Li S da Graca | PubMed Health

PubMed2006

Caspase-9 holoenzyme is a specific and optimal procaspase-3 processing machine.

Caspase-9 activation is critical for intrinsic cell death. The activity of caspase-9 is increased dramatically upon association with the apoptosome, and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo). In this study, we use quantitative enzymatic assays to fully characterize C9Holo and a leucine-zipper-linked dimeric caspase-9 (LZ-C9). We surprisingly show that LZ-C9 is more active than C9Holo for the optimal caspase-9 peptide substrate LEHD-AFC but is much less active than C9Holo for the physiological substrate procaspase-3. The measured Km values of C9Holo and LZ-C9 for LEHD-AFC are similar, demonstrating that dimerization is sufficient for catalytic activation of caspase-9. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. However, the measured Km of C9Holo for procaspase-3 is much lower than that of LZ-C9. Therefore, in addition to dimerization, the apoptosome activates caspase-9 by enhancing its affinity for procaspase-3, which is important for procaspase-3 activation at the physiological concentration.

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PubMed2003

DAF-5 is a Ski oncoprotein homolog that functions in a neuronal TGF beta pathway to regulate C. elegans dauer development.

An unconventional TGF beta superfamily pathway plays a crucial role in the decision between dauer diapause and reproductive growth. We have studied the daf-5 gene, which, along with the daf-3 Smad gene, is antagonized by upstream receptors and receptor-regulated Smads. We show that DAF-5 is a novel member of the Sno/Ski superfamily that binds to DAF-3 Smad, suggesting that DAF-5, like Sno/Ski, is a regulator of transcription in a TGF beta superfamily signaling pathway. However, we present evidence that DAF-5 is an unconventional Sno/Ski protein, because DAF-5 acts as a co-factor, rather than an antagonist, of a Smad protein. We show that expressing DAF-5 in the nervous system rescues a daf-5 mutant, whereas muscle or hypodermal expression does not. Previous work suggested that DAF-5 and DAF-3 function in pharyngeal muscle to regulate gene expression, but our analysis of regulation of a pharynx specific promoter suggests otherwise. We present a model in which DAF-5 and DAF-3 control the production or release of a hormone from the nervous system by either regulating the expression of biosynthetic genes or by altering the connectivity or the differentiated state of neurons.

Acyltransferases↗