RESUMEN
Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.
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Acetilcolina , Cloruros , Células Epiteliales , Mucosa Intestinal , Animales , Acetilcolina/metabolismo , Ratones , Cloruros/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Células Epiteliales/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitología , Intestino Delgado/inmunología , Intestino Delgado/parasitología , Intestino Delgado/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Células en PenachoRESUMEN
Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.
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Microscopía por Crioelectrón , Fimbrias Bacterianas , Sistemas de Secreción Tipo IV , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/genética , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/química , Modelos Moleculares , Conjugación Genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Escherichia coli/metabolismo , Escherichia coli/genética , Conformación ProteicaRESUMEN
The evolutionary conserved YopJ family comprises numerous type-III-secretion system (T3SS) effectors of diverse mammalian and plant pathogens that acetylate host proteins to dampen immune responses. Acetylation is mediated by a central acetyltransferase domain that is flanked by conserved regulatory sequences, while a nonconserved N-terminal extension encodes the T3SS-specific translocation signal. Bartonella spp. are facultative-intracellular pathogens causing intraerythrocytic bacteremia in their mammalian reservoirs and diverse disease manifestations in incidentally infected humans. Bartonellae do not encode a T3SS, but most species possess a type-IV-secretion system (T4SS) to translocate Bartonella effector proteins (Beps) into host cells. Here we report that the YopJ homologs present in Bartonellae species represent genuine T4SS effectors. Like YopJ family T3SS effectors of mammalian pathogens, the "Bartonella YopJ-like effector A" (ByeA) of Bartonella taylorii also targets MAP kinase signaling to dampen proinflammatory responses, however, translocation depends on a functional T4SS. A split NanoLuc luciferase-based translocation assay identified sequences required for T4SS-dependent translocation in conserved regulatory regions at the C-terminus and proximal to the N-terminus of ByeA. The T3SS effectors YopP from Yersinia enterocolitica and AvrA from Salmonella Typhimurium were also translocated via the Bartonella T4SS, while ByeA was not translocated via the Yersinia T3SS. Our data suggest that YopJ family T3SS effectors may have evolved from an ancestral T4SS effector, such as ByeA of Bartonella. In this evolutionary scenario, the signal for T4SS-dependent translocation encoded by N- and C-terminal sequences remained functional in the derived T3SS effectors due to the essential role these sequences coincidentally play in regulating acetyltransferase activity.
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Proteínas Bacterianas , Bartonella , Sistemas de Secreción Tipo IV , Bartonella/metabolismo , Bartonella/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Humanos , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/genética , Transporte de Proteínas , AnimalesRESUMEN
The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.
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Receptores de Antígenos de Linfocitos T , Linfocitos T , Péptidos/química , Antígenos de Histocompatibilidad/química , AntígenosRESUMEN
Newly synthesized secretory proteins are exported from the endoplasmic reticulum (ER) at specialized subcompartments called exit sites (ERES). Cargoes like procollagen are too large for export by the standard COPII-coated vesicle of 60 nm average diameter. We have previously suggested that procollagen is transported from the ER to the next secretory organelle, the ER-Golgi intermediate compartment (ERGIC), in TANGO1-dependent interorganelle tunnels. In the theoretical model presented here, we suggest that intrinsically disordered domains of TANGO1 in the ER lumen induce an entropic contraction, which exerts a force that draws procollagen toward the ERES. Within this framework, molecular gradients of pH and/or HSP47 between the ER and ERGIC create a force in the order of tens of femto-Newtons. This force is substantial enough to propel procollagen from the ER at a speed of approximately 1 nm · s-1. This calculated speed and the quantities of collagen secreted are similar to its observed physiological secretion rate in fibroblasts, consistent with the proposal that ER export is the rate-limiting step for procollagen secretion. Hence, the mechanism we propose is theoretically adequate to explain how cells can utilize molecular gradients and export procollagens at a rate commensurate with physiological needs.
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Colágeno , Procolágeno , Procolágeno/metabolismo , Transporte de Proteínas/fisiología , Colágeno/metabolismo , Transporte Biológico , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismoRESUMEN
Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.
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Cisteína , Mucinas , Mucinas/metabolismo , Cisteína/metabolismo , Transporte Biológico , Vesículas Secretoras/metabolismoRESUMEN
Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.
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Aurodox , Sistemas de Secreción Tipo III , Sistemas de Secreción Tipo III/metabolismo , Aurodox/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Gramnegativas/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.
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Saltamontes , Toxinas Biológicas , Sistemas de Secreción Tipo VII , Animales , Sistemas de Secreción Tipo VII/genética , CitoplasmaRESUMEN
Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.
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Pliegue de Proteína , Enfermedades Raras , Animales , Humanos , Enfermedades Raras/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Retículo Endoplásmico/metabolismo , Mutación , Mamíferos/metabolismo , Glucosiltransferasas/metabolismoRESUMEN
The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.
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Proteínas de Drosophila , Exocitosis , Miosina Tipo II , Proteínas de Unión al GTP rho , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Miosina Tipo II/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genética , Exocitosis/fisiología , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Larva/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/citología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Vesículas Secretoras/metabolismoRESUMEN
Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.
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Neuronas , Vesículas Sinápticas , Ratas , Animales , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Exocitosis/fisiología , Vesículas Secretoras/metabolismo , Estrés del Retículo EndoplásmicoRESUMEN
Many bacteria kill rival species by translocating toxic effectors into target cells. Effectors are often encoded along with cognate immunity proteins that could (i) protect against "friendly-fire" (trans-intoxication) from neighboring sister cells and/or (ii) protect against internal cis-intoxication (suicide). Here, we distinguish between these two mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS). We use a set of X. citri mutants lacking multiple effector/immunity protein (X-Tfe/X-Tfi) pairs to show that X-Tfis are not absolutely required to protect against trans-intoxication by wild-type cells. Our investigation then focused on the in vivo function of the lysozyme-like effector X-TfeXAC2609 and its cognate immunity protein X-TfiXAC2610. In the absence of X-TfiXAC2610, we observe X-TfeXAC2609-dependent and X-T4SS-independent accumulation of damage in the X. citri cell envelope, cell death, and inhibition of biofilm formation. While immunity proteins in other systems have been shown to protect against attacks by sister cells (trans-intoxication), this is an example of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication.
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Proteínas Bacterianas , Xanthomonas , Humanos , Proteínas Bacterianas/metabolismo , Xanthomonas/metabolismo , Sistemas de Secreción Tipo IV/metabolismo , Antibacterianos/metabolismoRESUMEN
Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.
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Proteoma , Trombina , Proteoma/metabolismo , Trombina/farmacología , Trombina/metabolismo , Glicosilación , Plaquetas/metabolismo , Activación PlaquetariaRESUMEN
The hormone leptin, primarily secreted by adipocytes, plays a crucial role in regulating whole-body energy homeostasis. Homozygous loss-of-function mutations in the leptin gene (LEP) cause hyperphagia and severe obesity, primarily through alterations in leptin's affinity for its receptor or changes in serum leptin concentrations. Although serum concentrations are influenced by various factors (e.g., gene expression, protein synthesis, stability in the serum), proper delivery of leptin from its site of synthesis in the endoplasmic reticulum via the secretory pathway to the extracellular serum is a critical step. However, the regulatory mechanisms and specific machinery involved in this trafficking route, particularly in the context of human LEP mutations, remain largely unexplored. We have employed the Retention Using Selective Hooks system to elucidate the secretory pathway of leptin. We have refined this system into a medium-throughput assay for examining the pathophysiology of a range of obesity-associated LEP variants. Our results reveal that leptin follows the default secretory pathway, with no additional regulatory steps identified prior to secretion. Through screening of leptin variants, we identified three mutations that lead to proteasomal degradation of leptin and one variant that significantly decreased leptin secretion, likely through aberrant disulfide bond formation. These observations have identified novel pathogenic effects of leptin variants, which can be informative for therapeutics and diagnostics. Finally, our novel quantitative screening platform can be adapted for other secreted proteins.
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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.
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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.
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Autofagia , Neuronas , Proteína Sequestosoma-1 , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Neuronas/metabolismo , Ratones , Humanos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Ácido Glutámico/metabolismo , Beclina-1/metabolismo , Beclina-1/genética , Calcio/metabolismo , Línea Celular Tumoral , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Sirolimus/farmacologíaRESUMEN
The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors, like coat protein complexes (COPI and COPII), clathrin, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and GRASP55 (Golgi reassembly stacking protein of 55 kDa) that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders.
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Clatrina , Proteínas SNARE , Humanos , Acetilglucosamina/metabolismo , Clatrina/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas/fisiología , Proteínas SNARE/metabolismo , Animales , Acetilación , Glucosa/metabolismoRESUMEN
The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.
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Aminoaciltransferasas , Proteínas Bacterianas , Cisteína Endopeptidasas , Proteínas Fimbrias , Fimbrias Bacterianas , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/química , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Cristalografía por Rayos X , Actinomyces/metabolismo , Actinomyces/enzimología , Especificidad por Sustrato , Modelos MolecularesRESUMEN
Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing â¼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein IpaD. Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.
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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.