Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

The N2A region of titin has a unique structural configuration

The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca²⁺-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specifically, we characterized the structure of its Ig domains by elucidating the atomic structure of the I81-I83 tandem using x-ray crystallography and computing a homology model for I80. Structural data revealed these domains to present heterogeneous and divergent Ig folds, where I81 and I83 have unique loop structures. Notably, the I81-I83 tandem has a distinct rotational chain arrangement that confers it a unique multi-domain topography. However, we could not identify specific Ca²⁺-binding sites in these Ig domains, nor evidence of the association of titin N2A components with F-actin in transfected C2C12 myoblasts or C2C12-derived myotubes. In addition, F-actin cosedimentation assays failed to reveal binding to N2A. We conclude that N2A has a unique architecture that predictably supports its selective recruitment of binding partners in signaling, but that its mechanical role through interaction with F-actin awaits validation.     

Right Bundle Branch Block: A Predictor of Mortality in Early Systemic Sclerosis

Objective

To evaluate the prognostic significance of baseline electrocardiogram (ECG) abnormalities in a multiethnic cohort of patients with early systemic sclerosis (SSc) and to determine the serological, clinical, and echocardiogram correlates of ECG findings.

Methods

SSc patients with disease duration of≤5 years were enrolled in the GENISOS (Genetics versus Environment in Scleroderma Outcome Study) cohort. At the first visit, a standard 12 lead ECG was obtained along with demographic information, clinical data, and autoantibodies. The results of echocardiograms were also recorded. All ECGs were interpreted by a cardiologist unaware of the patients'' clinical data.

Results

Of 265 SSc patients with average disease duration at enrollment of 2.5 years, 140 (52.8%) had abnormal ECG findings. These findings were not associated with SSc disease type or autoantibody profile but were associated with more severe heart and lung involvement. A total of 75 patients (28.3%) died over a follow up time of 9.9 years. Complete right bundle branch block (± left anterior hemiblock) on ECG, present in 7 (2.6%) patients, predicted a higher risk of mortality (HR: 5.3; 95% CI: 2.1 to 13.4; p<0.001). The predictive significance of right bundle branch block was independent of age at enrollment, gender, ethnicity and risk factors for coronary artery disease.

Conclusion

ECG abnormalities are common in patients with early SSc and are associated with the severity of lung and heart involvement. Right bundle branch block is an independent predictor of mortality, and should be considered a marker of disease severity in SSc.     

CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems

Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein–binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap.     

Surfactant metabolism and anti-oxidative capacity in hyperoxic neonatal rat lungs: effects of keratinocyte growth factor on gene expression in vivo

Development of preterm infant lungs is frequently impaired resulting in bronchopulmoary dysplasia (BPD). BPD results from interruption of physiologic anabolic intrauterine conditions, the inflammatory basis and therapeutic consequences of premature delivery, including increased oxygen supply for air breathing. The latter requires surfactant, produced by alveolar type II (AT II) cells to lower surface tension at the pulmonary air:liquid interface. Its main components are specific phosphatidylcholine (PC) species including dipalmitoyl-PC, anionic phospholipids and surfactant proteins. Local antioxidative enzymes are essential to cope with the pro-inflammatory side effects of normal alveolar oxygen pressures. However, respiratory insufficiency frequently requires increased oxygen supply. To cope with the injurious effects of hyperoxia to epithelia, recombinant human keratinocyte growth factor (rhKGF) was proposed as a surfactant stimulating, non-catabolic and epithelial-protective therapeutic. The aim of the present study was to examine the qualification of rhKGF to improve expression parameters of lung maturity in newborn rats under hyperoxic conditions (85 % O₂ for 7 days). In response to rhKGF proliferating cell nuclear antigen mRNA, as a feature of stimulated proliferation, was elevated. Similarly, the expressions of ATP-binding cassette protein A3 gene, a differentiation marker of AT II cells and of peroxiredoxin 6, thioredoxin and thioredoxin reductase, three genes involved in oxygen radical protection were increased. Furthermore, mRNA levels of acyl-coA:lysophosphatidylcholine acyltransferase 1, catalyzing dipalmitoyl-PC synthesis by acyl remodeling, and adipose triglyceride lipase, considered as responsible for fatty acid supply for surfactant PC synthesis, were elevated. These results, together with a considerable body of other confirmative evidence, suggest that rhKGF should be developed into a therapeutic option to treat preterm infants at risk for impaired lung development.     

Improved chemical shift based fragment selection for CS-Rosetta using Rosetta3 fragment picker

A new fragment picker has been developed for CS-Rosetta that combines beneficial features of the original fragment picker, MFR, used with CS-Rosetta, and the fragment picker, NNMake, that was used for purely sequence based fragment selection in the context of ROSETTA de-novo structure prediction. Additionally, the new fragment picker has reduced sensitivity to outliers and other difficult to match data points rendering the protocol more robust and less likely to introduce bias towards wrong conformations in cases where data is bad, missing or inconclusive. The fragment picker protocol gives significant improvements on 6 of 23 CS-Rosetta targets. An independent benchmark on 39 protein targets, whose NMR data sets were published only after protocol optimization had been finished, also show significantly improved performance for the new fragment picker (van der Schot et al. in J Biomol NMR, 2013).