca²⁺ entry. A stretch of basic residues within the Tarbp2 protein is required for these actions. Tarbp2 binding to and modulation of TRPC4 occurs in the presence of endogenously expressed Dicer but is no longer detectable when the Dicer cDNA is overexpressed. Dicer activity in crude cell lysates is increased in the presence of Ca²⁺, most probably by Ca²⁺-dependent proteolytic activation of Dicer. Apparently, Tarbp2 binding to TRPC4 promotes changes of cytosolic Ca²⁺ and, thereby, leads to a dynamic regulation of Dicer activity, essentially at low endogenous Dicer concentrations.     

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin–Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.     

Temperature-Dependent Inhibition of Opportunistic Vibrio Pathogens by Native Coral Commensal Bacteria

Bacteria living within the surface mucus layer of corals compete for nutrients and space. A number of stresses affect the outcome of this competition. The interactions between native microorganisms and opportunistic pathogens largely determine the coral holobiont's overall health and fitness. In this study, we tested the hypothesis that commensal bacteria isolated from the mucus layer of a healthy elkhorn coral, Acropora palmata, are capable of inhibition of opportunistic pathogens, Vibrio shiloi AK1 and Vibrio coralliilyticus. These vibrios are known to cause disease in corals and their virulence is temperature dependent. Elevated temperature (30 °C) increased the cell numbers of one commensal and both Vibrio pathogens in monocultures. We further tested the hypothesis that elevated temperature favors pathogenic organisms by simultaneously increasing the fitness of vibrios and decreasing the fitness of commensals by measuring growth of each species within a co-culture over the course of 1 week. In competition experiments between vibrios and commensals, the proportion of Vibrio spp. increased significantly under elevated temperature. We finished by investigating several temperature–dependent mechanisms that could influence co-culture differences via changes in competitive fitness. The ability of Vibrio spp. to utilize glycoproteins found in A. palmata mucus increased or remained stable when exposed to elevated temperature, while commensals' tended to decrease utilization. In both vibrios and commensals, protease activity increased at 30 °C, while chiA expression increased under elevated temperatures for Vibrio spp. These results provide insight into potential mechanisms through which elevated temperature may select for pathogenic bacterial dominance and lead to disease or a decrease in coral fitness.     

Influence of Shrub Encroachment on the Soil Microbial Community Composition of Remnant Hill Prairies

Hill prairies are remnant grasslands perched on the bluffs of major river valleys, and because their steep slopes make them unsuitable for traditional row crop agriculture, they have some of the lowest levels of anthropogenic disturbance of any prairie ecosystems in the Midwestern USA. However, many decades of fire suppression have allowed for shrub encroachment from the surrounding forests. While shrub encroachment of grasslands can modify soil respiration rates and nutrient storage, it is not known whether shrubs also alter the community composition of soil microorganisms. We conducted transect sampling of nine different hill prairie remnants showing varying degrees of shrub encroachment, and we used DNA-based community profiling (automated ribosomal intergenic spacer analysis) to characterize the composition of bacterial and fungal communities in the open prairie habitat, the shrub-encroached border, and the surrounding forest. While both bacterial and fungal communities showed statistically significant variation across these habitats, their predominant patterns were different. Bacterial communities of forest soils were distinct from those of the open prairie and the shrub-encroached areas, while fungal communities of the open prairie were distinct from those of the forest and the shrub-encroached border. Shrub encroachment significantly altered the community composition of soil fungal communities. Furthermore, fungal communities of heavily encroached prairie remnants more closely resembled those of the surrounding forest than those of lightly encroached prairies. Thus, shrub encroachment can cause soil fungi to shift from a “grassland” community to a “woody” community, with potential consequences for soil processes and plant-microbe interactions.     

A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy^P2A complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9–16 nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500 nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10–12 nm comparable to the size of single TatAy^P2A complexes. The combined data indicate that the mutation leads to super-assembly of TatAy^P2A complexes and we propose that an individual TatAy^P2A complex assembles initially with a TatAy^P2ACy complex, after which further TatAy^P2A complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.