Insights into the structure and function of the RNA ligase RtcB.

To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5'-3' and 3'-5' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3'-5' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3'-5' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.

Endoplasmic Reticulum Stress-Regulated Chaperones as a Serum Biomarker Panel for Parkinson's Disease.

Examination of post-mortem brain tissues has previously revealed a strong association between Parkinson's disease (PD) pathophysiology and endoplasmic reticulum (ER) stress. Evidence in the literature regarding the circulation of ER stress-regulated factors released from neurons provides a rationale for investigating ER stress biomarkers in the blood to aid diagnosis of PD. The levels of ER stress-regulated proteins in serum collected from 29 PD patients and 24 non-PD controls were measured using enzyme-linked immunosorbent assays. A panel of four biomarkers, protein disulfide-isomerase A1, protein disulfide-isomerase A3, mesencephalic astrocyte-derived neurotrophic factor, and clusterin, together with age and gender had higher ability (area under the curve 0.64, sensitivity 66%, specificity 57%) and net benefit to discriminate PD patients from the non-PD group compared with other analyzed models. Addition of oligomeric and total α-synuclein to the model did not improve the diagnostic power of the biomarker panel. We provide evidence that ER stress-regulated proteins merit further investigation for their potential as diagnostic biomarkers of PD.

New insights into IRE1α activation and function in anti-tumor immunity.

Logue, Gorman, and Samali highlight a study by Guttman and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202111068) that shows exogenous antigen peptides imported into the ER can activate the ER stress sensor IRE1α, attenuating cross-presentation by dendritic cells.

Regulated IRE1α-dependent decay (RIDD)-mediated reprograming of lipid metabolism in cancer.

IRE1α is constitutively active in several cancers and can contribute to cancer progression. Activated IRE1α cleaves XBP1 mRNA, a key step in production of the transcription factor XBP1s. In addition, IRE1α cleaves select mRNAs through regulated IRE1α-dependent decay (RIDD). Accumulating evidence implicates IRE1α in the regulation of lipid metabolism. However, the roles of XBP1s and RIDD in this process remain ill-defined. In this study, transcriptome and lipidome profiling of triple negative breast cancer cells subjected to pharmacological inhibition of IRE1α reveals changes in lipid metabolism genes associated with accumulation of triacylglycerols (TAGs). We identify DGAT2 mRNA, encoding the rate-limiting enzyme in TAG biosynthesis, as a RIDD target. Inhibition of IRE1α, leads to DGAT2-dependent accumulation of TAGs in lipid droplets and sensitizes cells to nutritional stress, which is rescued by treatment with the DGAT2 inhibitor PF-06424439. Our results highlight the importance of IRE1α RIDD activity in reprograming cellular lipid metabolism.

The stressosome, a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner.

Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the 'stressosome'. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1ß-converting enzyme-inhibitory protein (cFLIPL ), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.

Regulation of lipid metabolism by the unfolded protein response.

The endoplasmic reticulum (ER) is the site of protein folding and secretion, Ca2+ storage and lipid synthesis in eukaryotic cells. Disruption to protein folding or Ca2+ homeostasis in the ER leads to the accumulation of unfolded proteins, a condition known as ER stress. This leads to activation of the unfolded protein response (UPR) pathway in order to restore protein homeostasis. Three ER membrane proteins, namely inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6), sense the accumulation of unfolded/misfolded proteins and are activated, initiating an integrated transcriptional programme. Recent literature demonstrates that activation of these sensors can alter lipid enzymes, thus implicating the UPR in the regulation of lipid metabolism. Given the presence of ER stress and UPR activation in several diseases including cancer and neurodegenerative diseases, as well as the growing recognition of altered lipid metabolism in disease, it is timely to consider the role of the UPR in the regulation of lipid metabolism. This review provides an overview of the current knowledge on the impact of the three arms of the UPR on the synthesis, function and regulation of fatty acids, triglycerides, phospholipids and cholesterol.

An Emerging Role for the Unfolded Protein Response in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and one of the leading causes of cancer-associated deaths in the world. It is characterised by dismal response rates to conventional therapies. A major challenge in treatment strategies for PDAC is the presence of a dense stroma that surrounds the tumour cells, shielding them from treatment. This unique tumour microenvironment is fuelled by paracrine signalling between pancreatic cancer cells and supporting stromal cell types including the pancreatic stellate cells (PSC). While our molecular understanding of PDAC is improving, there remains a vital need to develop effective, targeted treatments. The unfolded protein response (UPR) is an elaborate signalling network that governs the cellular response to perturbed protein homeostasis in the endoplasmic reticulum (ER) lumen. There is growing evidence that the UPR is constitutively active in PDAC and may contribute to the disease progression and the acquisition of resistance to therapy. Given the importance of the tumour microenvironment and cytokine signalling in PDAC, and an emerging role for the UPR in shaping the tumour microenvironment and in the regulation of cytokines in other cancer types, this review explores the importance of the UPR in PDAC biology and its potential as a therapeutic target in this disease.

Local intracerebral inhibition of IRE1 by MKC8866 sensitizes glioblastoma to irradiation/chemotherapy in vivo.

Glioblastoma multiforme (GBM) is the most severe primary brain cancer. Despite an aggressive treatment comprising surgical resection and radio/chemotherapy, patient's survival post diagnosis remains short. A limitation for success in finding novel improved therapeutic options for such dismal disease partly lies in the lack of a relevant animal model that accurately recapitulates patient disease and standard of care. In the present study, we have developed an immunocompetent GBM model that includes tumor surgery and a radio/chemotherapy regimen resembling the Stupp protocol and we have used this model to test the impact of the pharmacological inhibition of the endoplasmic reticulum (ER) stress sensor IRE1, on treatment efficacy.

The IRE1 and PERK arms of the unfolded protein response promote survival of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma, is associated with a low 5-year survival and harsh treatment side effects, underscoring an urgent need for therapy. The unfolded protein response (UPR) is activated in response to endoplasmic reticulum (ER) stress, where three ER stress receptors, IRE1, PERK and ATF6, aim to restore cellular homeostasis. The UPR is pro-tumourigenic in many cancers. In this study, we investigate basal UPR activity in RMS. Basal activation of IRE1 and PERK was observed in RMS cell lines, which was diminished upon addition of the IRE1 RNase inhibitor, MKC8866, or PERK inhibitor, AMGEN44. UPR inhibition caused a reduction in cell viability, cell proliferation and inhibition of long-term colony formation in both subtypes of RMS. Alveolar RMS (ARMS) subtype was highly sensitive to IRE1 inhibition, whereas embryonal RMS (ERMS) subtypes responded more markedly to PERK inhibition. Further investigation revealed a robust activation of senescence upon UPR inhibition. For the first time, the UPR is implicated in RMS biology and phenotype, and inhibition of UPR signalling reduces cell growth, suggesting that the UPR may be a promising target in RMS.

Novel Pt(IV) Prodrugs Displaying Antimitochondrial Effects.

The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that binds to the translocator protein (TSPO) at the outer mitochondrial membrane. The cytotoxicities of cis,cis,trans-[Pt(NH3)2Cl2(L3)(OH)] (C-Pt1), cis,cis,trans-[Pt(NH3)2Cl2(L3)(BZ)] (C-Pt2), trans-[Pt(DACH)(OX)(L3)(OH)] (C-Pt3), and trans-[Pt(DACH)(OX)(L3)(BZ)] (C-Pt4) (DACH: R,R-diaminocyclohexane, BZ: benzoate, OX: oxalate) in MCF-7 breast cancer and noncancerous MCF-10A epithelial cells were assessed and compared with those of cisplatin, oxaliplatin, and the free ligand L3. Moreover, the cellular uptake, ROS generation, DNA damage, and the effect on the mitochondrial function, mitochondrial membrane potential, and morphology were investigated. Molecular interactions of L3 in the TSPO binding site were studied using molecular docking. The results showed that complex C-Pt1 is the most effective Pt(IV) complex and exerts a multimodal mechanism involving DNA damage, potent ROS production, loss of the mitochondrial membrane potential, and mitochondrial damage.

Tumour Cell Secretome in Chemoresistance and Tumour Recurrence.

Chemoresistance is a major factor driving tumour relapse and the high rates of cancer-related deaths. Understanding how cancer cells overcome chemotherapy-induced cell death is critical in promoting patient survival. One emerging mechanism of chemoresistance is the tumour cell secretome (TCS), an array of protumorigenic factors released by tumour cells. Chemotherapy exposure can also alter the composition of the TCS, known as therapy-induced TCS, and can promote tumour relapse and the formation of an immunosuppressive tumour microenvironment (TME). Here, we outline how the TCS can protect cancer cells from chemotherapy-induced cell death. We also highlight recent evidence describing how therapy-induced TCS can impact cancer stem cell (CSC) expansion and tumour-associated immune cells to enable tumour regrowth and antitumour immunity.

Correction: Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Time and region-dependent manner of increased brain derived neurotrophic factor and TrkB in rat brain after binge-like methamphetamine exposure.

Methamphetamine (MA), a synthetic derivate of amphetamine, has become a major drug of abuse worldwide. This study investigated the effect of binge-like MA dosing (4 x 4 mg/kg, s.c., 2 h (h) apart) at a range of different time points (from 2 h to 7 days after treatment) on brain-derived neurotrophic factor (BDNF) levels and its receptors, TrkB and p75NTR. BDNF levels were significantly increased in the frontal cortex from 2 to 36 h after treatment, returning to normal within 48 h after treatment. In the striatum, BDNF expression was increased at 12 and 24 h after binge-like MA treatment and had returned to normal at 36 h. Increased expression of the TrkB receptor was observed in the frontal cortex at 2, 24 and 48 h after MA treatment and in the striatum at 24 and 48 h after the MA regimen. A significant increase in the p75NTR receptor was also noted in the striatum but not the frontal cortex, and it was less pronounced than the effect on TrkB receptor expression. These findings show that the binge-like regimen of MA affects expression of BDNF and its receptors, particularly the TrkB receptor, in a time and region dependent manner, and highlights the importance of the frontal cortex and the striatum in the response following MA binge-like dosing.

Application of a New Multiplexed Array for Rapid, Sensitive, Simultaneous and Quantitative Assessment of Spliced and Unspliced XBP1.

BACKGROUND: IRE1α-mediated unconventional splicing of XBP1 is emerging as a biomarker in several disease states and is indicative of activation of the unfolded protein response sensor IRE1. Splicing of XBP1 mRNA results in the translation of two distinct XBP1 protein isoforms (XBP1s and XBP1u) which, due to post-translational regulation, do not correlate with mRNA levels. As both XBP1 isoforms are implicated in pathogenic or disease progression mechanisms there is a need for a reliable, clinically applicable method to detect them. METHODS: A multiplexed isoform-specific XBP1 array utilising Biochip array technology (BAT™) was assessed for specificity and suitability when using cell protein lysates. The array was applied to RIPA protein lysates from several relevant pre-clinical models with an aim to quantify XBP1 isoforms in comparison with RT-PCR or immunoblot reference methods. RESULTS: A novel reliable, specific and sensitive XBP1 biochip was successfully utilised in pre-clinical research. Application of this biochip to detect XBP1 splicing at the protein level in relevant breast cancer models, under basal conditions as well as pharmacological inhibition and paclitaxel induction, confirmed the findings of previous studies. The biochip was also applied to non-adherent cells and used to quantify changes in the XBP1 isoforms upon activation of the NLRP3 inflammasome. CONCLUSIONS: The XBP1 biochip enables isoform specific quantification of protein level changes upon activation and inhibition of IRE1α RNase activity, using a routine clinical methodology. As such it provides a research tool and potential clinical tool with a quantified, simultaneous, rapid output that is not available from any other published method.

Effect of Kinase Inhibiting RNase Attenuator (KIRA) Compounds on the Formation of Face-to-Face Dimers of Inositol-Requiring Enzyme 1: Insights from Computational Modeling.

Inositol-requiring enzyme 1α (IRE1α) is a transmembrane dual kinase/ribonuclease protein involved in propagation of the unfolded protein response (UPR). Inositol-requiring enzyme 1α is currently being explored as a potential drug target due to the growing evidence of its role in variety of disease conditions. Upon activation, IRE1 cleaves X-box binding protein 1 (XBP1) mRNA through its RNase domain. Small molecules targeting the kinase site are known to either increase or decrease RNase activity, but the allosteric relationship between the kinase and RNase domains of IRE1α is poorly understood. Subsets of IRE1 kinase inhibitors (known as "KIRA" compounds) bind to the ATP-binding site and allosterically impede the RNase activity. The KIRA compounds are able to regulate the RNase activity by stabilizing the monomeric form of IRE1α. In the present work, computational analysis, protein-protein and protein-ligand docking studies, and molecular dynamics simulations were applied to different IRE1 dimer systems to provide structural insights into the perturbation of IRE1 dimers by small molecules kinase inhibitors that regulate the RNase activity. By analyzing structural deviations, energetic components, and the number of hydrogen bonds in the interface region, we propose that the KIRA inhibitors act at an early stage of IRE1 activation by interfering with IRE1 face-to-face dimer formation thus disabling the activation of the RNase domain. This work sheds light on the mechanism of action of KIRA compounds and may assist in development of further compounds in, for example, cancer therapeutics. The work also provides information on the sequence of events and protein-protein interactions initiating the unfolded protein response.

Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

The inflammasome is a multiprotein complex assembled in response to Pathogen Associated Molecular Patterns (PAMPs) and Danger Associated Molecular Patterns (DAMPs). Inflammasome activation occurs through a two-step mechanism, with the first signal facilitating priming of inflammasome components while the second signal triggers complex assembly. Once assembled, the inflammasome recruits and activates pro-caspase-1, which in turn processes pro-interleukin (IL)-18 and pro-IL-1ß into their bio-active forms. Owing to its key role in the regulation of innate immune responses, the inflammasome has emerged as a therapeutic target for the treatment of inflammatory conditions. In this study we demonstrate that IRE1α, a key component of the Unfolded Protein Response, contributes to assembly of the NLRP3 inflammasome. Blockade of IRE1α RNase signaling lowered NLRP3 inflammasome assembly, caspase-1 activation and pro-IL-1ß processing. These results underscore both the importance and potential therapeutic relevance of targeting IRE1α signaling in conditions of excessive inflammasome formation.

Merits and pitfalls of conventional and covalent docking in identifying new hydroxyl aryl aldehyde like compounds as human IRE1 inhibitors.

IRE1 is an endoplasmic reticulum (ER) bound transmembrane bifunctional kinase and endoribonuclease protein crucial for the unfolded protein response (UPR) signaling pathway. Upon ER stress, IRE1 homodimerizes, oligomerizes and autophosphorylates resulting in endoribonuclease activity responsible for excision of a 26 nucleotide intron from the X-box binding protein 1 (XBP1) mRNA. This unique splicing mechanism results in activation of the XBP1s transcription factor to specifically restore ER stress. Small molecules targeting the reactive lysine residue (Lys907) in IRE1α's RNase domain have been shown to inhibit the cleavage of XBP1 mRNA. Crystal structures of murine IRE1 in complex with covalently bound hydroxyl aryl aldehyde (HAA) inhibitors show that these molecules form hydrophobic interactions with His910 and Phe889, a hydrogen bond with Tyr892 and an indispensable Schiff-base with Lys907. The availability of such data prompted interest in exploring structure-based drug design as a strategy to develop new covalently binding ligands. We extensively evaluated conventional and covalent docking for drug discovery targeting the catalytic site of the RNase domain. The results indicate that neither computational approach is fully successful in the current case, and we highlight herein the potential and limitations of the methods for the design of novel IRE1 RNase binders.

Molecular modeling provides a structural basis for PERK inhibitor selectivity towards RIPK1.

Protein kinases are crucial drug targets in cancer therapy. Kinase inhibitors are promiscuous in nature due to the highly conserved nature of the kinase ATP binding pockets. PERK has emerged as a potential therapeutic target in cancer. However, PERK inhibitors GSK2606414 and GSK2656157 also target RIPK1 whereas AMG44 is more specific to PERK. To understand the structural basis for the selectivity of PERK ligands to RIPK1 we have undertaken a detailed in silico analysis using molecular docking followed by molecular dynamics simulations to explore the selectivity profiles of the compounds. Although the binding sites of PERK and RIPK1 are similar, their binding response to small molecules is different. The docking models revealed a common binding mode for GSK2606414 and GSK2656157 in the RIPK1 binding site, similar to its cognate ligand. In contrast, AMG44 had a strikingly different predicted binding profile in the RIPK1 binding site with both rigid docking and induced fit docking settings. Our study shows a molecular mechanism responsible for dual targeting by the GSK ligands. More broadly, this work illustrates the potential of molecular docking to correctly predict the binding towards different kinase structures, and will aid in the design of selective PERK kinase inhibitors.

Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications.

The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.