Research Background

Endoplasmic Reticulum Stress

ER stress: The endoplasmic reticulum (ER) is the eukaryotic cellular organelle responsible for protein folding, maturation, quality control and trafficking of secretory and transmembrane proteins. Physiological or pathological processes that lead to the failure, overload or malfunction of ER may provoke a condition known as ‘ER stress’.

When the ER is stressed it is unable to cope with the load of proteins it has to fold and therefore unfolded proteins accumulate triggering an adaptive response called the “Unfolded Protein Response” (UPR).

The UPR activates a series of molecular pathways that attempt to clear the unfolded proteins and increase the capacity of the ER to fold proteins. ER stress is known to play a key role in human diseases including cancer, neurodegenerative and cardiovascular diseases, inflammation, diabetes and obesity, and can negatively impact therapeutic outcome. All of these diseases are of growing prevalence in the Western world and, as such, are very relevant to the future health of the European population.

UPR

Unfolded Protein Response

The Unfolded Protein Response (UPR) is an adaptive response triggered by the accumulation of unfolded proteins in the ER.

The UPR aims to resolve the stress through a multi-pronged approach by expanding the cellular protein-folding apparatus, decreasing the load of newly synthesized proteins, enhancing the clearance of unfolded proteins from the ER by a process termed ER-associated degradation (ERAD), and by inducing autophagy. However, when ER stress cannot be resolved, cell death ensues.

The molecular mechanisms involved in transition of the UPR from a protective to a death response are incompletely understood, but are fundamental to developing strategies to limit or avoid the pathological consequences of ER stress. Therefore understanding and exploiting the ER stress response has the potential to allow us to tackle many ER stress-associated diseases.

UPRadaptation

ER stress and the UPR

Conditions such as hypoxia, elevated levels of fatty acids or cholesterol, oxidative stress, variant proteins, high or low glucose levels, and inflammatory cytokines induce ER stress and activation of the UPR.

Despite the complexity in the signalling pathways activated during the response to ER stress, ultimately they all converge to modulate a core set of central signalling hubs which govern ER stress responses. In mammals, the major ER stress-sensing hubs are three ER transmembrane proteins (Fig. 1): PERK (PKR-like ER kinase), IRE1 (ERN1, endoplasmic reticulum-to-nucleus signalling 1) and ATF6 (activating transcription factor 6).

These proteins are maintained in an inactive state through interaction of their ER-lumenal domains with the ER chaperone GRP78. Upon accumulation of unfolded proteins in the ER lumen, GRP78 dissociates from these molecules, leading to their activation and initiation of the UPR. IRE1 is an ER transmembrane protein whose cytoplasmic part possess both kinase and endonuclease activity.

ERstressSignalling

IRE1 is a core component of a large protein complex, termed the UPRosome, which plays an important role in modulating UPR signalling outputs. Accumulation of unfolded proteins in the ER activates IRE1. The endoribonuclease activity of IRE1 can (1) unconventionally splice Xbp1 mRNA to produce a potent transcription factor (XBP1s) that mediates cytoprotection during ER stress, and (2) degrade specific mRNAs through an activity termed Regulated IRE1-Dependent mRNA Decay (RIDD)

Low levels of RIDD that occur during transient weak ER stress may help restore ER homeostasis by controlling the amount of mRNA to be translated into protein, however during persistent, high intensity ER stress RIDD is thought to induce cell death, possibly by reducing levels of critical pro-survival proteins. PERK is an ER transmembrane serine/threonine protein kinase that, when activated, phosphorylates eukaryotic initiation factor 2α (eIF2α) to inhibit the initiation of mRNA translation thus reducing the demand on the protein folding machinery.

Paradoxically, eIF2α phosphorylation promotes translation of the transcription factor ATF4 which induces the pro-apoptotic transcription factor, CHOP. ATF4 also mediates transcriptional induction of autophagy genes. ATF6 is an ER-localised transcription factor. Upon ER stress it translocates to the Golgi, where it is processed by site 1 and site 2 proteases (S1P, S2P) to render it active. It then translocates to the nucleus to induce UPR gene expression.

Depending on the particular physiological and pathophysiological context and the interactions of multiple pathways, the UPR can have different signalling outputs including pro-survival and pro-death, or it can influence other cellular processes such as immunity and metabolism. Therefore drugs that modulate the signals received by the UPR and signalling outputs of the UPR could potentially be used to:

(1) Stimulate protein folding and survival to evoke therapeutic benefits in neurodegenerative diseases
(2) Promote cell death as an anti-cancer strategy
(3) Alter inflammatory and metabolic processes as potential therapeutics for diseases like diabetes and arthritis.
In addition, patterns of expression of ER stress regulators can be used for prognosis/prediction of disease progression/treatment outcome.

ER stress and Disease

Under normal conditions cells that synthesise large amounts of proteins and lipids for export are particularly subject to physiological ER stress and include immune cells such as macrophages and plasma cells, as well as cells regulating metabolism such as hepatocytes, pancreatic β-cells, adipocytes, and mucosal epithelial cells. In addition environmental conditions such as low nutrient and oxygen levels or developmental cues stress the ER.

The UPR provides a flexible system that allows cells to cope with a wide range of physiological demands on the ER. However many studies have now implicated ER stress and disrupted UPR functioning in the development, progression and pathology of diverse diseases including neoplastic, inflammatory, metabolic and neurodegenerative diseases.

Metabolic: Constant and excessive exposure to nutrients places a high demand on adaptive responses from metabolic networks. While the ER can adapt to short-term metabolic demands it appears less flexible in managing chronic and escalating metabolic changes. Accumulating evidence suggests that disruption of ER homeostasis is intimately involved in the mechanisms underlying obesity, diabetes, cardiovascular diseases and fatty liver disease and that the IRE1α/ XBP1 pathway plays a critical role in glucose and lipid metabolism as well as insulin function.

Inflammation: Many studies suggest that unresolved ER stress due to improper functioning of the UPR contributes to inflammatory pathology and underlies many chronic low-grade inflammatory conditions such as inflammatory bowel disease and diabetes. All three branches of the UPR intersect with a variety of inflammatory and stress signaling systems. The UPR induced inflammatory response can vary in longevity, intensity and the type of immune response elicited, which influences the pathogenesis and/or progression of diseases such as Crohn’s disease, IBD, COPD, asthma, cystic fibrosis and sepsis.

Cancer: During tumorigenesis environmental fluctuations coupled with inadequate vascularisation lead to decreased levels of oxygen, nutrients, and glucose and thereby induce ER stress. In addition the rapid growth of tumor cells requires major metabolic changes putting further demand on the ER. Studies suggest that cancer cells selectively activate the pro-survival adaptive mechanisms of the UPR for continued growth and survival in these otherwise toxic conditions. All branches of the UPR appear to contribute to the development of cancer by affecting diverse aspects of the disease including cell transformation, tumor angiogenesis, cell differentiation, cell migration, tumour growth and the immune microenvironment.

Neurodegeneration: Disruption of ER function is associated with the accumulation of misfolded proteins which is a characteristic occurrence in many neurodegenerative diseases (including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Huntington’s disease, prion-related disorders, and demyelinating neurodegenerative diseases such as multiple sclerosis). Abnormal protein aggregation alters essential cellular functions leading to neurological impairment and in many cases neuronal loss. Signs of ER stress have been detected in most experimental models of neurological disorders and more recently in brain samples from human patients with neurodegenerative disease.