Search

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 ΔF508-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. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

To function properly, neurons must maintain a proteome that differs in their somatodendritic and axonal domain. This requires the polarized sorting of newly synthesized secretory and transmembrane proteins into different vesicle populations as they traverse the secretory pathway. Although the trans-Golgi-network is generally considered to be the main sorting hub, this sorting process may already begin at the ER and continue through the Golgi cisternae. At each step in the sorting process, specificity is conferred by adaptors, GTPases, tethers, and SNAREs. Besides this, local synthesis and unconventional protein secretion may contribute to the polarized proteome to enable rapid responses to stimuli. For some transmembrane proteins, some of the steps in the sorting process are well-studied. These will be highlighted here. The universal rules that govern polarized protein sorting remain unresolved, therefore we emphasize the need to approach this problem in an unbiased, top-down manner. Unraveling these rules will contribute to our understanding of neuronal development and function in health and disease. [ABSTRACT FROM AUTHOR]

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.

ABSTRACTUnconventional protein secretion (UPS) plays important roles in cell physiology. In contrast to canonical secretory routes, UPS does not generally require secretory signal sequences and often bypasses secretory compartments such as the ER and the Golgi apparatus. Giardia lamblia is a protist parasite with reduced subcellular complexity which releases several proteins, some of them virulence factors, without canonical secretory signals. This implicates UPS at the parasite–host interface. No dedicated machinery nor mechanism(s) for UPS in Giardia are currently known, although speculations on the involvement of endocytic organelles called PV/PECs, have been put forth. To begin to address the question of whether PV/PECs are implicated in virulence-associated UPS and to define the composition of molecular machinery involved in protein release, we employed affinity purification and mass spectrometry, coupled to microscopy-based subcellular localization and signal correlation quantification to investigate the interactomes of 11 reported unconventionally secreted proteins, all predicted to be cytosolic. A subset of these are associated with PV/PECs. Extended and validated interactomes point to a core PV/PECs-associated UPS machinery, which includes uncharacterized and Giardia-specific coiled-coil proteins and NEK kinases. Finally, a subset of the alpha-giardin protein family was enriched in all PV/PECs-associated protein interactomes, highlighting a previously unappreciated role for these proteins at PV/PECs and in UPS. Taken together, our results provide the first characterization of a virulence-associated UPS protein complex in Giardia lamblia at PV/PECs, suggesting a novel link between these primarily endocytic and feeding organelles and UPS at the parasite–host interface.

Recent findings demonstrate that cytoplasmic effectors from fungal and oomycete pathogens enter plant cells via clathrin-mediated endocytosis (CME). This raises several questions: Does effector secretion pathway facilitate host uptake? How is CME triggered in host cells? How are the effectors released from endosomal compartments to reach diverse subcellular destinations? [ABSTRACT FROM AUTHOR]

Neurodegenerative disease-causing proteins such as alpha-synuclein, tau, and huntingtin are known to traverse across cells via exosomes, extracellular vesicles and tunneling nanotubes (TNTs). There seems to be good synergy between exosomes and TNTs in intercellular communication. Interestingly, many of the known major neurodegenerative proteins/proteolytic products are leaderless and are also reported to be secreted out of the cell via unconventional protein secretion. Such classes contain intrinsically disordered proteins and regions (IDRs) within them. The dynamic behavior of these proteins is due to their heterogenic conformations that is exhibited owing to various factors that occur inside the cells. The amino acid sequence along with the chemical modifications has implications on the functional roles of IDRs inside the cells. Proteins that form aggregates resulting in neurodegeneration become resistant to degradation by the processes of autophagy and proteasome system thus leading to Tunneling nanotubes, TNT formation. The proteins that traverse across TNTs may or may not be dependent on the autophagy machinery. It is not yet clear whether the conformation of the protein plays a crucial role in its transport from one cell to another without getting degraded. Although there is some experimental data, there are many grey areas which need to be revisited. This review provides a different perspective on the structural and functional aspects of these leaderless proteins that get secreted outside the cell. In this review, attention has been focused on the characteristic features that lead to aggregation of leaderless secretory proteins (from structuralfunctional aspect) with special emphasis on TNTs. [ABSTRACT FROM AUTHOR]

Unconventional protein secretion (UPS) is a crucial mechanism controlling the localization of cytosolic proteins lacking signal peptides and is implicated in inflammation, neurodegenerative diseases, and cancer. Several previous studies on immune cells have demonstrated the mechanisms of UPS. In cancer, the active secretion of several cytosolic proteins, including PKCδ and nucleolin, has been described. Moreover, we have recently demonstrated that extended synaptotagmin 1, one of the membrane proteins of the endoplasmic reticulum, plays a critical role in UPS in liver cancer cells. Importantly, UPS in cancer cells shows characteristics that are markedly different from those of the previously known UPS, and therefore, we categorize them as cancer-related UPS (CUPS). In this article, we provide an overview of UPS mechanisms and discuss the process that leads to the naming of cancer-specific UPS as CUPS. [ABSTRACT FROM AUTHOR]

Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives controlled at 5%, and are also able to classify the limited number of established plant LSPs (SPLpred (3/4, LSPpred 4/4). Until such time as a larger set of bona fide (independently experimentally validated) LSPs is established using imaging technologies (light/fluorescence/electron microscopy) to confirm sub-cellular location, these tools represent a bridging method for predicting and identifying plant putative LSPs for subsequent experimental validation. [ABSTRACT FROM AUTHOR]

Neurodegenerative disease-causing proteins such as alpha-synuclein, tau, and huntingtin are known to traverse across cells via exosomes, extracellular vesicles and tunneling nanotubes (TNTs). There seems to be good synergy between exosomes and TNTs in intercellular communication. Interestingly, many of the known major neurodegenerative proteins/proteolytic products are leaderless and are also reported to be secreted out of the cell via unconventional protein secretion. Such classes contain intrinsically disordered proteins and regions (IDRs) within them. The dynamic behavior of these proteins is due to their heterogenic conformations that is exhibited owing to various factors that occur inside the cells. The amino acid sequence along with the chemical modifications has implications on the functional roles of IDRs inside the cells. Proteins that form aggregates resulting in neurodegeneration become resistant to degradation by the processes of autophagy and proteasome system thus leading to Tunneling nanotubes, TNT formation. The proteins that traverse across TNTs may or may not be dependent on the autophagy machinery. It is not yet clear whether the conformation of the protein plays a crucial role in its transport from one cell to another without getting degraded. Although there is some experimental data, there are many grey areas which need to be revisited. This review provides a different perspective on the structural and functional aspects of these leaderless proteins that get secreted outside the cell. In this review, attention has been focused on the characteristic features that lead to aggregation of leaderless secretory proteins (from structural-functional aspect) with special emphasis on TNTs.

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.

Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1β) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1β) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1β is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1β processing in these cells. We found that although caspase-1 is required for IL-1β secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1β processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1β secretion. [ABSTRACT FROM AUTHOR]

Unconventional protein export/secretion (UPE/UPS), in contrast to the classical ER-Golgi-dependent export/secretion of proteins with a leader sequence (signal peptide), employs multiple means to release leaderless cargoes (and in some special cases, cargoes with a leader sequence) to the extracellular space. By far, two major types of UPE have been classified, vesicle-independent UPE and vesicle-dependent UPE. In the former, UPE cargoes can directly translocate across the plasma membrane from the cytoplasm without the assistance of a vesicle carrier. In the latter, UPE cargoes translocate into the lumen of a vesicle which then delivers them out of the cell through membrane trafficking. Both types of UPE require multiple unconventional solutions to complete secretion. Here, we briefly discuss the multiple strategies for a UPE cargo release, focusing on two key steps of leaderless cargoes release in UPE: protein translocation and membrane trafficking.

Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1β) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1β) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1β is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1β processing in these cells. We found that although caspase-1 is required for IL-1β secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1β processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1β secretion.

Intracellular membrane protein trafficking is crucial for both normal cellular physiology and cell-cell communication. The conventional secretory route follows transport from the Endoplasmic reticulum (ER) to the plasma membrane via the Golgi apparatus. Alternative modes of secretion which can bypass the need for passage through the Golgi apparatus have been collectively termed as Unconventional protein secretion (UPS). UPS can comprise of cargo without a signal peptide or proteins which escape the Golgi in spite of entering the ER. UPS has been classified further depending on the mode of transport. Type I and Type II unconventional secretion are non-vesicular and non-SNARE protein dependent whereas Type III and Type IV dependent on vesicles and on SNARE proteins. In this review, we focus on the Type III UPS which involves the import of cytoplasmic proteins in membrane carriers of autophagosomal/endosomal origin and release in the extracellular space following SNARE-dependent intracellular membrane fusion. We discuss the role of vesicular SNAREs with a strong focus on VAMP7, a vesicular SNARE involved in exosome, lysosome and autophagy mediated secretion. We further extend our discussion to the role of unconventional secretion in health and disease with emphasis on cancer and neurodegeneration.

Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives controlled at 5%, and are also able to classify the limited number of established plant LSPs (SPLpred (3/4, LSPpred 4/4). Until such time as a larger set of bona fide (independently experimentally validated) LSPs is established using imaging technologies (light/fluorescence/electron microscopy) to confirm sub-cellular location, these tools represent a bridging method for predicting and identifying plant putative LSPs for subsequent experimental validation.

Heat shock proteins (HSPs) are highly conserved stress proteins known as molecular chaperones, which are considered to be cytoplasmic proteins with functions restricted to the intracellular compartment, such as the cytoplasm or cellular organelles. However, an increasing number of observations have shown that HSPs can also be released into the extracellular matrix and can play important roles in the modulation of inflammation and immune responses. Recent studies have demonstrated that extracellular HSPs (eHSPs) were involved in many human diseases, such as cancers, neurodegenerative diseases, and kidney diseases, which are all diseases that are closely linked to inflammation and immunity. In this review, we describe the types of eHSPs, discuss the mechanisms of eHSPs secretion, and then highlight their functions in the modulation of inflammation and immune responses. Finally, we take cancer as an example and discuss the possibility of targeting eHSPs for human disease therapy. A broader understanding of the function of eHSPs in development and progression of human disease is essential for developing new strategies to treat many human diseases that are critically related to inflammation and immunity. [ABSTRACT FROM AUTHOR]

Several proteins are secreted outside the cell, and in many cases, they may be identified by a characteristic signal peptide. However, more and more studies point to the evidence for an “unconventional” secretion, where proteins without a hitherto unknown signal are secreted, possibly in conditions of starvation. In this work, we analyse a set of 202 RNA binding mammalian proteins, whose unconventional secretion has recently been established. Analysis of these proteins secreted by LC3 mediation, the largest unconventionally secreted dataset to our knowledge, identifies the role of KKX motif as well as triacidic amino acid motif in unconventional secretion, the latter being an extension of the recent implicated diacidic amino acid motif. Further data analysis evolves a hypothesis on the sequence or structural proximity of the triacidic or KKX motifs to the LC3 interacting region, and a phosphorylatable amino acid such as serine as a statistically significant feature among these unconventionally secreted proteins. This hypothesis, although needs to be validated in experiments that challenge the specific details of each of these aspects, appears to be one of the early steps in defining what may be a plausible signal for unconventional protein secretion.

In eukaryotes, many proteins contain an N-terminal signal peptide that allows their translocation into the endoplasmic reticulum followed by secretion outside the cell according to the classical secretory system. However, an increasing number of secreted proteins lacking the signal peptide sequence are emerging. These proteins, secreted in several alternative ways collectively known as unconventional protein secretion (UPS) pathways, exert extracellular functions including cell signaling, immune modulation, as well as moonlighting activities different from their well-described intracellular functions. Pathways for UPS include direct transfer across the plasma membrane, secretion from endosomal/multivesicular body-related components, release within plasma membrane-derived microvesicles, or use of elements of autophagy. In this review we describe the mammals and plants UPS pathways identified so far highlighting commonalities and differences.

Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that belongs to a subgroup of extracellular proteins lacking N-terminal signal peptides. Whereas this phenomenon was already recognized in the early 1990s, detailed insights into the molecular mechanisms underlying alternative pathways of protein secretion from eukaryotic cells were obtained only recently. Today, we know about a number of alternative secretory mechanisms, collectively termed unconventional protein secretion (UPS). FGF2 belongs to a subgroup of cargo proteins secreted by direct translocation across the plasma membrane. This feature has been classified as type I UPS and is shared with other unconventionally secreted proteins, such as HIV-Tat and Tau. FGF2 translocation across the membrane is initiated through sequential interactions with the Na,K-ATPase, Tec kinase, and phosphoinositide PI(4,5)P2 at the inner plasma membrane leaflet. Whereas the first two are auxiliary factors of this pathway, the interaction of FGF2 with PI(4,5)P2 triggers the core mechanism of FGF2 membrane translocation. It is based on a lipidic membrane pore that is formed by PI(4,5)P2-induced oligomerization of FGF2. Membrane-inserted FGF2 oligomers are recognized as translocation intermediates that are resolved at the outer plasma membrane leaflet by glypican-1, a heparan sulfate proteoglycan that captures and disassembles FGF2 oligomers on cell surfaces. Here, we discuss recent findings suggesting the molecular machinery mediating FGF2 membrane translocation to be highly organized in liquid-ordered plasma membrane nanodomains, the core process underlying this unusual pathway of protein secretion.

For a long time, leaderless secreted proteins (LLSP) were neglected as artifacts derived from dying cells. It is now generally accepted that secretion of LLSP–as a part of the collective term unconventional protein secretion (UPS) - is an evolutionarily conserved process and that these LLSP are actively and selectively secreted from living cells bypassing the classical endoplasmic reticulum-Golgi pathway. However, the mechanism of UPS pathways, as well as the number of LLSP and which part of a protein is involved in the selection of LLSPs for secretion, are still enigmatic and await clarification. Secretomics-a proteomics-based approach to identify and quantify all proteins secreted by a cell-is inherently unbiased toward a particular secretion pathway and offers the opportunity to shed light on the UPS. Here, we will evaluate and present recent results of proteomic workflows allowing to obtain high-confident secretome data. Additionally, we address that cell culture conditions largely affect the composition of the secretome. This has to be kept in mind to control cell culture induced artifacts and adaptation stress in serum free conditions. Evaluation of click chemistry for secretome analysis of cells under serum-containing conditions showed a significant change in the cellular proteome with longer incubation time upon treatment with non-canonical amino acid azidohomoalanine. Finally, we showed that the number of LLSP far exceeds the number of secreted proteins annotated in Uniprot and ProteinAtlas. Thus, secretomics in combination with sophisticated microbioanalytical and sample preparation methods is well suited to provide a comprehensive picture of UPS.

Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that is transported into the extracellular space by an unconventional secretory mechanism. Cell surface heparan sulfate proteoglycans are known to play an essential role in this process. Unexpectedly, we found that among the diverse subclasses consisting of syndecans, perlecans, glypicans, and others, Glypican-1 (GPC1) is the principle and rate-limiting factor that drives unconventional secretion of FGF2. By contrast, we demonstrate GPC1 to be dispensable for FGF2 signaling into cells. We provide first insights into the structural basis for GPC1-dependent FGF2 secretion, identifying disaccharides with N-linked sulfate groups to be enriched in the heparan sulfate chains of GPC1 to which FGF2 binds with high affinity. Our findings have broad implications for the role of GPC1 as a key molecule in tumor progression.

The participation of amyloids in neurodegenerative diseases and functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. The autofluorescence from the extended β-sheets of amyloids is here used to track fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS), and here its participation also in heat shock response (HSR) is suggested. Fluorescence Lifetime Imaging (FLIM) is used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress is further supported by size exclusion chromatography and ultracentrifugation. The data show for the first time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR. [ABSTRACT FROM AUTHOR]

The Golgi complex is an essential organelle of the eukaryotic exocytic pathway. A subfamily of Golgi matrix proteins, called GRASPs, is central in stress-induced unconventional secretion, Golgi dynamics during mitosis/apoptosis, and Golgi ribbon formation. The Golgi ribbon is vertebrate-specific and correlates with the appearance of two GRASP paralogues and two Golgins (GM130/Golgin45), which form specific GRASP-Golgin pairs. The molecular details of their appearance only in Metazoans are unknown. Moreover, despite new functionalities supported by GRASP paralogy, little is known about their structural and evolutionary differences. Here, we used ancestor sequence reconstruction and biophysical/biochemical approaches to assess the evolution of GRASPs structure/dynamics, fibrillation, and how they started anchoring their Golgin partners. Our data showed that a GRASP ancestor anchored Golgins before gorasp gene duplication in Metazoans. After gene duplication, variations within the GRASP binding pocket determined which paralogue would recruit which Golgin. These interactions are responsible for their specific Golgi location and Golgi ribbon appearance. We also suggest that GRASPs have a long-standing capacity to form supramolecular structures, affecting their participation in stress-induced processes. [ABSTRACT FROM AUTHOR]

The pathogenesis of Parkinson's disease (PD), which is a progressive neurodegenerative disease, is associated with the formation of protein inclusion bodies called Lewy bodies (LB) or Lewy neurites (LN). α‐Synuclein (α‐Syn) is a major component of LB and LN. The formation of LB or LN is mediated by formation of α‐Syn fibrils, which are formed from α‐Syn monomers and oligomers. Additionally, intercellular α‐Syn propagation has been proposed to be important for the progression of PD. Thus, various studies have focused on elucidating the role of α‐Syn propagation in the pathogenesis of PD. Previous studies have reported that α‐Syn species are released from the cells through various pathways, including the unconventional secretion pathways. The released α‐Syn species are internalized by the cells through multiple mechanisms, including receptor‐mediated endocytosis. Some molecular processes involved in intercellular α‐Syn propagation have been recently elucidated. This review discusses the current studies on the molecular mechanisms underlying the release and uptake of α‐Syn and their physiological relevance. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2024 The Protein Society.)

Nuclear protein HMGB1 is secreted in response to various stimuli and functions as a danger-associated molecular pattern. Extracellular HMGB1 induces inflammation, cytokine production, and immune cell recruitment via activation of various receptors. As HMGB1 does not contain an endoplasmic reticulum-targeting signal peptide, HMGB1 is secreted via the endoplasmic reticulum-Golgi independently via an unconventional secretion pathway. However, the mechanism underlying HMGB1 secretion remains largely unknown. Here, we investigated the role of secretory autophagy machinery and vesicular trafficking in HMGB1 secretion. We observed that HSP90AA1 (heat shock protein 90 alpha family class A member 1), a stress-inducible protein, regulates the translocation of HMGB1 from the nucleus to the cytoplasm and its secretion through direct interaction. Additionally, geldanamycin, an HSP90AA1 inhibitor, reduced HMGB1 secretion. GORASP2/GRASP55 (golgi reassembly stacking protein 2), ARF1Q71L (ADP ribosylation factor 1), and SAR1AT39N (secretion associated Ras related GTPase 1A), which promoted unconventional protein secretion, increased HMGB1 secretion. HMGB1 secretion was inhibited by an early autophagy inhibitor and diminished in ATG5-deficient cells even when GORASP2 was overexpressed. In contrast, a late autophagy inhibitor increased HMGB1 secretion under the same conditions. The multivesicular body formation inhibitor GW4869 dramatically decreased HMGB1 secretion under HMGB1 secretion-inducing conditions. Thus, we demonstrated that secretory autophagy and multivesicular body formation mediate HMGB1 secretion. [ABSTRACT FROM AUTHOR]

The best-known appetite-regulating factors identified in rodents are leptin, an appetite inhibitor, and ghrelin, an appetite stimulator. Rare cases of loss-of-functions mutations affecting leptin and its receptor, as well as polymorphisms concerning ghrelin and its receptor, have been documented in human obesity, apparently validating the relevance of leptin and ghrelin for human physiology. Paradoxically, however, the overwhelming majority of obese individuals manifest high leptin and low ghrelin plasma levels, suggesting that both factors are not directly disease-relevant. We recently discovered that acyl-CoA-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), acts as an efficient lipogenic and appetite stimulator in mice. Indeed, in response to starvation, ACBP/DBI is released from tissues in an autophagy-dependent fashion and increases in the plasma. Intravenous injection of ACBP/DBI stimulates feeding behavior through a reduction of circulating glucose levels, and consequent activation of orexigenic neurons in the hypothalamus. In contrast, neutralization of ACBP/DBI abolishes the hyperphagia observed after starvation of mice. Of note, ACBP/DBI is increased in the plasma of obese persons and mice, pointing to a convergence (rather than divergence) between its role in appetite stimulation and human obesity. Based on our results, we postulate a novel ‘hunger reflex’ in which starvation induces a surge in extracellular ACBP/DBI, which in turn stimulates feeding behavior. Thus, ACBP/DBI might be the elusive ‘hunger factor’ that explains increased food uptake in obesity.

Main conclusion: Secretome analysis of a salt-tolerant and control Chlamydomonas reinhardtii revealed 514 differentially expressed proteins. Membrane transport and trafficking, signal transduction and channel proteins were up-regulated in the ST secretome. Salinity is a major abiotic stress that limits crop production worldwide. Multiple adverse effects have been reported in many living organisms exposed to high-saline concentrations. Chlamydomonas reinhardtii is known for secreting proteins in response to many environmental stresses. A salinity-tolerant (ST) strain of Chlamydomonas has been developed, whose cells were able to grow at 300 mM NaCl. The current study analyzed the secretomes of ST grown in TAP medium supplemented with 300 mM NaCl and the laboratory strain CC-503 grown in TAP medium without NaCl supplement. In total, 514 secreted proteins were identified of which 203 were up-regulated and 110 were down-regulated. Bioinformatic analysis predicted 168 proteins to be secreted or in the conventional secretory pathway. Out of these, 70 were up-regulated, while 51 proteins were down-regulated. Proteins involved in membrane transport and trafficking, signal transduction and channel proteins were altered in their expression in the ST secretome, suggesting the response of saline stress acts toward not only the intracellular pool of proteins but also the extracellular proteins. This also suggested that the secreted proteins might have roles in the extracellular space. Signal peptide (SP) prediction revealed that almost 40% of the predicted secreted proteins contained a signal peptide; however, a high proportion of proteins lacked an SP, suggesting that these proteins might be secreted through an unconventional protein secretion pathway. [ABSTRACT FROM AUTHOR]

Unconventional protein secretion (UCPS) of leaderless proteins bypasses the conventional endoplasmic reticulum (ER)-Golgi route. The proportion of UCPS in the secretome varies tremendously across eukaryotes. Interestingly, macroautophagy, an intracellular recycling process that is generally involved in cargo degradation, also participates in UCPS. This emerging field of secretory mode of autophagy is underexplored and has several unanswered questions regarding the composition of players, cargo, and the mechanisms that drive it. As secretomes vary considerably across cell types and physiological conditions, the contribution of secretory autophagy in healthy and pathophysiological states remain to be elucidated. Recent studies have begun to shed light on this enigmatic process.

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 PIP 2 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., Competing Interests: Competing interests: All authors declare no competing interests.

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.

Golgi Reassembly and Stacking Proteins (GRASPs) were firstly described as crucial elements in determining the structure of the Golgi complex. However, data have been accumulating over the years showing GRASPs can participate in various cell processes beyond the Golgi maintenance, including cell adhesion and migration, autophagy and unconventional secretion of proteins. A comprehensive understanding of the GRASP functions requires deep mechanistic knowledge of its structure and dynamics, especially because of the unique structural plasticity observed for many members of this family coupled with their high promiscuity in mediating protein-protein interactions. Here, we critically review data regarding the structural biophysics of GRASPs in the quest for understanding the structural determinants of different functionalities. We dissect GRASP structure starting with the full-length protein down to its separate domains (PDZ1, PDZ2 and SPR) and outline some structural features common to all members of the GRASP family (such as the presence of many intrinsically disordered regions). Although the impact of those exquisite properties in vivo will still require further studies, it is possible, from our review, to pinpoint factors that must be considered in future interpretation of data regarding GRASP functions, thus bringing somewhat new perspectives to the field. [ABSTRACT FROM AUTHOR]

It has been demonstrated that two Golgi stacking proteins, GRASP55 and GRASP65, self-interact to form trans -oligomers that tether adjacent Golgi membranes into stacks and ribbons in mammalian cells. This ensures proper functioning of the Golgi apparatus in protein trafficking and processing. More recently, GRASP proteins have drawn extensive attention from researchers due to their diverse and essential roles in and out of the Golgi in different organisms. In this review, we summarize their established roles in Golgi structure formation and function under physiological conditions. We then highlight the emerging and divergent roles for individual GRASP proteins, focusing on GRASP65 in cell migration and apoptosis and GRASP55 in unconventional protein secretion and autophagy under stress or pathological conditions. GRASP55 and GRASP65 play complementary roles in maintaining the Golgi structure by forming trans -oligomers in mammalian cells. Depletion of GRASP55 and GRASP65 in cells impairs Golgi functions in protein trafficking, sorting and glycosylation, and reduces cell adhesion and cell migration. GRASP-interacting proteins may help GRASPs in Golgi structure formation and function. GRASP65 is the target of signaling events leading to Golgi reorientation during cell migration and breakdown during apoptosis. GRASP55 participates in unconventional protein secretion of both cytosolic and transmembrane proteins in response to certain stresses. GRASP55 facilitates autophagosome maturation under starvation conditions. [ABSTRACT FROM AUTHOR]

The progressive accumulation and spread of misfolded tau protein in the nervous system is the hallmark of tauopathies, progressive neurodegenerative diseases with only symptomatic treatments available. A growing body of evidence suggests that spreading of tau pathology can occur via cell-to-cell transfer involving secretion and internalization of pathological forms of tau protein followed by templated misfolding of normal tau in recipient cells. Several studies have addressed the cell biological mechanisms of tau secretion. It now appears that instead of a single mechanism, cells can secrete tau via three coexisting pathways: (1) translocation through the plasma membrane; (2) membranous organelles-based secretion; and (3) ectosomal shedding. The relative importance of these pathways in the secretion of normal and pathological tau is still elusive, though. Moreover, glial cells contribute to tau propagation, and the involvement of different cell types, as well as different secretion pathways, complicates the understanding of prion-like propagation of tauopathy. One of the important regulators of tau secretion in neuronal activity, but its mechanistic connection to tau secretion remains unclear and may involve all three secretion pathways of tau. This review article summarizes recent advancements in the field of tau secretion with an emphasis on cell biological aspects of the secretion process and discusses the role of neuronal activity and glial cells in the spread of pathological forms of tau.

Proteins to be secreted through so-called “conventional mechanisms” are characterized by the presence of an N-terminal peptide that is a leader or signal peptide, needed for access to the endoplasmic reticulum and the Golgi apparatus for further secretion. However, some relevant cytosolic proteins lack of this signal peptides and should be secreted by different unconventional or “non-canonical” processes. One form of this unconventional secretion was named secretory autophagy (SA) because it is specifically associated with the autophagy pathway. It is defined by ATG proteins that regulate the biogenesis of the autophagosome, its representative organelle. The canonical macroautophagy involves the fusion of the autophagosomes with lysosomes for content degradation, whereas the SA pathway bypasses this degradative process to allow the secretion. ATG5, as well as other factors involved in autophagy such as BCN1, are also activated as part of the secretory pathway. SA has been recognized as a new mechanism that is becoming of increasing relevance to explain the unconventional secretion of a series of cytosolic proteins that have critical biological importance. Also, SA may play a role in the release of aggregation-prone protein since it has been related to the autophagosome biogenesis machinery. SA requires the autophagic pathway and both, secretory autophagy and canonical degradative autophagy are at the same time, integrated and highly regulated processes that interact in ultimate cross-talking molecular mechanisms. The potential implications of alterations in SA, its cargos, pathways, and regulation in human diseases such as metabolic/aging pathological processes are predictable. Further research of SA as potential target of therapeutic intervention is deserved.

Fibroblast growth factor 2 (FGF2) and interleukin 1β (IL-1β) were among the earliest examples of a subclass of proteins with extracellular functions that were found to lack N-terminal secretory signal peptides and were shown to be secreted in an ER- and Golgi-independent manner. Many years later, a number of alternative secretory pathways have been discovered, processes collectively termed unconventional protein secretion (UPS). In the course of these studies, unconventional secretion of FGF2 and IL-1β were found to be based upon distinct pathways, mechanisms and molecular machineries. Following a concise introduction into various pathways mediating unconventional secretion and transcellular spreading of proteins, this Cell Science at a Glance poster article aims at a focused analysis of recent key discoveries providing unprecedented detail about the molecular mechanisms and machineries driving FGF2 and IL-1β secretion. These findings are also highly relevant for other unconventionally secreted cargoes that, like FGF2 and IL1β, exert fundamental biological functions in biomedically relevant processes, such as tumor-induced angiogenesis and inflammation. [ABSTRACT FROM AUTHOR]

The progressive accumulation and spread of misfolded tau protein in the nervous system is the hallmark of tauopathies, progressive neurodegenerative diseases with only symptomatic treatments available. A growing body of evidence suggests that spreading of tau pathology can occur via cell-to-cell transfer involving secretion and internalization of pathological forms of tau protein followed by templated misfolding of normal tau in recipient cells. Several studies have addressed the cell biological mechanisms of tau secretion. It now appears that instead of a single mechanism, cells can secrete tau via three coexisting pathways: (1) translocation through the plasma membrane; (2) membranous organelles-based secretion; and (3) ectosomal shedding. The relative importance of these pathways in the secretion of normal and pathological tau is still elusive, though. Moreover, glial cells contribute to tau propagation, and the involvement of different cell types, as well as different secretion pathways, complicates the understanding of prion-like propagation of tauopathy. One of the important regulators of tau secretion in neuronal activity, but its mechanistic connection to tau secretion remains unclear and may involve all three secretion pathways of tau. This review article summarizes recent advancements in the field of tau secretion with an emphasis on cell biological aspects of the secretion process and discusses the role of neuronal activity and glial cells in the spread of pathological forms of tau. [ABSTRACT FROM AUTHOR]

Background: The secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight β-glycosidases in a comparative cultivation approach. Results: A low sized β-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized β-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR–Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkatoNPGal/Lmedium, while a volumetric activity of 2.98 µkatoNPGal/Lmedium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica. Conclusion: The results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further. [ABSTRACT FROM AUTHOR]

Proteins to be secreted through so-called "conventional mechanisms" are characterized by the presence of an N-terminal peptide that is a leader or signal peptide, needed for access to the endoplasmic reticulum and the Golgi apparatus for further secretion. However, some relevant cytosolic proteins lack of this signal peptides and should be secreted by different unconventional or "non-canonical" processes. One form of this unconventional secretion was named secretory autophagy (SA) because it is specifically associated with the autophagy pathway. It is defined by ATG proteins that regulate the biogenesis of the autophagosome, its representative organelle. The canonical macroautophagy involves the fusion of the autophagosomes with lysosomes for content degradation, whereas the SA pathway bypasses this degradative process to allow the secretion. ATG5, as well as other factors involved in autophagy such as BCN1, are also activated as part of the secretory pathway. SA has been recognized as a new mechanism that is becoming of increasing relevance to explain the unconventional secretion of a series of cytosolic proteins that have critical biological importance. Also, SA may play a role in the release of aggregation-prone protein since it has been related to the autophagosome biogenesis machinery. SA requires the autophagic pathway and both, secretory autophagy and canonical degradative autophagy are at the same time, integrated and highly regulated processes that interact in ultimate cross-talking molecular mechanisms. The potential implications of alterations in SA, its cargos, pathways, and regulation in human diseases such as metabolic/aging pathological processes are predictable. Further research of SA as potential target of therapeutic intervention is deserved. [ABSTRACT FROM AUTHOR]

Accumulation of misfolded and aggregated forms of tau protein in the brain is a neuropathological hallmark of tauopathies, such as Alzheimer's disease and frontotemporal lobar degeneration. Tau aggregates have the ability to transfer from one cell to another and to induce templated misfolding and aggregation of healthy tau molecules in previously healthy cells, thereby propagating tau pathology across different brain areas in a prion-like manner. The molecular mechanisms involved in cell-to-cell transfer of tau aggregates are diverse, not mutually exclusive and only partially understood. Intracellular accumulation of misfolded tau induces several mechanisms that aim to reduce the cellular burden of aggregated proteins and also promote secretion of tau aggregates. However, tau may also be released from cells physiologically unrelated to protein aggregation. Tau secretion involves multiple vesicular and non-vesicle-mediated pathways, including secretion directly through the plasma membrane. Consequently, extracellular tau can be found in various forms, both as a free protein and in vesicles, such as exosomes and ectosomes. Once in the extracellular space, tau aggregates can be internalized by neighboring cells, both neurons and glial cells, via endocytic, pinocytic and phagocytic mechanisms. Importantly, accumulating evidence suggests that prion-like propagation of misfolding protein pathology could provide a general mechanism for disease progression in tauopathies and other related neurodegenerative diseases. Here, we review the recent literature on cellular mechanisms involved in cell-to-cell transfer of tau, with a particular focus in tau secretion. [ABSTRACT FROM AUTHOR]