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.

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.

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.

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.

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.

Crosstalk between inflammatory mediators and endoplasmic reticulum stress in liver diseases.

An excessive inflammatory response is frequently associated with cellular dysfunction and cell death. The latter may cause single and multiple organ failure. The most susceptible organs are liver, lung, kidney, heart and intestine. This review will focus on the liver as a target organ for an excessive inflammatory response. It is commonly accepted that organ failure is caused by the action of inflammatory cytokines released in excess during the inflammatory response. It has been suggested that inflammation mediated liver failure is not due to an increased death rate of parenchymal cells, but due to an intracellular metabolic disorder. This metabolic disorder is associated with mitochondrial and endoplasmic reticulum (ER) dysfunction during the acute phase response elicited by systemic inflammation. An overproduction of acute phase proteins in the liver as well as elevated reactive oxygen species (ROS) generation induce ER stress, triggering the unfolded protein response (UPR), which may initiate or aggravate inflammation. It is known that certain inflammatory mediators, such as the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α induce ER stress. These findings suggest that ER stress and the subsequent UPR on the one hand, and the inflammatory response on the other create a kind of feed forward loop, which can be either beneficial (e.g., elimination of the pathogen and restoration of tissue homeostasis) or deleterious (e.g., excessive cell dysfunction and cell death). This review aims to unfurl the different pathways contributing to this loop and to highlight the relevance of UPR signaling (IRE1α, ATF6, and PERK) and mediators of the inflammatory response (NF-κB, STAT3, IL-1ß, IL-6, TLR) which have a particular role as pathophysiological triggers in the liver.

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.

Generation of rationally-designed nerve growth factor (NGF) variants with receptor specificity.

Nerve growth factor (NGF) is the prototypic member of the neurotrophin family and binds two receptors, TrkA and the 75 kDa neurotrophin receptor (p75NTR), through which diverse and sometimes opposing effects are mediated. Using the FoldX protein design algorithm, we generated eight NGF variants with different point mutations predicted to have altered binding to TrkA or p75NTR. Of these, the I31R NGF variant exhibited specific binding to p75NTR. The generation of this NGF variant with selective affinity for p75NTR can be used to enhance understanding of neurotrophin receptor imbalance in diseases and identifies a key targetable residue for the development of small molecules to disrupt binding of NGF to TrkA with potential uses in chronic pain.

RIP2 enhances cell survival by activation of NF-ĸB in triple negative breast cancer cells.

Receptor-interacting protein 2 (RIP2) is an essential mediator of inflammation and innate immunity, but little is known about its role outside the immune system. Recently, RIP2 has been linked to chemoresistance of triple negative breast cancer (TNBC), the most aggressive breast cancer subtype for which there is an urgent need for targeted therapies. In this study we show that high expression of RIP2 in breast tumors correlates with a worse prognosis and a higher risk of recurrence. We also demonstrate that RIP2 confers TNBC cell resistance against paclitaxel and ceramide-induced apoptosis. Overexpression of RIP2 lead to NF-κB activation, which contributed to higher expression of pro-survival proteins and cell survival. Conversely, RIP2 knockdown inhibited NF-κB signaling, reduced levels of anti-apoptotic proteins and sensitized cells to drug treatment. Together, these data show that RIP2 promotes survival of breast cancer cells through NF-κB activation and that targeting RIP2 may be therapeutically beneficial for treatment of TNBC.

The integrated stress response.

In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.

Homology model of the human tRNA splicing ligase RtcB.

RtcB is an essential human tRNA ligase required for ligating the 2',3'-cyclic phosphate and 5'-hydroxyl termini of cleaved tRNA halves during tRNA splicing and XBP1 fragments during endoplasmic reticulum stress. Activation of XBP1 has been implicated in various human tumors including breast cancer. Here we present, for the first time, a homology model of human RtcB (hRtcB) in complex with manganese and covalently bound GMP built from the Pyrococcus horikoshii RtcB (bRtcB) crystal structure, PDB ID 4DWQA. The structure is analyzed in terms of stereochemical quality, folding reliability, secondary structure similarity with bRtcB, druggability of the active site binding pocket and its metal-binding microenvironment. In comparison with bRtcB, loss of a manganese-coordinating water and movement of Asn226 (Asn202 in 4DWQA) to form metal-ligand coordination, demonstrates the uniqueness of the hRtcB model. Rotation of GMP leads to the formation of an additional metal-ligand coordination (Mn-O). Umbrella sampling simulations of Mn binding in wild type and the catalytically inactive C122A mutant reveal a clear reduction of Mn binding ability in the mutant, thus explaining the loss of activity therein. Our results furthermore clearly show that the GTP binding site of the enzyme is a well-defined pocket that can be utilized as target site for in silico drug discovery.

Neurotrophins and B-cell malignancies.

Neurotrophins and their receptors act as important proliferative and pro-survival factors in a variety of cell types. Neurotrophins are produced by multiple cell types in both pro- and mature forms, and can act in an autocrine or paracrine fashion. The p75(NTR) and Trk receptors can elicit signalling in response to the presence or absence of their corresponding neurotrophin ligands. This signalling, along with neurotrophin and receptor expression, varies between different cell types. Neurotrophins and their receptors have been shown to be expressed by and elicit signalling in B lymphocytes. In general, most neurotrophins are expressed by activated B-cells and memory B-cells. Likewise, the TrkB95 receptor is seen on activated B-cells, while TrkA and p75(NTR) are expressed by both resting and active B-cells as well as memory B-cells. Nerve growth factor stimulates B-cell proliferation, memory B-cell survival, antibody production and CD40 expression. Brain-derived neurotrophic factor is involved in B-cell maturation in the bone marrow through TrkB95. Overall neurotrophins and their receptors have been shown to be involved in B-cell proliferation, development, differentiation, antibody secretion and survival. As well as expression and activity in healthy B-cells, the neurotrophins and their receptors can contribute to B-cell malignancies including acute lymphoblastic leukaemia, diffuse large B-cell lymphoma, Burkitt's lymphoma and multiple myeloma. They are involved in B-cell malignancy survival and potentially in drug resistance.

Nerve growth factor (NGF)-mediated regulation of p75(NTR) expression contributes to chemotherapeutic resistance in triple negative breast cancer cells.

Triple negative breast cancer [TNBC] cells are reported to secrete the neurotrophin nerve growth factor [NGF] and express its receptors, p75 neurotrophin receptor [p75(NTR)] and TrkA, leading to NGF-activated pro-survival autocrine signaling. This provides a rationale for NGF as a potential therapeutic target for TNBC. Here we show that exposure of TNBC cells to NGF leads to increased levels of p75(NTR), which was diminished by NGF-neutralizing antibody or NGF inhibitors [Ro 08-2750 and Y1086]. NGF-mediated increase in p75(NTR) levels were partly due to increased transcription and partly due to inhibition of proteolytic processing of p75(NTR). In contrast, proNGF caused a decrease in p75(NTR) levels. Functionally, NGF-induced increase in p75(NTR) caused a decrease in the sensitivity of TNBC cells to apoptosis induction. In contrast, knock-down of p75(NTR) using shRNA or small molecule inhibition of NGF-p75(NTR) interaction [using Ro 08-2750] sensitized TNBC cells to drug-induced apoptosis. In patient samples, the expression of NGF and NGFR [the p75(NTR) gene] mRNA are positively correlated in several subtypes of breast cancer, including basal-like breast cancer. Together these data suggest a positive feedback loop through which NGF-mediated upregulation of p75(NTR) can contribute to the chemo-resistance of TNBC cells.

A close connection between the PERK and IRE arms of the UPR and the transcriptional regulation of autophagy.

Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation.

Stress-induced self-cannibalism: on the regulation of autophagy by endoplasmic reticulum stress.

Macroautophagy (autophagy) is a cellular catabolic process which can be described as a self-cannibalism. It serves as an essential protective response during conditions of endoplasmic reticulum (ER) stress through the bulk removal and degradation of unfolded proteins and damaged organelles; in particular, mitochondria (mitophagy) and ER (reticulophagy). Autophagy is genetically regulated and the autophagic machinery facilitates removal of damaged cell components and proteins; however, if the cell stress is acute or irreversible, cell death ensues. Despite these advances in the field, very little is known about how autophagy is initiated and how the autophagy machinery is transcriptionally regulated in response to ER stress. Some three dozen autophagy genes have been shown to be required for the correct assembly and function of the autophagic machinery; however; very little is known about how these genes are regulated by cellular stress. Here, we will review current knowledge regarding how ER stress and the unfolded protein response (UPR) induce autophagy, including description of the different autophagy-related genes which are regulated by the UPR.

The eIF2α kinases: their structures and functions.

Cell signaling in response to an array of diverse stress stimuli converges on the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2). Phosphorylation of eIF2α on serine 51 results in a severe decline in de novo protein synthesis and is an important strategy in the cell's armory against stressful insults including viral infection, the accumulation of misfolded proteins, and starvation. The phosphorylation of eIF2α is carried out by a family of four kinases, PERK (PKR-like ER kinase), PKR (protein kinase double-stranded RNA-dependent), GCN2 (general control non-derepressible-2), and HRI (heme-regulated inhibitor). Each primarily responds to a distinct type of stress or stresses. Thus, while significant sequence similarity exists between the eIF2α kinases in their kinase domains, underlying their common role in phosphorylating eIF2α, additional unique features determine the regulation of these four proteins, that is, what signals activate them. This review will describe the structure of each eIF2α kinase and discuss how this is linked to their activation and function. In parallel to the general translational attenuation elicited by eIF2α kinase activation the translation of stress-induced mRNAs, most notably activating transcription factor 4 (ATF4) is enhanced and these set in motion cascades of gene expression constituting the integrated stress response (ISR), which seek to remediate stress and restore homeostasis. Depending on the cellular context and concurrent signaling pathways active, however, translational attenuation can also facilitate apoptosis. Accordingly, the role of the kinases in determining cell fate will also be discussed.

The unfolded protein response at the crossroads of cellular life and death during endoplasmic reticulum stress.

One of the early cellular responses to endoplasmic reticulum (ER) stress is the activation of the unfolded protein response (UPR). ER stress and the UPR are both implicated in numerous human diseases and pathologies. In spite of this, our knowledge of the molecular mechanisms that regulate cell fate following ER stress is limited. The UPR is initiated by three ER transmembrane receptors: PKR-like ER kinase (PERK), activating transcription factor (ATF) 6 and inositol-requiring enzyme 1 (IRE1). These proteins sense the accumulation of unfolded proteins and their activation triggers specific adaptive responses to resolve the stress. Intriguingly, the very same receptors can initiate signalling pathways that lead to apoptosis when the attempts to resolve the ER stress fail. In this review, we describe the known pro-apoptotic signalling pathways emanating from activated PERK, ATF6 and IRE1 and discuss how their signalling switches from an adaptive to a pro-apoptotic response.

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.