bims-ershed Biomed News
on ER Stress in Health and Diseases
Issue of 2021‒05‒30
fourteen papers selected by
Matías Eduardo González Quiroz
Worker’s Hospital

  1. Autophagy. 2021 May 24. 1-23
      Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition.Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN-induced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1.
    Keywords:  Mitophagy; PINK1; Parkinson disease; XBP1; phosphorylation; transcription; unfolded protein response
  2. Prog Mol Subcell Biol. 2021 ;59 197-214
      The endoplasmic reticulum, as the site of synthesis for proteins in the secretory pathway has evolved select machineries to ensure the correct folding and modification of proteins. However, sometimes these quality control mechanisms fail and proteins are misfolded. Other factors, such as nutrient deprivation, hypoxia or an increased demand on protein synthesis can also cause the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum. There are mechanisms that recognise and deal with this accumulation of protein through degradation and/or export. Many diseases are associated with aberrant quality control mechanisms, and among these, cancer has emerged as a group of diseases that rely on endoplasmic reticulum homeostasis to sustain development and growth. The knowledge of how protein quality control operates in cancer has identified opportunities for these pathways to be pharmacologically targeted, which could lead to newer or more effective treatments in the future.
    Keywords:  Endoplasmic reticulum; cancer; proteostasis; quality control
  3. Prog Mol Subcell Biol. 2021 ;59 115-143
      Protein aggregation is now a common hallmark of numerous human diseases, most of which involve cytosolic aggregates including Aβ (AD) and ⍺-synuclein (PD) in Alzheimer's disease and Parkinson's disease. However, it is also evident that protein aggregation can also occur in the lumen of the endoplasmic reticulum (ER) that leads to specific diseases due to loss of protein function or detrimental effects on the host cell, the former is inherited in a recessive manner where the latter are dominantly inherited. However, the mechanisms of protein aggregation, disaggregation and degradation in the ER are not well understood. Here we provide an overview of factors that cause protein aggregation in the ER and how the ER handles aggregated proteins. Protein aggregation in the ER can result from intrinsic properties of the protein (hydrophobic residues in the ER), oxidative stress or nutrient depletion. The ER has quality control mechanisms [chaperone functions, ER-associated protein degradation (ERAD) and autophagy] to ensure only correctly folded proteins exit the ER and enter the cis-Golgi compartment. Perturbation of protein folding in the ER activates the unfolded protein response (UPR) that evolved to increase ER protein folding capacity and efficiency and degrade misfolded proteins. Accumulation of misfolded proteins in the ER to a level that exceeds the ER-chaperone folding capacity is a major factor that exacerbates protein aggregation. The most significant ER resident protein that prevents protein aggregation in the ER is the heat shock protein 70 (HSP70) homologue, BiP/GRP78, which is a peptide-dependent ATPase that binds unfolded/misfolded proteins and releases them upon ATP binding. Since exogenous factors can also reduce protein misfolding and aggregation in the ER, such as chemical chaperones and antioxidants, these treatments have potential therapeutic benefit for ER protein aggregation-associated diseases.
    Keywords:  Amyloid; BiP/GRP78; CANX/CALR; CFTR; Clotting factor VIII; Proinsulin; UPR
  4. Front Mol Biosci. 2021 ;8 668821
      DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. To protect genomic stability and ensure cell homeostasis, cells mount a complex signaling-based response that not only coordinates the repair of the broken DNA strand but also activates cell cycle checkpoints and, if necessary, induces cell death. The last decade has seen a flurry of studies that have identified RNA-binding proteins (RBPs) as novel regulators of the DSB response. While many of these RBPs have well-characterized roles in gene expression, it is becoming increasingly clear that they also have non-canonical functions in the DSB response that go well beyond transcription, splicing and mRNA processing. Here, we review the current understanding of how RBPs are integrated into the cellular response to DSBs and describe how these proteins directly participate in signal transduction, amplification and repair at damaged chromatin. In addition, we discuss the implications of an RBP-mediated DSB response for genome instability and age-associated diseases such as cancer and neurodegeneration.
    Keywords:  DNA damage; DNA double strand break; DNA repair; DSB response; RNA-binding protein; genome stability; non-coding RNA; phase separation
  5. Biochem Biophys Res Commun. 2021 May 23. pii: S0006-291X(21)00809-3. [Epub ahead of print]562 69-75
      XBP1 is a basic leucine zipper (bZIP) transcription factor and a key mediator of the endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR). XBP1-mediated transcription facilitates cell adaptation to ER stress and also promotes tumor progression, while suppressing anti-tumor immunity. Here we report a novel XBP1 variant, namely XBP1 variant 1 (XBP1v1, Xv1 for short), that is specifically required for survival of cancer cells. Xv1 contains a cryptic first exon that is conserved only in humans and great apes. Comparing to XBP1, Xv1 encodes a protein with a different N-terminal sequence containing 25 amino acids. Analysis of RNAseq database reveals that Xv1 is broadly expressed across cancer types but almost none in normal tissues. Elevated Xv1 expression is associated with poor survival of patients with several types of cancer. Knockdown of Xv1 induces death of multiple cancer cell lines but has little effect on non-cancerous cells in vitro. Moreover, knockdown of Xv1 also inhibits growth of a xenograft breast tumor in mice. Together, our results indicate that Xv1 is essential for survival of cancer cells.
    Keywords:  Cancer cell survival; XBP1; XBP1v1; Xv1
  6. Methods Mol Biol. 2021 ;2274 305-314
      Bioluminescence resonance energy transfer (BRET) is an energy transfer phenomenon from a luciferase donor to a fluorescence acceptor and serves as an indicator of protein-protein interaction or protein proximity. BRET imaging is a powerful tool in the investigation of signaling proteins because it enables spatial analysis of such protein interactions. Here, we describe a method exerting high-resolution BRET imaging by combining bright-light output luciferases, such as NanoLuc , photon-counting EM-CCD, and unique algorithms for image correction and denoising.
    Keywords:  BRET; EM-CCD; Fluorescent protein; Imaging; Luciferase; Luminescence; Microscopy; Photon counting
  7. Sci Signal. 2021 May 25. pii: eaaz4401. [Epub ahead of print]14(684):
      During cellular stress in the budding yeast Saccharomyces cerevisiae, an endoplasmic reticulum (ER)-resident dual kinase and RNase Ire1 splices an intron from HAC1 mRNA in the cytosol, thereby releasing its translational block. Hac1 protein then activates an adaptive cellular stress response called the unfolded protein response (UPR) that maintains ER homeostasis. The polarity-inducing protein kinases Kin1 and Kin2 contribute to HAC1 mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediated HAC1 mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3' untranslated region (3'UTR) of HAC1 mRNA, and a yeast strain lacking both Pal1 and Pal2 was deficient in HAC1 mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a nonphosphorylatable Pal2 mutant could not rescue the UPR defect in a pal1Δ pal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinates HAC1 mRNA processing and ER homeostasis.
  8. Nat Metab. 2021 May;3(5): 682-700
      It is known that β cell proliferation expands the β cell mass during development and under certain hyperglycemic conditions in the adult, a process that may be used for β cell regeneration in diabetes. Here, through a new high-throughput screen using a luminescence ubiquitination-based cell cycle indicator (LUCCI) in zebrafish, we identify HG-9-91-01 as a driver of proliferation and confirm this effect in mouse and human β cells. HG-9-91-01 is an inhibitor of salt-inducible kinases (SIKs), and overexpression of Sik1 specifically in β cells blocks the effect of HG-9-91-01 on β cell proliferation. Single-cell transcriptomic analyses of mouse β cells demonstrate that HG-9-91-01 induces a wave of activating transcription factor (ATF)6-dependent unfolded protein response (UPR) before cell cycle entry. Importantly, the UPR wave is not associated with an increase in insulin expression. Additional mechanistic studies indicate that HG-9-91-01 induces multiple signalling effectors downstream of SIK inhibition, including CRTC1, CRTC2, ATF6, IRE1 and mTOR, which integrate to collectively drive β cell proliferation.
  9. J Invest Dermatol. 2021 May 25. pii: S0022-202X(21)01243-4. [Epub ahead of print]
      Melanoma cells are relatively resistant to ER stress, which contributes to tumor progression under stressful conditions and renders tolerance to ER stress-inducing therapeutic agents. Mitochondria are tightly interconnected with ER. However, whether mitochondria play a role in regulating ER stress resistance in melanoma remains elusive. Herein, we reported that the XBP1-MARCH5-MFN2 axis conferred ER stress resistance by coordinating mitochondrial fission and mitophagy in melanoma. Our integrative bioinformatics first revealed that the down-regulation of mitochondrial genes was highly correlated with UPR activation in melanoma. Then we proved that mitochondrial fission and mitophagy were prominently induced to contribute to ER stress resistance both in vitro and in vivo by maintaining mitochondrial function. Mechanistically, the activation of IRE1α/ATF6-XBP1 branches of UPR promoted the transcription of E3 ligase MARCH5 to facilitate the ubiquitination and degradation of MFN2, which thereby triggered mitochondrial fission and mitophagy under ER stress. Together, our findings demonstrate a regulatory axis that links mitochondrial fission and mitophagy to the resistance to ER stress. Targeting mitochondrial quality control machinery can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.
    Keywords:  ER stress; MFN2; melanoma; mitochondrial fission; mitophagy
  10. STAR Protoc. 2021 Jun 18. 2(2): 100468
      A guiding principle of STAR Protocols is that we make researchers' lives easier by publishing robust and usable protocols. We leverage the strength of peer review to help authors improve their protocol. This Backstory details the transformation of a bench protocol to a published protocol, highlighting the improvements to the article through the drafting, review, and revision stages. This underscores the value of the peer review process in general and the collaborative peer review philosophy at STAR Protocols specifically. For complete details, please refer to Chhoy et al. (2021).
  11. Immunol Cell Biol. 2021 May 27.
      Checkpoint signalling in the context of a functional DNA damage response is crucial for the prevention of oncogenic transformation of cells. Our immune system, though, takes the risk of attenuated checkpoint responses during immunoglobulin diversification. B cells undergo continuous DNA damage and error-prone repair of their immunoglobulin genes during the process of somatic hypermutation. An accompanying attenuation of the DNA damage response via the ATR/Chk1 axis in B cells is believed to allow for a better DNA damage tolerance and for evasion of apoptosis, so as to ensure mutations to be passed on. We sought to determine whether the downregulation of Chk1 could also directly influence the process of hypermutation in vivo by altering the relative activity of error-prone DNA repair pathways. We analysed the humoral response and the hypermutation process in mice whose B cells express reduced levels of the Chk1 protein. We found that Chk1 heterozygosity limits the accumulation of mutations in the immunoglobulin loci, likely by impacting on the survival of B cells as they accumulate DNA damage. Nevertheless, we unveiled an unanticipated role for Chk1 downregulation in favoring A/T mutagenesis at the antibody-variable regions during hypermutation. Even though immunoglobulin mutagenesis was found to be reduced, Chk1 signalling attenuation allows for sustained mutagenesis outside the immunoglobulin loci. Our study thus reveals that a proper Chk1 dosage is crucial for adequate somatic hypermutation in B cells.
    Keywords:  Checkpoint Kinase 1; Class Switch Recombination; Germinal Center; Somatic Hypermutation; non-canonical Mismatch Repair
  12. Methods Mol Biol. 2021 ;2255 43-53
      Phenotypic analysis of the effects of a gene of interest may be limited because stable expression of some genes leads to adverse consequences in cell survival, such as disturbance of cell cycle progression, senescence, autophagy, and programmed cell death. One of the best examples is tumor suppressor p53. p53 functions as a tumor suppressor by inducing cell cycle arrest and apoptosis in response to genotoxic and environmental insults. The choice and timing of either pathways induced by p53 depend on cellular context, cell types, and the degree of cellular/genomic damage (For review, see (Chen J, Cold Spring Harb Perspect Med 6:a026104, 2016)). The uncertainty makes the studies on the long-term effects of p53 in cells challenging. This chapter describes a method of flow cytometric analysis of ectopic expression of p53 to better quantify cell cycle distribution and apoptosis in cells treated with DNA damaging agents. The method can be easily adapted to other genes of interest to study their contributions to the fate of variety of cell types in response to endogenous or exogenous stresses.
    Keywords:  Apoptosis; Cell cycle arrest; DNA damage; Flow cytometry; Green fluorescent protein; Reporter plasmid; p53
  13. Mol Oncol. 2021 May 26.
      Glioblastoma is the most frequently diagnosed type of primary brain tumour in adults. These aggressive tumours are characterised by inherent treatment resistance and disease progression, contributing to ~190,000 brain-tumour related deaths globally each year. Current therapeutic interventions consist of surgical resection followed by radiotherapy and temozolomide chemotherapy, but average survival is typically around 1 year, with less than 10% of patients surviving more than 5 years. Recently, a fourth treatment modality of intermediate-frequency low-intensity electric fields [called tumour-treating fields ( TTFields)] was clinically approved for glioblastoma in some countries after it was found to increase median overall survival rates by ~5 months in a phase III randomised clinical trial. However, beyond these treatments, attempts to establish more effective therapies have yielded little improvement in survival for patients over the last 50 years. This is in contrast to many other types of cancer and highlights glioblastoma as a recognised tumour of unmet clinical need. Previous work has revealed that glioblastomas contain stem-cell-like subpopulations that exhibit heightened expression of DNA damage response (DDR) factors, contributing to therapy resistance and disease relapse. Given that radiotherapy, chemotherapy and TTFields-based therapies all impact DDR mechanisms, this Review will focus on our current knowledge of the role of the DDR in glioblastoma biology and treatment. We also discuss the potential of effective multi-modal targeting of the DDR combined with standard-of-care therapies, as well as emerging therapeutic targets, in providing much-needed improvements in survival rates for patients.
    Keywords:  Chemotherapy; DNA damage response (DDR); Glioblastoma; Radiotherapy; Synthetic lethality; Tumour treating fields (TTFields)
  14. Curr Sex Health Rep. 2019 Dec;11 320-330
      Purpose of Review: Sexual well-being and intimacy are critical to overall quality of life and retain a high degree of significance for aging individuals, even though these considerations are often overlooked in older populations. Sexual health may be particularly impacted in older individuals living with HIV, especially women, as a result of both physical and psychosocial disease-specific factors. Despite this, sexuality research related to HIV has traditionally focused on risk reduction, rather than on other elements of sexual wellness. In this review, we examine several aspects of sexual well-being that may be important to older women living with HIV (OWLH).Recent Findings: This review summarizes existing literature on sexuality in OWLH over the age of 50 and explores five themes related to sexual health: physical and emotional intimacy, desire/interest, satisfaction/pleasure, frequency of sexual activity, and abstinence. Reduced intimacy among OWLH was reported across most studies, due to stigma and disclosure concerns, lack of opportunity for relationships, and difficulty communicating sexual preferences. Data on sexual desire/interest and satisfaction/pleasure among OWLH were mixed. Frequency of sexual activity varied widely across studies, and abstinence emerged as both an intentional and inadvertent decision for OWLH. Factors related to menopause as it relates to sexuality and HIV are also discussed.
    Summary: Sexual health and well-being are important to women living with HIV over 50, though key components such as intimacy, desire, and pleasure remain poorly understood. As this population continues to grow, comprehensive and age-specific interventions are needed to examine positive aspects of sexuality and promote sexual wellness among OWLH.
    Keywords:  HIV; Intimacy; Older women; Sexual function; Sexuality