The unfolded protein response (UPR) is a conserved stress‐signaling pathway activated after accumulation of unfolded proteins within the endoplasmic reticulum (ER). Active UPR signaling leads to ...unconventional, enzymatic splicing of XBP1 mRNA enabling expression of the transcription factor XBP1s to control ER homeostasis. While IRE1 has been identified as the endoribonuclease required for cleavage of this mRNA, the corresponding ligase in mammalian cells has remained elusive. Here, we report that RTCB, the catalytic subunit of the tRNA ligase complex, and its co‐factor archease mediate XBP1 mRNA splicing both in vitro and in vivo. Depletion of RTCB in plasma cells of Rtcbfl/fl Cd23‐Cre mice prevents XBP1s expression, which normally is strongly induced during plasma cell development. RTCB‐depleted plasma cells show reduced and disorganized ER structures as well as severe defects in antibody secretion. Targeting RTCB and/or archease thus represents a promising strategy for the treatment of a growing number of diseases associated with elevated expression of XBP1s.
Synopsis
The capacity of B cells to differentiate into plasma cells and secrete high amounts of antibodies upon activation relies on the efficient splicing of the XBP1 mRNA, catalyzed by the tRNA ligase complex and its co‐factor archease during the unfolded protein response.
The tRNA ligase RTCB and its co‐factor archease co‐localize at the endoplasmic reticulum to catalyze unconventional XBP1 mRNA splicing during the unfolded protein response.
The presence of archease is needed to sustain full activity of the tRNA ligase complex and support XBP1 mRNA ligation.
Depletion of RTCB in a conditional B‐cell‐specific knockout mouse model (Rtcbfl/fl Cd23‐Cre) prevents XBP1s expression, which in normal conditions is strongly induced during plasma cell development.
Ex vivo stimulated RTCB‐depleted B cells show distorted ER structures and severe defects in antibody secretion.
In vivo, RTCB‐deficient mice show significantly reduced levels of serum immunoglobulins after immunization.
The capacity of B cells to differentiate into plasma cells and secrete antibodies upon activation relies on the efficient splicing of the XBP1 mRNA, catalyzed by the tRNA ligase complex and its co‐factor archease during the unfolded protein response.
Broadly neutralizing anti–HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy ...of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH inNicotiana benthamiana.Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells (CHOPG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated inN. benthamianaare capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed forCHOPG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti–HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.
Transcription factor activity and turnover are functionally linked, but the global patterns by which DNA-bound regulators are eliminated remain poorly understood. We established an assay to define ...the chromosomal location of DNA-associated proteins that are slated for degradation by the ubiquitin-proteasome system. The genome-wide map described here ties proteolysis in mammalian cells to active enhancers and to promoters of specific gene families. Nuclear-encoded mitochondrial genes in particular correlate with protein elimination, which positively affects their transcription. We show that the nuclear receptor corepressor NCoR1 is a key target of proteolysis and physically interacts with the transcription factor CREB. Proteasome inhibition stabilizes NCoR1 in a site-specific manner and restrains mitochondrial activity by repressing CREB-sensitive genes. In conclusion, this functional map of nuclear proteolysis links chromatin architecture with local protein stability and identifies proteolytic derepression as highly dynamic in regulating the transcription of genes involved in energy metabolism.
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•An activity-based assay localizes degradation of DNA-bound proteins•Proteolysis is dominant at enhancers and at promoters of distinct gene ontologies•Steady expression of mitochondrial genes requires degradation of NCoR1•Ubiquitin ligase Siah2 mediates derepression especially at CREB target promoters
A genome-wide proteolysis map shows a correlation between DNA-bound protein degradation and active gene promoters. In particular, continual removal of the corepressor NCoR1 from nuclear-encoded mitochondrial genes is required for optimal mitochondrial function.
* 27. Mai 1809 in Magdeburg, † 28. Dezember 1889 in Dresden, Komponist, Musikschriftsteller und Herausgeber. Carl, Sohn von Johann Carl Heinrich (1772–1842, Domvikar in Magdeburg), studierte bei Carl ......
The tobacco‐related species Nicotiana benthamiana has recently emerged as a promising host for the manufacturing of protein therapeutics. However, the production of recombinant proteins in N. ...benthamiana is frequently hampered by undesired proteolysis. Here, we show that the expression of the human anti‐HIV antibodies 2F5, 2G12, and PG9 in N. benthamiana leaves leads to the accumulation of discrete heavy chain‐derived degradation products of 30–40 kDa. Incubation of purified 2F5 with N. benthamiana intercellular fluid resulted in rapid conversion into the 40‐kDa fragment, whereas 2G12 proved largely resistant to degradation. Such a differential susceptibility to proteolytic attack was also observed when these two antibodies were exposed to various types of proteinases in vitro. While serine and cysteine proteinases are both capable of generating the 40‐kDa 2F5 fragment, the 30‐kDa polypeptide is most readily obtained by treatment with the latter class of enzymes. The principal cleavage sites reside within the antigen‐binding domain, the VH–CH1 linker segment and the hinge region of the antibodies. Collectively, these results indicate that down‐regulation of endogenous serine and cysteine proteinase activities could be used to improve the performance of plant‐based expression platforms destined for the production of biopharmaceuticals.
Monoclonal antibodies are important biopharmaceuticals. Their production in plant‐based expression platforms is frequently accompanied by unwanted proteolysis. The results of this study show that antibody fragmentation in Nicotiana benthamiana is mainly caused by serine and cysteine proteinases, indicating that down‐regulation of endogenous serine and cysteine proteinase activities could improve the performance of plant‐based expression platforms destined for the production of biopharmaceuticals.
To determine the effectiveness of a multidimensional neonatal simulation-based medical education training programme on direct and indirect patient outcome parameters.
This was a retrospective ...analytical study with a historical control group in a level II neonatal care unit (1,700 births per year). A multidimensional interdisciplinary training programme on neonatal resuscitation was implemented in 2015; pre-training (2012–2014) and post-training (2015–2019) eras were compared in terms of mortality (direct outcome) and the received intervention level immediately after birth (indirect outcome). Intervention levels were defined as follows: A) short-term non-invasive ventilation, B) prolonged non-invasive ventilation (>5 inflation breaths), C) chest compressions.
Of 13,950 neonates born during the study period, 826 full-term newborns received one of the three intervention levels for adaptation after birth. A total of 284 (34.4%) patients received short-term non-invasive ventilation (A), 477 (57.8%) had prolonged ventilation (B), and 65 (7.9%) chest compressions (C), respectively. Comparing the pre- and post-training eras, there was no significant reduction in mortality, and no significant changes were found in groups A or B. However, the risk for chest compressions (group C) decreased significantly from 0.91% in the pre-training era to 0.20% in the post-training era (p < 0.001).
Although there was no significant effect on neonatal mortality, regular interdisciplinary simulation training decreased the number of administered chest compressions immediately after birth. Further studies are needed to test indirect outcome–related parameters, such as frequency of chest compressions as a measure of effectiveness and impact of medical training.
Squamous cell carcinoma (SCC) is a malignant tumor derived from squamous cells and can be found in different localizations. In the oral cavity especially, it represents the most common type of ...malignant tumor. First-line therapy for oral squamous cell carcinoma (OSCC) is surgery, including tumor resection, neck dissection, and maybe reconstruction. Although perioperative mortality is low, complications such as delirium are very common, and may have long-lasting consequences on the patient’s quality of life. This study examines if excessive fluid administration, among other parameters, is an aggravating factor for the development of postoperative delirium. A total of 198 patients were divided into groups concerning the reconstruction technique used: group A for primary wound closure or reconstruction with a local flap, and group B for microsurgical reconstruction. The patients with and without delirium in both groups were compared regarding intraoperative fluid administration, fluid balance, and other parameters, such as blood loss, duration of surgery and overall ventilation, alcohol consumption, and creatinine, albumin, natrium, and hematocrit levels. The logistic regression for group A shows that fluid intake (p = 0.02, OR = 5.27, 95% CI 1.27–21.8) and albumin levels (p = 0.036, OR = 0.22, CI 0.054–0.908) are independent predictors for the development of delirium. For group B, gender (p = 0.026, OR = 0.34, CI 0.133–0.879) with a protective effect for females, fluid intake (p = 0.003, OR = 3.975, CI 1.606–9.839), and duration of ventilation (p = 0.025, OR = 1.178, CI 1.021–1.359) are also independent predictors for delirium. An intake of more than 3000 mL for group A, and 4150 mL for group B, increases the risk of delirium by approximately five and four times, respectively. Fluid management should be considered carefully in patients with OSCC, in order to reduce the occurrence of postoperative delirium. Different factors may become significant for the development of delirium regarding different surgical procedures.
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