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1.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32725137

RESUMO

Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. During the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. Therefore, secretory pathway-mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. Here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of SARS-CoV-2 and their relevance for potential antiviral targeting. The fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for SARS-CoV-2 biology that could also be targeted with therapeutic objectives. Finally, we provide an overview of recent evidence implicating proteins of the early secretory pathway as potential antiviral targets with effective therapeutic applications.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Via Secretória/fisiologia , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Via Secretória/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
2.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G74-G86, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538138

RESUMO

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the ß-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.


Assuntos
Células Acinares/metabolismo , Movimento Celular/fisiologia , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Amilases/metabolismo , Animais , Células Cultivadas , Exocitose/fisiologia , Glândula Parótida/metabolismo
3.
Ann Biol Clin (Paris) ; 78(3): 231-242, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32540812

RESUMO

The identification of leptin allowed the discovery of a new endocrine system. This major adipokine controlling energy homeostasis is also involved in the regulation of neuroendocrine function and fertility. Unfortunately, leptin is not able to treat common obesity, which associates hyperleptinemia and resistance to the hormone. Conversely, treatment with recombinant leptin is effective in situations of leptin deficiency. Several pathophysiological situations associated with adipose tissue dysfunctions and abnormal regulation of leptin secretion are discussed in this review. The advantage of the potential use of the leptin assay in some pathophysiological conditions is proposed.


Assuntos
Leptina/fisiologia , Adipocinas/fisiologia , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiopatologia , Animais , Homeostase/fisiologia , Humanos , Obesidade/metabolismo , Obesidade/fisiopatologia , Via Secretória/fisiologia
4.
J Biotechnol ; 312: 11-22, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32114154

RESUMO

An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.


Assuntos
Células CHO , Engenharia Celular/métodos , Engenharia Genética/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO/metabolismo , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Via Secretória/genética , Via Secretória/fisiologia
5.
J Mol Histol ; 51(1): 99-107, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32095972

RESUMO

Tooth formation is accomplished under strict genetic control procedures. Therefore, exploring the gene network system of tooth development has a very positive practical significance for the study of tooth tissue regeneration and the prevention and treatment of tooth abnormalities. Early bell stage is the initial phase of odontoblast formation and dentin matrix deposition in the process of tooth development. Through RNA sequencing and differential gene analysis of the rat tooth germ samples at cap stage and early bell stage, we found that the bile secretion pathway was the most significant difference signal pathway during the development between cap stage and bell stage, which mainly included ABCC3, AQP4, SLC10A1, SLC2A1, SLC4A4, ADCY5, AQP9, CFTR, ATP1A2, ATP1B1 and ATP1A1, totally 11genes. Immunostaining revealed that SLC2A1, SLC4A4, ADCY5 and ATP1B1were mainly expressed in epithelium in bud stage and inner and outer enamel epithelium during the embryonic phase. In the postnatal 1 and postnatal 7, SLC2A1, SLC4A4 and ABCC3 were highly expressed in ameloblasts and odontoblasts while ADCY5, ATP1B1 and SLC10A1was expressed moderately only in odontoblasts. This finding illustrated that the bile secretion pathway related genes may participate in the development of tooth germ.


Assuntos
Bile , Proteínas de Transporte/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Odontogênese , Via Secretória/fisiologia , Germe de Dente/embriologia , Animais , Ratos , Ratos Sprague-Dawley , Germe de Dente/citologia
6.
PLoS Biol ; 18(1): e3000599, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31945054

RESUMO

The senescence-associated secretory phenotype (SASP) has recently emerged as a driver of and promising therapeutic target for multiple age-related conditions, ranging from neurodegeneration to cancer. The complexity of the SASP, typically assessed by a few dozen secreted proteins, has been greatly underestimated, and a small set of factors cannot explain the diverse phenotypes it produces in vivo. Here, we present the "SASP Atlas," a comprehensive proteomic database of soluble proteins and exosomal cargo SASP factors originating from multiple senescence inducers and cell types. Each profile consists of hundreds of largely distinct proteins but also includes a subset of proteins elevated in all SASPs. Our analyses identify several candidate biomarkers of cellular senescence that overlap with aging markers in human plasma, including Growth/differentiation factor 15 (GDF15), stanniocalcin 1 (STC1), and serine protease inhibitors (SERPINs), which significantly correlated with age in plasma from a human cohort, the Baltimore Longitudinal Study of Aging (BLSA). Our findings will facilitate the identification of proteins characteristic of senescence-associated phenotypes and catalog potential senescence biomarkers to assess the burden, originating stimulus, and tissue of origin of senescent cells in vivo.


Assuntos
Envelhecimento/metabolismo , Biomarcadores/metabolismo , Senescência Celular/fisiologia , Proteoma/análise , Via Secretória/fisiologia , Biomarcadores/análise , Células Cultivadas , Bases de Dados de Proteínas , Exossomos/química , Exossomos/metabolismo , Feminino , Humanos , Fenótipo , Proteoma/metabolismo , Proteômica
7.
Ann Endocrinol (Paris) ; 81(1): 11-17, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31982107

RESUMO

OBJECTIVE: The aim of this study was to describe endocrinological outcome in patients operated on for acromegaly. METHODS: A retrospective study included 167 patients. Patients were assessed in the early postoperative period (EPP), at 3 months (M3), at 1 year (Y1), and then annually. They were classified as grade I (IGF-1 level normal-for-age and positive GH response on oral glucose tolerance test [nadir <0.4ng/L]); grade II (discordant); or grade III or IV (acromegaly, controlled or uncontrolled under medical therapy, respectively). RESULTS: Taking all patients with all grades, 35% changed grades between EPP and M3, 26% between M3 and Y1 and 9% after Y1. In grade I, respectively 22%, 15% and 2% of patients changed grades between EPP and M3, between M3 and Y1, and after Y1, compared to 31%, 6% and 6% in grade IV. Respectively 57%, 67%, and 47% of grade II patients changed grades between EPP and M3, between M3 and Y1, and after Y1; between EPP or M3 and last follow-up (>1 year), respectively 74% and 75% of grade II patients changed grades. Knosp category, resection quality and abnormal GH response (vs. abnormal IGF-1) significantly impacted grade II patients' outcome. CONCLUSIONS: Whereas outcome in grades I and III-IV seems to be determined by 1 year, grade II discordant patients' outcome remains uncertain even after 1 year.


Assuntos
Acromegalia/metabolismo , Acromegalia/cirurgia , Hormônio do Crescimento Humano/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Acromegalia/diagnóstico , Acromegalia/patologia , Adenoma/diagnóstico , Adenoma/metabolismo , Adenoma/patologia , Adenoma/cirurgia , Adulto , Idoso , Feminino , Seguimentos , Teste de Tolerância a Glucose , Adenoma Hipofisário Secretor de Hormônio do Crescimento/diagnóstico , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/cirurgia , Hormônio do Crescimento Humano/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Prognóstico , Recidiva , Estudos Retrospectivos , Via Secretória/fisiologia , Resultado do Tratamento
8.
Nat Cell Biol ; 22(1): 74-86, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907414

RESUMO

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.


Assuntos
Relógios Circadianos/fisiologia , Colágeno/metabolismo , Homeostase/fisiologia , Via Secretória/fisiologia , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/efeitos dos fármacos , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Carbazóis/farmacologia , Colágeno/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Matriz Extracelular/metabolismo , Camundongos Transgênicos , Pirrolidinas/farmacologia , Canais de Translocação SEC/efeitos dos fármacos , Canais de Translocação SEC/metabolismo , Via Secretória/genética , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo
9.
Biochim Biophys Acta Mol Cell Res ; 1867(1): 118567, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676354

RESUMO

Acinar cell exocytosis requires spatiotemporal Ca2+ signals regulated through endoplasmic reticulum (ER) stores, Ca2+ATPases, and store-operated Ca2+ entry (SOCE). The secretory pathway Ca2+ATPase 2 (SPCA2) interacts with Orai1, which is involved in SOCE and store independent Ca2+ entry (SICE). However, in the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. The goal of this study was to determine if SPCA2C effects Ca2+ homeostasis in a similar fashion to the full-length SPCA2. Using epitope-tagged SPCA2C (SPCA2CFLAG) expressed in HEK293A cells and Fura2 imaging, cytosolic [Ca2+] was examined during SICE, SOCE and secretagogue-stimulated signaling. Exogenous SPCA2C expression increased resting cytosolic [Ca2+], Ca2+ release in response to carbachol, ER Ca2+ stores, and store-mediated and independent Ca2+ influx. Co-IP detected Orai1-SPCA2C interaction, which was altered by co-expression of STIM1. Importantly, SPCA2C's effects on store-mediated Ca2+ entry were independent of Orai1. These findings indicate SPCA2C influences Ca2+ homeostasis through multiple mechanisms, some of which are independent of Orai1, suggesting novel and possibly cell-specific Ca2+ regulation.


Assuntos
Sinalização do Cálcio/fisiologia , ATPases Transportadoras de Cálcio/fisiologia , Cálcio/metabolismo , Pâncreas/metabolismo , Canais de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Homeostase , Humanos , Proteína ORAI2/genética , Proteína ORAI2/metabolismo , Especificidade de Órgãos/genética , Isoformas de Proteínas/fisiologia , Via Secretória/fisiologia
10.
Semin Reprod Med ; 37(3): 109-118, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31869838

RESUMO

The tachykinin family of peptides, composed of the neurokinins A and B (NKA, NKB) and substance P are involved in the central control of gonadotropin-releasing hormone (GnRH) release through a variety of neuronal circuitries that mediate the activation of Kiss1 neurons and the synchronization of their activity within the arcuate nucleus. The major outcome of this role is the precise regulation of the pulsatile pattern of GnRH release. In addition, tachykinins are involved in the maturation of the reproductive axis by determining the optimal timing of puberty onset, as well as in the timing of the preovulatory luteinizing hormone surge in females. Therefore, the action of tachykinins in reproduction appears to extend to all the critical aspects required for the successful attainment and maintenance of fertility. In this review, we summarize the latest advances in our understanding of the biology of tachykinins in the control of GnRH release, addressing the existing controversies, open questions, and future perspectives.


Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Taquicininas/fisiologia , Animais , Feminino , Fertilidade/efeitos dos fármacos , Fertilidade/fisiologia , Humanos , Neurônios/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/fisiologia , Via Secretória/efeitos dos fármacos , Via Secretória/fisiologia , Maturidade Sexual/fisiologia , Taquicininas/farmacologia
11.
Cells ; 8(11)2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671908

RESUMO

: Discrimination between properly folded proteins and those that do not reach this state is necessary for cells to achieve functionality. Eukaryotic cells have evolved several mechanisms to ensure secretory protein quality control, which allows efficiency and fidelity in protein production. Among the actors involved in such process, both endoplasmic reticulum (ER) and the Golgi complex play prominent roles in protein synthesis, biogenesis and secretion. ER and Golgi functions ensure that only properly folded proteins are allowed to flow through the secretory pathway while improperly folded proteins have to be eliminated to not impinge on cellular functions. Thus, complex quality control and degradation machineries are crucial to prevent the toxic accumulation of improperly folded proteins. However, in some instances, improperly folded proteins can escape the quality control systems thereby contributing to several human diseases. Herein, we summarize how the early secretory pathways copes with the accumulation of improperly folded proteins, and how insufficient handling can cause the development of several human diseases. Finally, we detail the genetic and pharmacologic approaches that could be used as potential therapeutic tools to treat these diseases.


Assuntos
Proteostase/fisiologia , Via Secretória/fisiologia , Animais , Retículo Endoplasmático/metabolismo , Células Eucarióticas/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Humanos , Biossíntese de Proteínas/fisiologia , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo
12.
Sci Rep ; 9(1): 16892, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729431

RESUMO

α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity. Surprisingly, several reports show that CRYAB exerts a protective role also extracellularly, and it has been recently demonstrated that CRYAB is secreted from human retinal pigment epithelial cells by an unconventional secretion pathway that involves multi-vesicular bodies. Here we show that autophagy is crucial for this unconventional secretion pathway and that phosphorylation at serine 59 residue regulates CRYAB secretion by inhibiting its recruitment to the autophagosomes. In addition, we found that autophagosomes containing CRYAB are not able to fuse with lysosomes. Therefore, CRYAB is capable to highjack and divert autophagosomes toward the exocytic pathway, inhibiting their canonical route leading to the lysosomal compartment. Potential implications of these findings in the context of disease-associated mutant proteins turn-over are discussed.


Assuntos
Autofagossomos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Via Secretória/fisiologia , Cadeia B de alfa-Cristalina/metabolismo , Animais , Autofagia/fisiologia , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Proteínas Mutantes/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/genética
13.
Scand J Gastroenterol ; 54(12): 1448-1451, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31725337

RESUMO

Background: Randomized and controlled trials of glucagon-like peptide-1 (GLP-1) derived drugs have shown that the most frequent adverse symptoms are gastrointestinal. Some of the side effects such as dyspepsia, nausea and upper abdominal pain may well be of gastric origin. Since the antral hormone gastrin regulates gastric secretion of acid and enzymes and contributes to the regulation of gastric motility, we examined the effect of GLP-1 on the secretion of gastrin in normal subjects and diabetes patients.Method: Plasma was sampled from ten healthy subjects and ten patients with diabetes mellitus type 1 with glucose clamped between 6 and 9 mM. GLP-1 or saline were infused for 4 h during and after a meal. Plasma concentrations of gastrin and GLP-1 were measured using specific radioimmunoassays.Results: Basal plasma concentrations of gastrin were similar in controls and patients. After the meal, the gastrin concentrations rose significantly during saline infusion, whereas the GLP-1 infusion suppressed the secretion of gastrin significantly, most pronounced in the diabetes patients.Conclusions: The results show that GLP-1 infusion suppresses the postprandial secretion of gastrin in normal subjects and even more so in the diabetes patients. The results may therefore shed further light on the upper gastrointestinal side effects of GLP-1-derived drugs in diabetic patients.


Assuntos
Diabetes Mellitus Tipo 1 , Gastrinas , Fármacos Gastrointestinais , Peptídeo 1 Semelhante ao Glucagon , Período Pós-Prandial , Estômago , Adulto , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Gastrinas/sangue , Gastrinas/metabolismo , Fármacos Gastrointestinais/metabolismo , Fármacos Gastrointestinais/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Humanos , Incretinas/metabolismo , Incretinas/farmacologia , Masculino , Período Pós-Prandial/efeitos dos fármacos , Período Pós-Prandial/fisiologia , Projetos de Pesquisa , Via Secretória/efeitos dos fármacos , Via Secretória/fisiologia , Estômago/efeitos dos fármacos , Estômago/enzimologia , Estômago/fisiopatologia
14.
Sci Adv ; 5(10): eaax0821, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31663020

RESUMO

Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.


Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/patogenicidade , Transporte Proteico/fisiologia , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Receptores CCR1/metabolismo , Receptores CXCR4/metabolismo , Via Secretória/fisiologia
15.
PLoS Genet ; 15(9): e1008351, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527874

RESUMO

Wnt proteins are secreted signaling factors that regulate cell fate specification and patterning decisions throughout the animal kingdom. In the Drosophila wing epithelium, Wingless (Wg, the homolog of Wnt1) is secreted from a narrow strip of cells at the dorsal-ventral boundary. However, the route of Wg secretion in polarized epithelial cells remains poorly understood and key proteins involved in this process are still unknown. Here, we performed an in vivo RNAi screen and identified members of the exocyst complex to be required for apical but not basolateral Wg secretion. Specifically blocking the apical Wg secretion leads to reduced downstream signaling. Using an in vivo 'temporal-rescue' assay, our results further indicate that apically secreted Wg activates target genes that require high signaling activity. In conclusion, our results demonstrate that the exocyst is required for an apical route of Wg secretion from polarized wing epithelial cells.


Assuntos
Proteínas de Drosophila/metabolismo , Via Secretória/fisiologia , Via de Sinalização Wnt/fisiologia , Proteína Wnt1/metabolismo , Animais , Padronização Corporal/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Ligantes , Transdução de Sinais , Asas de Animais/embriologia , Asas de Animais/metabolismo , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Proteína Wnt1/genética , Proteína Wnt1/fisiologia
16.
Cells ; 8(10)2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31546993

RESUMO

The HtrA4 human protease is crucial in placentation and embryo implantation, and its altered level is connected with pre-eclampsia. The meta-analyses of microarray assays revealed that the HtrA4 level is changed in brain tumors and breast and prostate cancers, which suggests its involvement in oncogenesis. In spite of the HtrA4 involvement in important physiological and pathological processes, its function in the cell is poorly understood. In this work, using lung and breast cancer cell lines, we showed for the first time that the full-length HtrA4 and its N-terminally deleted variant promote cancer cell death induced by chemotherapeutic drugs by enhancing apoptosis. The effect is dependent on the HtrA4 proteolytic activity, and the N-terminally deleted HtrA4 is more efficient in the cell death stimulation. Furthermore, HtrA4 increases the effect of chemotherapeutics on the clonogenic potential and motility of cancer cells, and it increases cell cycle arrest at the G2/M phase. HtrA4 may modulate cell death by degrading the anti-apoptotic XIAP protein and also by proteolysis of the executioner pro-caspase 7 and cytoskeletal proteins, actin and ß-tubulin. These findings provide new insight into the mechanism of the HtrA4 protease function in cell death and oncogenesis, and they may help to develop new anti-cancer therapeutic strategies.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias/patologia , Serina Proteases/fisiologia , Células A549 , Morte Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Células MCF-7 , Mitocôndrias/genética , Mitocôndrias/metabolismo , Células PC-3 , Via Secretória/fisiologia , Serina Proteases/metabolismo
17.
Blood ; 134(12): 979-982, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31262780

RESUMO

Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular hemostasis and local inflammation. Endothelial activation triggers rapid exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet capture (von Willebrand factor [VWF]). Despite its role as an important secretory organelle, a comprehensive compilation of factors associated with WPB has not been carried out. We addressed this via a proximity proteomics approach employing the peroxidase APEX2 coupled with 2 known WPB-associated proteins: the Rab GTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB of primary endothelial cells, and that proteins in their close proximity can be biotinylated through the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. Whereas these include factors reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. Among them, the SNARE-interacting protein Munc13-2 was shown here to specifically localize to WPB and to serve as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion. Thus, APEX2-based proximity proteomics can be used to specifically identify novel organelle-associated factors in primary endothelial cells.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteômica/métodos , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Biotinilação , Células Cultivadas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Exocitose/fisiologia , Humanos , Enzimas Multifuncionais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Via Secretória/fisiologia
18.
J Alzheimers Dis ; 70(3): 667-680, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256134

RESUMO

Increased levels of total tau (t-tau) and hyperphosphorylated tau (p-tau) proteins in the cerebrospinal fluid of Alzheimer's disease (AD) patients are well documented and strongly correlate with AD pathology. Recent studies have further shown that human tau can be released into the extracellular space and transferred to nascent neurons. However, because the tau protein has no signal peptide identity, the mechanisms underlying its secretion remain poorly understood. In the present study, we confirmed that tau protein secretion was promoted by autophagy inducers and downregulated by beclin1 knockdown or autophagy inhibitors derived from human wild type tau (wt-tau)-overexpressing SH-SY5Y cells. Moreover, both t-tau and p-tau secretion were increased by autophagy activation. Furthermore, we identified that six isoforms of tau protein are secreted in an autophagy-dependent manner. These results indicate that both normal and pathological tau are secreted via an autophagy-mediated secretory pathway in neurons. Understanding this new pathway for tau secretion may provide critical future insights into tau pathologies, such as AD.


Assuntos
Doença de Alzheimer , Neurônios/metabolismo , Via Secretória/fisiologia , Proteínas tau , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/metabolismo , Animais , Autofagia/fisiologia , Sobrevivência Celular , Células Cultivadas , Camundongos , Emaranhados Neurofibrilares/metabolismo , Fosforilação , Transporte Proteico/fisiologia , Proteínas tau/líquido cefalorraquidiano , Proteínas tau/metabolismo
19.
PLoS Biol ; 17(6): e3000060, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233488

RESUMO

Apicomplexan parasites invade host cells in an active process involving their ability to move by gliding motility. While the acto-myosin system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions regarding the involvement of other mechanisms in parasite motility. In many eukaryotes, a secretory-endocytic cycle leads to the recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility, and generation of retrograde membrane flow. Here, we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, because inhibition of endocytosis blocks retrograde flow and motility. We demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as labelled phospholipids, nanogold particles (NGPs), antibodies, and Concanavalin A (ConA). Using labelled phospholipids, we observed that the endocytic and secretory pathways of the parasite converge, and endocytosed lipids are subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings, and we propose an additional model, working in parallel to the acto-myosin motor, that reconciles parasite motility with observations in other eukaryotes: an apicomplexan fountain-flow-model for parasite motility.


Assuntos
Movimento Celular/fisiologia , Endocitose/fisiologia , Toxoplasma/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Proteínas de Membrana/metabolismo , Miosinas/metabolismo , Parasitos , Proteínas de Protozoários/metabolismo , Via Secretória/fisiologia , Toxoplasma/fisiologia
20.
Exp Cell Res ; 381(2): 265-279, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31128105

RESUMO

The RNaseA superfamily member Angiogenin (ANG) is a secreted protein involved in neovascularization, cell proliferation and stress response. Dysregulation of ANG expression is found in many cancers with poor prognosis and mutations in ANG are associated with neurodegenerative diseases. While the uptake and nuclear translocation of ANG is relatively well characterised, little is known about how it reaches the plasma membrane and its mode of secretion. We generated SH-SY5Y neuroblastoma cell lines constitutively expressing wild type (WT) Hemagglutinin (HA) epitope tagged mouse Ang1 (mAng1), and two amyotrophic lateral sclerosis associated ANG variants (C39W and K40I). Herein, we show that these cell lines secrete mAng1 into the culture media. Using small molecule inhibitors we probed the route taken between the endoplasmic reticulum and trans-Golgi network during secretion and have characterised it as COPII and microtubule dependent. In addition, we show that disruption by the PI3-kinase inhibitor wortmannin of the later stages of transit to the plasma membrane leads to mAng1 trafficking to lysosomal compartments. This suggests an autophagy dependent regulation of secretion.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Microtúbulos/fisiologia , Ribonuclease Pancreático/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Autofagia/fisiologia , Diferenciação Celular/genética , Proliferação de Células/genética , Humanos , Camundongos , Neurônios Motores/metabolismo , Proteínas Mutantes/metabolismo , Transporte Proteico , Ribonuclease Pancreático/genética , Via Secretória/fisiologia , Células Tumorais Cultivadas
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