ER Stress and the Unfolded Protein Response

Unit 03: ER Stress and the Unfolded Protein Response

Overview of the Endoplasmic Reticulum (ER)

  • The endoplasmic reticulum (ER) is essential for:

    • Synthesis, folding, and structural maturation of proteins

    • Approximately one-third of all proteins made in cells

  • Types of ER:

    • Rough ER:

    • Ribosome-associated, responsible for biosynthesis of secretory and membrane proteins.

    • Post-translational modifications include glycosylation, maturation, and folding

    • Rough ER properly modifies and folds around 30% of total proteins

    • Proteins are transported to the Golgi apparatus in vesicles for secretion or membrane integration

    • Smooth ER:

    • Lacks ribosomes, involved in lipid biosynthesis

    • Acts as a major intracellular calcium reservoir

    • Stores calcium ions in ER and mitochondria; responsible for biogenesis of peroxisomes and autophagosomes.

ER Stress

  • Defined as a cell stress condition caused by the accumulation of misfolded or unfolded proteins in the ER lumen.

  • Key factors and mechanisms contributing to ER stress:

    • Chemical stimuli such as biochemical inhibitors affecting:

      • N-linked protein glycosylation

      • Calcium homeostasis

      • Vesicular transport

      • Cellular redox properties

    • Viral infections can overload the ER with virus-encoded proteins

    • Associated with diseases involving oxidative stress, hypoxia, and cellular homeostasis disruptions.

Common Drugs Inducing ER Stress

  • Tunicamycin:

    • A competitive inhibitor of N-linked protein glycosylation.

    • Isolated from Streptomyces sp.; mimics UDP-GlcNAc

    • Interrupts synthesis of core oligosaccharides needed for N-glycosylation

    • Also induces apoptosis via alterations in cell surface receptors

  • Thapsigargin:

    • A non-competitive inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPases (SERCAs)

    • Disrupts calcium ion gradient essential for ER protein folding, leading to accumulation of misfolded proteins

    • Used to study cellular redox homeostasis and ER stress process

  • Brefeldin A (BFA):

    • A macrocyclic lactone from Penicillium brefeldianum

    • Inhibits vesicular transport between ER and Golgi by blocking ARF1 recruitment

    • Prevents return of essential ER proteins, increasing unfolded protein accumulation

ER Stress-Related Diseases

  • ER stress impacts overall cellular health and is associated with various diseases:

    • Neurodegeneration

    • Stroke

    • Bipolar disorder

    • Cardiac disease

    • Cancer

    • Diabetes

    • Muscle degeneration

  • Understanding ER stress regulation could lead to innovative drug development strategies

Unfolded Protein Response (UPR)

  • The UPR is a series of cellular responses activated by the accumulation of unfolded proteins in the ER.

  • The aim is to restore ER homeostasis through:

    • Transcriptional and translational adjustments

    • Inducing cell survival mechanisms or, under severe stress, triggering apoptosis

Review of UPR Effectors and Their Functions

  • Key UPR Effectors:

    • IRE1 (inositol-requiring enzyme 1):

    • Initiates a pathway to splicing XBP1 mRNA, vital for UPR signaling

    • Exists as a bifunctional enzyme with a kinase and an endoribonuclease domain

    • Activates genes needed for ER function through splicing

    • PERK (protein kinase RNA-like ER kinase):

    • Upon activation, phosphorylates eIF2α, decreasing global protein synthesis

    • Promotes translation of ATF4, leading to expression of stress response genes including CHOP

    • ATF6 (activating transcription factor 6):

    • Activates genes by travelling from the ER to the Golgi, where it is processed to release its active cytosolic domain

    • Upregulates UPR target genes responsible for mitigating UPR-induced stress

Mechanisms of UPR Signal Sensing

  • BiP (Binding immunoglobulin protein):

    • An Hsp70 chaperone that plays a critical role in sensing ER stress; it binds to misfolded proteins and releases IRE1, PERK, and ATF6 to activate UPR.

Detailed Signaling Pathways and Functions

  • Signaling by IRE1:

    • Under stress, it dimerizes and undergoes trans-autophosphorylation, activating its endoribonuclease function which leads to XBP1 mRNA splicing; spliced XBP1 protein activates UPR genes.

    • Under severe stress, oligomerized IRE1 induces apoptosis through various pathways.

  • Signaling by PERK:

    • Activates ATF4 and leads to selective upregulation of stress-induced genes while inhibiting general protein synthesis via eIF2α phosphorylation.

  • Signaling by ATF6:

    • Relies on BiP release to translocate to the Golgi, where it is cleaved to release ATF6f, a transcription factor that activates UPR-stress response genes.

Techniques for Monitoring ER Stress and UPR

  • RT-PCR for XBP1 mRNA:

    • Distinguishes between unspliced and spliced XBP1 mRNA to measure UPR activation.

    • Primers can be used to amplify different lengths of PCR products, allowing visualization of ER stress through gel electrophoresis.

  • Western Blotting for UPR Proteins:

    • Used to determine protein levels of UPR target proteins such as BiP, CHOP, phosphorylated eIF2α.

    • Involves separating proteins by size and identifying them through specific antibodies.

  • Immunostaining Techniques:

    • Utilize antibodies for direct or indirect detection of UPR proteins in cells or tissue sections, measuring their localization and abundance under stress conditions.