45 research outputs found
Sindbis viral vector induced apoptosis requires translational inhibition and signaling through Mcl-1 and Bak
<p>Abstract</p> <p>Background</p> <p>Sindbis viral vectors are able to efficiently target and kill tumor cells <it>in vivo</it>, as shown using pancreatic and ovarian cancer models. Infection results in apoptosis both <it>in vitro </it>and <it>in vivo</it>. Sindbis vector uptake is mediated by the LAMR, which is upregulated on a number of different tumor types, thus conferring specificity of the vector to a wide range of cancers. In this study we elucidate the mechanism of apoptosis in two tumor cell lines, MOSEC, derived from the ovarian epithelium and Pan02, derived from a pancreatic adenocarcinoma. A comprehensive understanding of the mechanism of apoptosis would facilitate the design of more effective vectors for cancer therapy.</p> <p>Results</p> <p>The initial phase of Sindbis vector induced apoptosis in MOSEC and Pan02 models reconfirms that viral infection is sensed by PKR due to double-stranded RNA intermediates associated with genomic replication. PKR activation results in translation inhibition through eIF2Ξ± phosphorylation and initiation of the stress response. Our studies indicate that the roles of two proteins, Mcl-1 and JNK, intimately link Sindbis induced translational arrest and cellular stress. Translational arrest inhibits the synthesis of anti-apoptotic Bcl-2 protein, Mcl-1. JNK activation triggers the release of Bad from 14-3-3, which ultimately results in apoptosis. These signals from translational arrest and cellular stress are propagated to the mitochondria where Bad and Bik bind to Bcl-xl and Mcl-1 respectively. Formation of these heterodimers displaces Bak, which results in caspase 9 cleavage and signaling through the mitochondrial pathway of apoptosis.</p> <p>Conclusion</p> <p>The host cell response to Sindbis is triggered through PKR activation. Our studies demonstrate that PKR activation and subsequent translational arrest is linked to both cellular stress and apoptosis. We have also found the linkage point between translational arrest and apoptosis to be Mcl-1, a protein whose constant translation is required for inhibition of apoptosis. With this information vectors can be designed, which express or repress proteins implicated in this study, to enhance their therapeutic potential.</p
Lack of class I H-2 antigens in cells transformed by radiation leukemia virus is associated with methylation and rearrangement of H-2 DNA
Transformation of murine thymocytes by
radiation leukemia virus is associated with reduced expression
of the class I antigens encoded in the major histocompatibility
complex (MHC) and increased methylation and altered restriction
enzyme patterns of MHC DNA. These changes may play
a role in host susceptibility to virus-induced leukemogenesis
and accord with the notion that viral genomes play a regulatory
function when they integrate adjacent to histocompatibiity
genes
Activation of Cytotoxic and Regulatory Functions of NK Cells by Sindbis Viral Vectors
Oncolytic viruses (OVs) represent a relatively novel anti-cancer modality. Like other new cancer treatments, effective OV therapy will likely require combination with conventional treatments. In order to design combinatorial treatments that work well together, a greater scrutiny of the mechanisms behind the individual treatments is needed. Sindbis virus (SV) based vectors have previously been shown to target and kill tumors in xenograft, syngeneic, and spontaneous mouse models. However, the effect of SV treatment on the immune system has not yet been studied. Here we used a variety of methods, including FACS analysis, cytotoxicity assays, cell depletion, imaging of tumor growth, cytokine blockade, and survival experiments, to study how SV therapy affects Natural Killer (NK) cell function in SCID mice bearing human ovarian carcinoma tumors. Surprisingly, we found that SV anti-cancer efficacy is largely NK cell-dependent. Furthermore, the enhanced therapeutic effect previously observed from Sin/IL12 vectors, which carry the gene for interleukin 12, is also NK cell dependent, but works through a separate IFNΞ³-dependent mechanism, which also induces the activation of peritoneal macrophages. These results demonstrate the multimodular nature of SV therapy, and open up new possibilities for potential synergistic or additive combinatorial therapies with other treatments
Interactions Between Laminin Receptor and the Cytoskeleton During Translation and Cell Motility
Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration
A role for elevated H-2 antigen expression in resistance to neoplasia caused by radiation-induced leukemia virus. Enhancement of effective tumor surveillance by killer lymphocytes
Resistance to radiation-leukemia virus-induced leukemia is mediated by gene(s) in the H-2D region of the major murine histocompatibifity complex (1). The mechanisms by which gene(s) in this region affect leukemogenesis are hitherto unknown. However, previous observations (2) suggest a role in disease resistance for changes in H-2 expression occurring immediately after virus inoculation. For example, gene products (antigens) of the H-2D region show the most marked and prolonged changes in expression after virus inoculation. In addition, elevated H-2D antigen expression after virus inoculation occurs for thymocytes of resistant but not susceptible mice. Furthermore, there is an inverse relation between expression of H-2D and viral antigens. Viral antigen expression is greater in susceptible animals, whereas H-2D antigen expression is maximal for virus-infected resistant mice. Finally, H-2 antigens usually disappear from the surface of radiation-induced leukemia virus (RadLV) l transformed cultures when overt leukemia develops. Thus, resistance to the disease is associated with increased H-2 antigenic expression, and the onset of leukemia is associated with disappearance of these antigens
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Computational prediction and analysis of protein structure
Identifying polymer-forming SAM domainsSterile Alpha Motif (SAM) domains are common protein modules in eukaryotic cells. It has not been possible to assign functions to uncharacterized SAM domains because they have been found to participate in diverse functions ranging from protein-protein interactions to RNA binding. Here we computationally identify likely members of the subclass of SAM domains that form polymers. Sequences were virtually threaded onto known polymer structures and then evaluated for compatibility with the polymer. We find that known SAM polymers score better than the vast majority of known non-polymers: 100% (7 of 7) of known polymers and only 8% of known non- polymers (1 of 12) score above a defined threshold value. Of 2901 SAM family members, we find 694 that score above the threshold and are likely polymers, including SAM domains from the proteins Lethal Malignant Brain Tumor, Bicaudal-C, Liprin-beta, Adenylate Cyclase and Atherin. In polymerization experiments, all of these predictions (except Adenylate Cyclase) were confirmed. As a result, the original SAM database was updated and additional predictions were obtained.TMKink: A method to predict transmembrane helix kinksA hallmark of membrane protein structure is the large number of distorted transmembrane helices. Because of the prevalence of bends, it is important to not only understand how they are generated but also to learn how to predict their occurrence. Here, we find that there are local sequence preferences in kinked helices, most notably a higher abundance of proline, which can be exploited to identify bends from sequence information. A neural network predictor identifies over two-thirds of all bends (sensitivity 0.70) with high reliability (specificity 0.89). It is likely that more structural data will allow for better helix distortion predictors with increased coverage in the future. The kink predictor, TMKink, is available at http://tmkinkpredictor.mbi.ucla.edu/.Structural differences between mesophilic and thermophilic membrane proteinsProtein thermostability remains a focal point of interest for protein scientists. The differences in thermostability between mesophilic and thermophilic soluble proteins have been extensively studied. No differences in packing values have been found in soluble proteins. Membrane protein packing is different from soluble protein packing; thermophilic adaptation may be different as a result. Surprisingly, burial and packing values appear to be shared between mesophiles and thermophiles in both soluble and membrane proteins. We created a non-redundant database of unpaired and paired structures for the study of thermophile-mesophile structural differences in membrane proteins. We found little or no differences in burial or packing values in both the soluble and transmembrane regions of membrane proteins
From Sequence to Function through Secondary Structure Kinetics of RNA and DNA
A number of efforts to determine function from sequence of RNA and DNA have
been made with varying success. Here we study the determination of function from
sequence of DNA and RNA through their secondary structure kinetics, specifically
the series of transitions between secondary structures. This series of transitions or
microscopic structure can be described by a system of ordinary differential equations
that can be approximating using balanced truncation to determine the macroscopic
structure. By doing so, we have been able to identify signature topological features of
microscopic structure and mathematically characterize the corresponding classes of
macroscopic structure. Thus we are now able to take large, complex systems, reduce
them, and understand their behavior. In the future, we hope to be able to identify
small microscopic changes that lead to large macroscopic changes and possibly phasetransition
like conditions between secondary states. Ultimately, this may lead to the
development of a secondary-structure kinetics theory describing how one or more
strands of DNA pair with one another to form different secondary structures and its
potential future experimental verification
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Computational prediction and analysis of protein structure
Identifying polymer-forming SAM domainsSterile Alpha Motif (SAM) domains are common protein modules in eukaryotic cells. It has not been possible to assign functions to uncharacterized SAM domains because they have been found to participate in diverse functions ranging from protein-protein interactions to RNA binding. Here we computationally identify likely members of the subclass of SAM domains that form polymers. Sequences were virtually threaded onto known polymer structures and then evaluated for compatibility with the polymer. We find that known SAM polymers score better than the vast majority of known non-polymers: 100% (7 of 7) of known polymers and only 8% of known non- polymers (1 of 12) score above a defined threshold value. Of 2901 SAM family members, we find 694 that score above the threshold and are likely polymers, including SAM domains from the proteins Lethal Malignant Brain Tumor, Bicaudal-C, Liprin-beta, Adenylate Cyclase and Atherin. In polymerization experiments, all of these predictions (except Adenylate Cyclase) were confirmed. As a result, the original SAM database was updated and additional predictions were obtained.TMKink: A method to predict transmembrane helix kinksA hallmark of membrane protein structure is the large number of distorted transmembrane helices. Because of the prevalence of bends, it is important to not only understand how they are generated but also to learn how to predict their occurrence. Here, we find that there are local sequence preferences in kinked helices, most notably a higher abundance of proline, which can be exploited to identify bends from sequence information. A neural network predictor identifies over two-thirds of all bends (sensitivity 0.70) with high reliability (specificity 0.89). It is likely that more structural data will allow for better helix distortion predictors with increased coverage in the future. The kink predictor, TMKink, is available at http://tmkinkpredictor.mbi.ucla.edu/.Structural differences between mesophilic and thermophilic membrane proteinsProtein thermostability remains a focal point of interest for protein scientists. The differences in thermostability between mesophilic and thermophilic soluble proteins have been extensively studied. No differences in packing values have been found in soluble proteins. Membrane protein packing is different from soluble protein packing; thermophilic adaptation may be different as a result. Surprisingly, burial and packing values appear to be shared between mesophiles and thermophiles in both soluble and membrane proteins. We created a non-redundant database of unpaired and paired structures for the study of thermophile-mesophile structural differences in membrane proteins. We found little or no differences in burial or packing values in both the soluble and transmembrane regions of membrane proteins