Method for identifying inhibitors of cytokinesis

Details Method for identifying inhibitors of cytokinesis Method for identifying inhibitors of cytokinesis

2000-12-07

2002-11-26

Gitomer, Ralph (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S018000, C435S004000

Reexamination Certificate

active

06485924

ABSTRACT:

The present invention relates to a therapy interfering with cell division, in particular tumor therapy.
The septins are an evolutionary conserved family of proteins required for cytokinesis (reviewed in Cooper and Kiehart, 1996); Longtine et al., 1996; Field and Kellogg 1999). The septins were first described in S. cerevisiae, where mutants in any of the genes CDC3, 10, 11 and 12 are unable to complete cytokinesis, giving rise to multinucleate cells (Hartwell, 1971). Sequence analysis of these four genes by Pringle and co-workers (GenBank accession numbers L16548-L16551) revealed that they encode proteins with similar primary structure, defining the septin family. The known septins range in size from 30 to 60 kDa and contain sequences characteristic of the GTPase superfamily of proteins. In yeast, the septins localize at the site of bud emergence, and indirect but compelling evidence indicates that the septins are components of the neck filaments, a structure previously described by electron microscopy as an ordered array of filaments in close association with the membrane of the bud (Byers and Goetsch, 1976). Neck filaments have only been observed in yeast cells so far. However, and in spite of the differences in the mechanism of cytokinesis between yeast and animal cells, septins were later found to be widespread in higher eukaryotes. A Drosophila septin mutant, pnut, is defective in cytokinesis, and the Peanut protein localizes to the cleavage furrow of dividing cells (Neufeld and Rubin, 1994). Septins with similar localization patterns have also been described in amphibia and mammals (Kinoshita et al., 1997; Xie et al., 1999), and inactivation of septin function by antibody microinjection in cultured mammalian cells and in Xenopus embryos results in cytokinesis defects (Kinoshita et al., 1997; Xie et al., 1999)). Thus, it appears that the septins are involved in an aspect of cell division that has been conserved from yeast to animal cells. Several yeast proteins required for cytokinesis and bud site selection are recruited to the cell division site in a septin-dependent manner (Chant, et al., 1995; Sanders and Herskowitz 1996; DeMarini et al., 1997; Bi et al., 1998), suggesting that the septins can work as a scaffold that directs the correct localization of other proteins. However, the molecular mechanism of septin function in animal cells is still unclear.
Biochemical experiments have revealed that the septins exist as an heteromultimeric complex containing three (in Drosophila), four (in yeast) or more (in mammals) different septin polypeptides (Field et al., 1996; Frazier et al., 1998; Hsu et al., 1998). Moreover, septin complexes can be purified in a filamentous state (Field et al., 1996; Frazier et al., 1998; Hsu et al., 1998). Thus, it is likely that the septins can form filaments in vivo, even if septin-containing filamentous structures (like the neck filaments in yeast) have not yet been described in animal cells. The mechanism and regulation of septin filament assembly remain nevertheless mysterious, as well as many of the physico-chemical properties of the filaments. For instance, it is not known whether the coiled coil domain is involved in polymerization, or whether septin filaments are polar structures. Several septin proteins have been shown to bind, or bind and hydrolyse, guanine nucleotide (Kinoshita et al., 1997; Field et al., 1996; Beites et al., 1999), and mutations that inhibit nucleotide binding also affect septin localization in mammalian cells (Kinoshita et al., 1997; Field et al., 1996). Inter alia, it was shown that the GTP-binding activity of the mammalian septin Nedd5 is necessary for its normal localization (Kinoshita et al., 1997).
However, the precise role of GTP (guanosine triphosphate) binding and hydrolysis in filament formation has not been elucidated.
It was an object of the invention to elucidate the mechanisms involved in septin filament formation in order to provide a novel approach for therapy, in particular cancer therapy, that is based on modulating septin filament formation and thus interfering with cytokines is.
To solve the problem underlying the present invention, septin filament assembly was reconstituted in vitro using a recombinant septin. It was shown that a septin protein can assemble into filaments in a nucleotide-dependent fashion, and that these polymers, like actin filaments and microtubules, are polar structures that assemble with a nucleation mechanism.
The results obtained in the experiments of the present invention show that GTP binding and hydrolysis regulate the filament assembly of a septin protein, in addition, they present a kinetic analysis of septin polymerization.
Thus, the present invention provides the first direct evidence of nucleotide-dependent filament assembly of a septin protein. Based on these observations, it may also be assumed that XSepA filaments assemble with a nucleation mechanism, and that filament growth and stability is regulated by the state of bound guanine nucleotide. In addition, due to the similarity of the septin, both on the sequence level and in terms of their function, it may be assumed that an essentially identical mechanism operates at the level of heteromultimeric septin filaments.
Kinetic analysis of the polymerization reaction has revealed the existence of a lag phase in septin filament assembly FIG.
4
A. This feature is indicative of nucleated polymerization, whereby initiation of filaments is energetically unfavored, but under sufficiently high monomer concentrations, nuclei can form. Addition of monomers to these nuclei to yield long filaments subsequently takes place with a higher association constant than the one required for filament initiation (Cantor and Schimmel; 1980)). Actin and tubulin follow such a polymerization mechanism. The existence of a critical concentration (the concentration below which polymerization cannot occur) is a consequence of the kinetic barrier to nucleation. As shown in
FIG. 4B
, a critical concentration of approximately 0.5 mg/ml, or ~12 &mgr;M (in comparison with ~0.2 &mgr;M for actin (Mitchison, 1992), or 14 &mgr;M for pure tubulin in glycerol buffer (Mitchison and Kirschner, 1984) for XSepA polymerization exists. These data indicate that XSepA filaments are nucleated polymers. On the other hand, a mechanism of linear polymerization (where monomers associate end-to-end with an affinity constant independent of polymer length) has been proposed for septin filaments, based on the length distribution of immunopurified septin complexes (Field et al., 1996); Frazier et al., 1998). It is very likely, however, that in the absence of polymer-stabilising conditions long filaments would not have survived the purification procedure. This could be due to simple mechanical breakage and/or to depolymerization caused by either depletion of nucleotide, or to effects of dilution of the septin complex below the putative critical concentration. In fact, only short filaments (up to 350 nm) were observed in these cases (Field et al., 1996; Frazier et al. 1998) rendering ambiguous a kinetic interpretation of length distribution data.
Two additional properties of actin filaments and microtubules seem to be shared by XSepA filaments. The polymerization dynamics of XSepA in the presence of the slowly hydrolysable GTP analogue, GTP-&ggr;-S suggest a role of nucleotide hydrolysis in the destabilisation of the filament structure (FIGS.
4
A-
4
C). Likewise, a number of experimental approaches has established that NTP hydrolysis is linked to destabilisation of the microtubule and actin filament lattices (Mitchison, 1992). Another important feature of these cytoskeletal elements is filament polarity, since it plays a key role in the organization of higher order structures and in the directional transport of molecules along filamentous tracks (Mitchison, 1992); Kirschner and Mitchison, 1986). Using fluorescence microscopy to visualise elongation of pre-assembled septin filaments, it could be observed that the two ends grow with different kinetics (FIGS.
5
A and
5
B).

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