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Effect of glutamine limitation on the behavior of Sp2/0-Ag14 mouse hybridoma cells.
No full textCancer cells often display a dependence toward the amino acid L-glutamine for their
survival, a phenomenon termed glutamine addiction. The mouse hybridoma Sp2/0-Ag14 (Sp2/0)
undergoes rapid apoptotic cell death upon glutamine deprivation, making it a useful model for
uncovering the molecular and cellular processes through which glutamine controls cell survival.
This work was aimed at gaining a better understanding of the molecular and cellular events
triggered when Sp2/0 cells are exposed to limiting amounts of glutamine.
First, the effect of glutamine limitation on Sp2/0 cell behavior was investigated. We
found that a threshold concentration of 100 μM glutamine exists where Sp2/0 cell density does
not increase, but cells remain viable. Under this threshold, Sp2/0 cells underwent apoptosis, but
in a more protracted fashion than under conditions of acute glutamine deprivation.
Unexpectedly, I found that exposure of Sp2/0 cells to 25μM glutamine triggered a biphasic
activation of caspase-3. Interestingly, glutamine limitation, but not acute glutamine deprivation, was sufficient to maintain intact mitochondria for several hours and to trigger the expression of the stress-related transcription factor GADD-153. My results raise the possibility that glutamine limitation triggers a stress response which could enable Sp2/0 cells to adapt to its environment. Using microscopic and biochemical techniques, I also provided evidence for a reduction in autophagic processes in Sp2/0 cells exposed to glutamine-limiting conditions. Chemical inhibitors of autophagy caused Sp2/0 cell death even in the presence of adequate supply of glutamine. On the other hand, rapamycin, a known activator of autophagy, improved Sp2/0 viability under glutamine limitation conditions. Therefore, the loss of Sp2/0 cell viability when exposed to limiting amounts of glutamine could be the result, at least in part, of a reduction in the cell’s autophagic capabilities. Finally, I explored the effect of ammonium ions, a product of glutamine metabolism, on
the behavior of Sp2/0 cells exposed to limiting amounts of glutamine. Ammonium ions treatment
rescued Sp2/0 cell viability and proliferation in Sp2/0 cells cultured in glutamine-limiting conditions. Interestingly, ammonium acetate, but not ammonium chloride, caused a reduction in caspase-3 activity in Sp2/0 cells maintained under limiting glutamine conditions. Finally, my data suggest that ammonium salts led to a partial restoration of the autophagy process in Sp2/0 cells exposed to limiting amounts of glutamine, providing a potential explanation for the beneficial effect of ammonium ions on cell viability. All together, the results obtained in the course my studies argue in favor of a mechanistic link between autophagy and ammonium ions in the modulation of the viability of a Sp2/0 cells exposed to glutamine-limiting conditions
Identifying Escherichia Coli factors that selectively bind the mRNA of secreted proteins
No full textIt is well accepted that the majority of secreted proteins are targeted to the secretory pathway through amino acid signal sequences located at the N–terminus of the pre-protein during the initial stages of translation on the ribosome. This is true in both eukaryotic and prokaryotic systems. These signal sequences display distinct structural features such as charged/hydrophilic residues at each termini and a continuous hydrophobic stretch of amino acids between them. The
signal-recognition particle (SRP), which targets the ribosome to the membrane translocon for secretion of the protein, is hypothesized to recognize and these features and therefore differentiate these proteins from non-secretory proteins. Recent work in other laboratories has suggested a role for the mRNA itself, rather than only the amino acid sequence of the N–terminus of the pre-protein as playing a role in targeting the pre-protein-ribosome complex to the
translocon within the membrane. To test this hypothesis, direct interaction between mRNAs
encoding secreted proteins and the E. coli SRP equivalent (Ffh) was pursued using pull down assays. The mRNA’s used as bait corresponded to the N-terminal 40 amino acids of secreted and cytosolic proteins including periplasmic propyl isomerase chaperone SurA (as a model secretory
protein with a cleavable signal peptide) and the cytoplasmic protein 3-isopropyl malate
dehydrogenase (IsodH). Additionally, the mRNA of two other proteins, PhoA (secreted) and GMP (cytoplasmic), were used but in these mRNA the 5' UTR were also included in case these regions were involved in SRP recognition. Following extensive optimizations and modifications
of these experiments, the Ffh protein (the Escherichia coli SRP homolog) could not be isolated from cytoplasmic extracts of E. coli with the pull down assays. One interesting finding however was that the mRNA of the IsodH protein was pulled down using its cognate mRNA transcript as
bait. This implies a role for this enzyme in regulating its own levels in the cell by binding to and potentially modulating its translation. Other factors involved in DNA and RNA binding were also isolated and include RNase and ribosomal proteins, amongst others. It can therefore be
concluded that under these experimental conditions, the mRNA hypothesis for targeting protein secretion could not be supported
