scholarly journals Global Proteotoxicity Caused by Human β2 Microglobulin Variants Impairs the Unfolded Protein Response in C. elegans

2021 ◽  
Vol 22 (19) ◽  
pp. 10752
Author(s):  
Sarah C. Good ◽  
Katherine M. Dewison ◽  
Sheena E. Radford ◽  
Patricija van Oosten-Hawle
Keyword(s):  
Er Stress ◽  
Molecular Mechanisms ◽  
Amyloid Fibrils ◽  
Signal Sequence ◽  
Systemic Amyloidosis ◽  
Wild Type ◽  
Β2 Microglobulin ◽  
C Elegans

Aggregation of β2 microglobulin (β2m) into amyloid fibrils is associated with systemic amyloidosis, caused by the deposition of amyloid fibrils containing the wild-type protein and its truncated variant, ΔN6 β2m, in haemo-dialysed patients. A second form of familial systemic amyloidosis caused by the β2m variant, D76N, results in amyloid deposits in the viscera, without renal dysfunction. Although the folding and misfolding mechanisms of β2 microglobulin have been widely studied in vitro and in vivo, we lack a comparable understanding of the molecular mechanisms underlying toxicity in a cellular and organismal environment. Here, we established transgenic C. elegans lines expressing wild-type (WT) human β2m, or the two highly amyloidogenic naturally occurring variants, D76N β2m and ΔN6 β2m, in the C. elegans bodywall muscle. Nematodes expressing the D76N β2m and ΔN6 β2m variants exhibit increased age-dependent and cell nonautonomous proteotoxicity associated with reduced motility, delayed development and shortened lifespan. Both β2m variants cause widespread endogenous protein aggregation contributing to the increased toxicity in aged animals. We show that expression of β2m reduces the capacity of C. elegans to cope with heat and endoplasmic reticulum (ER) stress, correlating with a deficiency to upregulate BiP/hsp-4 transcripts in response to ER stress in young adult animals. Interestingly, protein secretion in all β2m variants is reduced, despite the presence of the natural signal sequence, suggesting a possible link between organismal β2m toxicity and a disrupted ER secretory metabolism.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach ◽  
Telomeric Protein ◽  
Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

scholarly journals Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

2021 ◽  
pp. 1-9
Author(s):  
Dayana Torres Valladares ◽  
Sirisha Kudumala ◽  
Murad Hossain ◽  
Lucia Carvelli
Keyword(s):  
Caenorhabditis Elegans ◽  
Molecular Mechanisms ◽  
Drugs Of Abuse ◽  
Genetic Model ◽  
In Vivo Model ◽  
C Elegans ◽  
Hyperactivity Disorder ◽  
In Vitro Data

Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in <i>Caenorhabditis elegans</i> to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that <i>C. elegans</i> is a suitable system to study tolerance to drugs of abuse such as amphetamines.

Methods to study the structure of misfolded protein states in systemic amyloidosis

2021 ◽  
Vol 49 (2) ◽  
pp. 977-985
Author(s):  
Marcus Fändrich ◽  
Matthias Schmidt
Keyword(s):  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Systemic Amyloidosis ◽  
Structural Basis ◽  
Amyloid A ◽  
Serum Amyloid A Protein ◽  
Proteolytic Stability

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2020 ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Binding Proteins ◽  
Protein Complexes ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach

AbstractTelomeres are bound by dedicated protein complexes, like shelterin in mammals, which protect telomeres from DNA damage. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 are expressed in the germline and during embryogenesis. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp-1; tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. Furthermore, we find that POT-1 bridges the double- stranded binders TEBP-1 and TEBP-2, with the single-stranded binders POT-2 and MRT-1. These results describe the first telomere-binding complex in C. elegans, with TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility and that mediate opposite telomere dynamics.

Expression Of Mutant Spliceosomal Protein SF3B1 Results In Dysregulated Hematopoietic Maturation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2773-2773
Author(s):  
Alexander C. Minella ◽  
Oscar Ramirez ◽  
Yanfei Xu ◽  
Tushar Murthy ◽  
Xiaodong Yang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Retroviral Vectors ◽  
Peptide Sequence ◽  
Causative Role ◽  
Wild Type ◽  
Erythroid Maturation

Abstract Whole genome sequencing has recently revealed the prevalence of mutations in proteins directing splicing of RNA in up to half of the patients with Myelodysplastic Syndrome (MDS). Mutations in the protein SF3B1 are particularly common in MDS patients with the phenotypic abnormality termed ring sideroblasts (dysplastic erythroid precursors with perinculear rings formed by iron-laden mitochondria). The most common SF3B1 mutation in MDS patients results in a change from lysine to glutamic acid at amino acid position 700 (K700E). Given that splicing of RNA is a ubiquitous phenomenon, it is unclear how these mutations result in clonal proliferation and dysplastic hematopoiesis; two hallmark features of MDS. Furthermore, direct experimental evidence demonstrating a causative role for SF3B1 mutations in MDS-related phenotypes is lacking. To better understand how mutations of spliceosomal proteins contribute to MDS pathogenesis, we sought to define how expression of mutant SF3B1 changes erythroid maturation in vitro and in vivo. Native SF3B1 cDNA constructs are not amenable to bacterial propagation due to toxicity of its HEAT-domain repeats. We overcame this problem by codon optimization (changing the DNA sequence while preserving the native peptide sequence). Human cord blood derived CD34+ cells were transduced with retroviral vectors to express either the wild-type or K700E mutant of SF3B1. After a week of expansion in cytokines (IL-3, SCF and IL6), cells were induced to erythroid differentiation by addition of erythropoietin (EPO) and analyzed for surface markers of erythroid differentiation (CD 71, CD117, CD105, CD45 and CD235A) at regular intervals. K700E mutant expressing cells were found to have significantly reduced expression of CD105 when compared to wild-type SF3B1-expressing cells (average 50% recuction, n =8). CD105 or endoglin is a TGF-beta receptor accessory receptor expressed at high levels during intermediate stages of erythroid maturation. A more modest reduction of CD71 expression was also noted in K700E-SF3B1 cells. MDS bone marrow is known to express low levels of both CD105 and CD71 making our results clinically relevant. To further characterize how mutant SF3B1 may cause dysplastic hematopoiesis, we studied transduced and transplanted murine progenitor cells in vivo and in colony forming assays. Murine data demonstrate significantly reduced K700E-transduced hematopoietic progenitors (as defined by flow-cytometry) in vivo and impaired erythroid colony formation in vitro. Together, our results suggest that enforced expression of K700E-SF3B1 induces aberrant erythroid maturation and impairs homeostasis of hematopoietic precursor cells. Thus, we provide direct evidence that MDS-associated SF3B1 mutations perturb normal hematopoiesis and offer rationale for using our complementary experimental approach as a platform for elucidating the molecular mechanisms through which mutations in RNA splicing factors promote hematologic disease. Disclosures: No relevant conflicts of interest to declare.

The Ets-1 transcription factor is required for Stat1-mediated T-bet expression and IgG2a class switching in mouse B cells

Blood ◽  
2012 ◽  
Vol 119 (18) ◽  
pp. 4174-4181 ◽  
Author(s):  
Hai Vu Nguyen ◽  
Enguerran Mouly ◽  
Karine Chemin ◽  
Romain Luinaud ◽  
Raymonde Despres ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
Molecular Mechanisms ◽  
Wild Type ◽  
Transcriptional Enhancer ◽  
T Box ◽  
Class Switch ◽  
Ifn Γ

Abstract In response to antigens and cytokines, mouse B cells undergo class-switch recombination (CSR) and differentiate into Ig-secreting cells. T-bet, a T-box transcription factor that is up-regulated in lymphocytes by IFN-γ or IL-27, was shown to regulate CSR to IgG2a after T cell–independent B-cell stimulations. However, the molecular mechanisms controlling this process remain unclear. In the present study, we show that inactivation of the Ets-1 transcription factor results in a severe decrease in IgG2a secretion in vivo and in vitro. No T-bet expression was observed in Ets-1–deficient (Ets-1−/−) B cells stimulated with IFN-γ and lipopolysaccharide, and forced expression of T-bet in these cells rescued IgG2a secretion. Furthermore, we identified a transcriptional enhancer in the T-bet locus with an activity in B cells that relies on ETS-binding sites. After IFN-γ stimulation of Ets-1−/− B cells, activated Stat1, which forms a complex with Ets-1 in wild-type cells, no longer binds to the T-bet enhancer or promotes histone modifications at this site. These results demonstrate that Ets-1 is critical for IgG2a CSR and acts as an essential cofactor for Stat1 in the regulation of T-bet expression in B cells.

scholarly journals Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dong Hoon Lee ◽  
Go Woon Kim ◽  
Jung Yoo ◽  
Sang Wu Lee ◽  
Yu Hyun Jeon ◽  
...  
Keyword(s):  
Tumor Suppressor Genes ◽  
Therapeutic Strategy ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Histone Demethylase ◽  
Wild Type ◽  
P53 Target Gene ◽  
The Stability

AbstractGlioblastoma is the most lethal brain tumor and its pathogenesis remains incompletely understood. KDM4C is a histone H3K9 demethylase that contributes to epigenetic regulation of both oncogene and tumor suppressor genes and is often overexpressed in human tumors, including glioblastoma. However, KDM4C’s roles in glioblastoma and the underlying molecular mechanisms remain unclear. Here, we show that KDM4C knockdown significantly represses proliferation and tumorigenesis of glioblastoma cells in vitro and in vivo that are rescued by overexpressing wild-type KDM4C but not a catalytic dead mutant. KDM4C protein expression is upregulated in glioblastoma, and its expression correlates with c-Myc expression. KDM4C also binds to the c-Myc promoter and induces c-Myc expression. Importantly, KDM4C suppresses the pro-apoptotic functions of p53 by demethylating p53K372me1, which is pivotal for the stability of chromatin-bound p53. Conversely, depletion or inhibition of KDM4C promotes p53 target gene expression and induces apoptosis in glioblastoma. KDM4C may serve as an oncogene through the dual functions of inactivation of p53 and activation of c-Myc in glioblastoma. Our study demonstrates KDM4C inhibition as a promising therapeutic strategy for targeting glioblastoma.

scholarly journals Deficiency of MicroRNA-181a Results in Transcriptome-Wide Cell Specific Changes in the Kidney that Lead to Elevated Blood Pressure and Salt Sensitivity

2021 ◽  
Author(s):  
Madeleine R. Paterson ◽  
Kristy L. Jackson ◽  
Malathi I. Dona ◽  
Gabriella E. Farrugia ◽  
Bruna Visniauskas ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Salt Sensitivity ◽  
Juxtaglomerular Cells ◽  
Renal Cells ◽  
Wild Type ◽  
Knockout Mouse Model

AbstractMicroRNA miR-181a is down-regulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a knockout mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radio-telemetry probes were implanted in twelve-week-old C57BL/6J wild-type and miR-181a/b-1 knockout mice. Systolic and diastolic BP were 4-5mmHg higher in knockout compared with wild-type mice over 24-hours (P<0.01). Compared with wild-type mice, renal renin was higher in the juxtaglomerular cells of knockout mice. BP was similar in wild-type mice on a high (3.1%) versus low (0.3%) sodium diet (+0.4±0.8mmHg) but knockout mice showed salt sensitivity (+3.3±0.8mmHg, P<0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA-sequencing in 6,699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4, Col4a1, Cd81, Flt3l, Cxcl16, Smad4. We observed up-regulation of pathways related to the immune system, inflammatory response, reactive oxygen species and nerve development, consistent with higher tyrosine hydroxylase. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension.

scholarly journals Ononin Alleviates Doxorubicin-Induced Cardiotoxicity by Inhibiting ER Stress Through Activation of SIRT3

2021 ◽  
Author(s):  
Shimin Sun ◽  
Jingfan Weng ◽  
Qi Yang ◽  
Xingxiao Huang ◽  
Hanlin Zhang ◽  
...  
Keyword(s):  
Er Stress ◽  
Myocardial Injury ◽  
Molecular Mechanisms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Protective Effects ◽  
H9c2 Cells ◽  
Rat Cardiomyocytes

Abstract Introduction Doxorubicin (DOX) is a powerful anthracycline antineoplastic drug, but the clinical application of DOX is seriously limited by its dose-dependent cardiotoxicity. Ononin is a natural isoflavone glycoside and plays a key role in modulating apoptosis related signaling pathways. The aim of this study was to assess the possible cardioprotective effects of Ononin in DOX-induced cardiotoxicity and the underlying molecular mechanisms. Materials and methods Wistar rats were treated with normal saline, DOX with or without Ononin. After the last administration, cardiac function was evaluated by echocardiography. Rats were then sacrificed for histological and TUNEL analyses, with immunological detection for β-actinin, Bax, Bcl-2, GRP78, CHOP and SIRT3. An enzyme-linked immunosorbent assay was performed to assess the myocardial injury markers. H9C2 cells were treated with vehicle, DOX with or without Ononin. Then, 3-TYP was used to find out the relationship between ER stress and SIRT3. Results Ononin treatment ameliorated DOX-induced myocardial injury as demonstrated by echocardiography. Ononin partially restored DOX-induced cardiac dysfunction, both LVEF and LVFS were increased under the cotreatment of Ononin. Ononin also inhibited DOX-induced ER stress and apoptosis in rat cardiomyocytes and H9C2 cells. DOX group had a higher Bax/Bcl-2 ratio, GRP78 and CHOP expression then control group, but Ononin treatment improved these results. This effect was associated with SIRT3 activity, moreover, selective inhibition of SIRT3 blocked the protective effects of Ononin. Conclusion In the present study, we tested the hypothesis that Ononin may protect against DOX-induced cardiomyopathy through ER stress both in vitro and in vivo. Ononin is able to protect against DOX-induced cardiotoxicity by inhibiting ER stress and apoptosis, this effect may via stimulation of the SIRT3 pathway.

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