Biomedical Engineering Reference
In-Depth Information
GEF Dbs. Nonprenylated Cdc42, which does not associated with the membrane, was
unaffected by treatment with secramine, suggesting that the probe did not inhibit
nucleotide exchange or interfere with GEF. Secramine also reduced the membrane
association of prenylated Cdc42 with PIP2 liposomes in the form of a Cdc42(GDP)-
RhoGDI1 complex, as determined by sedimentation of PIP2 liposomes after incuba-
tion with Cdc42-RhoGDI1 complex. Based on the combined results, it was concluded
that secramine inhibits Cdc42's membrane association in a RhoGDI1-dependent
manner.
In addition to being useful as a tool for probing the secretory pathway, secramine
has been used to study a variety of other Cdc42-related biological processes, such
as cAMP-induced K
+
conductance [62], cell shape and growth control in anaplastic
large cell lymphoma [63], and Cdc42-dependent Golgi polarization in migrating cells
[7b].
18.4 EXPANDING THE COLLECTION OF IMPORTANT
BIOLOGICAL PROBES
18.4.1
Inhibitors of Heat Shock Protein 70: Probes with Higher Potency
Members of the heat shock protein 70 (Hsp70) family are operative in a wide range
of protein folding and transport processes. Hsp70 is characterized by an N-terminal
nucleotide-binding domain that binds ATP, a substrate-binding domain that associates
with hydrophobic regions of client polypeptides, and a C-terminal helical lid that
regulates access to the peptide-binding domain. Upon hydrolysis of ATP in the
nucleotide-binding domain, a conformational change in the helical lid traps client
proteins, allowing them to fold and preventing aggregate formation [64]. Hydrolysis
of ATP is accelerated by the association of Hsp70 and a co-chaperone, heat shock
protein 40 (Hsp40), through the J-domain of Hsp40 [65]. The large T antigen (TAg)
of simian virus 40 (SV40) contains a similar J-domain that also can stimulate Hsp70's
activity [49]. It has been reported that elevated Hsp70 expression is associated with
metastasis and poor prognosis in patients after combined chemotherapy and radiation
therapy treatment. The role of these heat shock proteins in oncogenesis has made
them attractive targets in the development of anticancer drugs [66].
To discover new modulators of Hsp70, the Wipf and Brodsky groups assembled a
library of 31 compounds based on two small molecules (deoxyspergualin and NSC-
630668-R/1) known to modulate the ATPase activity of Hsp70 [49]. In assembling
the library, compounds were drawn from the Developmental Therapeutics Program
of the National Cancer Institute and the University of Pittsburgh Center for Chemical
Methodologies and Library Development Program (UPCMLD, a DOS library). To
assess the compounds' activities toward Hsp70, a thin-layer chromatography (TLC)
assay was developed using a pre-formed complex of the yeast Hsp70 Ssa1p and [
-
32
P]ATP. The complexwas incubatedwith compound and aliquots were removed from
the mixture and quenched at specified time points. Separation of [
-
32
P]ATP from
[
-
32
P]ADP in each aliquot was accomplished by TLC, and the percentages of ATP
