scholarly journals SEC24A facilitates colocalization and Ca2+ flux between the endoplasmic reticulum and mitochondria

2021 ◽  
Vol 134 (6) ◽  
Author(s):  
Tamutenda Chidawanyika ◽  
Rajarshi Chakrabarti ◽  
Kathryn S. Beauchemin ◽  
Henry N. Higgs ◽  
Surachai Supattapone
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Fold Increase ◽  
Cellular Mechanism ◽  
Autophagic Flux ◽  
Wild Type ◽  
Genome Wide ◽  
Fold Reduction ◽  
A Genome ◽  
Indicator Dyes

ABSTRACT A genome-wide screen recently identified SEC24A as a novel mediator of thapsigargin-induced cell death in HAP1 cells. Here, we determined the cellular mechanism and specificity of SEC24A-mediated cytotoxicity. Measurement of Ca2+ levels using organelle-specific fluorescent indicator dyes showed that Ca2+ efflux from endoplasmic reticulum (ER) and influx into mitochondria were significantly impaired in SEC24A-knockout cells. Furthermore, SEC24A-knockout cells also showed ∼44% less colocalization of mitochondria and peripheral tubular ER. Knockout of SEC24A, but not its paralogs SEC24B, SEC24C or SEC24D, rescued HAP1 cells from cell death induced by three different inhibitors of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCA) but not from cell death induced by a topoisomerase inhibitor. Thapsigargin-treated SEC24A-knockout cells showed a ∼2.5-fold increase in autophagic flux and ∼10-fold reduction in apoptosis compared to wild-type cells. Taken together, our findings indicate that SEC24A plays a previously unrecognized role in regulating association and Ca2+ flux between the ER and mitochondria, thereby impacting processes dependent on mitochondrial Ca2+ levels, including autophagy and apoptosis.

scholarly journals RACK1 modulates polyglutamine-induced neurodegeneration by promoting ERK degradation in Drosophila

PLoS Genetics ◽  
2021 ◽  
Vol 17 (5) ◽  
pp. e1009558
Author(s):  
Jun Xie ◽  
Yongchao Han ◽  
Tao Wang
Keyword(s):  
Cell Death ◽  
Drug Targets ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Wild Type ◽  
Genome Wide ◽  
A Genome ◽  
Potent Drug ◽  
Biochemical Analyses ◽  
Polyq Diseases

Polyglutamine diseases are neurodegenerative diseases caused by the expansion of polyglutamine (polyQ) tracts within different proteins. Although multiple pathways have been found to modulate aggregation of the expanded polyQ proteins, the mechanisms by which polyQ tracts induced neuronal cell death remain unknown. We conducted a genome-wide genetic screen to identify genes that suppress polyQ-induced neurodegeneration when mutated. Loss of the scaffold protein RACK1 alleviated cell death associated with the expression of polyQ tracts alone, as well as in models of Machado-Joseph disease (MJD) and Huntington’s disease (HD), without affecting proteostasis of polyQ proteins. A genome-wide RNAi screen for modifiers of this rack1 suppression phenotype revealed that knockdown of the E3 ubiquitin ligase, POE (Purity of essence), further suppressed polyQ-induced cell death, resulting in nearly wild-type looking eyes. Biochemical analyses demonstrated that RACK1 interacts with POE and ERK to promote ERK degradation. These results suggest that RACK1 plays a key role in polyQ pathogenesis by promoting POE-dependent degradation of ERK, and implicate RACK1/POE/ERK as potent drug targets for treatment of polyQ diseases.

BIOM-32. ENDOPLASMIC RETICULUM PROTEIN SSR3 DETERMINES AND PREDICTS RESPONSE TO PACLITAXEL IN BREAST CANCER AND GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi17-vi18
Author(s):  
Crismita Dmello ◽  
Aarón Sonabend ◽  
Víctor Arrieta ◽  
Daniel Zhang ◽  
Deepak Kanojia ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Endoplasmic Reticulum ◽  
Glioma Cells ◽  
Predictive Biomarkers ◽  
Predictive Biomarker ◽  
Precision Oncology ◽  
Patient Response ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome

Abstract Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Given the potency of this drug for glioblastomas (GBM) several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for this disease. In spite of the efficacy of PTX, individual tumors exhibit variable susceptibility to this drug, with response rate in the range of 30%-60%. To identify predictive biomarkers for response to PTX, we performed a genome-wide CRISPR knock-out screen using human glioma cells. The most enriched genes in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort. This led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with response to PTX in breast cancer cells, glioma cells, in multiple intracranial glioma xenografts and in GBM patient derived explant cultures. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. In gliomas, SSR3-mediated susceptibility to PTX relates to modulation of phosphorylation of ER stress sensor IRE1α. Thus, by using genome-wide screen combined with patient response data, we discovered a biomarker that demonstrates causal and correlative relationship with response to PTX in breast cancer and GBM. Prospective validation of this biomarker is warranted for its broad implementation for precision oncology.

scholarly journals Genome-wide polygenic score to identify a monogenic risk-equivalent for coronary disease

2017 ◽  
Author(s):  
Amit V. Khera ◽  
Mark Chaffin ◽  
Krishna G. Aragam ◽  
Connor A. Emdin ◽  
Derek Klarin ◽  
...  
Keyword(s):  
Coronary Disease ◽  
Fold Increase ◽  
Predictive Capacity ◽  
Cumulative Impact ◽  
Polygenic Score ◽  
Genome Wide ◽  
A Genome ◽  
Increased Risk ◽  
Polygenic Scores ◽  
Artery Disease

AbstractIdentification of individuals at increased genetic risk for a complex disorder such as coronary disease can facilitate treatments or enhanced screening strategies. A rare monogenic mutation associated with increased cholesterol is present in ~1:250 carriers and confers an up to 4-fold increase in coronary risk when compared with non-carriers. Although individual common polymorphisms have modest predictive capacity, their cumulative impact can be aggregated into a polygenic score. Here, we develop a new, genome-wide polygenic score that aggregates information from 6.6 million common polymorphisms and show that this score can similarly identify individuals with a 4-fold increased risk for coronary disease. In >400,000 participants from UK Biobank, the score conforms to a normal distribution and those in the top 2.5% of the distribution are at 4-fold increased risk compared to the remaining 97.5%. Similar patterns are observed with genome-wide polygenic scores for two additional diseases – breast cancer and severe obesity.One Sentence SummaryA genome-wide polygenic score identifies 2.5% of the population born with a 4-fold increased risk for coronary artery disease.

scholarly journals SEC24A identified as an essential mediator of thapsigargin-induced cell death in a genome-wide CRISPR/Cas9 screen

2018 ◽  
Vol 4 (1) ◽  
Author(s):  
Tamutenda Chidawanyika ◽  
Elizabeth Sergison ◽  
Michael Cole ◽  
Kenneth Mark ◽  
Surachai Supattapone
Keyword(s):  
Cell Death ◽  
Genome Wide ◽  
A Genome

scholarly journals Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+ Homeostasis To Promote Cell Death

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e02250-20
Author(s):  
Kathrin Stelzner ◽  
Ann-Cathrin Winkler ◽  
Chunguang Liang ◽  
Aziza Boyny ◽  
Carsten P. Ade ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Death ◽  
Host Cell ◽  
Intracellular Bacterium ◽  
Tissue Destruction ◽  
Content Type ◽  
Host Cell Death ◽  
Genome Wide ◽  
A Genome ◽  
Spread Of Infection

ABSTRACTThe opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.

scholarly journals Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy

2020 ◽  
Vol 12 (536) ◽  
pp. eaay0271 ◽  
Author(s):  
Angela Lek ◽  
Yuanfan Zhang ◽  
Keryn G. Woodman ◽  
Shushu Huang ◽  
Alec M. DeSimone ◽  
...  
Keyword(s):  
Cell Death ◽  
Muscular Dystrophy ◽  
Phenotypic Selection ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Loss Of Function ◽  
Hypoxia Response ◽  
Genome Wide ◽  
A Genome ◽  
Structural And Functional Properties ◽  
Cellular Hypoxia

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

scholarly journals Genome-Wide Identification of CsATGs in Tea Plant and the Involvement of CsATG8e in Nitrogen Utilization

2020 ◽  
Vol 21 (19) ◽  
pp. 7043
Author(s):  
Wei Huang ◽  
Dan-Ni Ma ◽  
Hong-Ling Liu ◽  
Jie Luo ◽  
Pu Wang ◽  
...  
Keyword(s):  
Camellia Sinensis ◽  
Woody Species ◽  
Nutrient Utilization ◽  
Tea Plant ◽  
Plant Genome ◽  
Wild Type ◽  
Genome Wide ◽  
A Genome ◽  
Phosphorus And Potassium ◽  
Stage 1

Nitrogen (N) is a macroelement with an indispensable role in the growth and development of plants, and tea plant (Camellia sinensis) is an evergreen perennial woody species with young shoots for harvest. During senescence or upon N stress, autophagy has been shown to be induced in leaves, involving a variety of autophagy-related genes (ATGs), which have not been characterized in tea plant yet. In this study, a genome-wide survey in tea plant genome identified a total of 80 Camellia Sinensis autophagy-related genes, CsATGs. The expression of CsATG8s in the tea plant showed an obvious increase from S1 (stage 1) to S4 (stage 4), especially for CsATG8e. The expression levels of AtATGs (Arabidopsis thaliana) and genes involved in N transport and assimilation were greatly improved in CsATG8e-overexpressed Arabidopsis. Compared with wild type, the overexpression plants showed earlier bolting, an increase in amino N content, as well as a decrease in biomass and the levels of N, phosphorus and potassium. However, the N level was found significantly higher in APER (aerial part excluding rosette) in the overexpression plants relative to wild type. All these results demonstrated a convincing function of CsATG8e in N remobilization and plant development, indicating CsATG8e as a potential gene for modifying plant nutrient utilization.

Different Isoforms of the B-Cell Mutator Activation-Induced Cytidine Deaminase (AID) Are Aberrantly Over-Expressed in BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL) Patients and Promote Genetic Instability.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1497-1497
Author(s):  
Ilaria Iacobucci ◽  
Annalisa Lonetti ◽  
Anna Ferrari ◽  
Simona Soverini ◽  
Emanuela Ottaviani ◽  
...  
Keyword(s):  
Genetic Instability ◽  
De Novo ◽  
Splice Variants ◽  
Cytidine Deaminase ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Wild Type ◽  
Genome Wide ◽  
A Genome ◽  
Activation Induced Cytidine Deaminase

Abstract Background: BCR-ABL1-positive ALL is the most frequent and prognostically most unfavorable subtype of adult ALL. The main reason for the poor clinical outcome of BCR-ABL1-positive ALL is genetic instability. However, how normal B-cell precursor cells acquire the genetic changes that lead to transformation and progression has not been completely defined. Activation-induced cytidine deaminase (AID) produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig). Aim: Since at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. Patients and methods: We analyzed 61 adult de novo Ph+ ALL patients (pts) and 60 CML pts (chronic phase and myeloid/lymphoid blast crisis). AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5′ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results: On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 41 (67%). AID expression correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. Moreover, AID expression was also found in lymphoid blast crisis CML (60%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were expressed. In 10/61 (16%) BCR-ABL1-positive ALL pts the full-length isoform (GeneBank accession number NM_020661) was identified, in 16/61 (26%) the co-expression of the wild-type isoform and of different AID splice variants was found and in 15/61 (25%) only the expression of splice variants was found. These can result from retention of intron 4 (Variant A), omission of exon 4 (Variant B) and 3 (Variant C), and from a deletion of 30 bp in the initial portion of exon 4 (Variant D). In the wild-type mRNA, codon 148 spans exons 3 and 4. In both variants A and B, mRNA splicing disrupts this codon and causes a frameshift, which results in a premature stop codon. If translated, the splice variants produce truncated proteins of 187 and 145 amino acids, respectively. However, the putative deaminase active site, encoded by exon 3, is preserved in both splice variants, but is lacking in the variant C. Since enforced expression of Pax5 induces endogenous AID gene expression, we analyzed the expression levels of Pax5 in all pts (ΔΔCt method, GAPDH as control gene). As expected, we found a very strong difference (p<0.0001) between chronic phase CML and BCR-ABL1-positive ALL, but total Pax5-transcripts did not differ significantly when BCR-ABL1-positive ALL/AID+ and BCR-ABL1-positive ALL/AID− were compared. To investigate whether AID introduces DNA-single strand breaks in BCR-ABL1-positive ALL, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array (Affymetrix Inc., USA). We identified a region of high level amplification and homozygous deletion in all patients. Patients who expressed wild-type AID had a higher number of alterations compared to AID-negative patients (median copy number alteration of 14, range 5–27, versus 4, range 1–6, respectively, p<0.03). Recurring copy number abnormalities were identified in genes with an established role in leukemogenesis, such as IKZF1, CDKN2A, CDKN2B, PAX5, MELK, BTG1 and MDS1. AID consensus motifs (DGYW/WRCH) were mapped very close to the breakpoint cluster regions. Conclusions: Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as a mutator outside the Ig gene loci in promoting genetic instability in leukemia cells.

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3′-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5′ of the stop codon, six nucleotides 3′ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3′-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3′-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 and mRNA secondary structure in the 3′-UTR had milder effects. Additionally, we found low readthrough genes to have shorter 3′-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

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