scholarly journals Improvement of Foreign-Protein Production in Aspergillus niger var. awamori by Constitutive Induction of the Unfolded-Protein Response

2003 ◽  
Vol 69 (12) ◽  
pp. 6979-6986 ◽  
Author(s):  
Mari Valkonen ◽  
Michael Ward ◽  
Huaming Wang ◽  
Merja Penttilä ◽  
Markku Saloheimo
Keyword(s):  
Aspergillus Niger ◽  
Unfolded Protein Response ◽  
Protein Production ◽  
Production Systems ◽  
Foreign Protein ◽  
Mrna Levels ◽  
Unfolded Protein ◽  
Novel Strategy ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Unfolded-protein response (UPR) denotes the upregulation of endoplasmic reticulum (ER)-resident chaperone and foldase genes and numerous other genes involved in secretory functions during the accumulation of unfolded proteins into the ER. Overexpression of individual foldases and chaperones has been used in attempts to improve protein production in different production systems. We describe here a novel strategy to improve foreign-protein production. We show that the constitutive induction of the UPR pathway in Aspergillus niger var. awamori can be achieved by expressing the activated form of the transcription factor hacA. This induction enhances the production of Trametes versicolor laccase by up to sevenfold and of bovine preprochymosin by up to 2.8-fold in this biotechnically important fungus. The regulatory range of UPR was studied by analyzing the mRNA levels of novel A. niger var. awamori genes involved in different secretory functions. This revealed both similarities and differences to corresponding studies in Saccharomyces cerevisiae.

XBP-1 Levels Predict Sensitivity of Myelomas to Proteasome Inhibitor Bortezomib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1473-1473
Author(s):  
Silvia C. Ling ◽  
Edwin Lau ◽  
Lye L. Ho ◽  
Joy Ho ◽  
Douglas E. Joshua ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Myeloma Cell ◽  
26S Proteasome ◽  
Mrna Levels ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Background: Proteasome inhibitors (PI) are remarkably effective in relapsed and refractory myeloma but the origin of this peculiar sensitivity remains unclear. Myeloma is dependent on the unfolded protein response (UPR) and its regulator, transcription factor XBP-1. PI perturbs the unfolded protein response (UPR) by inhibition of the 26S proteasome-the main pathway for protein degradation. We hypothesize that the dependence on the UPR and XBP-1 mediates sensitivity to PI and the level of XBP-1 correlates with sensitivity to PI. The aim of this study is to correlate Bortezomib sensitivity with XBP-1 in vitro and in myeloma patients; to check the effect of manipulating XBP-1 on Bortezomib sensitivity and develop Bortezomib-resistant myeloma cell lines to ascertain the effects on XBP-1 and the UPR. Methods and Results: Sensitivity to Bortezomib was measured by growth inhibition assay. XBP-1 mRNA levels and its isoforms were measured by a two-step quantitative QPCR assay, in 6 myeloma cell lines and 17 other cancer cell lines. There is a strong inverse correlation in myeloma cell lines between total or unspliced XBP-1 with Bortezomib sensitivity (r = −0.9) but not in other cancer cell lines. 23 marrow biopsies from 11 Bortezomib-treated myeloma patients were analysed for XBP-1 expression. Myeloma cells (CD38 hi, CD14 lo, kappa or lambda light chain +ve) were purified by flow cytometry. XBP-1 levels in myeloma cell lines were manipulated by shRNA-mediated knockdown and overexpression by retroviral transduction and had little effect on Bortezomib sensitivity. Bortezomib-resistant myeloma lines were developed. The mechanism of resistance was elucidated (XBP-1, ATF6, P-EIF2a, P58 INK and immunogloblin production). Marked downregulation of XBP-1 was demonstrated. Conclusion: XBP-1 is a surrogate marker of Bortezomib sensitivity and its clinical utility is being tested now. Sensitivity to PI is related to the dependence on the UPR, reflected in the level of XBP-1. Bortezomib resistance is mediated by downregulation of the UPR.

HIV protease inhibitors activate the unfolded protein response and disrupt lipid metabolism in primary hepatocytes

2006 ◽  
Vol 291 (6) ◽  
pp. G1071-G1080 ◽  
Author(s):  
Huiping Zhou ◽  
Emily C. Gurley ◽  
Sirikalaya Jarujaron ◽  
Hong Ding ◽  
Youwen Fang ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Unfolded Protein Response ◽  
Protease Inhibitors ◽  
Hiv Protease ◽  
Hiv Protease Inhibitors ◽  
Mrna Levels ◽  
Primary Hepatocytes ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Treatment of human immunodeficiency virus (HIV)-infected patients with HIV protease inhibitors (PIs) has been associated with serious lipid disturbances. However, the incidence and degree of impaired lipid metabolism observed in the clinic vary considerably between individual HIV PIs. Our previous studies demonstrated that HIV PIs differ in their ability to increase the levels of transcriptionally active sterol regulatory element-binding proteins (SREBPs), activate the unfolded protein response (UPR), induce apoptosis, and promote foam cell formation in macrophages. In the present study, we examined the effects of three HIV PIs, including amprenavir, atazanavir, and ritonavir, on the UPR activation and the expression of key genes involved in lipid metabolism in primary rodent hepatocytes. Both atazanavir and ritonavir activated the UPR, induced apoptosis, and increased nuclear SREBP levels, but amprenavir had no significant effect at the same concentrations. In rat primary hepatocytes, cholesterol 7α-hydroxylase (CYP7A1) mRNA levels were significantly decreased by atazanavir (38%) and ritonavir (56%) but increased by amprenavir (90%); 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase mRNA levels were increased by amprenavir (23%) but not by ritonavir and atazanavir; low-density lipoprotein receptor mRNA was increased by atazanavir (20%) but not by amprenavir and ritonavir. Similar results were obtained in mouse primary hepatocytes. Atazanavir and ritonavir also decreased CYP7A1 protein levels and bile acid biosynthesis, while amprenavir had no significant effect. The current results may help provide a better understanding of the cellular mechanisms of HIV PI-induced dyslipidemia and also provide useful information to help predict clinical adverse effects in the development of new HIV PIs.

scholarly journals Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae

2003 ◽  
Vol 69 (4) ◽  
pp. 2065-2072 ◽  
Author(s):  
Mari Valkonen ◽  
Merja Penttilä ◽  
Markku Saloheimo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Unfolded Protein Response ◽  
Protein Production ◽  
Active Form ◽  
Constitutive Expression ◽  
Amylase Secretion ◽  
Unfolded Protein ◽  
Foreign Proteins ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens α-amylase and Trichoderma reesei endoglucanase EGI, were studied. Disruption of HAC1 caused decreases in the secretion of both α-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain. Constitutive overexpression of HAC1 caused a 70% increase in α-amylase secretion but had no effect on EGI secretion. The invertase levels were twofold higher in the strain overexpressing HAC1. Also, the effect of the active form of T. reesei hac1 was tested in S. cerevisiae. hac1 expression caused a 2.4-fold increase in the secretion of α-amylase in S. cerevisiae and also slight increases in invertase and total protein production. Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation.

scholarly journals EPO and TMBIM3/GRINA Promote the Activation of the Adaptive Arm and Counteract the Terminal Arm of the Unfolded Protein Response after Murine Transient Cerebral Ischemia

2019 ◽  
Vol 20 (21) ◽  
pp. 5421 ◽  
Author(s):  
Pardes Habib ◽  
Ann-Sophie Stamm ◽  
Joerg B. Schulz ◽  
Arno Reich ◽  
Alexander Slowik ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Unfolded Protein Response ◽  
Calcium Release ◽  
Mrna Levels ◽  
Mixed Cell ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
The Impact

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis leading to impairment of endoplasmic reticulum (ER) function. The unfolded protein response (UPR) is an ER-located and cytoprotective pathway that aims to resolve ER stress. Transmembrane BAX inhibitor-1 motif-containing (TMBIM) protein family member TMBIM3/GRINA is highly expressed in the brain and mostly located at the ER membrane suppressing ER calcium release by inositol-1,4,5-trisphosphate receptors. GRINA confers neuroprotection and is regulated by erythropoietin (EPO) after murine cerebral ischemia. However, the role of GRINA and the impact of EPO treatment on the post-ischemic UPR have not been elucidated yet. We subjected GRINA-deficient (Grina−/−) and wildtype mice to transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 6 h or 72 h of reperfusion. We administered EPO or saline 0, 24 and 48 h after tMCAo/sham surgery. Oxygen–glucose deprivation (OGD) and pharmacological stimulation of the UPR using Tunicamycin and Thapsigargin were carried out in primary murine cortical mixed cell cultures. Treatment with the PERK-inhibitor GSK-2606414, IRE1a-RNase-inhibitor STF-083010 and EPO was performed 1 h prior to either 1 h, 2 h or 3 h of OGD. We found earlier and larger infarct demarcations in Grina−/− mice compared to wildtype mice, which was accompanied by a worse neurological outcome and an abolishment of EPO-mediated neuroprotection after ischemic stroke. In addition, GRINA-deficiency increased apoptosis and the activation of the corresponding PERK arm of the UPR after stroke. EPO enhanced the post-ischemic activation of pro-survival IRE1a and counteracted the pro-apoptotic PERK branch of the UPR. Both EPO and the PERK-inhibitor GSK-2606414 reduced cell death and regulated Grina mRNA levels after OGD. In conclusion, GRINA plays a crucial role in post-ischemic UPR and the use of both GSK-2606414 and EPO might lead to neuroprotection.

scholarly journals HacA-Independent Induction of Chaperone-Encoding Gene bipA in Aspergillus niger Strains Overproducing Membrane Proteins

2006 ◽  
Vol 72 (1) ◽  
pp. 953-955 ◽  
Author(s):  
Anoushka Davé ◽  
David J. Jeenes ◽  
Donald A. Mackenzie ◽  
David B. Archer
Keyword(s):  
Aspergillus Niger ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Unfolded Protein Response ◽  
Mrna Levels ◽  
Unfolded Protein ◽  
Protein Overproduction ◽  
Encoding Gene ◽  
Protein Response

ABSTRACT Transcription of two unfolded protein response genes, hacA and bipA, was examined in Aspergillus niger strains overproducing membrane proteins. Despite elevated bipA mRNA levels, no 5′-truncated hacA transcript was detected, raising the possibility of a hacA-independent induction of bipA mRNA under the stress of membrane protein overproduction in A. niger.

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