CELL BIOLOGY OF EFFECTOR PROTEINS 
Linda J. Van Eldik, Ph.D., Associate Investigator 
One of the long-term goals of the research in Dr. 
Van Eldik's laboratory is to increase understanding 
of the molecular mechanisms controlling the path- 
ways by which the brain develops from a single cell 
into a complex organ capable of functions such as 
learning, memory, motor control, and perception. A 
growing body of evidence suggests that an impor- 
tant aspect of regulation of neuronal development 
and maintenance involves soluble neurotrophic fac- 
tors, i.e., molecules that enhance neuronal survival 
and/or stimulate neurite outgrowth. The prototypi- 
cal neurotrophic factor is nerve growth factor, 
whose effects include maintenance of the differenti- 
ated state of mature sympathetic and sensory neu- 
rons, enhancement of neuronal survival, and pro- 
motion and guidance of neurite outgrowth. 
Dr. Van Eldik's laboratory has recently shown 
that a form of S 100(3, a protein found in high levels 
in glial cells, has neurotrophic activity on central 
nervous system (CNS) neurons, specifically on 
neurons of the cerebral cortex and spinal cord. 
SIOOP belongs to a family of proteins with primary 
sequence similarities. The SlOO family includes 
SlOOa and SIOOP; calpactin light chain; SIOOL; pro- 
teins that are elevated in patients with cystic fibro- 
sis (CP) and rheumatoid arthritis (CP antigen, MRP- 
8, and MRP- 14); and predicted protein sequences 
deduced from RNAs that are expressed in high lev- 
els in differentiated, transformed, or growth factor- 
treated cells (calcyclin, 18A2, 42A and 42C, p9Ka, 
pEL98). The observation that expression of mem- 
bers of the SlOO family is altered during cell 
growth, differentiation, and in certain diseases sug- 
gests that they may play regulatory roles in these 
processes. 
Although little information is available about the 
in vivo roles of many of the members of the SlOO 
family, a disulfide form of SIOOP has been shown to 
stimulate neurite outgrowth in primary cultures of 
cortical neurons, in a neuroblastoma cell line, and 
in organotypic cultures of spinal cord/ganglia. The 
form of S 100(3 with neurotrophic activity is referred 
to here as NEF (neurite extension factor). The ob- 
servation that NEF has neurotrophic activity in 
vitro, coupled with the presence of extracellular 
SlOO in brain and glial cell cultures, suggests that 
during development of the nervous system, NEF 
might be released from glial cells and act in a 
paracrine fashion to stimulate neurite outgrowth. 
The potential importance of this protein as a neu- 
rotrophic factor is also indicated by its localization 
in the developing nervous system during the time 
of elongation of neuronal processes. Altogether, 
the data suggest that NEF may be a neurotrophic 
factor synthesized and released by glial cells for 
neurons of the CNS. 
To develop the necessary reagents for addressing 
the long-term question of how NEF may be in- 
volved in neuronal development and maintenance. 
Dr. Van Eldik's laboratory previously synthesized a 
gene coding for SIOOP, expressed the gene in Es- 
cherichia colt, and produced protein (VUSB-l) by 
recombinant DNA technology. VUSB-l preparations 
have neurotrophic activity on embryonic chick cor- 
tical neurons, whereas preparations ofE. coli trans- 
formed with vector lacking the S 100(3 gene do not 
have activity. VUSB-l enhanced cell maintenance in 
culture as well as stimulated neurite extension. In 
addition, in control experiments, the neurite exten- 
sion activity was reduced by preincubation with an- 
tibodies made against bovine brain S 100(3. Finally, 
VUSB-l preparations lose activity when treated 
with reducing agents or when purifications are 
done in the presence of reducing agents; this is 
consistent with the idea that the activity of NEF re- 
quires a disulfide form of the protein. This appar- 
ent requirement for a disulfide linkage in order to 
function as a neurotrophic factor is intriguing in 
light of the fact that SlOOp proteins isolated from 
different species and tissues have invariant cys- 
teines at residues 68 and 84. In addition, S 100(3 is 
the only member of the SlOO family of proteins that 
has cysteines at both of these positions in the 
amino acid sequence; all other members have a va- 
line at the position analogous to Cys-68. 
Dr. Van Eldik's recent studies using site-directed 
mutagenesis/protein engineering approaches have 
shown that both cysteine residues of VUSB-l are 
important for neurite extension and cell survival ac- 
tivity but that the relative position of the two cys- 
teines can be altered without loss of activity Specif- 
ically, the relative contribution of Cys-68 and Cys-84 
to the NEF activity of VUSB-l was tested by prepar- 
ing mutant VUSB-l proteins with Cys-68 changed 
to alanine, serine, or valine; Cys-84 was changed to 
alanine or serine; or both cysteines were changed 
(Cys-68 to valine and Cys-84 to alanine or serine). 
Mutant proteins lacking either Cys-68 or Cys-84 
were unable to stimulate neurite extension or en- 
hance neuronal survival. These data indicate that 
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