CRTC-CREB Axis as a Sensor of Proteotoxic Stress in Drosophila

Study Background and Research Question

The cAMP Response Element-Binding Protein (CREB) is an evolutionarily conserved transcription factor that governs diverse cellular processes, including metabolism, development, and stress response. Its activity is tightly regulated by phosphorylation events mediated by upstream kinases responding to signals such as cAMP, calcium, and growth factors. The CREB-regulated transcriptional coactivator (CRTC) further modulates CREB’s nuclear function. In mammals, phosphorylation of CREB at Ser133 is crucial for its transcriptional activation. In Drosophila, the homologous site (Ser231 of dCREB2) is constitutively phosphorylated, suggesting alternative mechanisms of CREB activation, likely involving nuclear accumulation and CRTC engagement. The ubiquitin-proteasome system (UPS) is fundamental for protein quality control, and its dysfunction can lead to the accumulation of misfolded proteins, a hallmark of neurodegenerative and age-related diseases. However, how the CREB pathway senses and responds to proteasome inhibition and proteotoxic stress in a multicellular context remained unclear. The reference study (Yin et al., 2022) addresses this gap by investigating the molecular mechanisms by which CREB and CRTC coordinate protective responses to proteasome inhibition in Drosophila.

Key Innovation from the Reference Study

The pivotal innovation of the Yin et al. study lies in the discovery that the CRTC-CREB axis serves as a transcriptional sensor for proteotoxic and oxidative stress induced by proteasome inhibition. Leveraging a high-throughput compound screen in adult flies, the authors found that all tested proteasome inhibitors, including MLN2238, robustly activate CREB-dependent transcription. Mechanistically, this activation is mediated by reactive oxygen species (ROS) generated as a consequence of proteasome inhibition, which in turn activate the c-Jun N-terminal kinase (JNK) pathway. This signaling cascade culminates in enhanced CREB phosphorylation and the upregulation of genes involved in redox balance and proteostasis. The study further demonstrates that overexpression of CRTC in specific tissues, such as muscle, can restore protein folding capacity, proteasome function, and mitigate pathogenesis in a Drosophila Huntington’s disease (HD) model, highlighting translational relevance for neurodegenerative and aging research (Yin et al., 2022).

Methods and Experimental Design Insights

The authors implemented several methodological advances to overcome technical barriers in compound delivery and in vivo screening. Notably, they utilized the U-GLAD (U-shape Gum Arabic Liquid Assisted Drug delivery) system, enabling effective administration of poorly soluble proteasome inhibitors like MLN2238 to adult Drosophila. CREB activity was monitored using reporter assays, and a combination of genetic, pharmacological, and transcriptomic approaches dissected the signaling events downstream of proteasome inhibition. ROS levels were measured following inhibitor treatment, and the requirement for JNK signaling was validated using both fly and mammalian (293T) cell models. For functional studies, the team overexpressed CRTC in intestinal and muscle tissues, evaluating the impact on gene expression, protein aggregation, motility, and lifespan, especially in the context of a Drosophila model of Huntington’s disease. Transcriptome analyses of fly intestines revealed gene networks upregulated by CRTC overexpression, particularly those linked to redox homeostasis and protein folding.

Core Findings and Why They Matter

The reference study provides several key findings:

• Proteasome inhibition triggers CREB activation via ROS/JNK signaling: Treatment with proteasome inhibitors, including the potent proteasome β5 subunit inhibitor MLN2238, induces CREB-dependent gene expression in adult flies by elevating ROS, which subsequently activate JNK and enhance CREB phosphorylation both in Drosophila and human cell lines (Yin et al., 2022).
• CRTC overexpression augments proteostasis and redox genes: Transcriptomic profiling revealed that CRTC upregulation in fly intestines boosts the expression of genes involved in managing oxidative stress and proteotoxicity, indicating that the CRTC-CREB axis orchestrates a comprehensive adaptive response.
• Functional rescue in neurodegenerative models: In a Drosophila Huntington’s disease model, muscle-specific CRTC overexpression reduced protein aggregates, improved motility, and extended lifespan, demonstrating a protective role against proteotoxic stress driven by protein misfolding.
• Relevance to aging: The study found that CREB activity naturally increases with age and that further boosting this pathway can suppress age-associated protein aggregation in muscle tissue.

These findings position the CRTC-CREB axis as a central, evolutionarily conserved node that integrates proteasome dysfunction, oxidative stress, and gene expression, providing new targets and readouts for research on aging, neurodegeneration, and cellular stress adaptation.

Comparison with Existing Internal Articles

Several internal resources contextualize the broader utility of MLN2238 and related proteasome inhibitors in stress response and oncology research. For example, the article CRTC-CREB Axis Senses Proteotoxic Stress via Proteasome Inhibition highlights how proteasome inhibition activates conserved transcriptional programs through redox signaling, aligning with the reference study's mechanistic findings. Similarly, internal resources such as MLN2238: Proteasome β5 Subunit Inhibitor in Oncology Workflows and MLN2238: Reversible 20S Proteasome β5 Subunit Inhibitor detail the compound’s nanomolar potency, selectivity for chymotrypsin-like proteasome activity, and its translational value in multiple myeloma and lymphoma models. While these articles focus on oncology and drug resistance, the reference study extends MLN2238’s relevance to basic mechanisms of proteostasis, aging, and neurodegeneration through the lens of CRTC-CREB signaling. Together, these resources illustrate the versatility of proteasome β5 subunit inhibitors for dissecting both disease-specific and conserved cellular stress pathways.

Limitations and Transferability

Despite its broad implications, the study has notable limitations. The primary findings are derived from Drosophila models, necessitating further validation in mammalian systems to confirm the universality of the CRTC-CREB axis in sensing proteotoxic stress. While the authors demonstrate that MLN2238-induced JNK/CREB activation occurs in human 293T cells, the physiological and pathological relevance in mammalian tissues, particularly in the context of neurodegenerative diseases or aging, remains to be defined. Additionally, the delivery method (U-GLAD) used for compound administration may not be directly translatable to mammalian in vivo models. Finally, chronic activation of stress-response pathways like CREB could have pleiotropic effects, potentially promoting both adaptive and maladaptive outcomes depending on cellular context.

Protocol Parameters

• Compound administration in Drosophila: Proteasome inhibitors such as MLN2238 were delivered to adult flies using the U-GLAD system to overcome solubility challenges.
• Dose and duration: MLN2238 was applied at concentrations sufficient to induce measurable CREB activation and ROS production; precise values may require pilot titration depending on species and delivery system.
• Readout assays: CREB activity was monitored via reporter constructs, while ROS levels and JNK activation were assessed using established staining and immunoblot protocols.
• Tissue-specific overexpression: UAS-CRTC constructs were used for targeted overexpression in intestine and muscle, with phenotypic assessment in neurodegenerative disease models.

Research Support Resources

Researchers studying proteasome inhibition, chymotrypsin-like proteasome inhibition, or the cellular stress response can leverage MLN2238 (SKU A4008), a reversible β5 subunit inhibitor with nanomolar IC₅₀ and Ki values, in both oncology and proteostasis workflows. According to the product information, MLN2238 is active against the chymotrypsin-like site and can be used to model proteasome inhibition in preclinical systems, including studies of multiple myeloma, lymphoma, and proteotoxic stress. As always, protocol optimization is recommended to match the specific requirements of each experimental model. For further workflow insights, the internal articles above provide additional protocol guidance and troubleshooting strategies.