Bachelor of Science, S.U.N.Y. State University at Stony Brook (2012)
Doctor of Philosophy, S.U.N.Y. State University at Stony Brook (2018)
A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites.
2019; 17 (2): e3000136
Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid-the apicoplast-which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.
View details for DOI 10.1371/journal.pbio.3000136
View details for PubMedID 30726238
- Molecular mechanisms of regulation of sphingosine kinase 1 BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS 2018; 1863 (11): 1413–22
Discovery of new sphingosine kinase 1 inhibitors
AMER CHEMICAL SOC. 2018
View details for Web of Science ID 000447600003043
An intrinsic lipid-binding interface controls sphingosine kinase 1 function
JOURNAL OF LIPID RESEARCH
2018; 59 (3): 462–74
Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.
View details for DOI 10.1194/jlr.M081307
View details for Web of Science ID 000426357600007
View details for PubMedID 29326159
View details for PubMedCentralID PMC5832922
Novel sphingosine kinase-1 inhibitor, LCL351, reduces immune responses in murine DSS-induced colitis.
Prostaglandins & other lipid mediators
2017; 130: 47–56
Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite which has been implicated in many diseases including cancer and inflammatory diseases. Recently, sphingosine kinase 1 (SK1), one of the isozymes which generates S1P, has been implicated in the development and progression of inflammatory bowel disease (IBD). Based on our previous work, we set out to determine the efficacy of a novel SK1 selective inhibitor, LCL351, in a murine model of IBD. LCL351 selectively inhibits SK1 both in vitro and in cells. LCL351, which accumulates in relevant tissues such as colon, did not have any adverse side effects in vivo. In mice challenged with dextran sodium sulfate (DSS), a murine model for IBD, LCL351 treatment protected from blood loss and splenomegaly. Additionally, LCL351 treatment reduced the expression of pro-inflammatory markers, and reduced neutrophil infiltration in colon tissue. Our results suggest inflammation associated with IBD can be targeted pharmacologically through the inhibition and degradation of SK1. Furthermore, our data also identifies desirable properties of SK1 inhibitors.
View details for DOI 10.1016/j.prostaglandins.2017.03.006
View details for PubMedID 28377281
View details for PubMedCentralID PMC5509055
CHK1 regulates NF-κB signaling upon DNA damage in p53- deficient cells and associated tumor-derived microvesicles.
2016; 7 (14): 18159–70
The recently discovered CHK1-Suppressed (CS) pathway is activated by inhibition or loss of the checkpoint kinase CHK1, promoting an apoptotic response to DNA damage mediated by caspase-2 in p53-deficient cells. Although functions of the CS-pathway have been investigated biochemically, it remains unclear whether and how CHK1 inhibition can be regulated endogenously and whether this constitutes a key component of the DNA damage response (DDR). Here, we present data that define the first endogenous activation of the CS-pathway whereby, upon DNA damage, wild type p53 acts as an endogenous regulator of CHK1 levels that modulates caspase-2 activation. Moreover, we demonstrate that persistence of CHK1 levels in response to DNA damage in p53-deficient cancer cells, leads to CHK1-mediated activation of NF-κB and induction of NF-κB-regulated genes in cells and in associated tumor-derived microvesicles (TMVs), both of which are abrogated by loss or inhibition of CHK1. These data define a novel role for CHK1 in the DDR pathway as a regulator NF-κB activity. Our data provide evidence that targeting CHK1 in p53-deficient cancers may abrogate NF-κB signaling that is associated with increased cellular survival and chemoresistance.
View details for DOI 10.18632/oncotarget.7566
View details for PubMedID 26921248
View details for PubMedCentralID PMC4951279
A novel role of sphingosine kinase-1 in the invasion and angiogenesis of VHL mutant clear cell renal cell carcinoma.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2015; 29 (7): 2803–13
Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was ∼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an ∼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an ∼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.
View details for DOI 10.1096/fj.15-270413
View details for PubMedID 25805832
View details for PubMedCentralID PMC4478804
Structural Basis for Ceramide Recognition and Hydrolysis by Human Neutral Ceramidase.
Structure (London, England : 1993)
2015; 23 (8): 1482–91
Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate. As an enzyme regulating the balance of ceramide and sphingosine-1-phosphate, nCDase is emerging as a therapeutic target for cancer. Here, we present the 2.6-Å crystal structure of human nCDase in complex with phosphate that reveals a striking, 20-Å deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases. Utilizing flexible ligand docking, we predict a likely binding mode for ceramide that superimposes closely with the crystallographically observed transition state analog phosphate. Our results suggest that nCDase uses a new catalytic strategy for Zn(2+)-dependent amidases, and generates ceramide specificity by sterically excluding sphingolipids with bulky headgroups and specifically recognizing the small hydroxyl head group of ceramide. Together, these data provide a foundation to aid drug development and establish common themes for how proteins recognize the bioactive lipid ceramide.
View details for DOI 10.1016/j.str.2015.06.013
View details for PubMedID 26190575
View details for PubMedCentralID PMC4830088
Intracellular sphingosine kinase 2-derived sphingosine-1-phosphate mediates epidermal growth factor-induced ezrin-radixin-moesin phosphorylation and cancer cell invasion.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2015; 29 (11): 4654–69
The bioactive sphingolipid sphingosine-1-phosphate (S1P) mediates cellular proliferation, mitogenesis, inflammation, and angiogenesis. These biologies are mediated through S1P binding to specific GPCRs [sphingosine-1-phosphate receptor (S1PR)1-5] and some other less well-characterized intracellular targets. Ezrin-radixin-moesin (ERM) proteins, a family of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, are emerging as critical regulators of cancer invasion via regulation of cell morphology and motility. Recently, we identified S1P as an acute ERM activator (via phosphorylation) through its action on S1PR2. In this work, we dissect the mechanism of S1P generation downstream of epidermal growth factor (EGF) leading to ERM phosphorylation and cancer invasion. Using pharmacologic inhibitors, small interfering RNA technologies, and genetic approaches, we demonstrate that sphingosine kinase (SK)2, and not SK1, is essential and sufficient in EGF-mediated ERM phosphorylation in HeLa cells. In fact, knocking down SK2 decreased ERM activation 2.5-fold. Furthermore, we provide evidence that SK2 is necessary to mediate EGF-induced invasion. In addition, overexpressing SK2 causes a 2-fold increase in HeLa cell invasion. Surprisingly, and for the first time, we find that this event, although dependent on S1PR2 activation, does not generate and does not require extracellular S1P secretion, therefore introducing a potential novel model of autocrine/intracrine action of S1P that still involves its GPCRs. These results define new mechanistic insights for EGF-mediated invasion and novel actions of SK2, therefore setting the stage for novel targets in the treatment of growth factor-driven malignancies.
View details for DOI 10.1096/fj.15-274340
View details for PubMedID 26209696
View details for PubMedCentralID PMC4608912
Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles.
Nature chemical biology
2012; 8 (4): 366–74
Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently identified, in a DNA-templated macrocycle library, inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated 'gatekeeper' mutant. We solved two cocrystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bisubstrate-competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance.
View details for DOI 10.1038/nchembio.792
View details for PubMedID 22344177
View details for PubMedCentralID PMC3307835