Student News and Views
Suppressor of TCR signaling (Sts-1, and -2) knockout mice offer insight on host-virus response
Reported by Nadia Jaber, Ph.D. candidate in Molecular and Cellular Biology (MCB)
Emerging interests in host-virus response among Stony Brook investigators Dr. Laurie Krug and
Dr. Nicholas Carpino has brought about a successful scientific collaboration. Dr. Carpino studies the role
of Suppressor of TCR signaling -1 and -2 (Sts-1 and -2) and discovered that genetic deletion of this
suppressor leads to hyper-activation of T-cells. Dr. Krug’s lab is interested in gammaherpesvirus, which
establishes life-long infections and can lead to serious malignancies. Gammaherpesviruses establish
latency in host cells for many years before reactivating and manifesting an infection. T-cells have a wellappreciated
role in controlling gammaherpesvirus infections during both the acute and latent phases of
Graduate student Brandon Cieniewicz characterized the Sts-1/Sts-2 double knockout (dKO) mice
infected with murine gammaherpesvirus 68, which has pathological similarities to human disease.
Brandon found that T-cells from Sts dKO mice
increased interferon-γ production (Figure 1), and
degranulation in response to infected cells. Brandon
hypothesized that the enhanced activity of T-cells in
Sts dKO mice may increase clearance of the
gammaherpesvirus infection. In addition, the dKO Tcells
may require less stimulation for activation and
may be better equipped to recognize latent
infections. Contrary to his hypothesis, Brandon found
no effect of Sts dKO on acute viral replication,
establishment of latency, or reactivation from latency
in vivo. He observed increased levels of total CD8+
T-cells in Sts dKO, but not of the virus-specific pool.
Brandon and colleagues concluded that deletion of Sts-1 and -2 does not confer a greater ability
to clear gammaherpesvirus infections. Although the Sts molecules are implicated for their role in T-cell
receptor signaling, it is plausible that they play a similar role in other signaling pathways, and that its
deletion leads to effects that offset the hyper-activity of T-cells. The investigators believe a CD4+ or CD8+
T-cell-specific deletion may offer more insight. Still, the investigators believe the hyper-responsive Sts
dKO T-cells could be harnessed for therapies involving adoptive transfer, such as cancer immunotherapy.
In addition, Brandon thinks the Sts dKO system could be used to study other infectious diseases. Another
collaboration with the Konopka lab shows promising results for the clearance of yeast infections.
To read the full publication, please see
Cieniewicz B, Carpino N, Krug LT (2014) Enhanced Response of T-Cells from Murine Gammaherpesvirus 68-Infected Mice Lacking the Suppressor of T-Cell
Receptor Signaling Molecules Sts-1 and Sts-2. PLoS ONE 9(2): e90196.
Author Highlight: Brandon Cieniewicz
Brandon is a sixth year Ph.D. student in the laboratory of Dr. Laurie Krug. When he’s
not in the lab, Brandon spends his time brewing beer for the annual Oktoberfest
party, trying new vegetarian recipes and hiking in the woods.
A novel role for cytosolic Phospholipase Cβ in reversal of siRNA hydrolysis by C3PO
Reported by: Yang Liu, Ph.D. Candidate Molecular and Cellular Biology (MCB)
Phospholipase Cβ (PLCβ) 1-4 is a family of Gα downstream signaling enzymes that elicit increased levels
of intracellular calcium and mediate PKC activation. Although the majority of the PLCβ resides on the plasma
membrane where it interacts with Gαq , the function of the cytosolic pool of PLCβ is elusive. Recent work in the
laboratory of Dr. Suzanne Scarlata showed that cytosolic PLCβ strongly binds to the endonuclease translinassociated
factor X (TRAX) and reverses siRNA mediated down regulation by interfering with the function of the
RNA silencing promoter C3PO. In a recent publication in the Journal of Biological Chemistry, graduate student
Shriya Sahu and her colleague Dr. Finly Philip, characterized how the association between PLCβ and C3PO
affects the hydrolysis of certain siRNAs.
While PLCβ reverses silencing by siRNAs against GAPDH but
not Hsp90, the explanation for such a phenomenon is unclear. To rule
out the possibility that it is merely due to differences in the
compartmentalization of the cytosolic PLCβ pool and TRAX population,
Sahu visualized fluorescently labeled proteins in the cytosol using
FRET, and identified the consistent association of PLCβ with TRAXcontaining
C3PO complexes. By analyzing the aggregation states of
the purified translin, TRAX and C3PO, the authors showed that C3PO
is octameric in solution. Further study of the association of PLCβ with
C3PO complex using FRET, suggested a model in which a single
molecule of PLCβ binds to C3PO on an external site on the TRAX
subunits without affecting the assembly of C3PO complexes (Figure 1).
To study how PLCβ affects C3PO’s function, they measured the displacement of GAPDH and Hsp90
siRNAs labeled with a fluorescence probe from C3PO and C3PO-PLCβ with the addition of increasing amounts of
ssDNA. Interestingly, they found that siRNA (Hsp90) binds to C3PO complexes more strongly than siRNA
(GAPDH), even though PLCβ slightly weakened the binding of the both siRNA species. Sahu and coworkers
proposed that the differences in the binding reflect differences in nucleotide structure. Fluorescence quenching
studies supported this idea by suggesting a less stable structure of Hsp90 siRNA. However similar protection was
seen for both siRNAs on binding to C3PO, and similar decreases in protection were seen in the presence of
PLCβ. These results suggest that the effect of PLCβ on C3PO function may lie in the nature of the microRNAs. If
this is the case, then differences in the effect of PLCβ on C3PO-mediated siRNA degradation could be due to the
differences in siRNA’s vulnerability to hydrolysis. When they measured the hydrolysis rates of siRNAs by C3PO,
they consistently observed that GAPDH siRNAs was hydrolyzed faster than Hsp90 siRNAs and PLCβ binding only
reduced the rate of hydrolysis of siRNA (GAPDH) but not siRNA (Hsp90). Therefore, the reason why PLCβ
reverses degradation by siRNA(GAPDH) but not the Hsp90 could be because of intrinsic differences in hydrolysis
This work supports the existence of a novel function of cytosolic PLCβ in RNA interference and gene
regulation in addition to its traditional role in generating calcium response by G-protein signaling. Moreover, the
molecular mechanism underlying the function of PLCβ in reversal of siRNA mediated gene silencing adds another
interesting dimension to the way gene expression might be controlled.
To read the full article, please see:
Sahu, S, et al. (2014). Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing
Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ. Journal of Biological
Chemistry 289(8): 5134-5144.
Author Highlight: Shriya Sahu
Shriya is a PhD candidate in the MCB program and works in the laboratory of Dr. Suzanne
Scarlata at Stony Brook University. Her thesis research involved characterizing the interaction
of PLCβ with C3PO and uncovering the molecular mechanism of C3PO nuclease activity.
Aside from scientific research, Shriya’s interests include traveling to new places, hiking,
dancing and cooking.
Lysosomal protease inhibitor helps tumor cells deal with stress
Reported by: Jennifer L. DeLeon, Ph.D. Candidate Molecular and Cellular Biology (MCB)
Squamous cell carcinoma antigen, or SCCA, was identified as a serum biomarker in several cancer types
including cervical, lung, head and neck, and liver. However, the mechanism by which upregulation of SCCA contributes to tumorigenesis has been poorly understood. Recent work in the laboratory of Dr. Wei-Xing Zong showed that SCCA
overexpression is associated with poorly differentiated and aggressive cancers of the breast, colon and pancreas. In
normal tissue, SCCA functions as a protease inhibitor to block cytosolic leakage of lysosomal proteases and promote
cell survival in the event of lysosomal damage. MCB alum Dr. Namratha Sheshadri and colleagues uncovered a tumorpromoting
role of SCCA, which was recently published in Cancer Research. Dr. Sheshadri discovered that the
upregulation of SCCA promotes the unfolded protein response and activation of pro-inflammatory signaling through the
cytokine IL-6 in cell lines and a mouse model of mammary tumorigenesis.
||Figure 1: SCCA promotes EMT
To study the tumor promoting function of SCCA, Sheshadri et al.
overexpressed SCCA in the “normal” non-neoplastic human breast
epithelial cell line MCF10A. Upon SCCA overexpression, they observed
phenotypic changes resembling epithelial to mesenchymal transition (EMT);
a change of cultured cells from a cobblestone-like monolayer to a spindle
morphology (Figure 1). Likewise, transcription analysis showed an increase
in EMT promoting markers like ZEB1 and vimentin, and the loss of
epithelial markers like E-cadherin. SCCA overexpression leads to increased
migration, anchorage independent colony formation, and growth factor
autonomous cell proliferation and survival.
To elucidate the molecular mechanism for the cellular EMT like phenotype, Sheshadri screened several EMTpromoting
growth factors and identified a significant increase in IL-6 transcription and secretion in SCCA expressing
cells. Interestingly, pharmacological inhibition or genetic knockdown of IL-6 abrogated several of the transformation
phenotypes induced by SCCA. IL-6 has been well appreciated as a potent cytokine for cell signaling, inflammation, and
tumorigenesis, however, the question still remained, how does SCCA promotes IL-6 expression?
In a previous study, SCCA overexpression was shown to block lysosomal and proteasomal protein turnover,
thus disrupting normal protein homeostasis. Sheshadri hypothesized that SCCA may potentiate the build-up of
misfolded and ubiquitinated proteins and trigger the cellular stress response termed the unfolded-protein response
(UPR). Indeed, SCCA transformed cells have increased levels of several UPR markers and display a chronic but nonlethal
endoplasmic reticulum stress response. By genetic knockdown of these ER stress receptors, Sheshadri
demonstrated a decrease in IL-6 transcription. Thus, the impaired protein degradation caused by elevated SCCA
expression may lead to UPR, which has been implicated in pro-inflammatory response in numerous human diseases
This work establishes an important example where disruption of protein homeostasis can contribute to EMT and
tumorigenesis via the unfolded protein response. Importantly, it also identifies the Achilles heel for tumors that exhibit high expression of SCCA such that they are sensitized to ER stress-inducing pharmacological agents; and thus may be
an advantageous therapeutic target in human cancers.
To read the full article please see:
Sheshadri, N., et al. SCCA1/SERPINB3 Promotes Oncogenesis and Epithelial-Mesenchymal Transition via the
Unfolded Protein Response. Cancer Research. 2014 Sep 11.
Author Highlight: Dr. Namratha Sheshadri, Ph.D.
Namratha graduated from Stony Brook University with a Ph.D. in Molecular and Cell biology
from the laboratory of Dr. Wei-Xing Zong in August 2014. Her thesis research uncovered the
cellular mechanism of lysosomal protease inhibitor SCCA to initiate breast cancer
tumorigenesis. Namratha recently obtained a post-doctoral position at the National Cancer
Institute (NCI). Aside from scientific research, Namratha’s interests include bird watching,
gardening, hiking and traveling.
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