CRTC-CREB Axis as a Sensor of Proteotoxic Stress via Proteasome Inhibition

Study Background and Research Question

The maintenance of protein homeostasis is essential for cellular health, with the ubiquitin-proteasome system (UPS) serving as a principal pathway for protein turnover and degradation. Disruption of proteasomal function can lead to the accumulation of misfolded proteins and proteotoxic stress, processes implicated in neurodegenerative and age-related diseases. The cAMP response element-binding protein (CREB) is a conserved transcription factor known to regulate diverse cellular functions, but its role as a sensor and effector in proteotoxic stress has remained insufficiently understood. The current study by Yin et al. (Cell Death and Disease, 2022) addresses how CREB and its co-activator CRTC (CREB-regulated transcriptional coactivator) respond to proteasome inhibition, and explores the underlying molecular signaling events.

Key Innovation from the Reference Study

The key innovation of Yin et al.'s work lies in the unexpected discovery that proteasome inhibitors, such as MLN2238, robustly enhance CREB activity in adult Drosophila. This effect is mediated by reactive oxidative species (ROS) generated in response to proteasome inhibition, which then activate the c-Jun N-terminal kinase (JNK) pathway. Activated JNK, in turn, increases CREB phosphorylation, linking the UPS, redox homeostasis, and stress response transcriptional programs for the first time in an in vivo model. Furthermore, the study demonstrates the protective function of the CRTC-CREB axis against proteotoxic and oxidative stress, positioning it as a transcriptional sensor and potential therapeutic target in protein aggregation-associated pathologies.

Methods and Experimental Design Insights

The investigators employed a multifaceted experimental approach, leveraging both genetic and pharmacological tools in Drosophila and mammalian cell models:

• High-throughput compound screening in adult flies using the U-GLAD drug delivery system enabled efficient administration of poorly soluble compounds, including various proteasome inhibitors.
• Genetic manipulation of CRTC and CREB (overexpression and loss-of-function) in specific tissues (muscle, intestine) allowed dissection of their roles in stress responses.
• ROS levels and JNK signaling were measured in response to proteasome inhibition, and pharmacological inhibition of JNK was used to delineate pathway specificity.
• Transcriptome analysis of fly intestines after CRTC overexpression identified downstream genetic programs associated with redox and proteostasis.
• A Drosophila Huntington's disease (HD) model was used to assess the functional consequences of modulating the CRTC-CREB axis on protein aggregation, motility, and lifespan.
• Validation in HEK293T cells demonstrated conservation of the ROS/JNK/CREB pathway in mammalian systems, with MLN2238 used to induce proteasome inhibition and monitor CREB phosphorylation at Ser133.

Core Findings and Why They Matter

The study's findings are significant for several reasons:

• Proteasome β5 subunit inhibition drives CREB activation: Inhibition of the proteasome, including chymotrypsin-like proteasome inhibition by MLN2238, sharply increased CREB-driven transcription in vivo. This response was dependent on the generation of ROS and subsequent activation of JNK.
• CRTC-CREB as a protective axis: Overexpression of CRTC in muscle or intestine conferred robust resistance to proteotoxic and oxidative stress, restoring protein folding capacity and proteasomal activity. Notably, in a fly HD model, CRTC overexpression ameliorated pathological protein aggregation and improved organismal health metrics.
• Redox/proteostasis gene networks: Transcriptomic profiling revealed that CRTC overexpression upregulated genes involved in antioxidant defense and proteostasis, highlighting a transcriptional program that counters the deleterious effects of proteasome inhibition-induced ROS.
• Conservation across species: MLN2238-induced CREB phosphorylation required JNK activation in both Drosophila and HEK293T cells, suggesting evolutionary conservation of this stress-sensing mechanism.
• Relevance to aging and neurodegeneration: CREB activity was found to increase during aging, and further boosting its activity suppressed age- and disease-related protein aggregates, suggesting translational potential for interventions targeting neurodegenerative disorders.

Together, these results elucidate a direct mechanistic link between reversible 20S proteasome inhibition (with selectivity toward the β5 subunit) and the activation of a protective transcriptional axis, with broad implications for multiple myeloma research, lymphoma research, and neurodegenerative disease models.

Comparison with Existing Internal Articles

Several recent reviews and research workflows have highlighted the importance of MLN2238 as a potent proteasome β5 subunit inhibitor in hematologic malignancy research. For example, the article "MLN2238: Redefining Proteasome β5 Subunit Inhibition in Hematologic Malignancies" discusses the intersection between proteasome inhibition and CREB signaling, aligning with the current study's demonstration of ROS/JNK/CREB axis activation following MLN2238 treatment. Similarly, "MLN2238: Potent Reversible 20S Proteasome β5 Subunit Inhibitor" details MLN2238's selectivity, nanomolar potency, and utility in bortezomib-resistant models—attributes leveraged in the reference paper to study stress signaling and apoptosis in both fly and mammalian systems. The current study advances this field by providing direct in vivo evidence of how β5-selective proteasome inhibition dynamically engages the CRTC-CREB transcriptional sensor, a finding previously hypothesized but not empirically validated.

Limitations and Transferability

While the research establishes a mechanistic framework linking proteasome inhibition to CREB activation via ROS/JNK signaling, several limitations should be noted:

• Most experiments were conducted in Drosophila, with only selected validation in mammalian cells. Extrapolation to human pathophysiology, especially in the context of cancer or neurodegeneration, requires additional study.
• The precise downstream effectors of the CRTC-CREB axis in different tissues and disease contexts remain to be comprehensively mapped.
• Although MLN2238 and related inhibitors robustly activate CREB signaling, the long-term effects of chronic proteasome inhibition on cellular health and viability need careful consideration, particularly in aged or diseased tissues.
• Potential compensatory mechanisms or feedback loops may modulate the observed effects in more complex mammalian systems.

Protocol Parameters

• Proteasome inhibitor administration in Drosophila: Use of the U-GLAD delivery system to achieve robust compound uptake, especially for poorly soluble agents like MLN2238 (Yin et al., 2022).
• MLN2238 concentration for proteasome β5 subunit inhibition: In vitro, nanomolar IC₅₀ values (3.4 nM for β5) are effective, as reported in the product information.
• ROS/JNK pathway interrogation: Employ specific ROS scavengers and JNK inhibitors to delineate pathway involvement, as per the reference protocol.
• Gene expression analysis: Tissue-specific overexpression or knockdown of CRTC and/or CREB, followed by transcriptomic profiling, to map downstream response networks.
• Protein aggregation assays: Use established disease models (e.g., Huntington’s in Drosophila) to evaluate functional consequences of CRTC-CREB modulation.

Research Support Resources

Researchers aiming to replicate or extend these workflows may consider using MLN2238 (SKU A4008), a reversible and selective proteasome β5 subunit inhibitor, for studies on proteotoxic stress, apoptosis induction, and stress signaling. APExBIO provides detailed handling and storage guidance to optimize compound solubility and reproducibility in experimental protocols. As always, MLN2238 is intended strictly for scientific research and not for diagnostic or therapeutic use.