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1.
Sci Rep ; 12(1): 17805, 2022 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-36284196

RESUMEN

SMER28 originated from a screen for small molecules that act as modulators of autophagy. SMER28 enhanced the clearance of autophagic substrates such as mutant huntingtin, which was additive to rapamycin-induced autophagy. Thus, SMER28 was established as a positive regulator of autophagy acting independently of the mTOR pathway, increasing autophagosome biosynthesis and attenuating mutant huntingtin-fragment toxicity in cellular- and fruit fly disease models, suggesting therapeutic potential. Despite many previous studies, molecular mechanisms mediating SMER28 activities and its direct targets have remained elusive. Here we analyzed the effects of SMER28 on cells and found that aside from autophagy induction, it significantly stabilizes microtubules and decelerates microtubule dynamics. Moreover, we report that SMER28 displays neurotrophic and neuroprotective effects at the cellular level by inducing neurite outgrowth and protecting from excitotoxin-induced axon degeneration. Finally, we compare the effects of SMER28 with other autophagy-inducing or microtubule-stabilizing drugs: whereas SMER28 and rapamycin both induce autophagy, the latter does not stabilize microtubules, and whereas both SMER28 and epothilone B stabilize microtubules, epothilone B does not stimulate autophagy. Thus, the effect of SMER28 on cells in general and neurons in particular is based on its unique spectrum of bioactivities distinct from other known microtubule-stabilizing or autophagy-inducing drugs.


Asunto(s)
Neuroprotección , Fármacos Neuroprotectores , Fármacos Neuroprotectores/farmacología , Neurotoxinas/farmacología , Autofagia , Serina-Treonina Quinasas TOR/metabolismo , Sirolimus/farmacología , Microtúbulos/metabolismo
2.
J Cell Sci ; 135(15)2022 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-35971979

RESUMEN

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP.


Asunto(s)
Actinas , Seudópodos , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Movimiento Celular/fisiología , Seudópodos/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo
3.
Cells ; 11(10)2022 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-35626685

RESUMEN

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here, we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and, consequently, growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction in phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3Kδ and to a lesser extent p110γ. The biological relevance of our observations was underscored by SMER28 interfering with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor tyrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited. Lastly, in B cell lymphoma cells, which predominantly depend on tonic signaling through PI3Kδ, apoptosis upon SMER28 treatment is profound in comparison to non-hematopoietic cells. This indicates SMER28 as a possible drug candidate for the treatment of diseases that derive from aberrant PI3Kδ activity.


Asunto(s)
Fosfatidilinositol 3-Quinasas , Serina-Treonina Quinasas TOR , Autofagia , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo
4.
Curr Biol ; 31(10): 2051-2064.e8, 2021 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-33711252

RESUMEN

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbß3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Adhesión Celular , Movimiento Celular , Integrinas/metabolismo , Macrófagos/metabolismo , Fagocitosis , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Quinasa 1 de Adhesión Focal/metabolismo , Masculino , Ratones , Paxillin/metabolismo , Fosforilación , Seudópodos
5.
Elife ; 92020 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-32960172

RESUMEN

Exploring the complexity of host-pathogen communication is vital to understand why microbes persist within a host, while others are cleared. Here, we employed a dual-sequencing approach to unravel conversational turn-taking of dynamic host-pathogen communications. We demonstrate that upon hitting a host cell, motile Pseudomonas aeruginosa induce a specific gene expression program. This results in the expression of spermidine on the surface, which specifically activates the PIP3-pathway to induce phagocytic uptake into primary or immortalized murine cells. Non-motile bacteria are more immunogenic due to a lower expression of arnT upon host-cell contact, but do not produce spermidine and are phagocytosed less. We demonstrate that not only the presence of pathogen inherent molecular patterns induces immune responses, but that bacterial motility is linked to a host-cell-induced expression of additional immune modulators. Our results emphasize on the value of integrating microbiological and immunological findings to unravel complex and dynamic host-pathogen interactions.


Asunto(s)
Interacciones Huésped-Patógeno , Fagocitosis , Pseudomonas aeruginosa/metabolismo , Espermidina/metabolismo , Animales , Transporte Biológico , Ratones , Células RAW 264.7
6.
Sci Immunol ; 5(49)2020 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-32646852

RESUMEN

The WAVE regulatory complex (WRC) is crucial for assembly of the peripheral branched actin network constituting one of the main drivers of eukaryotic cell migration. Here, we uncover an essential role of the hematopoietic-specific WRC component HEM1 for immune cell development. Germline-encoded HEM1 deficiency underlies an inborn error of immunity with systemic autoimmunity, at cellular level marked by WRC destabilization, reduced filamentous actin, and failure to assemble lamellipodia. Hem1-/- mice display systemic autoimmunity, phenocopying the human disease. In the absence of Hem1, B cells become deprived of extracellular stimuli necessary to maintain the strength of B cell receptor signaling at a level permissive for survival of non-autoreactive B cells. This shifts the balance of B cell fate choices toward autoreactive B cells and thus autoimmunity.


Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Proteínas de la Membrana/inmunología , Animales , Enfermedades Autoinmunes/genética , Trasplante de Médula Ósea , Línea Celular , Niño , Citoesqueleto , Femenino , Humanos , Lactante , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunología
7.
Int J Cancer ; 138(8): 1982-93, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26619320

RESUMEN

The importance of tumor associated neutrophils (TANs) in cancer development is in the meantime well established. Numerous of clinical data document the adverse prognostic effects of neutrophil infiltration in solid tumors. However, certain tumor therapies need functional neutrophils to be effective, suggesting altered neutrophil polarization associated with different outcomes for cancer patients. Therefore, modulation of neutrophilic phenotypes represents a potent therapeutic option, but factors mediating neutrophil polarization are still poorly defined. In this manuscript we provide evidence that type I IFNs alter neutrophilic phenotype into anti-tumor, both in mice and human. In the absence of IFN-ß, pro-tumor properties, such as reduced tumor cytotoxicity with low neutrophil extracellular traps (NETs) expression, low ICAM1 and TNF-α expression, dominated neutrophil phenotypes in primary lesion and premetastatic lung. Interestingly, such neutrophils have significantly prolonged life-span. Notably, interferon therapy in mice altered TAN polarization towards anti-tumor N1. Similar changes in neutrophil activation could be observed in melanoma patients undergoing type I IFN therapy. Altogether, these data highlight the therapeutic potential of interferons, suggesting optimization of its clinical use as potent anti-tumor agent.


Asunto(s)
Interferón Tipo I/inmunología , Melanoma/inmunología , Neoplasias Experimentales/inmunología , Neutrófilos/inmunología , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Separación Celular , Femenino , Citometría de Flujo , Humanos , Interferón Tipo I/uso terapéutico , Melanoma/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias Experimentales/patología , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , Fenotipo
8.
Hepatology ; 59(6): 2110-20, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24425003

RESUMEN

UNLABELLED: Gene therapy has become an accepted concept for the treatment of a variety of different diseases. In contrast to preclinical models, subjects enrolled in clinical trials, including gene therapy, possess a history of infection with microbes that may influence its safety and efficacy. Especially, viruses that establish chronic infections in the liver, one of the main targets for in vivo gene therapy, raise important concerns. Among them is the hepatitis B virus (HBV), which has chronically infected more than 350 million people worldwide. Here, we investigated the effect of HBV on adeno-associated viral (AAV) vectors, the most frequently applied gene transfer vehicles for in vivo gene therapy. Unexpectedly, we found that HBV greatly improved AAV transduction in cells replicating HBV and identified HBV protein x (HBx) as a key factor. Whereas HBV-positive and -negative cells were indistinguishable with respect to cell-entry efficiency, significantly higher numbers of AAV vector genomes were successfully delivered to the nucleus in the presence of HBV. The HBV-promoting effect was abolished by inhibitors of phosphatidylinositol 3-kinase (PI3K). PI3K was required for efficient trafficking of AAV to the nucleus and was enhanced in HBV-replicating cells and upon HBx expression. Enhancement of AAV transduction was confirmed in vivo using HBV transgenic mice and could successfully be applied to inhibit HBV progeny release. CONCLUSION: Our results demonstrate that acute, as well as chronic, infections with unrelated viruses change the intracellular milieu, thereby likely influencing gene therapy outcomes. In the case of HBV, HBx-mediated enhancement of AAV transduction is an advantage that could be exploited for development of novel treatments of HBV infection.


Asunto(s)
Dependovirus/genética , Predisposición Genética a la Enfermedad , Virus de la Hepatitis B/genética , Hepatitis B/virología , Animales , Activación Enzimática/genética , Femenino , Vectores Genéticos , Células HEK293 , Hepatitis B/genética , Hepatitis B/terapia , Humanos , Interferón gamma/genética , Masculino , Ratones , Ratones Transgénicos , ARN Interferente Pequeño/genética , Transactivadores/genética , Transducción Genética , Proteínas Reguladoras y Accesorias Virales , Replicación Viral/genética
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