SGTA interacts with the proteasomal ubiquitin receptor Rpn13 via a carboxylate clamp mechanism

YesThe fate of secretory and membrane proteins that mislocalize to the cytosol is decided by a collaboration between cochaperone SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the BAG6 complex, whose operation relies on multiple transient and subtly discriminated interactions with diverse binding partners. These include chaperones, membrane-targeting proteins and ubiquitination enzymes. Recently a direct interaction was discovered between SGTA and the proteasome, mediated by the intrinsic proteasomal ubiquitin receptor Rpn13. Here, we structurally and biophysically characterize this binding and identify a region of the Rpn13 C-terminal domain that is necessary and sufficient to facilitate it. We show that the contact occurs through a carboxylate clamp-mediated molecular recognition event with the TPR domain of SGTA, and provide evidence that the interaction can mediate the association of Rpn13 and SGTA in a cellular context.RLI was supported by MRC New Investigator Research Grant: G0900936. RLI and SH are funded by BBSRC grants: BB/L006952/1 and BB/L006510/1 respectively. RLI is funded by BBSRC grant: BB/N006267/1. AT is funded by BBSRC grant: BB/J014567/1. ILT was the recipient of a Wellcome Trust Vacation Scholarship 2015. NMR experiments were performed at the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trus

DNA G-segment bending is not the sole determinant of topology simplification by type II DNA topoisomerases

DNA topoisomerases control the topology of DNA. Type II topoisomerases exhibit topology simplification, whereby products of their reactions are simplified beyond that expected based on thermodynamic equilibrium. The molecular basis for this process is unknown, although DNA bending has been implicated. To investigate the role of bending in topology simplification, the DNA bend angles of four enzymes of different types (IIA and IIB) were measured using atomic force microscopy (AFM). The enzymes tested were Escherichia coli topo IV and yeast topo II (type IIA enzymes that exhibit topology simplification), and Methanosarcina mazei topo VI and Sulfolobus shibatae topo VI (type IIB enzymes, which do not). Bend angles were measured using the manual tangent method from topographical AFM images taken with a novel amplitude-modulated imaging mode: small amplitude small set-point (SASS), which optimises resolution for a given AFM tip size and minimises tip convolution with the sample. This gave improved accuracy and reliability and revealed that all 4 topoisomerases bend DNA by a similar amount: ~120° between the DNA entering and exiting the enzyme complex. These data indicate that DNA bending alone is insufficient to explain topology simplification and that the ‘exit gate’ may be an important determinant of this process

Relationships between self-reported potentially traumatizing events, psychoform and somatoform dissociation, and absorption, in two non-clinical populations

Contains fulltext : 54627.pdf ( ) (Open Access)Objective: Some authors have suggested that the personality characteristic 'fantasy proneness' may mediate the correlation between reported potentially traumatizing events and dissociative symptoms. Other authors question the reported magnitude of this correlation in non-clinical samples, because these are usually derived from student samples and may therefore suffer from a restriction of range. The primary aim of this study is to assess the relationship between a self-report measure of traumatization and psychoform dissociation as well as somatoform dissociation in a non-clinical population, while accounting for the influence of fantasy proneness. Method: Two random non-clinical samples, that is, a student and an adult non-student sample, completed a range of relevant self-report questionnaires. Absorption was used as an index of fantasy proneness. Results: The range of reported potentially traumatizing events was restricted in students, compared to non-students. In both samples a significant correlation was found between reported potentially traumatizing events and dissociation. After partialling out absorption, the relationship between reported potential traumatization and psychoform dissociation diminished substantially in both samples. The magnitude of the correlation with somatoform dissociation decreased to a lesser degree, so that it remained significant in both samples. Conclusions: The correlation between somatoform dissociation and reported traumatization, after partialling out absorption, gives a reliable estimate of the magnitude of the relationships between potentially traumatizing events and dissociation. Findings regarding traumatization and dissociation in students should be generalized to the general population cautiously.6 p

Quantifying the heterogeneity of macromolecular machines by mass photometry

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization