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1.
J Bacteriol ; 193(19): 5465-76, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21803990

RESUMO

In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease composed of ClpY (HslU), an ATPase with unfolding activity, and ClpQ (HslV), a peptidase. In the ClpYQ proteolytic complex, the hexameric rings of ClpY (HslU) are responsible for protein recognition, unfolding, and translocation into the proteolytic inner chamber of the dodecameric ClpQ (HslV). Each of the three domains, N, I, and C, in ClpY has its own distinct activity. The double loops (amino acids [aa] 137 to 150 and 175 to 209) in domain I of ClpY are necessary for initial recognition/tethering of natural substrates such as SulA, a cell division inhibitor protein. The highly conserved sequence GYVG (aa 90 to 93) pore I site, along with the GESSG pore II site (aa 265 to 269), contribute to the central pore of ClpY in domain N. These two central loops of ClpY are in the center of its hexameric ring in which the energy of ATP hydrolysis allows substrate translocation and then degradation by ClpQ. However, no data have been obtained to determine the effect of the central loops on substrate binding or as part of the processivity of the ClpYQ complex. Thus, we probed the features of ClpY important for substrate engagement and protease processivity via random PCR or site-specific mutagenesis. In yeast two-hybrid analysis and pulldown assays, using isolated ClpY mutants and the pore I or pore II site of ClpY, each was examined for its influence on the adjoining structural regions of the substrates. The pore I site is essential for the translocation of the engaged substrates. Our in vivo study of the ClpY mutants also revealed that an ATP-binding site in domain N, separate from its role in polypeptide (ClpY) oligomerization, is required for complex formation with ClpQ. Additionally, we found that the tyrosine residue at position 408 in ClpY is critical for stabilization of hexamer formation between subunits. Therefore, our studies suggest that stepwise activities of the ClpYQ protease are necessary to facilitate the processive degradation of its natural substrates.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Western Blotting , Endopeptidase Clp/química , Endopeptidase Clp/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , Técnicas do Sistema de Duplo-Híbrido , Tirosina/química , Tirosina/genética
2.
J Bacteriol ; 191(13): 4218-31, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19395483

RESUMO

In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and cpsB::lacZ assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Sítios de Ligação/genética , Western Blotting , Endopeptidase Clp/genética , Proteínas de Escherichia coli/genética , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Técnicas do Sistema de Duplo-Híbrido
3.
Methods Mol Biol ; 1493: 209-222, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27787853

RESUMO

Primary tissue culture is an invaluable technique in cell biology and has a long history in demonstrating its versatility in characterizing cellular morphology, function, and behavior. Here, we describe a modified, low density, long-term, primary neuron culture system to characterize dendritic morphology and synaptic spine organization in developing mouse cortical neurons. While this method can be applied to investigate the signaling pathways of a range of extracellular cues' effect on neuronal development, we focus on how distinct secreted semaphorins regulate dendritic elaboration and spine morphogenesis in deep layer cortical neurons.


Assuntos
Dendritos/metabolismo , Semaforinas/fisiologia , Animais , Células Cultivadas , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Morfogênese , Neurônios/citologia , Neurônios/metabolismo , Semaforinas/metabolismo
4.
Sci Signal ; 7(316): ra24, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24619647

RESUMO

Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains--C1, Hinge/RBD, and C2--and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4-deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A-induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.


Assuntos
Cones de Crescimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/embriologia , Receptores de Superfície Celular/metabolismo , Semaforina-3A/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Gânglios Espinais/citologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Células Receptoras Sensoriais/citologia
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