Intracellular protein trafficking and secretion of proteins into the extracellular environment are sequential and tightly regulated processes in eukaryotic cells. Conventionally, proteins that are bound for secretion harbor an N-terminal signal peptide that guides their movement from the endoplasmic reticulum (ER) and Golgi apparatus to the exterior of the cell. However, some proteins can bypass this system using unconventional mechanisms, including direct translocation across the plasma membrane, transporter-mediated secretion, and intracellular vesicle-mediated exocytosis. Unconventionally secreted proteins have been implicated in inflammation, neurodegeneration, and cancer. Understanding the mechanisms that drive unconventional protein secretion can, therefore, reveal novel therapeutic targets and approaches.
Autophagy, traditionally known for degrading and recycling cytoplasmic components to maintain cellular homeostasis, has recently emerged as a novel route for the unconventional secretion of leaderless proteins. In a previous study published in Autophagy , researchers from Doshisha University, Japan, revealed that PARK7/DJ-1—a PD-associated protein renowned for its antioxidative function and mitochondrial protection—utilizes an autophagy-based mechanism for stress-induced secretion. Despite this breakthrough, the molecular events and regulatory mechanisms governing this unconventional secretion pathway remained largely undefined.
To address these gaps, the same research team has uncovered critical insights into the intracellular trafficking and extracellular release of PARK7. Their latest work delineates how this multifunctional protein, expressed across various tissues, is directed to the extracellular environment in response to cellular stress. The study was conducted by Research Assistant Dr. Biplab Kumar Dash, Professor Yasuomi Urano, and Professor Noriko Noguchi of the Graduate School of Life and Medical Sciences at Doshisha University, highlighting their continued contribution to unravelling the complexities of autophagy-mediated protein secretion in neurodegenerative disease contexts.
Giving further insight into their pioneering work, Dr. Dash explains, "Our study reveals a unique PARK7 secretion mechanism, which relies on the coordinated actions of both macroautophagy and chaperone-mediated autophagy (CMA). Under oxidative stress induced by 6-hydroxydopamine (6-OHDA), macroautophagy facilitates a robust autophagic flux that generates autophagosomes and supplies a pool of lysosomes. Simultaneously, CMA selectively translocates PARK7 to a specialized subset of these lysosomes—a critical early step in the secretion process. We propose that these CMA-enriched lysosomes subsequently fuse with autophagosomes to form 'secretory autolysosomes'. Within this compartment, PARK7 evades degradation and is instead secreted. Although CMA traditionally functions independently of autophagosomes or autolysosomes, our findings highlight its functional integration with macroautophagy during oxidative stress. This interplay between CMA-driven protein targeting and macroautophagy-mediated vesicle dynamics provides a novel mechanistic framework for the unconventional secretion of PARK7." Their research findings were published in Volume 122, Issue 19 of the Proceedings of the National Academy of Sciences (PNAS) on May 6, 2025.
The researchers found that 6-OHDA treatment in human cervical carcinoma cells induced a dose-dependent increase in PARK7 secretion. Notably, this increase was unaffected by blocking the conventional ER-to-Golgi trafficking protein pathway and the exosomal release pathway, thus confirming that PARK7 was released via an unconventional mechanism. Additionally, 6-OHDA treatment also led to a dose-dependent increase in the autophagosomal marker (LC3B) and a corresponding decrease in the autophagic substrate (SQSTM1), indicating activation of autophagy.
The researchers used fluorescently tagged LC3 to track the autophagy process, and demonstrated that 6-OHDA treatment increased autophagosome formation and enhanced autophagosome-lysosome fusion, ultimately increasing autophagic flux. Notably, blocking the early stage of autophagy significantly reduced 6-OHDA-induced LC3B-II formation and PARK7 secretion.
Additionally, treatment with an antioxidant agent nullified 6-OHDA treatment-induced oxidative stress, thereby, reducing autophagy induction and PARK7 secretion. Conversely, treatment with rapamycin, an inducer of autophagy, increased LC3B-II levels and enhanced PARK7 secretion, indicating that autophagy induction was essential for PARK7 secretion. Notably, inhibiting other protein degradation pathways, such as the ubiquitin–proteasome system, did not affect PARK7 secretion.
Delving deeper, the researchers found that blocking autophagosome-lysosome fusion and autolysosomal degradation (the downstream steps of autophagy) abrogated 6-OHDA-induced PARK7 secretion, suggesting that lysosomal activity is crucial to PARK7 secretion. Further experiments revealed that a dedicated soluble-N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex, a group of membrane-bound proteins that mediate vesicle fusion in endocytic and secretory pathways, was required for the 6-OHDA-induced extracellular secretion of PARK7. The researchers also identified specific KFERQ-like motifs in PARK7 that were selectively recognized by chaperones, thereby recruiting PARK7 to intact lysosomes, which ultimately fused with autophagosomes to form "secretory autolysosomes."
Overall, these findings provide novel insights into unconventional mechanisms that drive protein secretion. The study opens avenues for developing targeted therapies that can regulate the levels of PARK7 in PD and related conditions. Furthermore, PARK7 and related proteins hold promise as biomarkers for early disease detection, enabling timely interventions before symptoms progress.
Further, Dr. Dash concludes, "The adaptation of cells to stress while maintaining homeostasis always intrigued me to explore their deeper connections in diseases like Parkinson's. Through this research, our team hopes for drug development efforts focusing on secretory autolysosome-mediated unconventional secretion that may result in novel therapeutics enhancing cellular stress resilience and lysosomal function, leading to better patient outcomes and slowing disease progression."
About Dr. Biplab Kumar Dash from Doshisha University, Japan
Dr. Biplab Kumar Dash is a Research Assistant at Professor Noriko Noguchi's Lab at the Graduate School of Life and Medical Sciences, Doshisha University, Japan. His research focuses on autophagy-based unconventional mechanisms of protein secretion. He has authored more than 10 research articles with more than 250 citations and has been awarded the most prestigious Young Investigators Award by the Society for Free Radical Research-Asia (SFRR-Asia).
About Professor Yasuomi Urano from Doshisha University, Japan
Professor Yasuomi Urano is a distinguished researcher at Doshisha University's Graduate School of Life and Medical Sciences. He joined Doshisha University in 2009 as an Assistant Professor and was promoted to Associate Professor in 2018 and to Full Professor in 2024. His research focuses on cell death mechanisms, oxysterols, neuroscience, and lipid metabolism, with particular emphasis on cholesterols and their role in neuropathologies. He has published over peer-reviewed 40 research papers on these topics.
About Professor Noriko Noguchi from Doshisha University, Japan
Dr. Noriko Noguchi is a Professor at the Graduate School of Life and Medical Sciences at Doshisha University. She received her Ph.D. from The University of Tsukuba, Japan in 1987. Her research focuses on elucidating the molecular mechanisms of lifestyle-related diseases such as Alzheimer's disease, diabetes, and cancer, with a particular emphasis on oxidative stress. She has more than 38 years of experience as a researcher and has about 200 publications in peer-reviewed international journals.
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