Dear Editor,
We are profoundly grateful to Dr Seol Hee Park and Wonhyo Seo for their insightful editorial commentary on our recently published work in Clinical and Molecular Hepatology [
1]. We were particularly appreciative of their discussion regarding how the non-canonical RAB25/GCN1 signaling axis promotes endoplasmic reticulum (ER) stress, and their emphasis on this axis as a key integrative pathway connecting ER stress with mitochondrial metabolism and lipid synthesis in alcohol-associated liver disease (ALD) progression [
2]. We sincerely value this opportunity to engage in a scholarly dialogue that further refines our collective understanding of ER stress in ALD.
As mentioned in the editorial, excessive alcohol consumption initiates a spectrum of pathogenic processes, from oxidative stress to mitochondrial dysfunction; however, this current understanding remains insufficient to fully explain disease progression [
3]. Significantly, alcohol has been shown to directly induce hepatic ER stress, which in turn drives lipid metabolism disorders and acute liver injury [
4]. Growing evidence emphasizes the critical role of ER stress in the pathogenesis of ALD [
5].
The activation of the unfolded protein response (UPR) during ER stress affects hepatocyte fate and can contribute to the progression of liver injury [
6]. The editorial correctly emphasizes that the UPR sensors only partially explain the landscape of ER stress-mediated injury in ALD. This prompted our hypothesis that alternative, non-canonical pathways may be operative. We demonstrated ER enrichment of RAB25 using single-cell sequencing and histology. Furthermore, Gene set enrichment analysis of single-cell data stratified by RAB25 expression showed that high RAB25 levels in ALD strongly associate with gene signatures related to ER lumen, unfolded protein response, and stress (
Fig. 1A–
1D). These findings indicate that RAB25 promotes ALD progression by leveraging its intracellular trafficking capabilities from the ER to activate ER stress. This ER-localized activity positions RAB25 as a central pathophysiological mediator, revealing a critical functional coupling between intracellular trafficking and cellular stress-response signaling that was previously unrecognized. Furthermore, our analyses identified GCN1 as a major binding partner of RAB25. As highlighted in the editorial, the key mechanistic step involves RAB25-dependent stabilization of GCN1, which suppresses its K33-linked ubiquitination and degradation. This not only sustains GCN2 activation but, more importantly, triggers downstream ATF4-dependent ER stress signaling (
Fig. 1E,
1F). These findings suggest stress signaling plasticity in hepatocytes, where the source of eIF2α phosphorylation (via PERK or GCN2) may be determined by the nature and subcellular origin of the metabolic insult. This may explain the heterogeneous cellular responses observed within a stressed liver lobule.
We also wish to highlight the critical role of inter-organelle communication, particularly between the ER and mitochondria. Studies show that while mitochondria-associated ER membranes are crucial for physiological lipid metabolism and Ca²⁺ exchange, their pathological remodeling in ALD leads to excessive mitochondrial Ca²⁺ influx via disrupted calcium channels, thereby driving liver injury [
7]. Our study demonstrates that RAB25 acts not merely as an instigator of ER stress, but functions as a central node coordinating inter-organellar dysfunction. This is supported by the profound preservation of mitochondrial morphology, cristae integrity, and oxidative phosphorylation capacity observed upon RAB25 suppression. We propose that RAB25 overexpression promotes excessive and dysregulated ER-mitochondria tethering. This pathological tethering facilitates uncontrolled calcium flux from the ER into the mitochondria, overwhelming the mitochondrial calcium-buffering capacity and triggering the mitochondrial permeability transition, excessive reactive oxygen species generation, and ultimately, metabolic failure. RAB25 knockdown normalized these parameters highlighting its role as a master regulator of this deleterious cross-talk. Taken together, RAB25 emerges as a pivotal central regulator of pathological ER-mitochondria crosstalk in ALD.
Alcohol consumption disrupts endocrine signaling pathways that regulate metabolism and inflammation, thereby contributing to the development of hepatic steatosis in ALD [
8]. Furthermore, alcohol-exposed hepatocytes actively promote hepatic inflammation through the release of cytokines, chemokines, and damage-associated molecular patterns. As the editorialists point out, our study identifies RAB25 as a key driver of hepatic inflammation in ALD. Specifically, RAB25 enhances the expression of the chemokine CXCL12 via the ER stress–NLRP3 inflammatory axis [
9]. Activation of RAB25 leads to the upregulation of NLRP3, subsequent caspase-1 activation, and CXCL12 release, which collectively facilitate immune cell recruitment and amplify the inflammatory response. These findings indicate that RAB25 indirectly promotes CXCL12 production via the ER stress–NLRP3 pathway, thereby contributing to the inflammatory response in ALD. While CXCL12 appears to be a central mediator, the overall inflammatory environment likely results from a coordinated network of multiple chemokines, warranting further investigation. This remains a focus of our ongoing research.
Current ALD management employs mechanism-based pharmacotherapies (e.g., IL-1β inhibitors, CCR2/5 antagonists) and liver transplantation [
10]. Future directions focus on precision medicine guided by biomarkers and novel targets, such as deubiquitinating enzymes in the ubiquitin-specific protease (USP) family [
11]. USP inhibitors can modulate undruggable targets such as transcription factors and drug-resistant proteins, demonstrating unique potential to enhance the efficacy of targeted therapies, and to overcome drug resistance. For instance, the USP9X inhibitor WP1130 increases the effectiveness of doxorubicin, while the USP22 inhibitor S02 counteracts sorafenib resistance [
12]. Furthermore, co-delivery systems that combine a USP7 inhibitor with a PD-L1 antibody offer a novel approach for immune-combination therapy [
13]. Currently, the clinical translation of USP inhibitors is progressing, with several candidates having entered Phase I trials. Our study also highlights USP5-mediated deubiquitination as a potential target for intervention in ALD. Future research focused on developing combination therapies with existing treatments will be critical to advancing USP-targeted therapeutics for liver diseases.
In conclusion, we extend our deepest thanks to the editorial writers for their incisive and constructive analysis, which has significantly enriched the context, implications, and future directions stemming from our study. We appreciate the thoughtful commentary on the role of RAB25 in alcohol-related liver disease. We agree that RAB25 acts as a key molecular bridge connecting ER stress, mitochondrial dysfunction, and immune activation—a perspective that well aligns with evolving paradigms emphasizing organelle crosstalk in metabolic homeostasis. The editorial writers’ suggestion that elevated RAB25 could serve as a biomarker of active ER stress in alcohol-induced liver injury is particularly insightful. Furthermore, the proposed therapeutic strategies targeting the RAB25–GCN1 axis or USP5-mediated deubiquitination offer promising novel directions for intervention. We believe that elucidating such noncanonical ER stress pathways indeed expands the therapeutic landscape of ALD. Our ongoing work continues to explore these cellular stress networks, with the goal of identifying further mechanistic and translational opportunities in this field.
FOOTNOTES
-
Authors’ contribution
Manuscript drafting: Xue-wen Liu. Manuscript edition and final approval: all authors.
-
Acknowledgements
This work was supported by the National Natural Science Foundation of China (82200703).
-
Conflicts of Interest
The authors have no conflicts to disclose.
Figure 1.RAB25 interacts with GCN1 to mediate ER stress. (A–D) GSEA analysis of ER lumen (A), response to ER stress (B), intrinsic apoptotic pathway in response to ER stress (C) and ER unfolded protein response (D) signaling pathways. The enrichment plot shows the enriched pathway in the RAB25 high expression group. NES>1 and P<0.05 were considered significant. (E) Gene expression analysis in liver tissues from patients with ALD using GEO datasets. (F) Representative images of GCN1, BIP and ATF4 in liver tissues obtained from AAV-GFP and AAV-shRAB25 mice. Scale bars, 100 μm. The data are presented as mean±SEM, *P<0.05, **P<0.01 and ***P<0.001; n.s., not significant by Student’s t-test or two-way ANOVA.
Abbreviations
alcohol-associated liver disease
unfolded protein response
ubiquitin-specific protease
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Seol Hee Park, Wonhyo Seo
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