April 2011

Sphingolipids: always providing new scientific puzzles to solve

2011 ASBMB Avanti Young Investigator Award recipient Charles Chalfant talks research and mentoring.

Fig. 1. Structure of the cPLA2alphaC2 domain and full-length cPLA2alpha elucidating cationic residues involved in C1P binding. cPLA2alpha is shown to demonstrate the positioning of the C1P binding site (blue) and PIP2 binding site (red) in the full-length enzyme.


Products of arachidonic acid, eicosanoids, are well-established mediators of inflammatory responses with major roles in the pathogenesis of these disease states. The production of AA by phospholipases is the initial rate-limiting step in eicosanoid biosynthesis, and the major phospholipase that regulates eicosanoid synthesis in response to inflammatory agonists is group IVA cytosolic phospholipase A2 (cPLA2alpha).

The Chalfant laboratory at the Virginia Commonwealth University School of Medicine was the first to discover that ceramide-1-phosphate generated by ceramide kinase is a novel and specific activator (both in vitro and in cells) of group cPLA2alpha. Specifically, the Chalfant laboratory demonstrated that C1P can directly bind to cPLA2alpha in a Ca2+-dependant manner via the CaLB/C2 domain and that C1P also increased the enzymatic activity of cPLA2alpha in vitro. Recent findings demonstrated that the specific interaction site for C1P is localized to the calcium binding loop II of the C2 domain of cPLA2alpha specifically the cationic beta-groove (Fig. 1), making the laboratory group the first to fully characterize a specific interaction site for a bioactive sphingolipid.

Chalfant: committed to mentoring
By Laura Chalfant

Charles Chalfant does not like to take the credit for his scientific findings or any success that his research program has obtained. Instead, he gives most of the credit to his staff and his numerous mentors over the years. “One has to have dedicated graduate students and postdoctoral trainees to undertake this research. Furthermore, the mentoring provided by Denise (Cooper), Yusuf (Hannun), Lina (Obeid) and Sarah (Spiegel) has been invaluable,” says Chalfant.

As a result, Chalfant’s trainees have accumulated an impressive list of fellowships and awards under his tutelage. These include predoctoral and postdoctoral fellowships from the American Heart Association; T32 fellowships from the National Institutes of Health in wound healing, cancer signaling and lipidomics; and a career development award from the Veterans Administration, which will lead one of his senior postdoctoral fellows to an independent research career. Recently, Chalfant also was awarded the 2010 Outstanding Teaching Award in Biochemistry and Molecular Biology from the Virginia Commonwealth University School of Medicine.

Chalfant is committed to mentoring and credits his staff for pushing the research forward. “Honestly, I don’t know how my staff, or my wife for that matter, puts up with me!” says Chalfant. His mantra is, “Science can’t wait.” But if science can’t wait, it is apparent that Chalfant can’t wait for science either. His constant requests for data from the many graduate students and postdoctoral fellows that have walked through the Chalfant laboratory doors will not cease as long as science is on the move.

Laura, his wife, is just happy that she gets to see him home safe for dinner.

Laura Chalfant is an accomplished poet and writer of short stories.

Finally, the Chalfant laboratory came full circle and demonstrated that mutagenesis of amino acids critical for C1P interaction within this site inhibited the ability of cPLA2alpha to translocate to intracellular membranes in response to numerous inflammatory agonists. These findings, published in the Journal of Biological Chemistry (1), suggested that the C1P/cPLA2alpha interaction is a target for a new generation of therapies to combat inflammatory disorders, and a main thrust of the laboratory now is to determine whether modulation of this interaction in preclinical animal models affects inflammatory phenotypes.

A second major research focus for the Chalfant laboratory is the alternative splicing of caspase 9 and Bcl-x, major regulators of apoptosis and chemotherapy sensitivity. In previous studies by the laboratory, the generation of de novo ceramide and the activation of protein phosphatase-1 were defined as major components of the signal transduction pathway regulating both the 5' splice site selection of Bcl-x exon 2 and the inclusion of the exon 3,4,5,6 cassette of caspase 9 in a pro-apoptotic fashion. Recent endeavors of the laboratory in this area have been focused on the survival/oncogenic pathways that antagonize the ceramide pathway in regulating the alternative splicing in an anti-apoptotic fashion. Specifically, two recent reports by the Chalfant laboratory have linked the alternative splicing of caspase 9 to the AKT pathway, an antagonistic pathway to ceramide signaling (2, 3). These findings showed a pathway controlled by ceramide signaling that was dysregulated in NSCLC tumors favoring caspase-9b expression, which is the anti-apoptotic form of caspase-9 that promotes tumor formation, growth and maintenance. Further investigation discovered that the phosphorylation of both SRp30a and hnRNP L, RNA trans-factors, promoted the expression of caspase-9b.

“We’re dealing with an unexplored area of RNA trans-factors in relation to cancer and lipid signaling,” says Chalfant. “Before these studies, there had been very little evidence of an RNA splicing event modulated by a lipid signaling or an oncogenic pathway, let alone regulating a tumor biology significant to cancer development. This study points to caspase-9b being a very important target in the development of a durable therapy for nonsmall cell lung cancer, and our future research will focus on how ceramide signaling is blocking caspase 9b expression. In essence, we will now marry our early findings on de novo ceramide signaling with these findings on oncogenic/survival signaling and determine the merge point that acts as a biostat for programmed cell death and the sensitivity of cancer cells to chemotherapies like erlotinib/Tarceva.”

Click here to read the 2011 annual meeting thematic overviews.

For information on the annual meeting, click here.

References

1. Lamour, N. L., Wijesinghe, D. S., Subramanian, P., Stahelin, R. V., Bonventre, J. V., and Chalfant, C. E. (2009) Ceramide-1-phosphate is required for the translocation of group IVA cytosolicphospholipase A2 and prostaglandin synthesis. J. Biol. Chem. 284, 26897 – 26907.
2. Goehe, R. W., Shultz, J. C., Murudkar, C., Usanovic, S., Lamour, N. F., Massey, H. D., Zhang, L., Camidge, D. R., Shay, J. W., Minna, J. D., and Chalfant, C. E. (2010) hnRNP L regulates the tumor formation of human xenografts in mice via caspase 9 mRNA processing. J. Clin. Invest. 120, 3923 – 3939.
3. Shultz, J. C., Goehe, R.W., Wijesinghe, D. S., Murudkar, C., Hawkins, A. J., Shay, J. W., Minna, J. D., and Chalfant, C. E. (2010) The alternative splicing of Caspase 9 is modulated by the PI3K/Akt pathway via the phosphorylation of SRp30a. Cancer Res. 70, 9185 – 9196.

Charles Chalfant (cechalfant@vcu.edu and charles.chalfant@va.gov) currently is a tenured associate professor of biochemistry and molecular biology at Virginia Commonwealth University School of Medicine.


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