bims-ershed Biomed News
on ER Stress in Health and Diseases
Issue of 2022‒05‒15
fourteen papers selected by
Matías Eduardo González Quiroz
Worker’s Hospital

  1. FASEB J. 2022 May;36 Suppl 1
      Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by alveolar type II (AT2) cell dysfunction. Mutations in surfactant protein C (SP-C) are a high effect size, recognized etiological cause of IPF. The SP-C proprotein contains an ~100 KDa Bri2 chondromodulin-1 and prosurfactant protein C (BRICHOS) domain that acts as a molecular chaperone preventing protein aggregation. The presence of misfolded proteins in the ER can be sensed by three transmembrane sentinel proteins - activating transcription factor 6 (ATF6), protein kinase R-like ER kinase (PERK), and inositol-requiring enzyme 1α (IRE1α) - which trigger an intracellular signaling pathway called the unfolded protein response (UPR). Previous characterization of our in-vitro and in-vivo models has demonstrated that BRICHOS mutants activate all 3 arms of the UPR, activate a proinflammatory cascade, and drive AT2 cell death. In-vivo, their expression initiates pathological fibroproliferative lung remodeling, characteristic of human IPF. Using our in-vitro models of BRICHOS-driven ER stress, we explore potential IPF therapeutic intervention targeting two upstream sensors of the UPR, PERK and IRE-1. Mouse lung epithelial (MLE-12) cells were transfected with wildtype (WT) or BRICHOS mutant (C121G) SP-C plasmids in media containing inhibitors of either PERK signaling or IRE-1 RNase activity. Pathway readouts included: quantification of cell death via LDH cytotoxicity and flow cytometry, protein immunoblots to detect UPR activation and apoptotic signaling, QPCR of spliced X-Box Binding Protein 1 (XBP1) and UPR transcriptional targets, and high-resolution microscopy to observe altered SP-C trafficking. Within 48 hours of transfection of MLE-12 cells with SP-CC121G we observed activation of the 3 arms of the UPR, upregulated downstream targets, and initiation of cell death. Inhibition of PERK resulted in down-regulation of PERK pathway proteins and transcriptional targets, increased SP-C protein expression, and promoted increased XBP1 splicing. Inhibition of IRE1 RNase inhibited splicing of XBP1, reduced expression of XBP1 transcriptional targets, and increased JNK phosphorylation. Inhibition of either pathway alone did not reduce cell death, though partial reduction of cell death was noted with either small molecule targeted caspase 3/8 or necroptosis inhibition. Our data suggests that while intervention directed at one arm of the UPR may block proinflammatory signaling or transcriptomic reprogramming, this strategy may not be sufficient to completely inhibit cell death. These findings highlight the interconnectivity and redundancy of the cell quality control machinery and the emerging role of necroptosis due to chronic UPR stress-mediated cell death. Further characterization of this pathway is needed to improve IPF therapeutic outcomes.
  2. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: Liver injury activates Hepatic Stellate Cells (HSCs) which secrete fibrogenic proteins such as collagen I to promote scarring. Increased translation of the collagen I precursor procollagen I by HSCs causes ER stress due to 30% of nascent procollagen I failing to fold correctly, placing a burden on the ER; however, it is unclear how HSCs adapt to this stress. ER stress activates the Unfolded Protein Response (UPR) which signals through pathways mediated by Activating Transcription Factor 6α (ATF6α), Inositol Requiring Enzyme 1 (IRE1α), or Protein Kinase R-like ER kinase (PERK). ATF6α or IRE1α inhibition limits HSC activation and promotes apoptosis in vitro, while deletion of ATF6α or IRE1α limits fibrogenesis and reduces HSC number in vivo. While it is clear that the UPR plays a crucial role in HSC activation and survival, the mechanisms that facilitate this role are unknown. Recent work shows that misfolded procollagen I can undergo ER-phagy, where receptors on the ER recruit autophagic membranes to engulf portions of the ER containing misfolded proteins and target them for degradation. ER-phagy can be activated by ER stress, but the fibrogenic role and regulation of ER-phagy in HSCs is unknown. We hypothesized that UPR induction of ER-phagy targets misfolded procollagen I for degradation, thus promoting HSC survival and fibrogenesis.METHODS: Expression of ER-phagy receptors (Cell-cycle progression gene 1 (CCPG1), Family with sequence similarity 134B (FAM134B), and Atlastin 3 (ATL3)) was assessed in 1) livers from patients with advanced fibrosis or controls (GSE25097), 2) murine livers harvested from age- and sex-matched mice following bile-duct ligation (3 weeks) or sham controls; and 3) primary hHSCs or mHSCs, or immortalized hHSCs (LX-2) following TGFβ treatment (2ng/mL, 24h). ER-phagic flux was measured in LX-2 cells expressing a fluorescent ER-phagy reporter (RAMP4-GFP-mCherry), with RAMP4 as a known ER-phagic cargo. UPR signaling was disrupted using inhibitors targeting ATF6α (6µM Ceapin-A7) or IRE1α (0.5µM 4µ8C), or RNAi targeting PERK. CCPG1 or FAM134B were knocked out from LX-2 cells using CRISPR-Cas9. HSC activation and UPR signaling were measured by qPCR and Western blot.
    RESULTS: Expression of CCPG1 and ATL3 increased in fibrotic human livers compared to controls, while CCPG1 mRNA, and FAM134B protein and mRNA levels increased in fibrotic mouse livers compared to controls. In vitro activation of primary hHSCs or mHSCs also increased CCPG1, FAM134B, and ATL3 protein and mRNA levels, as well as increased ER-phagic flux in LX-2 cells. Regulation of ER-phagic flux and expression of ER-phagy receptors was UPR-dependent, with inhibition of ATF6α or IRE1α blocking TGFβ-induced ER-phagic flux, while PERK knockdown increased ER-phagic flux. Interestingly, CCPG1 or FAM134B loss did not impact HSC activation, or induce the pro-apoptotic UPR.
    CONCLUSIONS: ER-phagy receptors increased in fibrotic human and murine livers, and TGFβ upregulated ER-phagy receptors and increased ER-phagic flux in HSCs through UPR-dependent mechanisms. Deletion of CCPG1 or FAM134B did not impact HSC activation, suggesting redundancy between ER-phagic receptors. Future studies will focus on understanding the fibrogenic role of ER-phagy in HSCs.
  3. FASEB J. 2022 May;36 Suppl 1
      The integrated stress response (ISR) and the unfolded protein response (UPR) are conserved signaling networks governed by stress sensor kinases. Four ISR sensor kinases, GCN2, HRI, PERK, and PKR, phosphorylate eIF2α in response to multiple stresses, leading to a global protein synthesis shutdown coupled to the translation of select mRNAs, which results in an adaptive remodeling of the proteome. Besides PERK, which the ISR and the UPR share, the UPR relies on two additional ER stress sensors, the kinase/RNase IRE1 and the membrane-tethered transcription factor ATF6, to induce adaptive signaling programs that reinstate ER homeostasis. However, if the stress is unrelenting, both the ISR and the UPR can switch to drive cell death. The mechanistic commonalities and crosstalk between the ISR and UPR remain an active area of investigation. In this talk, I will share new results that support common signaling principles of the ISR and UPR sensor kinases and reveal a new layer of communication between the ISR and the UPR. Specifically, we found that dynamic clustering is a prominent feature of PKR activation reminiscent of the high-order assemblies of IRE1 and PERK observed during ER stress. Surprisingly, PKR clusters excluded eIF2α, and mutations in PKR that disrupt cluster assembly enhanced eIF2α phosphorylation, suggesting that PKR clusters act as enzyme sinks that control enzyme-substrate interactions by limiting PKR-eIF2α encounters. Moreover, stress-free activation of PKR induced a master cell death program dependent on ISR-driven expression of DR5 (death receptor 5), as occurs during unmitigable ER stress. Remarkably, stress-free activation of the ISR selectively activated IRE1 independent of sensing unfolded proteins in the ER lumen, and treatment with the small-molecule ISR inhibitor ISRIB reversed it. Our data provide new mechanistic insights into two fundamental aspects of stress sensor kinase signaling: (1) dynamic clustering of stress sensors may provide the means to fine-tune stress responses, and (2) the ISR selectively activates IRE1, thus coupling the ISR and the UPR outside their common node PERK.
  4. FASEB J. 2022 May;36 Suppl 1
      Despite its antitumor efficacy, use of Doxorubicin (Dox) is limited by its cardiotoxic side effects. In Dox-induced cardiomyopathy (DIC), a causal role of oxidative stress (OS) has gained a lot of traction. Among other subcellular changes, Dox causes vacuolization of the cytoplasm and dilation of endoplasmic reticulum (ER). Dox also causes an excessive production of reactive oxygen species as well as imbalance in redox state both of which can affect ER homeostasis and protein folding. An accumulation of unfolded/misfolded proteins triggers the unfolded protein response (UPR) in the ER, which activates transcriptional and translation pathways involving: a) Protein-kinase like endoplasmic reticulum kinase (PERK); b) activating transcription factor 6α (ATF6α); and c) inositol requiring kinase1α (IRE1α) (Fig.1). Thus leading to the activation of ER chaperons, protein foldases and induce the expression of various genes, including X-box binding protein 1 (XBP1). UPR plays an important role in the attenuation of protein translation, upregulation of chaperones and antioxidant expression, and thus maintains ER homeostasis. However, if UPR fails to restore ER homeostasis, ER stress-initiated apoptotic signaling pathways are activated via CCAAT/enhancer homologous protein (CHOP), caspase-12 activation, phosphorylation of c-JUN NH2-terminal kinase (JNK) and pro-apoptotic gene expression. Understanding the role of these pathways of the ER under Dox treatment will highlight potential targets for new interventions as an adjunct therapy to restore ER functioning. In this study on isolated rat cardiomyocytes, we analyzed ER-stress markers, spliced XBP1 mRNA, and ER-apoptotic proteins. We also assessed protective effects of Interleukin-10 (IL-10) against Dox initiated ER-induced apoptosis and overall ER-stress. Dox significantly decreased the viability of cardiomyocytes and upregulated ER stress markers (GRP78, GRP94, and PDI). Interestingly, the exogenous administration of IL-10, one hour prior to the exposure of cardiomyocytes to Dox for 24hrs, was found to be effective in suppressing the expressions of ER-stress markers as well as ER-related apoptotic proteins and preserved cell viability. Additionally, significant changes in downstream targets of IRE1α, XBP1 in IL-10 treatment were recorded. IL-10 significantly increased the expression of unspliced XBP1 (U), an inhibitor of spliced XBP1(S) particularly in Dox-treated cells. This was followed by a reduction in the UPR mediated cell death in cardiomyocytes confirmed by a decrease in expression of GRP78, GRP94, and spliced XBP1 (S). IRE1 α arm of UPR initiates phosphorylation of JNK, which was reduced by IL-10 in Dox treated cells. Dox also initiated activation of procaspase-12, which is known to activate caspase-3 and apoptosis. IL-10 significantly reduced the activation of procaspase-12 and cell death. These data suggest that IL-10 provides protection against Dox-induced cardiotoxicity by restoring ER homeostasis.
  5. FASEB J. 2022 May;36 Suppl 1
      Human immunodeficiency virus 1 (HIV-1) invades the central nervous system (CNS) early during infection and can persist in the CNS for life despite effective antiretroviral treatment. Infection and activation of residential glial cells leads to low viral replication and chronic inflammation, which damage neurons contributing to a spectrum of HIV-associated neurocognitive disorders (HAND). Astrocytes are the most numerous glial cells in the CNS and provide essential support to neurons. During a neuropathological challenge, such as HIV-1 infection, astrocytes can shift their neurotrophic functions to become neurotoxic and even serve as latent reservoirs for HIV-1 infection. Notably, substance use disorders, including methamphetamine (METH) are disproportionately elevated among people living with HIV-1. METH use can induce neurotoxic and neurodegenerative consequences, which can increase one's risk and severity of HAND. Thus, a better understanding of HIV-1 infection and METH exposure both alone and in combination on astrocyte function could help identify key cellular or molecular targets that can regulate astrocyte neuroprotective versus neurotoxic phenotypes to optimize astrocyte and neuronal coupling and combat CNS pathology. Direct contact sites between the endoplasmic reticulum (ER) and the mitochondria, termed mitochondria-associated ER membranes (MAMs), are central hubs for regulating several cellular processes, including inflammation and mitochondrial function and dynamics. In fact, the transfer of calcium from the ER to mitochondria is essential for mitochondrial bioenergetics. Interestingly, increasing evidence supports that the three arms of the unfolded protein response (UPR) are key cell signaling messengers within the ER-mitochondrial interface, beyond their classical ER stress functions. Briefly, protein kinase RNA-like endoplasmic reticulum kinase (PERK) has been determined as a regulator for MAM tethering and mitochondrial morphology. Inositol-requiring enzyme 1 alpha (IRE1α) is implicated in regulating MAM-mediated calcium transfer. Activating transcription factor 6 (ATF6) is suspected to participate in MAM formation as it is known to mediate ER elongation and lipid homeostasis. However, these regulatory mechanisms have not yet been fully elucidated. Our studies specifically highlight IRE1α as a key regulator of astrocyte metabolic and inflammatory phenotypes. Using primary human astrocytes infected with pseudotyped HIV and/or exposed to low doses of METH for seven days, astrocytes have increased protein expression of select UPR/MAM mediators. Under the same paradigms, we see increased cytosolic calcium flux and mitochondrial oxygen consumption rate, which were associated with increased mitochondria calcium uptake. Manipulation of IRE1α using both pharmacological inhibitors and an overexpression plasmid, confirms IRE1α modulates astrocyte calcium signaling, metabolic activity, glutamate clearance, and cytokine release. These findings identify a novel target for regulating astrocyte metabolic and inflammatory phenotypes, which could help combat astrocyte-mediated neurotoxicity and potentially promote a neurotrophic phenotype during CNS pathologies.
  6. Front Pharmacol. 2022 ;13 879204
      Pulmonary diseases are main causes of morbidity and mortality worldwide. Current studies show that though specific pulmonary diseases and correlative lung-metabolic deviance own unique pathophysiology and clinical manifestations, they always tend to exhibit common characteristics including reactive oxygen species (ROS) signaling and disruptions of proteostasis bringing about accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER). ER is generated by the unfolded protein response. When the adaptive unfolded protein response (UPR) fails to preserve ER homeostasis, a maladaptive or terminal UPR is engaged, leading to the disruption of ER integrity and to apoptosis, which is called ER stress. The ER stress mainly includes the accumulation of misfolded and unfolded proteins in lumen and the disorder of Ca2+ balance. ROS mediates several critical aspects of the ER stress response. We summarize the latest advances in of the UPR and ER stress in the pathogenesis of pulmonary disease and discuss potential therapeutic strategies aimed at restoring ER proteostasis in pulmonary disease.
    Keywords:  er stress; oxidative stress; pulmonary disease; reactive oxygen spieces; unfolded protein response
  7. FASEB J. 2022 May;36 Suppl 1
      The ER stress sensors IRE1α and IRE1β have dual kinase and endonuclease activities that mediate cellular proteostasis by splicing Xbp1 mRNA and degrading other ER-targeted transcripts. Although the two paralogues share a high degree of sequence homology, the epithelial cell-specific paralogue IRE1β, which is expressed at mucosal surfaces, has impaired kinase activity and phosphorylation that restrict stress-induced activation of the endonuclease domain. To uncover structural features that explain these differences, we used normal mode analysis to build dynamic correlation networks describing active and inactive conformations of IRE1 kinase-endonuclease domains. For human IRE1α, we identified a fingerprint of intra- and interdomain dynamic couplings that distinguish between the two conformations. Mutations that disrupted IRE1α endonuclease activity perturbed the dynamic networks of the active conformation and enhanced features of an inactive conformation, thus validating our approach. Strikingly, the dynamic networks of human IRE1β when modeled in an active conformation resembled the inactive IRE1α network. A set of non-conserved amino acids were found to distinguish between the dynamic networks linking kinase-endonuclease domains for the two IRE1 paralogues. In most cases, however, these divergent network-determining amino acids were found only in IRE1β sequences from mammals. In lower vertebrates, the key amino acids and the dynamic couplings of IRE1α active state were conserved in IRE1β. Thus, IRE1β evolved distinct conformational dynamics and enzymatic activity for a role in mucus barrier function that is unique to higher vertebrates.
  8. FASEB J. 2022 May;36 Suppl 1
      Pulmonary Fibrosis (PF), a devastating lung disease with rising prevalence, is defined by a physiologic defect in gas exchange owing to two cardinal pathological features: intrusion of myofibroblasts into the distal lung parenchyma and an alteration of normal alveolar epithelial composition most clearly defined by a loss of the squamous alveolar epithelial type 1 cell (AEC1) and the presence of a recently identified aberrant alveolar epithelial "transitional state" enriched in profibrotic mediators. While this transitional cell state has been identified transiently in the epithelium of murine lung injury models, the mechanism by which it arises in human fibrotic lung disease is unknown. We previously reported the in vivo modeling of PF utilizing inducible, knock-in expression of a clinical Surfactant Protein C (SP-C) mutation in alveolar type 2 cells (AEC2). Expression of the SP-C mutation in the adult mouse AEC2s led to activation of Unfolded Protein Response (UPR) signaling pathways, endoplasmic reticulum (ER) stress and spontaneous fibrosis providing proof of concept for disruption to proteostasis as a proximal driver of PF. We hypothesized that disruption of AEC2 protein quality control and specifically UPR signaling causes cell autonomous AEC2 reprogramming to the transitional state in the absence of an exogenous lung injury. Using two clinical SP-C mutation models we discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop the recently identified transitional cell state. Using single cell RNA sequencing of the epithelium in our murine models we identify that UPR activated AEC2s develop the transitional cell phenotype in the absence of an exogenous lung injury. Through organoid based modeling we validated that this state arises de novo from intrinsic AEC2 dysfunction. The cell autonomous AEC2 reprogramming is mediated through IRE1 signaling as use of a novel IRE1 inhibitor attenuated the development of the transitional cell state and diminished AEC2 driven recruitment of granulocytes, alveolitis, and lung injury arising from the loss of proteostasis. We further show that this transitional state persists into the fibrotic phase of our murine models and is enriched in pro-fibrotic mediators. These findings identify AEC2 proteostasis, and specifically IRE1 signaling, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.
  9. FASEB J. 2022 May;36 Suppl 1
      Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express IRE1β (Ern2), a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor IRE1α. How IRE1β functions at the host-environment interface and why a second IRE1 paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that IRE1β was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced IRE1β expression. IRE1β acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although IRE1α can also splice Xbp1 mRNA, it did not act redundantly to IRE1β in this context. By regulating assembly of the colon mucus layer, IRE1β further shaped the composition of the gut microbiota. Mice lacking IRE1β had a dysbiotic microbial community that failed to induce goblet cell development when transferred into germ-free wild type mice. These results show that IRE1β evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.
  10. J Cell Biol. 2022 Jun 06. pii: e202205019. [Epub ahead of print]221(6):
      Logue, Gorman, and Samali highlight a study by Guttman and colleagues (2022. J. Cell Biol. that shows exogenous antigen peptides imported into the ER can activate the ER stress sensor IRE1α, attenuating cross-presentation by dendritic cells.
  11. Toxicology. 2022 May 06. pii: S0300-483X(22)00105-6. [Epub ahead of print]473 153193
      Busulfan, a chemotherapeutic agent for cancer, has detrimental effects on germ cells and fertility, yet the specific mechanisms remain largely uncertain. The blood-testis barrier (BTB) maintains a suitable microenvironment for germ cells self-renewal and spermatogenesis by blocking the interference and damage of deleterious substances. Therefore, we hypothesized that BTB abnormalities might be involved in busulfan-induced oligospermia. To verify the hypothesis, thirty male Balb/c mice were randomly administered with busulfan (at a total dose of 40 mg/kg body weight) by intraperitoneal injection for 4 weeks to establish the model of oligospermia. The results displayed that busulfan caused testicular histopathological lesions and spermatogenesis disorder. Meanwhile, busulfan disrupted BTB integrity and lessened the expressions of BTB junction proteins, including Occludin, Claudin-11 and Connexin-43. Furthermore, busulfan activated the endoplasmic reticulum (ER) stress and PERK-eIF2α signaling pathway, reflected by the increased protein expressions of GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. Finally, to evaluate whether the ER stress is involved in busulfan-induced BTB destruction, the ER stress inhibitor 4-Phenylbutyric acid (4-PBA, 1 mM) was used to intervene in busulfan-exposed TM4 cells. The results displayed that inhibition of ER stress alleviated the reduction of BTB junction protein expressions induced by busulfan in TM4 cells. These data collectively indicated that busulfan-induced BTB impairment was mediated by triggering ER stress and activation of the PERK-eIF2α signaling pathway, thereby damaging the spermatogenesis, providing a new therapeutic target for male infertility induced by busulfan.
    Keywords:  BTB; Busulfan; ER stress; Oligospermia; PERK-eIF2α signaling pathway
  12. FASEB J. 2022 May;36 Suppl 1
      MS2 system is widely used to image mRNAs in living cells, where the spatial distribution of mRNAs regulates various aspects of cell biology, including translation and RNA decay. To image an mRNA of interest, the mRNA's 3' untranslated region (UTR) is genetically fused with an array of MS2 stem loops, which recruit fluorescently labeled MS2 coat proteins. And each mRNA molecule appears as a diffraction-limited spot under fluorescent microscope. Here, we report that the MS2 system renders some of the tagged mRNAs less stable and being degraded by the nonsense-mediated mRNA decay (NMD) pathway. To resolve this issue, we improved the MS2 system by fusing the MS2 coat protein with the translation termination factor eRF3, which protects the mRNA from being degraded through the NMD pathway. Our improved MS2 system faithfully reports the mRNAs' stability and localization, and will see wide applications in future studies of RNA biology.
  13. FASEB J. 2022 May;36 Suppl 1
      To maintain protein homeostasis (i.e., "proteostasis") and withstand the toxic effects brought about by the presence of misfolded proteins, eukaryotes have evolved a hierarchy of quality control checkpoints along the secretory pathway. The most prominent quality control step in this pathway, which acts during or soon after proteins are synthesized, is endoplasmic reticulum associated degradation (ERAD). The importance of this pathway is underscored by the fact that ~80 different protein substrates of the ERAD pathway have been linked to human disease. Although most misfolded proteins in the secretory pathway are eliminated by ERAD, others can exit the ER in COPII vesicles and are instead turned over by lysosomal proteases. This post-ER quality control event requires the ESCRT machinery. A different class of secreted proteins, particularly those that are aggregation-prone, can alternatively be degraded by ER-phagy. To date, it remains elusive how these diverse misfolded proteins--which can trigger various stress responses--are selected for different fates. However, by constructing a collection of model substrates and examining wild-type and disease-associated mutant forms of various proteins, we are beginning to define the requirements for the targeted selection of misfolded proteins in the secretory pathway for one fate versus another. This pursuit represents a vital step toward the development of pharmaceuticals that might one day repair folding-defective proteins. Indeed, a growing number of clinical and pre-clinical drugs that repair ERAD and other quality control substrates have shown efficacy in various disease models. In this presentation, each of these topics will be discussed and future research directions defined.
  14. FASEB J. 2022 May;36 Suppl 1
      Maintenance of genomic integrity requires functional repair mechanisms. Some mechanisms include the use of sumoylation, a process in which proteins are post-translationally modified with a small peptide SUMO (small ubiquitin-like modifier). In response to DNA damage, multiple proteins become sumoylated. Current evidence suggests that sumoylation in this context mediates protein-protein interactions to promote DNA damage repair. SUMO-interacting motifs (SIMs) play an essential role in SUMO-mediated protein-protein interactions. The SIM on a protein is a region that allows it to recognize and bind the SUMO attached to another protein. Previous studies have shown that inhibiting this type of interaction can lead to sensitized cancer cells by impairing DNA damage repair and response. Thus, this project aims to study a similar inhibition by overexpressing SIMs naturally found in two proteins, Slx5 and Sgs1, in the eukaryotic model organism, Saccharomyces cerevisiae. Both proteins are a part of their respective protein complexes, and their SIMs serve as a functional way for the complexes to interact with sumoylated proteins during DNA repair. We hypothesize that overexpressed SIMs will disrupt endogenous SUMO-SIM interactions important for DNA damage repair and that cells with overexpression of the SIM would be sensitized to DNA damaging agents. We observed that cells overexpressing SIMs from Slx5 exhibited a growth defect compared to cells without overexpression. However, when treated with DNA damaging agents, such as methyl methanesulfonate and UV, cells with SIM overexpression exhibited little to no sensitivity compared to the wild type. To further explore the effect of SUMO-SIM interaction, we have designed another protein containing the DNA-binding (SAP) domain of Siz2, a SUMO protein ligase specific to DNA damage, and the SUMO-binding domain of Ulp1, a SUMO isopeptidase, to potentially disrupt SUMO:SIM interactions involved in DNA repair. Inclusion of the SAP domain along with a nuclear localization signal (NLS) is hypothesized to increase the nuclear localization of the protein during DNA repair, which will be observed using fluorescent microscopy of a GFP tag incorporated into the protein. With increased nuclear localization and inclusion of the SUMO-binding domain, we hypothesize that there will be disruption to the SUMO:SIM interactions involved in DNA damage repair, resulting in an increase in the cells' sensitivity to DNA damaging agents.