Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress

BACKGROUND: Heat shock factor (HSF/HSF1) not only is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress but also coregulates other important signaling pathways. The factor mediates the stress-induced expression of heat shock or stress proteins (HSPs). HSF/HSF1 is inactive in unstressed cells and is activated during stress. Activation is accompanied by hyperphosphorylation of the factor. The regulatory importance of this phosphorylation has remained incompletely understood. Several previous studies on human HSF1 were concerned with phosphorylation on Ser(303), Ser(307 )and Ser(363), which phosphorylation appears to be related to factor deactivation subsequent to stress, and one study reported stress-induced phosphorylation of Ser(230 )contributing to factor activation. However, no previous study attempted to fully describe the phosphorylation status of an HSF/HSF1 in stressed cells and to systematically identify phosphoresidues involved in factor activation. The present study reports such an analysis for human HSF1 in heat-stressed cells. RESULTS: An alanine scan of all Ser, Thr and Tyr residues of human HSF1 was carried out using a validated transactivation assay, and residues phosphorylated in HSF1 were identified by mass spectrometry and sequencing. HSF1 activated by heat treatment was phosphorylated on Ser(121), Ser(230), Ser(292), Ser(303), Ser(307), Ser(314), Ser(319), Ser(326), Ser(344), Ser(363), Ser(419), and Ser(444). Phosphorylation of Ser(326 )but none of the other Ser residues was found to contribute significantly to activation of the factor by heat stress. Phosphorylation on Ser(326 )increased rapidly during heat stress as shown by experiments using a pSer(326 )phosphopeptide antibody. Heat stress-induced DNA binding and nuclear translocation of a S326A substitution mutant was not impaired in HSF1-negative cells, but the mutant stimulated HSP70 expression several times less well than wild type factor. CONCLUSION: Twelve Ser residues but no Thr or Tyr residues were identified that were phosphorylated in heat-activated HSF1. Mutagenesis experiments and functional studies suggested that phosphorylation of HSF1 residue Ser(326 )plays a critical role in the induction of the factor's transcriptional competence by heat stress. PhosphoSer(326 )also contributes to activation of HSF1 by chemical stress. To date, no functional role could be ascribed to any of the other newly identified phosphoSer residues

Spatiotemporal Control of Vascular Endothelial Growth Factor Expression Using a Heat-Shock-Activated, Rapamycin-Dependent Gene Switch

A major challenge in regenerative medicine is to develop methods for delivering growth and differentiation factors in specific spatial and temporal patterns, thereby mimicking the natural processes of development and tissue repair. Heat shock (HS)-inducible gene expression systems can respond to spatial information provided by localized heating, but are by themselves incapable of sustained expression. Conversely, gene switches activated by small molecules provide tight temporal control and sustained expression, but lack mechanisms for spatial targeting. Here we combine the advantages of HS and ligand-activated systems by developing a novel rapamycin-regulated, HS-inducible gene switch that provides spatial and temporal control and sustained expression of transgenes such as firefly luciferase and vascular endothelial growth factor (VEGF). This gene circuit exhibits very low background in the uninduced state and can be repeatedly activated up to 1 month. Furthermore, dual regulation of VEGF induction in vivo is shown to stimulate localized vascularization, thereby providing a route for temporal and spatial control of angiogenesis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140102/1/hgtb.2013.026.pd

Programmable and Scalable Software-Defined Networking Controllers

A major recent development in computer networking is the notion of Software-Defined Networking (SDN), which allows a network to customize its behaviors through centralized policies at a conceptually centralized network controller. The SDN architecture replaces closed, vertically-integrated, and fixed-function appliances with general-purpose packet processing devices, programmed through open, vendor-neutral APIs by control software executing on centralized servers. This open design exposes the capabilities of network devices and provides consumers with increased flexibility. Although several elements of the SDN architecture, notably the OpenFlow standards, have been developed, writing an SDN controller remains highly difficult. Existing programming frameworks require either explicit or restricted declarative specification of flow patterns and provide little support for maintaining consistency between controller and distributed switch state, thereby introducing a major source of complexity in SDN programming. In this dissertation, we demonstrate that it is feasible to use arguably the simplest possible programming model for centralized SDN policies, in which the programmer specifies the forwarding behavior of a network by defining a packet-processing function as an ordinary algorithm in a general-purpose language. This function, which we call an algorithmic policy, is conceptually executed on every packet in the network and has access to centralized network and policy state. This programming model eliminates the complex and performance-critical task of generating and maintaining sets of rules on individual, distributed switches. To implement algorithmic policies efficiently, we introduce Maple, an SDN programming framework that can be embedded into any programming language with appropriate support. We have implemented Maple in both Java and Haskell, including an optimizing compiler and runtime system with three novel components. First, Maple's optimizer automatically discovers reusable forwarding decisions from a generic running control program. Specifically, the optimizer observes algorithm execution traces, organizes these traces to develop a partial decision tree for the algorithm, called a trace tree, and incrementally compiles these trace trees into optimized flow tables for distributed switches. Second, Maple introduces state dependency localization and fast repair techniques to efficiently maintain consistency between algorithmic policy and distributed flow tables. Third, Maple includes the McNettle OpenFlow network controller that efficiently executes user-defined OpenFlow event handlers written in Haskell on multicore CPUs, supporting the execution of algorithmic policies that require the central controller to process many packets. Through efficient message processing and enhancements to the Glasgow Haskell Compiler runtime system, McNettle network controllers can scale to handle over 20 million OpenFlow events per second on 40 CPU cores

Nucleotide Sequence Analysis of the <i>Drosophila</i> Small Heat Shock Gene Cluster at Locus 67B

The four small heat shock protein genes of Drosophila melanogaster clustered at cytological locus 67B have been characterized by DNA sequencing. Over 6250 nucleotides, covering the 5′, protein-coding and 3′ regions of these genes have been determined together with their predicted amino acid sequences. Each gene possesses characteristic eukaryotic 5′ and 3′ sequence elements and a single uninterrupted protein-coding region. The four encoded polypeptides of 19,700, 20,600, 23,000 and 23,600 Mr share a homologous stretch of 108 amino acid residues, representing 51 to 62% of their lengths. This region is flanked by sequences of dissimilar length and amino acid composition, located mainly at the amino-terminal end, but also at the extreme carboxyl termini of these proteins. The first 14 amino acids exhibit a small degree of homology, both amongst themselves and with some signal peptides and a transmembrane protein. Investigation of the hydrophilic/hydrophobic characteristics of the (four polypeptides revealed, within the conserved 108 amino acid stretch, the presence of an α-helical region of very prominent local hydrophilicity, which probably represents a surface structural domain common to each protein. Sequence analysis with respect to transcription initiation and termination and possible regulatory signals is discussed together with some structural predictions for the four proteins

Multiple Layers of Regulation of Human Heat Shock Transcription Factor 1

Upon heat stress, monomeric human heat shock transcription factor 1 (hHSF1) is converted to a trimer, acquires DNA-binding ability, is transported to the nucleus, and becomes transcriptionally competent. It was not known previously whether these regulatory changes are caused by a single activation event or whether they occur independently from one another, providing a multilayered control that may prevent inadvertant activation of hHSF1. Comparison of wild-type and mutant hHSF1 expressed in Xenopus oocytes and human HeLa cells suggested that retention of hHSF1 in the monomeric form depends on hydrophobic repeats (LZ1 to LZ3) and a carboxy-terminal sequence element in hHSF1 as well as on the presence of a titratable factor in the cell. Oligomerization of hHSF1 appears to induce DNA-binding activity as well as to uncover an amino-terminally located nuclear localization signal. A mechanism distinct from that controlling oligomerization regulates the transcriptional competence of hHSF1. Components of this mechanism were mapped to a region, including LZ2 and nearby sequences downstream from LZ2, that is clearly separated from the carboxy-terminally located transcription activation domain(s). We propose the existence of a fold-back structure that masks the transcription activation domain in the unstressed cell but is opened up by modification of hHSF1 and/or binding of a factor facilitating hHSF1 unfolding in the stressed cell. Activation of hHSF1 appears to involve at least two independently regulated structural transitions.</p

Herpes Simplex Viruses Whose Replication Can Be Deliberately Controlled as Candidate Vaccines

Over the last few years, we have been evaluating a novel paradigm for immunization using viruses or virus-based vectors. Safety is provided not by attenuation or inactivation of vaccine viruses, but by the introduction into the viral genomes of genetic mechanisms that allow for stringent, deliberate spatial and temporal control of virus replication. The resulting replication-competent controlled viruses (RCCVs) can be activated to undergo one or, if desired, several rounds of efficient replication at the inoculation site, but are nonreplicating in the absence of activation. Extrapolating from observations that attenuated replicating viruses are better immunogens than replication-defective or inactivated viruses, it was hypothesized that RCCVs that replicate with wild-type-like efficiency when activated will be even better immunogens. The vigorous replication of the RCCVs should also render heterologous antigens expressed from them highly immunogenic. RCCVs for administration to skin sites or mucosal membranes were constructed using a virulent wild-type HSV-1 strain as the backbone. The recombinants are activated by a localized heat treatment to the inoculation site in the presence of a small-molecule regulator (SMR). Derivatives expressing influenza virus antigens were also prepared. Immunization/challenge experiments in mouse models revealed that the activated RCCVs induced far better protective immune responses against themselves as well as against the heterologous antigens they express than unactivated RCCVs or a replication-defective HSV-1 strain. Neutralizing antibody and proliferation responses mirrored these findings. We believe that the data obtained so far warrant further research to explore the possibility of developing effective RCCV-based vaccines directed to herpetic diseases and/or diseases caused by other pathogens