A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion.

Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.

Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment.

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.

Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

Cargo Capture and Bulk Flow in the Early Secretory Pathway.

Transport of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi complex is highly selective. As a general rule, such transport is limited to soluble and membrane-associated secretory proteins that have reached properly folded and assembled conformations. To secure the efficiency, fidelity, and control of this crucial transport step, cells use a combination of mechanisms. The mechanisms are based on selective retention of proteins in the ER to prevent uptake into transport vesicles, on selective capture of proteins in COPII carrier vesicles, on inclusion of proteins in these vesicles by default as part of fluid and membrane bulk flow, and on selective retrieval of proteins from post-ER compartments by retrograde vesicle transport.

An ADP-ribosyltransferase toxin kills bacterial cells by modifying structured non-coding RNAs.

ADP-ribosyltransferases (ARTs) were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they modify essential proteins, thereby inactivating cellular processes and promoting pathogenesis. Our understanding of ARTs has since expanded beyond protein-targeting toxins to include antibiotic inactivation and DNA damage repair. Here, we report the discovery of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. A structure of RhsP2 reveals that it resembles protein-targeting ARTs such as diphtheria toxin. Remarkably, however, RhsP2 ADP-ribosylates 2'-hydroxyl groups of double-stranded RNA, and thus, its activity is highly promiscuous with identified cellular targets including the tRNA pool and the RNA-processing ribozyme, ribonuclease P. Consequently, cell death arises from the inhibition of translation and disruption of tRNA processing. Overall, our data demonstrate a previously undescribed mechanism of bacterial antagonism and uncover an unprecedented activity catalyzed by ART enzymes.

The pathway of collagen secretion.

COPII vesicles mediate export of secretory cargo from the endoplasmic reticulum (ER). However, a standard COPII vesicle with a diameter of 60-90 nm is too small to export collagens that are composed of rigid triple helices of up to 400 nm in length. How do cells pack and secrete such bulky molecules? This issue is fundamentally important, as collagens constitute approximately 25% of our dry body weight and are essential for almost all cell-cell interactions. Recently, a potential mechanism for the biogenesis of mega-transport carriers was identified, involving packing collagens and increasing the size of COPII coats. Packing is mediated by TANGO1, which binds procollagen VII in the lumen and interacts with the COPII proteins Sec23/Sec24 on the cytoplasmic side of the ER. Cullin3, an E3 ligase, and its specific adaptor protein, KLHL12, ubiquitinate Sec31, which could increase the size of COPII coats. Recruitment of these proteins and their specific interactors into COPII-mediated vesicle biogenesis may be all that is needed for the export of bulky collagens from the ER. Nonetheless, we present an alternative pathway in which TANGO1 and COPII cooperate to export collagens without generating a mega-transport carrier.

Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage.

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. Molecular dynamics simulations indicate that this unique architecture generates specificity for SPs based on the length of their hydrophobic segments.

An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress.

Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.

Unconventional secretion mediated by direct protein self-translocation across the plasma membranes of mammalian cells.

In recent years, a surprisingly complex picture emerged about endoplasmic reticulum (ER)/Golgi-independent secretory pathways, and several routes have been discovered that differ with regard to their molecular mechanisms and machineries. Fibroblast growth factor 2 (FGF2) is secreted by a pathway of unconventional protein secretion (UPS) that is based on direct self-translocation across the plasma membrane. Building on previous research, a component of this process has been identified to be glypican-1 (GPC1), a GPI-anchored heparan sulfate proteoglycan located on cell surfaces. These findings not only shed light on the molecular mechanism underlying this process but also reveal an intimate relationship between FGF2 and GPC1 that might be of critical relevance for the prominent roles they both have in tumor progression and metastasis.

The Type VII Secretion System of Staphylococcus.

The type VII protein secretion system (T7SS) of Staphylococcus aureus is encoded at the ess locus. T7 substrate recognition and protein transport are mediated by EssC, a membrane-bound multidomain ATPase. Four EssC sequence variants have been identified across S. aureus strains, each accompanied by a specific suite of substrate proteins. The ess genes are upregulated during persistent infection, and the secretion system contributes to virulence in disease models. It also plays a key role in intraspecies competition, secreting nuclease and membrane-depolarizing toxins that inhibit the growth of strains lacking neutralizing immunity proteins. A genomic survey indicates that the T7SS is widely conserved across staphylococci and is encoded in clusters that contain diverse arrays of toxin and immunity genes. The presence of genomic islands encoding multiple immunity proteins in species such as Staphylococcus warneri that lack the T7SS points to a major role for the secretion system in bacterial antagonism.

Neuronal activity promotes secretory autophagy for the extracellular release of α-synuclein.

Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1+/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca2+-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.

Unveiling defects of secretion mechanisms in Parkinson's disease.

Neurodegenerative diseases are characterized by progressive dysfunction and loss of specific sets of neurons. While extensive research has focused on elucidating the genetic and epigenetic factors and molecular mechanisms underlying these disorders, emerging evidence highlights the critical role of secretion in the pathogenesis, possibly even onset, and progression of neurodegenerative diseases, suggesting the occurrence of non-cell-autonomous mechanisms. Secretion is a fundamental process that regulates intercellular communication, supports cellular homeostasis, and orchestrates various physiological functions in the body. Defective secretion can impair the release of neurotransmitters and other signaling molecules, disrupting synaptic transmission and compromising neuronal survival. It can also contribute to the accumulation, misfolding, and aggregation of disease-associated proteins, leading to neurotoxicity and neuronal dysfunction. In this review, we discuss the implications of defective secretion in the context of Parkinson's disease, emphasizing its role in protein aggregation, synaptic dysfunction, extracellular vesicle secretion, and neuroinflammation. We propose a multiple-hit model whereby protein accumulation and secretory defects must be combined for the onset and progression of the disease.

Transport of protein disulfide isomerase from the endoplasmic reticulum to the extracellular space without passage through the Golgi complex.

Protein disulfide isomerase-A1 (PDIA1) is a master regulator of oxidative protein folding and proteostasis in the endoplasmic reticulum (ER). However, PDIA1 can reach the extracellular space, impacting thrombosis and other pathophysiological phenomena. Whether PDIA1 is externalized via passive release or active secretion is not known. To investigate how PDIA1 negotiates its export, we generated a tagged variant that undergoes N-glycosylation in the ER (Glyco-PDIA1). Addition of N-glycans does not alter its enzymatic functions. Upon either deletion of its KDEL ER-localization motif or silencing of KDEL receptors, Glyco-PDIA1 acquires complex glycans in the Golgi and is secreted. In control cells, however, Glyco-PDIA1 is released with endoglycosidase-H sensitive glycans, implying that it does not follow the classical ER-Golgi route nor does it encounter glycanases in the cytosol. Extracellular Glyco-PDIA1 is more abundant than actin, lactate dehydrogenase, or other proteins released by damaged or dead cells, suggesting active transport through a Golgi-independent route. The strategy we describe herein can be extended to dissect how select ER-residents reach the extracellular space.

Cyclic AMP binding to a universal stress protein in Mycobacterium tuberculosis is essential for viability.

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP.

Distinct role of ERp57 and ERdj5 as a disulfide isomerase and reductase during ER protein folding.

Proteins entering the secretory pathway need to attain native disulfide pairings to fold correctly. For proteins with complex disulfides, this process requires the reduction and isomerisation of non-native disulfides. Two key members of the protein disulfide isomerase (PDI) family, ERp57 and ERdj5 (also known as PDIA3 and DNAJC10, respectively), are thought to be required for correct disulfide formation but it is unknown whether they act as a reductase, an isomerase or both. In addition, it is unclear how reducing equivalents are channelled through PDI family members to substrate proteins. Here, we show that neither enzyme is required for disulfide formation, but ERp57 is required for isomerisation of non-native disulfides within glycoproteins. In addition, alternative PDIs compensate for the absence of ERp57 to isomerise glycoprotein disulfides, but only in the presence of a robust reductive pathway. ERdj5 is required for this alternative pathway to function efficiently indicating its role as a reductase. Our results define the essential cellular functions of two PDIs, highlighting a distinction between formation, reduction and isomerisation of disulfide bonds.

Uncovering key steps in FGF12 cellular release reveals a common mechanism for unconventional FGF protein secretion.

FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which until recently were thought to be non-signaling intracellular proteins. Our recent studies have shown that although they lack a conventional signal peptide for secretion, they can reach the extracellular space, especially under stress conditions. Here, we unraveled that the long "a" isoform of FGF12 is secreted in a pathway involving the A1 subunit of Na(+)/K(+) ATPase (ATP1A1), Tec kinase and lipids such as phosphatidylinositol and phosphatidylserine. Further, we showed that the short "b" isoform of FGF12, which binds ATP1A1 and phosphatidylserine less efficiently, is not secreted from cells. We also indicated regions in the FGF12a protein sequence that are crucial for its secretion, including N-terminal fragment and specific residues, and proposed that liquid-liquid phase separation may be important in this process. Our results strongly suggest that the mechanism of this process is very similar for all unconventionally secreted FGF proteins.

The sorting platform in the type III secretion pathway: From assembly to function.

The type III secretion system (T3SS) is a specialized nanomachine that enables bacteria to secrete proteins in a specific order and directly deliver a specific set of them, collectively known as effectors, into eukaryotic organisms. The core structure of the T3SS is a syringe-like apparatus composed of multiple building blocks, including both membrane-associated and soluble proteins. The cytosolic components organize together in a chamber-like structure known as the sorting platform (SP), responsible for recruiting, sorting, and initiating the substrates destined to engage this secretion pathway. In this article, we provide an overview of recent findings on the SP's structure and function, with a particular focus on its assembly pathway. Furthermore, we discuss the molecular mechanisms behind the recruitment and hierarchical sorting of substrates by this cytosolic complex. Overall, the T3SS is a highly specialized and complex system that requires precise coordination to function properly. A deeper understanding of how the SP orchestrates T3S could enhance our comprehension of this complex nanomachine, which is central to the host-pathogen interface, and could aid in the development of novel strategies to fight bacterial infections.

Assembly and architecture of the type III secretion sorting platform.

Type III secretion systems are bacterial nanomachines specialized in protein delivery into target eukaryotic cells. The structural and functional complexity of these machines demands highly coordinated mechanisms for their assembly and operation. The sorting platform is a critical component of type III secretion machines that ensures the timely engagement and secretion of proteins destined to travel this export pathway. However, the mechanisms that lead to the assembly of this multicomponent structure have not been elucidated. Herein, employing an extensive in vivo cross-linking strategy aided by structure modeling, we provide a detailed intersubunit contact survey of the entire sorting platform complex. Using the identified cross-links as signatures for pairwise intersubunit interactions in combination with systematic genetic deletions, we mapped the assembly process of this unique bacterial structure. Insights generated by this study could serve as the bases for the rational development of antivirulence strategies to combat several medically important bacterial pathogens.

Proteo-genetic analysis reveals clear hierarchy of ESX-1 secretion in Mycobacterium marinum.

The ESX-1 (ESAT-6-system-1) system and the protein substrates it transports are essential for mycobacterial pathogenesis. The precise ways that ESX-1 substrates contribute to virulence remains unknown. Several known ESX-1 substrates are also required for the secretion of other proteins. We used a proteo-genetic approach to construct high-resolution dependency relationships for the roles of individual ESX-1 substrates in secretion and virulence in Mycobacterium marinum, a pathogen of humans and animals. Characterizing a collection of M. marinum strains with in-frame deletions in each of the known ESX-1 substrate genes and the corresponding complementation strains, we demonstrate that ESX-1 substrates are differentially required for ESX-1 activity and for virulence. Using isobaric-tagged proteomics, we quantified the degree of requirement of each substrate on protein secretion. We conclusively defined distinct contributions of ESX-1 substrates in protein secretion. Our data reveal a hierarchy of ESX-1 substrate secretion, which supports a model for the composition of the extracytoplasmic ESX-1 secretory machinery. Overall, our proteo-genetic analysis demonstrates discrete roles for ESX-1 substrates in ESX-1 function and secretion in M. marinum.

Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.