A transient homotypic interaction model for the influenza A virus NS1 protein effector domain

Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins

The Molecular Clockwork of a Protein-Based Circadian Oscillator

The circadian clock of the cyanobacterium Synechococcuselongatus PCC 7942 is governed by a core oscillator consisting of the proteins KaiA, KaiB, and KaiC. Remarkably, circadian oscillations in the phosphorylation state of KaiC can be reconstituted in a test tube by mixing the three Kai proteins and adenosine triphosphate. The in vitro oscillator provides a well-defined system in which experiments can be combined with mathematical analysis to understand the mechanism of a highly robust biological oscillator. In this Review, we summarize the biochemistry of the Kai proteins and examine models that have been proposed to explain how oscillations emerge from the properties of the oscillator’s constituents.Molecular and Cellular Biolog

Mathematical modeling of signaling pathway dynamics and stochastic gene expression

This thesis presents the development and analysis of stochastic and deterministic models of cellular biochemical networks, such as signaling pathways and gene regulatory networks. First, the model of the yeast pheromone response pathway is constructed. Stochastic modeling reveals that the biochemical steps that regulate activation of the mitogen-activated protein kinase Fus3 can account for the graded-to-binary conversion. The model is also used to investigate the effects of protein turnover on the response of the pathway. It is demonstrated that the inclusion of protein turnover can lead too sustained oscillations of protein concentration in the absence of feedback regulation, which indicates protein turnover as a important signaling regulation mechanism. Second, an engineered promoter that allowed the simultaneous repression and activation of gene expression in Escherichia coli was constructed and used to construct a stochastic model to study synthetic gene networks under increasingly complex conditions: unregulated, repressed, activated and simultaneously repressed and activated, and in the presence of positive feedback. The stochastic model quantitatively captures the means and distributions of the expression from the engineered promoter of this modular system and accurately predict the in vivo behavior of an expanded network that includes positive feedback. The model also reveals the counterintuitive prediction that noise in protein expression levels can increase upon arrest of cell division, which was confirmed experimentally

Concept and application of a computational vaccinology workflow

BACKGROUND : The last years have seen a renaissance of the vaccine area, driven by clinical needs in infectious diseases but also chronic diseases such as cancer and autoimmune disorders. Equally important are technological improvements involving nano-scale delivery platforms as well as third generation adjuvants. In parallel immunoinformatics routines have reached essential maturity for supporting central aspects in vaccinology going beyond prediction of antigenic determinants. On this basis computational vaccinology has emerged as a discipline aimed at ab-initio rational vaccine design.Here we present a computational workflow for implementing computational vaccinology covering aspects from vaccine target identification to functional characterization and epitope selection supported by a Systems Biology assessment of central aspects in host-pathogen interaction. We exemplify the procedures for Epstein Barr Virus (EBV), a clinically relevant pathogen causing chronic infection and suspected of triggering malignancies and autoimmune disorders. RESULTS : We introduce pBone/pView as a computational workflow supporting design and execution of immunoinformatics workflow modules, additionally involving aspects of results visualization, knowledge sharing and re-use. Specific elements of the workflow involve identification of vaccine targets in the realm of a Systems Biology assessment of host-pathogen interaction for identifying functionally relevant targets, as well as various methodologies for delineating B- and T-cell epitopes with particular emphasis on broad coverage of viral isolates as well as MHC alleles.Applying the workflow on EBV specifically proposes sequences from the viral proteins LMP2, EBNA2 and BALF4 as vaccine targets holding specific B- and T-cell epitopes promising broad strain and allele coverage. CONCLUSION : Based on advancements in the experimental assessment of genomes, transcriptomes and proteomes for both, pathogen and (human) host, the fundaments for rational design of vaccines have been laid out. In parallel, immunoinformatics modules have been designed and successfully applied for supporting specific aspects in vaccine design. Joining these advancements, further complemented by novel vaccine formulation and delivery aspects, have paved the way for implementing computational vaccinology for rational vaccine design tackling presently unmet vaccine challenges

Roles of GSK-3beta and PYK2 signaling pathways in synaptic plasticity

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.Cataloged from PDF version of thesis.Includes bibliographical references.Activity-dependent modification of synapses, as in long term potentiation (LTP) or long term depression (LTD), is widely believed to be a crucial mechanism for learning and memory. Molecular perturbations in these processes may underlie certain neuropsychiatric conditions. This thesis examines the role of two signaling pathways, glycogen synthase kinase 3 beta (GSK- 3beta) and proline-rich tyrosine kinase 2 (PYK2), in LTD at rat hippocampal synapses. GSK-3beta, a serine/threonine kinase implicated in the pathophysiology of schizophrenia, mood disorders, and Alzheimer's disease, is known to play a critical role in LTD. Here we report that GSK-3beta phosphorylates the postsynaptic scaffold protein PSD-95, a major determinant of synaptic strength, at the Thr- 19 residue. In hippocampal neurons, this promotes the activity-dependent dispersal of synaptic PSD-95 clusters. We found that overexpression of a phospho-null mutant (Ti 9A-PSD-95), but not a phospho-mimic mutant, blocks LTD without affecting basal synaptic function relative to wild type PSD-95 overexpression. Thus PSD-95 phosphorylation by GSK-3beta is a necessary step in LTD. [This project is a collaboration with Myung Jong Kim, and I am second author of the manuscript.] PYK2 is a calcium-dependent tyrosine kinase that is activated in cerebral ischemia and seizures. PYK2 is also known to bind PSD-95 at a region implicated in LTD signaling. Here we report a novel role for PYK2 in LTD. Chemical LTD treatment induces PYK2 phosphorylation at Tyr-402, and small hairpin RNA-mediated knockdown of PYK2 blocks LTD, but not LTP. We identify both enzymatic and non-enzymatic (scaffolding) roles for PYK2 in LTD, and find that PYK2 is required to suppress activity-dependent phosphorylation of the mitogen activated protein kinase ERK. ERK activity is believed to promote glutamate receptor insertion at synapses. Overexpression of WT-PYK2 further depresses activity-dependent ERK phosphorylation, and inhibits LTP, but not LTD. Our studies support a model whereby PYK2 antagonizes ERK signaling to promote LTD, at the expense of LTP, in hippocampal neurons. [This project is a collaboration with Myung Jong Kim and Chi-Fong Wang, and I am first author of the manuscript.]by Honor Hsin.Ph.D

Engineering Yeast to Evaluate Human Proteins Involved in Selective RNA Packaging During HIV Particle Production

Despite recent advances in antiretroviral therapy, nearly 37 million people continue to live with human immunodeficiency virus (HIV). Basic and applied research on the assembly of HIV could be enhanced by using a genetically tractable organism, such as yeast, rather than mammalian cells. While previous studies showed that expression of the HIV Gag polyprotein in Saccharomyces cerevisiae spheroplasts resulted in the production of virus-like particles (VLPs), many questions regarding the utility of yeast in HIV assembly remain uninvestigated. Here, we report use of S. cerevisiae for both the production of VLPs with selectively packaged RNA and to evaluate the human Y-box-binding protein 1 (YB-1) in selective RNA packaging into VLPs. Our data reveal: (1) When co-expressed alongside HIV-1 Gag, an RNA mammalian expression cassette is selectively encapsidated and released in VLPs produced from spheroplasts; (2) Inclusion of the 5’UTR-5’Gag RNA upstream of the mammalian expression cassette greatly increased the selectivity to which non-viral RNA was packaged into VLPs; and (3) heterologous expression of the human YB-1 protein in S. cerevisiae did not facilitate the selective packaging of viral RNA into VLPs, likely due to inability to bind upstream elements in the HIV-1 viral RNA. Overall, this research provides a key first step in the use of yeast for the production of viral vectors used in gene therapy, and lays a foundation for further experiments investigating the role of YB-1 and other host proteins in selective RNA packaging

Axonal Transport, Parkin, And Α-Synuclein; Novel Therapeutic Targets To Treat Methamphetamine Neurotoxicity

Methamphetamine (METH) is a commonly abuse psychostimulant. Exposure to chronic high doses of METH can result in neurotoxicity primarily characterized by damage to striatal dopaminergic (DAergic) axons. There are currently no therapeutic interventions for METH neurotoxicity. To some extent damage to striatal DAergic axons is reversible and DAergic axon function may return following abstinence from METH. The reversible nature of METH neurotoxicity suggests that normal striatal function could be restored following exposure to METH. However, potential targets to treat METH neurotoxicity are needed. Axonal transport is required for restoration of DAergic axon components damaged or lost following METH. Here we investigated several potential novel drug targets to treat METH neurotoxicity including with emphasis on targeting axonal transport. We also investigated the E3 ligase parkin and the aggregation prone nerve terminal protein α-synuclein. To investigate the role of axonal transport in METH neurotoxicity we treated a rat model of METH neurotoxicity with axonal transport enhancing drug, epothilone D. Results show that epothilone D could to some extent prevent METH-induced damage to DAergic axons in the striatum. To investigate parkin’s role in METH neurotoxicity we treated parkin knockout rats with a neurotoxic dose of METH. We found that parkin knockout rats were hypersensitive to the METH induced DAergic neurotoxicity, confirming the neuroprotective role of parkin for DAergic neurons. To investigate the role of α-synuclein in METH neurotoxicity we developed a novel method of measuring α-synuclein oligomerization in complex biological samples. In conclusion, here we lay the experimental foundation for three potential targets of METH neurotoxicity

Modeling a Snap-Action, Variable-Delay Switch Controlling Extrinsic Cell Death

When exposed to tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL), a closely related death ligand and investigational therapeutic, cells enter a protracted period of variable duration in which only upstream initiator caspases are active. A subsequent and sudden transition marks activation of the downstream effector caspases that rapidly dismantle the cell. Thus, extrinsic apoptosis is controlled by an unusual variable-delay, snap-action switch that enforces an unambiguous choice between life and death. To understand how the extrinsic apoptosis switch functions in quantitative terms, we constructed a mathematical model based on a mass-action representation of known reaction pathways. The model was trained against experimental data obtained by live-cell imaging, flow cytometry, and immunoblotting of cells perturbed by protein depletion and overexpression. The trained model accurately reproduces the behavior of normal and perturbed cells exposed to TRAIL, making it possible to study switching mechanisms in detail. Model analysis shows, and experiments confirm, that the duration of the delay prior to effector caspase activation is determined by initiator caspase-8 activity and the rates of other reactions lying immediately downstream of the TRAIL receptor. Sudden activation of effector caspases is achieved downstream by reactions involved in permeabilization of the mitochondrial membrane and relocalization of proteins such as Smac. We find that the pattern of interactions among Bcl-2 family members, the partitioning of Smac from its binding partner XIAP, and the mechanics of pore assembly are all critical for snap-action control

Modular Composition of Gene Transcription Networks

Predicting the dynamic behavior of a large network from that of the composing modules is a central problem in systems and synthetic biology. Yet, this predictive ability is still largely missing because modules display context-dependent behavior. One cause of context-dependence is retroactivity, a phenomenon similar to loading that influences in non-trivial ways the dynamic performance of a module upon connection to other modules. Here, we establish an analysis framework for gene transcription networks that explicitly accounts for retroactivity. Specifically, a module's key properties are encoded by three retroactivity matrices: internal, scaling, and mixing retroactivity. All of them have a physical interpretation and can be computed from macroscopic parameters (dissociation constants and promoter concentrations) and from the modules' topology. The internal retroactivity quantifies the effect of intramodular connections on an isolated module's dynamics. The scaling and mixing retroactivity establish how intermodular connections change the dynamics of connected modules. Based on these matrices and on the dynamics of modules in isolation, we can accurately predict how loading will affect the behavior of an arbitrary interconnection of modules. We illustrate implications of internal, scaling, and mixing retroactivity on the performance of recurrent network motifs, including negative autoregulation, combinatorial regulation, two-gene clocks, the toggle switch, and the single-input motif. We further provide a quantitative metric that determines how robust the dynamic behavior of a module is to interconnection with other modules. This metric can be employed both to evaluate the extent of modularity of natural networks and to establish concrete design guidelines to minimize retroactivity between modules in synthetic systems.United States. Air Force Office of Scientific Research (FA9550-12-1-0129