Structural Basis for Ovarian Tumor Domain-containing Protein 1 (OTU1) Binding to p97/Valosin-containing Protein (VCP)

Valosin-containing protein (VCP), also known as p97, is an AAA⁺ ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXL_OTU1) complexed to the N-terminal domain of VCP (N_VCP) at 1.8-Å resolution reveals that UBXL_OTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of N_VCP using the ³⁹GYPP⁴² loop of UBXL_OTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a K_D of 0.71 μm. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.     

Microtubule regulation of N-methyl-D-aspartate receptor channels in neurons

N-Methyl-D-aspartate (NMDA) receptors (NMDARs), which play a key role in synaptic plasticity, are dynamically regulated by many signaling molecules and scaffolding proteins. Although actin cytoskeleton has been implicated in regulating NMDAR stability in synaptic membrane, the role of microtubules in regulating NMDAR trafficking and function is largely unclear. Here we show that microtubule-depolymerizing agents inhibited NMDA receptor-mediated ionic and synaptic currents in cortical pyramidal neurons. This effect was Ca(2+)-independent, required GTP, and was more prominent in the presence of high NMDA concentrations. The NR2B subunit-containing NMDA receptor was the primary target of microtubules. The effect of microtubule depolymerizers on NMDAR currents was blocked by cellular knockdown of the kinesin motor protein KIF17, which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Neuromodulators that can stabilize microtubules, such as brain-derived neurotrophic factor, significantly attenuated the microtubule depolymerizer-induced reduction of NMDAR currents. Moreover, immunocytochemical studies show that microtubule depolymerizers decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Taken together, these results suggest that interfering with microtubule assembly suppresses NMDAR function through a mechanism dependent on kinesin-based dendritic transport of NMDA receptors.     

Transformation of the basidiomycete, Schizophyllum commune

Summary Protoplasts of aSchizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp⁺ with plasmid DNA containing the SchizophyllumTRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp⁺ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.     

Protein purification of Arthrospira platensis using aqueous two-phase system composed of polyethylene glycol and potassium phosphate/sodium citrate

Journal of Applied Phycology - Aqueous two-phase systems (ATPS) stand out as an alternative technique for recovering and concentrating proteins. However, the study of ATPS to recover Arthrospira...     

Quantitative multiplexed strategies for human Lyme disease serological testing

Lyme disease, which is primarily caused by infection with the bacterium Borrelia burgdorferi in the United States or other Borrelia species internationally, presents an ongoing challenge for diagnostics. Serological testing is the primary means of diagnosis but testing approaches differ widely, with varying degrees of sensitivity and specificity. Moreover, there is currently no reliable test to determine disease resolution following treatment. A distinct challenge in Lyme disease diagnostics is the variable patterns of human immune response to a plurality of antigens presented by Borrelia spp. during the infection. Thus, multiplexed testing approaches that capture these patterns and detect serological response against multiple antigens may be the key to prompt, accurate Lyme disease diagnosis. In this review, current state-of-the-art multiplexed diagnostic approaches are presented and compared with respect to their diagnostic accuracy and their potential for monitoring response to treatment.     

Differential herbivory of the specialist beetle Stolas punicea on chemical phenotypes of its host Mikania micrantha

We assessed the preference of two populations of the specialist beetle Stolas punicea, on plants from three phytochemically differentiated geographical mosaics of the American weed Mikania micrantha. Our results show a significant geographic variation in host preference in S. punicea that could affect its success as a biocontrol agent.     

Induction and cultivation of a stable L-form of Bacillus subtilis

The induction of L-forms of Bacillus subtilis from protoplasts is described. The method involved the frequent subculture of the unstable L-form on a growth medium supplemented with lysozyme and horse serum. A stable culture, which did not revert when lysozyme and horse serum were omitted from the medium, was obtained after 13 subcultures. This culture could be grown on solid and in liquid medium by routine microbiological methods. Long-term storage of these cells was achieved by freeze drying and maintenance in glycerol at −70°C. The cultural adaptability of the L-form is described and discussed with respect to methods of cultivation and growth.     

Coping with obesity stigma affects depressed mood in African-American and white candidates for bariatric surgery

Depressed mood in severely obese, bariatric surgery-seeking candidates is influenced by obesity stigma, yet the strategies for coping with this stigma are less well understood. This study hypothesized that coping strategies are significantly associated with depressed mood above and beyond demographic factors and frequency of weight-related stigma, with specific coping strategies differing between racial groups. Severely obese, bariatric surgery-seeking adults (N = 234; 91 African Americans) completed the Beck Depression Inventory (BDI) and Stigmatizing Situations Inventory (SSI). Two hierarchical linear regressions were conducted separately for African Americans and whites. For both racial groups, age, sex, BMI, years overweight, annual income, and education level did not account for a significant portion of the variance in BDI scores. The frequency of stigmatizing situations and coping strategies significantly explained 16.4% and 33.2%, respectively, of the variance for whites, and 25.9% and 25%, respectively, for African Americans (P < 0.001). Greater depressed mood in whites was associated with older age, lower education, fewer positive self-statements, and less self-love and more crying; while in African Americans greater depressed mood was associated only with ignoring the situation (P < 0.05). The study found that regardless of race, depressed mood in severely obese, bariatric surgery-seeking clients is related to the frequency of stigmatizing experiences and associated coping strategies. This suggests that efforts to reduce the deleterious effects of weight-related stigma need to focus both on reducing the frequency of stigmatization and on teaching effective coping strategies. These efforts also need to take into account the client's racial background.