DJ-1 interacts with and regulates paraoxonase-2, an enzyme critical for neuronal survival in response to oxidative stress.

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.

Interaction of the N- and C-terminal autoregulatory domains of FRL2 does not inhibit FRL2 activity.

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.

Evaluation of mammalian cell-free systems of nuclear disassembly and assembly.

Mammalian cell-free systems are very useful for the biochemical and structural study of nuclear disassembly and assembly. Through experimental manipulations, the role of specific proteins in these processes can be studied. Recently, we intended to examine the involvement of integral and peripheral inner nuclear membrane proteins in nuclear disassembly and assembly. However, we could not achieve proper disassembly when isolated interphase HeLa nuclei were exposed to mitotic soluble extracts obtained from the same cell line and containing cyclin B1. Homogenates of synchronized mitotic HeLa cells left to reassemble their nuclei generated incomplete nuclear envelopes on chromatin masses. Digitonin-permeabilized mitotic cells also assembled incomplete nuclei, generating a lot of cytoplasmic inclusions of inner nuclear membrane proteins as an intermediate. These results were therefore used as a basis for a critical evaluation of mammalian cell-free systems. We present here evidence that cell synchronization itself can interfere with the progress of nuclear assembly, possibly by causing aberrant nuclear disassembly and/or by inducing the formation of an abnormal number of mitotic spindles.