Secretory proteins, including plasma membrane proteins, are generally known to be transported to the plasma membrane through the endoplasmic reticulum- to-Golgi pathway. However, recent studies have revealed that several plasma membrane proteins and cytosolic proteins lacking a signal peptide are released via an unconventional protein secretion (UcPS) route, bypassing the Golgi during their journey to the cell surface. For instance, transmembrane proteins such as the misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein and the Spike protein of coronaviruses have been observed to reach the cell surface through a UcPS pathway under cell stress conditions. Nevertheless, the precise mechanisms of the UcPS pathway, particularly the molecular machineries involving cytosolic motor proteins, remain largely unknown. In this study, we identified specific kinesins, namely KIF1A and KIF5A, along with cytoplasmic dynein, as critical players in the unconventional trafficking of CFTR and the SARS-CoV-2 Spike protein. Gene silencing results demonstrated that knockdown of KIF1A, KIF5A, and the KIF-associated adaptor protein SKIP, FYCO1 significantly reduced the UcPS of △F508-CFTR. Moreover, gene silencing of these motor proteins impeded the UcPS of the SARS-CoV-2 Spike protein. However, the same gene silencing did not affect the conventional Golgimediated cell surface trafficking of wild-type CFTR and Spike protein. These findings suggest that specific motor proteins, distinct from those involved in conventional trafficking, are implicated in the stress-induced UcPS of transmembrane proteins.

Loss of phosphatase and tensin homolog (PTEN) has been linked to an immunosuppressive tumor microenvironment, but its underlying mechanisms remain largely enigmatic. Here, we report that PTEN can be secreted by the transmembrane emp24 domain-containing protein 10 (TMED10)-channeled protein secretion pathway. Inhibiting PTEN secretion from tumor cells contributes to immunosuppression and impairs the tumor-suppressive role of PTEN, while intratumoral injection of PTEN protein promotes antitumor immunity and suppresses tumor growth in mice. Mechanistically, extracellular PTEN binds to the plexin domain-containing protein 2 (PLXDC2) on macrophages, triggering subsequent activation of JAK2-STAT1 signaling, which switches tumor-associated macrophages (TAMs) from the immunosuppressive to inflammatory phenotype, leading to enhanced activation of CD8+ T and natural killer cells. Importantly, PTEN treatment also enhances the therapeutic efficacy of anti-PD-1 treatment in mice and reverses the immune-suppressive phenotype of patient-derived primary TAMs. These data identify a cytokine-like role of PTEN in immune activation and tumor suppression and demonstrate the therapeutic potential for extracellular administration of PTEN in cancer immunotherapy.

Newly synthesized proteins are delivered to the apoplast via conventional or unconventional protein secretion in eukaryotes. In plants, proteins are secreted to perform various biological functions. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been revealed in plants. In the conventional protein secretion pathway, secretory proteins with a signal peptide are translocated into the endoplasmic reticulum and transported to the extracellular region via the endomembrane system. On the contrary, unconventional protein secretion pathways have been demonstrated to mediate the secretion of the leaderless secretory proteins. In this chapter, we summarize the updated findings and provide a comprehensive overview of protein secretion pathways in plants.

Eukaryotic cells have developed intricate mechanisms for biomolecule transport, particularly in stressful conditions. This interdisciplinary study delves into unconventional protein secretion (UPS) pathways activated during starvation, facilitating the export of proteins bypassing most of the components of the classical secretory machinery. Specifically, we focus on the underexplored mechanisms of the GRASP\'s role in UPS, particularly in biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results show that liquid-liquid phase separation (LLPS) plays a key role in the coacervation of Grh1, the GRASP yeast homologue, under starvation-like conditions. This association seems a precursor to the Compartment for Unconventional Protein Secretion (CUPS) biogenesis. Grh1\'s self-association is regulated by electrostatic, hydrophobic, and hydrogen-bonding interactions. Importantly, our study demonstrates that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations. Additionally, we explore how the coacervate liquid-to-solid transition could impact cells\' ability to return to normal post-stress states. Our findings offer insights into intracellular protein dynamics and cell adaptive responses to stress.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis.
METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs.
RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs.
CONCLUSIONS: This study elucidates AGR2\'s pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy-mediated exocytosis of IL-1β, insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic β-cell \"Min-6\" and human lung cancer cell \"CL1-5-Q89L\" with high secretory autophagy tendency by LC-MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy-related vesicle trafficking. We used CL1-5-Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy-related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins.

The sleep-wake cycle regulates interstitial fluid and cerebrospinal fluid (CSF) tau levels in both mouse and human by mechanisms that remain unestablished. Here, we reveal a novel pathway by which wakefulness increases extracellular tau levels in mouse and humans. In mice, higher body temperature (BT) associated with wakefulness and sleep deprivation increased CSF tau. In vitro, wakefulness temperatures upregulated tau secretion via a temperature-dependent increase in activity and expression of unconventional protein secretion pathway-1 components, namely caspase-3-mediated C-terminal cleavage of tau (TauC3), and membrane expression of PIP2 and syndecan-3. In humans, the increase in both CSF and plasma tau levels observed post-wakefulness correlated with BT increase during wakefulness. Our findings suggest sleep-wake variation in BT may contribute to regulating extracellular tau levels, highlighting the importance of thermoregulation in pathways linking sleep disturbance to neurodegeneration, and the potential for thermal intervention to prevent or delay tau-mediated neurodegeneration.

The cellular secretome is pivotal in mediating intercellular communication and coordinating responses to stressors. Exosomes, initially recognized for their role in waste disposal, have now emerged as key intercellular messengers with significant therapeutic and diagnostic potential. Similarly, autophagy has transcended its traditional role as a waste removal mechanism, emerging as a regulator of intracellular communication pathways and a contributor to a unique autophagy-dependent secretome. Secretory authophagy, initiated by various stress stimuli, prompts the selective release of proteins implicated in inflammation, including leaderless proteins that bypass the conventional endoplasmic reticulum-Golgi secretory pathway. This reflects the significant impact of stress-induced autophagy on cellular secretion profiles, including the modulation of exosome release. The convergence of exosome biogenesis and autophagy is exemplified by the formation of amphisomes, vesicles that integrate autophagic and endosomal pathways, indicating their synergistic interplay. Regulatory proteins common to both pathways, particularly mTORC1, emerge as potential therapeutic targets to alter cellular secretion profiles involved in various diseases. This review explores the dynamic interplay between autophagy and exosome formation, highlighting the potential to influence the secretome composition. While the modulation of exosome secretion and cytokine preconditioning is well-established in regenerative medicine, the strategic manipulation of autophagy is still underexplored, presenting a promising but uncharted therapeutic landscape.

The Golgi Reassembly and Stacking Proteins (GRASPs) are engaged in various functions within the cell, both in unconventional secretion mechanisms and structuring and organizing the Golgi apparatus. Understanding their specific role in each situation still requires more structural and functional data at the molecular level. GRASP55 is one of the GRASP members in mammals, anchored to the membrane via the myristoylation of a Gly residue at its N-terminus. Therefore, co-translational modifications, such as myristoylation, are fundamental when considering a strategy to obtain detailed information on the interactions between GRASP55 and membranes. Despite its functional relevance, the N-terminal myristoylation has been underappreciated in the studies reported to date, compromising the previously proposed models for GRASP-membrane interactions. Here, we investigated the synergy between the presence of the membrane and the formation of oligomeric structures of myristoylated GRASP55, using a series of biophysical techniques to perform the structural characterization of the lipidated GRASP55 and its interaction with biological lipid model membranes. Our data fulfill an unexplored gap: the adequate evaluation of the presence of lipidations and lipid membranes on the structure-function dyad of GRASPs.Communicated by Ramaswamy H. Sarma.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.