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1.
BMC Mol Cell Biol ; 20(1): 33, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412778

RESUMO

BACKGROUND: The PINK1:Parkin pathway regulates the autophagic removal of damaged and dysfunctional mitochondria. While the response of this pathway to complete loss of ΔΨm, as caused by high concentrations of mitochondrial ionophores, has been well characterized, it remains unclear how the pathway makes coherent decisions about whether to keep or purge mitochondria in situations where ΔΨm is only partially lost or exhibits fluctuations, as has been observed in response to certain types of cellular stress. RESULTS: To investigate the responses of the PINK1:Parkin pathway to mitochondrial insults of different magnitude and duration, controlled titration of the mitochondrial protonophore, CCCP, was used to manipulate ΔΨm in live cells, and the dynamics of PINK1 and Parkin recruitment was measured by fluorescence microscopy. In contrast to the stable accumulation of PINK1 and Parkin seen at completely depolarized mitochondria, partial depolarization produced a transient pulse of PINK1 stabilization and rapid loss, which was driven by small fluctuations in ΔΨm. As the rate of Parkin dissociation from the mitochondria and phospho-polyubiquitin chain removal was comparatively slow, repetitive pulses of PINK1 were able to drive a slow step-wise accumulation of Parkin and phospho-polyubiquitin leading to deferred mitophagy. CONCLUSION: These data suggest that the PINK1:Parkin mitophagy pathway is able to exhibit distinct dynamic responses to complete and partial mitochondrial depolarization. In this way, the pathway is able to differentiate between irretrievably damaged mitochondria and those showing signs of dysfunction, promoting either rapid or delayed autophagy, respectively.


Assuntos
Mitocôndrias/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico , Ubiquitina-Proteína Ligases/metabolismo , Autofagia/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Poliubiquitina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fatores de Tempo
2.
Int Immunopharmacol ; 43: 116-128, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27988459

RESUMO

Suppressing cytokine responses has frequently been shown to have promising therapeutic effects for many chronic inflammatory and autoimmune diseases. However, the severe side effects associated with the long-term use of current treatments, such as allergic reactions and increased risk of stroke, have focused attention towards the targeting of intracellular signaling mechanisms, such as NF-κB, that regulate inflammation. We synthesized a series of non-natural aurone derivatives and investigated their ability to suppress pro-inflammatory signaling in human monocyte (THP-1) and murine macrophage-like (RAW 267.4) cell lines. One of these derivatives, (Z)-2-((5-(hydroxymethyl) furan-2-yl) methylene) benzofuran-3(2H)-one (aurone 1), was found to inhibit LPS-induced secretion of the pro-inflammatory cytokines, tumor-necrosis factor α (TNFα), interleukin 1ß (IL-1ß), and IL-8 by THP-1 cells. To investigate the mechanism, we probed the effect of aurone 1 on LPS-induced MAPK and NF-κB signaling in both THP-1 and RAW264.7. While aurone 1 pre-treatment had no effect on the phosphorylation of ERK, JNK, or p38 MAPK, it strongly suppressed activation of IKK-ß, as indicated by attenuation of Ser176/180 phosphorylation, resulting in decreased phosphorylation of p65 (ser536) as well as phosphorylation (ser32) and degradation of IκBα. Consistent with this, aurone 1 significantly reduced LPS-stimulated nuclear translocation of p65-containing NF-κB transcription factors and expression of an mCherry reporter of TNFα gene transactivation in RAW264.7 cells. Inhibition of TNFα expression at the transcription level was also demonstrated in THP-1 by qRT-PCR. In addition to its effects on cytokine expression, aurone 1 pre-treatment decreased expression of iNOS, a bona fide NF-κB target gene and marker of macrophage M1 polarization, resulting in decreased NO production in RAW264.7 cells. Together, these data indicate that aurone 1 may have the potential to function as a pharmacological agent for the treatment of chronic inflammation disorders.


Assuntos
Anti-Inflamatórios/uso terapêutico , Benzofuranos/farmacologia , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Benzofuranos/síntese química , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Camundongos , Monócitos/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética
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