Ovarian cancer is the most lethal gynecological cancer. Here we show that innate immune agonist, dsRNA, directly induces ovarian cancer cell death and identify biomarkers associated with responsiveness to this targeted treatment. Nuclear staining and MTT assays following dsRNA stimulation revealed two subpopulations, sensitive (OVCAR-3, CAOV-3; patient samples malignant 1 and 2) and resistant (DOV-13, SKOV-3). Microarray analysis identified 75 genes with differential expression that further delineated these two subpopulations. qPCR and immunoblot analyses showed increased dsRNA receptor expression after stimulation as compared to resistant and immortalized ovarian surface epithelial cells (e.g., 70-fold with malignant 2, 43-fold with OVCAR-3). Using agonists, antagonists, and shRNA-mediated knockdown of dsRNA receptors, we show that TLR3, RIG-I, and mda5 coordinated a caspase 8/9- and interferon-dependent cell death. In resistant cells, dsRNA receptor overexpression restored dsRNA sensitivity. When dsRNA was combined with carboplatin or paclitaxel, cell viability significantly decreased over individual treatments (1.5-to 7.5-fold). Isobologram analyses showed synergism in dsRNA combinations (CI=0.4–0.82) vs. an additive effect in carboplatin/paclitaxel treatment (CI= 1.5–2). Our data identify a predictive marker, dsRNA receptor expression, to target dsRNA responsive populations and show that, in dsRNA-sensitive cells, dsRNA induces apoptosis and enhances the potency of cytotoxic chemotherapeutics.—Van, D. N., Roberts, C. F., Marion, J. D., Lépine, S., Harikumar, K. B., Schreiter, J., Dumur, C. I., Fang, X., Spiegel, S., Bell, J. K. Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics. FASEB J. 26, 3188–3198 (2012). www.fasebj.org
The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.
Abstract Interest in allostery and drug development has significantly expanded with increasingly broad ranges of targets and diseases. Experimental and computational approaches have led to advances in understanding of the mechanisms and roles of protein dynamics and conformational states, and come together in the concept of conformational selection. The models of Monod, Wyman, and Changeux and Koshland, Nemethy, and Filmer provide conceptual explanations of homotropic cooperativity and heterotropic allostery, while conformational selection provides realistic detail that can be exploited in drug design. Distinction between allostery and cooperativity and development of approaches to identify cryptic sites on proteins significantly expands the range of targets for allosteric drug design. High‐throughput screening and SAR optimization remain a staple of drug design, however, increased understanding of the thermodynamics of protein–ligand interactions and conformational changes, and the mechanisms of information flow between existing allosteric sites and across subunit interfaces or between allosteric and active sites, rational design of ligands to interact with a cryptic “allosteric” site on any protein becomes possible. Recent “omics” approaches to identify druggable targets both genome wide and in vivo further enhance the range of targets for drug design, both covalent and noncovalent, exploiting allosteric and cooperative properties of proteins.
Innate immunity is the first line of defense against invading pathogens. Toll-like receptors (TLRs) act as sentinels of the innate immune system, sensing a variety of ligands from lipopolysaccharide to flagellin to dsRNA through their ligand-binding domain that is composed of leucine-rich repeats (LRRs). Ligand binding initiates a signaling cascade that leads to the up-regulation of inflammation mediators. In this study, we have expressed and crystallized the ectodomain (ECD) of human TLR3, which recognizes dsRNA, a molecular signature of viruses, and have determined the molecular structure to 2.4-Å resolution. The overall horseshoe-shaped structure of the TLR3-ECD is formed by 23 repeating LRRs that are capped at each end by specialized non-LRR domains. The extensive β-sheet on the molecule's concave surface forms a platform for several modifications, including insertions in the LRRs and 11 N -linked glycans. The TLR3-ECD structure indicates how LRR loops can establish distinct pathogen recognition receptors.
Novel therapeutic interventions for bacterial diseases have been of interest due to the rise in infections over the last decade. Much recent focus of drug design has been on “Allosteric” drugs targeting either existing or cryptic‐allosteric sites on proteins. Development of effective drugs depends in part on understanding fundamental features of protein structure and function, including binding site specificity and molecular mechanisms of allostery, including the transmission of signals from binding site to active site, a process that often involves relay of information across subunit interfaces. While a binding site contains “first sphere” residues (those touching the ligand) the transmission of information requires the involvement of “second sphere” residues. Second sphere residues may also govern the local polarity of first sphere residues contributing to binding and/or catalysis as well as providing triggers for subunit interactions. Glyoxysomal Malate Dehydrogenase (gMDH) catalyzes the reversible oxidation/reduction of substrates malate/oxaloacetate, coupled with cofactor conversion NAD+/NADH, a key stage in the Tricarboxylic Acid Cycle. MDH is known to be regulated via citrate and substrate inhibition with both processes thought to involve allosteric subunit communication. The active site contains a number of conserved. first sphere residues that contribute to catalysis and substrate binding including 3 conserved Arginines (R124, R130, R196) and the His‐Asp dyad (H220, D193) involved in catalysis.. To identify potential second sphere residues, High‐quality Protein Interactomes (HINT) tables were utilized to analyze interactions of H220 and D193 with nearby residues. HINT tables provide the types and magnitude of interactions occurring. HINT analysis was conducted on MDH with no ligand bound or with substrates This analysis identified four residues, V194, G218, Q251 and M271 as potential second sphere residues that might contribute to subunit interactions. Potential key interactions identified include V194 with D193 (active site), G218 with H220 (active site), Q251 with T255 (located on the interface loop containing S266). And M271 with close neighbor residues 272–276 and, when citrate is bound, across the subunit interface with D87 which is connected to the active site on the opposite subunit. Four mutants, V194E, G218W, M271Q and Q251A were constructed, using Quikchange mutagenesis, expressed and purified by NiNTA Chromatography. Mutants were characterized specific activity, by CD and Fluorescence based Thermal Shift assays to assess structure, stability and cofactor or citrate binding, and by kinetics assays to assess oxaloacetate and NADH interaction. All mutations resulted in altered specific activity. The Q251A mutation decreased Km for NADH while M271Q resulted in an increase in Km for NADH. With prior results on subunit interactions these observations give rise to a model of cofactor and substrate induced alterations at the subunit interface mediated by active site interactions and second sphere residues. Support or Funding Information This work was supported by NSF Grants 1726932 and 0448905.
Course-based Undergraduate Research Experiences (CUREs) have emerged as a well supported high-impact practice for improving both retention of students in science, technology, engineering, and mathematics (STEM) disciplines and overall achievement of students in STEM coursework. While considerable scholarship has been performed on the overall efficacy of CUREs as a teaching approach, comparatively little work has been done to uncover the more nuanced data on how CUREs serve students at different types of undergraduate institutions (community colleges, primarily-undergraduate institutions (PUIs), and research-intensive institutions) as well as students characterized as Persons Excluded due to Ethnicity or Race (PEER). In this study, we leverage the Malate Dehydrogenase CURE Community, a CURE network focused on bringing research experiences in protein biochemistry to undergraduates, to collect data on student achievement and attitudes. Using several validated assessment instruments, we considered how effective the MCC CURE experience was at each type of institution as well as for PEER students versus their White/Asian counterparts. Our data revealed that the CURE approach to instruction was especially effective at PUIs, with significantly-higher levels of student collaboration and overall positive attitudes about STEM careers found in comparison to community colleges and research-intensive institutions. Community colleges reported the most significant gains with respect to PEER student achievement relative to White/Asian students as well as enthusiasm among PEER students for conducting future research. Taken together, the data suggest that while all undergraduates may expect to benefit from exposure to CUREs, the specific benefits vary by the type of institution as well as the PEER status of student. The outcomes of this research will ultimately enable institutions to provide biochemistry-focused high-impact educational experiences for undergraduates tailored to the needs of these institutions to enable them to become a well-equipped next generation of life scientists.
Toll-like receptors (TLRs), type I integral membrane receptors, recognize pathogen associated molecular patterns (PAMPs). PAMP recognition occurs via the N-terminal ectodomain (ECD) which initiates an inflammatory response that is mediated by the C-terminal cytosolic signaling domain. To understand the molecular basis of PAMP recognition, we have begun to define TLR—ECD structurally. We have solved the structure of TLR3-ECD, which recognizes dsRNA, a PAMP associated with viral pathogens. TLR3-ECD is a horseshoe-shaped solenoid composed of 23 leucine-rich repeats (LRRs). The regular LRR surface is disrupted by two insertions at LRR12 and LRR20 and 11 N-linked carbohydrates. Of note, one side of the ECD is carbohydrate-free and could form an interaction interface. We have shown that TLR3-ECD binds directly to pI:pC, a synthetic dsRNA ligand, but not to p(dI):p(dC). Without a TLR3—dsRNA complex structure, we can only speculate how ligand binds. Analysis of the unliganded structure reveals two patches of basic residues and two binding sites for phosphate backbone mimics, sulfate ions, that may be capable of recognizing ligand. Mutational and co-crystallization studies are currently underway to determine how TLR3 binds its ligand at the molecular level.
Malate dehydrogenase specifically oxidizes malate to oxaloacetate. The specificity arises from three arginines in the active site pocket that coordinate the carboxyl groups of the substrate and stabilize the newly forming hydroxyl/keto group during catalysis. Here, the role of Arg-153 in distinguishing substrate specificity is examined by the mutant R153C. The x-ray structure of the NAD binary complex at 2.1 Å reveals two sulfate ions bound in the closed form of the active site. The sulfate that occupies the substrate binding site has been translated ∼2 Å toward the opening of the active site cavity. Its new location suggests that the low catalytic turnover observed in the R153C mutant may be due to misalignment of the hydroxyl or ketone group of the substrate with the appropriate catalytic residues. In the NAD·pyruvate ternary complex, the monocarboxylic inhibitor is bound in the open conformation of the active site. The pyruvate is coordinated not by the active site arginines, but through weak hydrogen bonds to the amide backbone. Energy minimized molecular models of unnatural analogues of R153C (Wright, S. K., and Viola, R. E. (2001) J. Biol. Chem. 276, 31151–31155) reveal that the regenerated amino and amido side chains can form favorable hydrogen-bonding interactions with the substrate, although a return to native enzymatic activity is not observed. The low activity of the modified R153C enzymes suggests that precise positioning of the guanidino side chain is essential for optimal orientation of the substrate.
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