Coupling of endoplasmic reticulum (ER) stress to dimerisation-dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex ...between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1
) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1
that mediated BiP-induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1
induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1
dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1
to initiate active repression of UPR signalling.
•Integrated process and product design method for cost and GWI minimization.•Multi-component biofuels designed for advanced, highly boosted SI engines.•Tailor-made fuels contain ethanol, isobutanol, ...2-butanone, 2-MF, cyclopentane.•Optimal fuel costs of 18–22 $/GJfuel and GWIs of 39–61 kgCO2eq./GJfuel.•Simultaneous design rigorously captures benefits of co-producing fuel components.
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Integrated product and process design aims at developing innovative products that provide a desired functionality and are produced efficiently. Tailor-made fuels from renewable feedstocks pose a prominent, societally-relevant example. We build upon the integrated design method from Dahmen and Marquardt (2017) and combine it with the production pathway screening tool from Ulonska et al. (2016). We thus design a tailor-made fuel and its optimal production process by minimizing economic and environmental criteria, i.e., cost and global warming impact (GWI). We consider the production of a tailor-made spark-ignition engine fuel from lignocellulosic biomass. Simultaneous process and product design yields optimal multi-component fuels that consist of ethanol, isobutanol, butanone, cyclopentane, and 2-methylfuran with production costs of 18–22 $ per GJfuel and GWI values of 38–61 kgCO2eq. per GJfuel. The proposed method and its solution strategies are, in principle, universal and thus also applicable to products other than fuels.
Intestinal goblet cells are secretory cells specialized in the production of mucins, and as such are challenged by the need for efficient protein folding. Goblet cells express Inositol-Requiring ...Enzyme-1β (IRE1β), a unique sensor in the unfolded protein response (UPR), which is part of an adaptive mechanism that regulates the demands of mucin production and secretion. However, how IRE1β activity is tuned to mucus folding load remains unknown. We identified the disulfide isomerase and mucin chaperone AGR2 as a goblet cell-specific protein that crucially regulates IRE1β-, but not IRE1α-mediated signaling. AGR2 binding to IRE1β disrupts IRE1β oligomerization, thereby blocking its downstream endonuclease activity. Depletion of endogenous AGR2 from goblet cells induces spontaneous IRE1β activation, suggesting that alterations in AGR2 availability in the endoplasmic reticulum set the threshold for IRE1β activation. We found that AGR2 mutants lacking their catalytic cysteine, or displaying the disease-associated mutation H117Y, were no longer able to dampen IRE1β activity. Collectively, these results demonstrate that AGR2 is a central chaperone regulating the goblet cell UPR by acting as a rheostat of IRE1β endonuclease activity.
Synopsis
IRE1β is specifically expressed in mucin-secreting goblet cells of the mammalian gut. Here, the mucin chaperone AGR2 is identified as master regulator of IRE1β oligomerization and endonuclease activity.
The protein disulfide isomerase AGR2 acts as a specific interactor of the unfolded protein response sensor IRE1β in colon epithelial cells.
AGR2 inhibits IRE1β endonuclease activity by promoting IRE1β monomerization.
The inflammatory bowel disease-associated AGR2
H117Y
mutant loses its repressive activity on IRE1β.
AGR2 regulates IRE1β UPR sensor activity by holding it in a monomeric inactive state.
AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER‐localised Fic protein, ...FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation‐competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide‐binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.
Synopsis
Regulation of the endoplasmic reticulum chaperone BiP involves AMPylation and deAMPylation, both of which are catalysed by the ER protein FICD. This study shows how transitions in FICD's oligomeric state enable switching of the activity of this bifunctional enzyme.
FICD forms high‐affinity dimers that exhibit BiP deAMPylation activity and repressed AMPylation activity.
Monomerisation of FICD allosterically releases AMPylation‐autoinhibition, permitting AMPylation‐competent ATP binding, whilst reciprocally inhibiting FICD's deAMPylation activity.
Changes in the ADP/ATP ratio influence FICD's monomer‐dimer equilibrium in vitro, suggesting a functional link between BiP modification and the energy state of the endoplasmic reticulum.
The bifunctional enzymatic activity of FICD in regulating AMPylation of the endoplasmic reticulum chaperone BiP is conferred by transitions of its oligomeric state.
Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the ...beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1β) with the luminal domain of IRE1β deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1β/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1β/α chimera. In vitro, AGR2 actively de-stabilised the IRE1β luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1β-AGR2 couple suggest that active repression of IRE1β by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.
Synopsis
Stress recognition by the unfolded protein response (UPR) of the ER is incompletely understood. Here, negative regulation of a goblet-cell selective isoform of the UPR transducer IRE1β by the mucin chaperone AGR2 shows how UPR signalling can be coupled to the load of abundant, cell-specific, secreted proteins via specialised chaperone titration.
In heterologous CHO cell cultures, the specialised UPR transducer IRE1β is constitutively active despite the absence of the ER client proteins normally co-expressed with it.
Coexpression of AGR2, a mucin chaperone normally present together with IRE1β, selectively represses the stress-sensing IRE1β luminal domain in CHO cells.
Introduction of the secreted client protein mucin into CHO cells reverses AGR2-mediated repression of IRE1β.
In vitro, AGR2 promotes a monomeric pool of IRE1β’s luminal stress-sensing domain.
Mucin-secreting goblet cells in the gut have evolved a specialised unfolded protein response signalling pathway.
Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected ...families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated.
Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history.
In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16-93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients <60 years was 30.2% (33.2%) versus 10.6% (18.9%) in patients ≥60 years. Family history was positive in 43% of all patients. Patients with a positive family history had a prevalence of deleterious variants of 31.6% (36.0%) versus 11.4% (17.6%) and histologic subtype of high grade serous ovarian cancer versus other showed a prevalence of deleterious variants of 23.2% (29.1%) and 10.2% (14.8%), respectively. Testing only for BRCA1/2 would miss in our series more than 5% of the patients with a deleterious variant in established risk genes.
26.4% of all patients harbor at least one deleterious variant in established risk genes. The threshold of 10% mutation rate which is accepted for reimbursement by health care providers in Germany was observed in all subgroups analyzed and neither age at primary diagnosis nor histo-type or family history sufficiently enough could identify a subgroup not eligible for genetic counselling and testing. Genetic testing should therefore be offered to every patient with invasive epithelial ovarian cancer and limiting testing to BRCA1/2 seems to be not sufficient.
During the COVID-19 pandemic, general practitioners (GPs) continued to be a main point of contact for patients. For GP practices, it was and still is a challenge to meet constantly changing ...requirements due to the various phases of the pandemic. The aim of the study is to explore retrospectively the subjective experience with supply and utilization of health care services from the perspective of general practitioners, medical practice assistants and patients, in particular regarding instances of underutilization of services for non-Covid related conditions, adjustments due to the pandemic, and the appropriateness of care.
The study is carried out within the RESPoNsE research practice network in three of Germany's federal states: Berlin, Brandenburg, and Thuringia (RESPoNsE-Research practice network east). The study follows a convergent mixed method design, and consists of the following sections: a) two anonymous paper-based questionnaires filled out by GPs and medical practice assistants (MPAs), at an interval of 12 to 18 months; b) in-depth qualitative interviews conducted among a subgroup of GPs and MPAs; c) anonymous paper-based questionnaires among patients of participating practices. The idea for the study was derived from discussions with the practice advisory board of the RESPoNsE network. The themes and issues to be explored in the surveys and interviews are developed and discussed in the practice advisory board, the patient advisory board, and with interested MPAs. The questionnaires will be analyzed descriptively, exploring the effect of demographic variables. Qualitative content analysis is used to analyze the data from the interviews and focus groups.
The study focuses on the conditions of GP care during the COVID-19 pandemic. A broad insight is provided as GPs and MPAs, as well as patients, are involved. It provides the opportunity to express needs and concerns. The results can support future discussions on lessons learned from the pandemic and necessary changes in health care delivery.
Trial registration at the German Clinical Trials Register: DRKS00028095.
Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial.
To ...assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants.
BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02-36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99-59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00-3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70-2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43-9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients.
To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded.
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