Interaction of Bacillus subtilis CsaA with SecA and precursor proteins

CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants. CsaA has chaperone-like activities in vivo and in vitro. To examine the role of CsaA in protein export in B. subtilis, expression of the csaA gene was repressed. While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion. CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E. coli and B. subtilis, and binds the B. subtilis preprotein prePhoB. Purified CsaA stimulates the translocation of prePhoB into E. coli membrane vesicles bearing the B. subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E. coli. The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B. subtilis.

Activity of transgene-produced B-domain–deleted factor VIII in human plasma following AAV5 gene therapy

Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain–deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19

Expression of protein synthesis elongation factors in winter wheat and oat in response to heat stress

The aim of our work was to examine the expression and accumulation of EF-Tu and eEF1A in grain filing stage of five genotypes of winter wheat and one oat genotype in conditions of heat stress. In addition, the correlation between accumulation of elongation factors eEF1A and EF-Tu, and yield components of cereals in the field was investigated. Flag leaf protein samples were analyzed by immunoblotting. Flag leaves were collected under conditions of moderate (23 degrees C; MT) and high air temperature (38 degrees C; HT) in a field experiment. After the harvest, grain yield was determined. The yield components, the weight of dry seed and grains number per spike, were assessed in the stage of full physiological maturity of investigated cultivars. Obtained results revealed a difference in the level of EF-Tu accumulation both under conditions of moderate air temperatures and conditions of heat stress among investigated cultivars. Cultivar Zvezdana was the only one that showed increase in EF-Tu accumulation under HT (25%) compared to MT. Immunoblot analysis indicated that the highest increase of eEF1A accumulation (43%) in relation to moderate temperature was detected in cultivar Talas. A significant, positive, linear correlation was found between the expression of eEF1A and small grains productivity under heatstress conditions