Lactacystin, Proteasome Function, and Cell Fate

Gabriel Fenteany,Stuart L Schreiber

doi:10.1074/jbc.273.15.8545

Gabriel Fenteany, Stuart L Schreiber

Open Access

https://doi.org/10.1074/jbc.273.15.8545

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Abstract

Lactacystin, Proteasome Function, and Cell Fate

Highlights

Lactacystin Inhibits Cell Cycle Progression in Different Cell Types and Induces Neurite Outgrowth in a Murine Neuroblastoma Cell Line The Streptomyces product lactacystin (Fig. 1) was discovered on the basis of its ability to inhibit cell growth and to induce neurite outgrowth in a murine neuroblastoma cell line, Neuro-2a [3]
The phosphatase inhibitors okadaic acid and calyculin A block lactacystin-induced bipolar morphology but not the formation of branched neurite networks after 3 days [6]. This implies that induction of bipolar morphology and subsequent formation of branched neurite networks are separable processes, with only the former being dependent on the activity of phosphatases
The predominantly bipolar morphology that follows treatment with lactacystin is distinct from the response to a number of other common treatments leading to morphological differentiation

Summary

Identification of the Target of Lactacystin and Mechanism of Action

Based on studies of neurite outgrowth in Neuro-2a cells and inhibition of cell cycle progression in MG-63 human osteosarcoma cells using a series of analogs of lactacystin, it was determined that an electrophilic carbonyl at the C-4 position was required for activity [4]. The C-4 carbonyls of both the thioester and the ␤-lactone are reactive electrophiles, whereas the carboxylate of the dihydroxy acid is essentially inert to nucleophilic attack. In order to identify the lactacystin-binding molecule, radioactive versions of lactacystin and the related ␤-lactone were synthesized [1], with the expectation that these compounds would serve as covalent affinity labels This strategy led to the identification of the 20 S proteasome as the sole observed target of lactacystin [1]. The ␤-lactone can undergo spontaneous hydrolysis with ring opening to form clasto-lactacystin dihydroxy acid, an inactive species (Fig. 1). The second is attack by the sulfhydryl of glutathione with ring opening to form a thioester adduct analogous to lactacystin; the resulting species does not directly react with the proteasome but can subsequently undergo recyclization to regenerate the active ␤-lactone [11]. Is there evidence to suggest that the ␤-lactone is the active intermediate in these reactions, but there is some evidence to suggest that only the ␤-lactone (and not lactacystin) can enter cultured mammalian cells [11]

Lactacystin as a Probe of Proteasome Function

The Proteasome and Cell Fate