RESUMO
It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells.
Assuntos
Catepsina B/imunologia , Catepsinas/imunologia , Macrófagos/imunologia , Proteólise , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 8/genética , Caspase 8/imunologia , Inibidores de Caspase/farmacologia , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
The Golgi apparatus is the central hub of intracellular trafficking and consists of tethered stacks of cis, medial, and trans cisternae. In mammalian cells, these cisternae are stitched together as a perinuclear Golgi ribbon, which is required for the establishment of cell polarity and normal subcellular organization. We previously identified FHDC1 (also known as INF1) as a unique microtubule-binding member of the formin family of cytoskeletal-remodeling proteins. We show here that endogenous FHDC1 regulates Golgi ribbon formation and has an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression results in defective Golgi assembly and suggests a role for FHDC1 in maintenance of the Golgi-derived microtubule network. Similarly, overexpression of FHDC1 induces dispersion of the Golgi ribbon into functional ministacks. This effect is independent of centrosome-derived microtubules and instead likely requires the interaction between the FHDC1 microtubule-binding domain and the Golgi-derived microtubule network. These effects also depend on the interaction between the FHDC1 FH2 domain and the actin cytoskeleton. Thus our results suggest that the coordination of actin and microtubule dynamics by FHDC1 is required for normal Golgi ribbon formation.
Assuntos
Actinas/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Citoesqueleto/metabolismo , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Transporte ProteicoAssuntos
Difusão de Inovações , Sistemas Computadorizados de Registros Médicos , Fidelidade a Diretrizes , Sistemas Computadorizados de Registros Médicos/legislação & jurisprudência , Sistemas Computadorizados de Registros Médicos/organização & administração , Sistemas Computadorizados de Registros Médicos/normas , Gerenciamento da Prática Profissional , Estados UnidosRESUMO
Cytoplasmic microtubules exist as distinct dynamic and stable populations within the cell. Stable microtubules direct and maintain cell polarity and it is thought that their stabilization is dependent on coordinative organization between the microtubule network and the actin cytoskeleton. A growing body of work suggests that some members of the formin family of actin remodeling proteins also regulate microtubule organization and stability. For example, we showed previously that expression of the novel formin INF1 is sufficient to induce microtubule stabilization and tubulin acetylation, but not tubulin detyrosination. An important issue with respect to the relationship between formins and microtubules is the determination of which formin domains mediate microtubule stabilization. INF1 has a distinct microtubule-binding domain at its C-terminus and the endogenous INF1 protein is associated with the microtubule network. Surprisingly, the INF1 microtubule-binding domain is not essential for INF1-induced microtubule acetylation. We show here that expression of the isolated FH1 + FH2 functional unit of INF1 is sufficient to induce microtubule acetylation independent of the INF1 microtubule-binding domain. It is not yet clear whether or not microtubule stabilization is a general property of all mammalian formins; therefore we expressed constitutively active derivatives of thirteen of the fifteen mammalian formin proteins in HeLa and NIH3T3 cells and measured their effects on stress fiber formation, MT organization and MT acetylation. We found that expression of the FH1 + FH2 unit of the majority of mammalian formins is sufficient to induce microtubule acetylation. Our results suggest that the regulation of microtubule acetylation is likely a general formin activity and that the FH2 should be thought of as a dual-function domain capable of regulating both actin and microtubule networks.
Assuntos
Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Animais , Proteínas Fetais/genética , Imunofluorescência , Forminas , Células HeLa , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Células NIH 3T3 , Proteínas do Tecido Nervoso , Proteínas Nucleares/genética , Proteínas/genética , Proteínas/metabolismo , Fibras de Estresse/metabolismo , TransfecçãoRESUMO
Formin proteins, characterized by the presence of conserved formin homology (FH) domains, play important roles in cytoskeletal regulation via their abilities to nucleate actin filament formation and to interact with multiple other proteins involved in cytoskeletal regulation. The C-terminal FH2 domain of formins is key for actin filament interactions and has been implicated in playing a role in interactions with microtubules. Inverted formin 1 (INF1) is unusual among the formin family in having the conserved FH1 and FH2 domains in its N-terminal half, with its C-terminal half being composed of a unique polypeptide sequence. In this study, we have examined a potential role for INF1 in regulating microtubule structure. INF1 associates discretely with microtubules, and this association is dependent on a novel C-terminal microtubule-binding domain. INF1 expressed in fibroblast cells induced actin stress fiber formation, coalignment of microtubules with actin filaments, and the formation of bundled, acetylated microtubules. Endogenous INF1 showed an association with acetylated microtubules, and knockdown of INF1 resulted in decreased levels of acetylated microtubules. Our data suggests a role for INF1 in microtubule modification and potentially in coordinating microtubule and F-actin structure.
Assuntos
Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Ativação Enzimática , Proteínas Fetais/genética , Forminas , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/classificação , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Nocodazol/metabolismo , Proteínas Nucleares/genética , Filogenia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tiazolidinas/metabolismo , Distribuição TecidualRESUMO
Formin homology proteins are a highly conserved family of cytoskeletal remodeling proteins best known for their ability to induce the formation of long unbranched actin filaments. They accomplish this by nucleating the de novo polymerization of F-actin and also by acting as F-actin barbed end "leaky cappers" that allow filament elongation while antagonizing the function of capping proteins. More recently, it has been reported that the FH2 domains of FRL1 and mDia2 and the plant formin AFH1 are able to bind and bundle actin filaments via distinct mechanisms. We find that like FRL1, FRL2 and FRL3 are also able to bind and bundle actin filaments. In the case of FRL3, this activity is dependent upon a proximal DAD/WH2-like domain that is found C-terminal to the FH2 domain. In addition, we show that, like other Diaphanous-related formins, FRL3 activity is subject to autoregulation mediated by the interaction between its N-terminal DID and C-terminal DAD. In contrast, the DID and DAD of FRL2 also interact in vivo and in vitro but without inhibiting FRL2 activity. These data suggest that current models describing DID/DAD autoregulation via steric hindrance of FH2 activity must be revised. Finally, unlike other formins, we find that the FH2 and N-terminal dimerization domains of FRL2 and FRL3 are able to form hetero-oligomers.