Phosphatase Specificity and Pathway Insulation in Signaling Networks

AbstractPhosphatases play an important role in cellular signaling networks by regulating the phosphorylation state of proteins. Phosphatases are classically considered to be promiscuous, acting on tens to hundreds of different substrates. We recently demonstrated that a shared phosphatase can couple the responses of two proteins to incoming signals, even if those two substrates are from otherwise isolated areas of the network. This finding raises a potential paradox: if phosphatases are indeed highly promiscuous, how do cells insulate themselves against unwanted crosstalk? Here, we use mathematical models to explore three possible insulation mechanisms. One approach involves evolving phosphatase KM values that are large enough to prevent saturation by the phosphatase’s substrates. Although this is an effective method for generating isolation, the phosphatase becomes a highly inefficient enzyme, which prevents the system from achieving switch-like responses and can result in slow response kinetics. We also explore the idea that substrate degradation can serve as an effective phosphatase. Assuming that degradation is unsaturatable, this mechanism could insulate substrates from crosstalk, but it would also preclude ultrasensitive responses and would require very high substrate turnover to achieve rapid dephosphorylation kinetics. Finally, we show that adaptor subunits, such as those found on phosphatases like PP2A, can provide effective insulation against phosphatase crosstalk, but only if their binding to substrates is uncoupled from their binding to the catalytic core. Analysis of the interaction network of PP2A’s adaptor domains reveals that although its adaptors may isolate subsets of targets from one another, there is still a strong potential for phosphatase crosstalk within those subsets. Understanding how phosphatase crosstalk and the insulation mechanisms described here impact the function and evolution of signaling networks represents a major challenge for experimental and computational systems biology

Qualification of Metallized Optical Fiber Connections for Chip-Level MEMS Packaging

A MEMS-based Safety and Arming (S&A) device is being developed for the next generation of Navy torpedoes. The MEMS-based S&A consists of a high aspect ratio MEMS chip fabricated by deep reactive ion etching (DRIE) of silicon on insulator substrates (SOI). The micro-machined structures, which include environmental sensors, actuators, and optical components, are susceptible to stiction related failures. A robust package is essential to transform the fragile MEMS S&A device into a rugged package capable of reliably functioning throughout the military stockpile to target sequence. To adequately protect the MEMS device from deleterious effects of the external environment, the package must be housed in a hermetic, organic-free package. This dissertation presents the design of, analyzes, and qualifies a die-level fluxless packaging concept. The die-level package consists of a metallized seal ring patterned around the perimeter of the chip, including the fiber groove, sidewalls, and base. The fiber grooves provide a fiber optic interconnect between the microstructure area and the macro-environment. A cap chip, with a matching seal ring, completes the clamshell package. Solder is deposited onto the seal ring and in the grooves at the wafer-level on the device and cap chips. A fluxless, and hence organic-free, soldering process joins and seals the fiber-chip assembly on the chip-level. The conditions that govern fluxless soldering are addressed and tailored for success in the developed design. Surface energy models are used to understand the fluxless soldering conditions and to study the geometric stability of fluid solder joints at the fiber to chip interface. Several techniques for fabrication of the chips and assembly of the packages are investigated. The effects of leak rate of the package seal on the internal package environment are discussed in detail to establish an acceptable leak rate of small volume MEMS packages. The calculations are then furthered to determine the acceptable leak path dimensions to ensure moisture does reach unacceptable levels during the package life. The presented work represents the first reported organic-free (fluxless) die-level package seal with optical fibers that cross the seal boundary

Crosstalk and the evolvability of intracellular communication

Metazoan signalling networks are complex, with extensive crosstalk between pathways. It is unclear what pressures drove the evolution of this architecture. We explore the hypothesis that crosstalk allows different cell types, each expressing a specific subset of signalling proteins, to activate different outputs when faced with the same inputs, responding differently to the same environment. We find that the pressure to generate diversity leads to the evolution of networks with extensive crosstalk. Using available data, we find that human tissues exhibit higher levels of diversity between cell types than networks with random expression patterns or networks with no crosstalk. We also find that crosstalk and differential expression can influence drug activity: no protein has the same impact on two tissues when inhibited. In addition to providing a possible explanation for the evolution of crosstalk, our work indicates that consideration of cellular context will likely be crucial for targeting signalling networks

Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association

Proteasome assembly is a complex process, requiring 66 subunits distributed over several subcomplexes to associate in a coordinated fashion. Ten proteasome-specific chaperones have been identified that assist in this process. For two of these, the Pba1-Pba2 dimer, it is well established that they only bind immature core particles (CPs) in vivo. In contrast, the regulatory particle (RP) utilizes the same binding surface but only interacts with the mature CP in vivo. It is unclear how these binding events are regulated. Here, we show that Pba1-Pba2 binds tightly to the immature CP, preventing RP binding. Changes in the CP that occur on maturation significantly reduce its affinity for Pba1-Pba2, enabling the RP to displace the chaperone. Mathematical modelling indicates that this ‘affinity switch’ mechanism has likely evolved to improve assembly efficiency by preventing the accumulation of stable, non-productive intermediates. Our work thus provides mechanistic insights into a crucial step in proteasome biogenesis

We Are History: The Outlines of a Quasi-Substantive Philosophy of History

In times of a felt need to justify the value of the humanities, the need to revisit and re-establish the public relevance of the discipline of history cannot come as a surprise. On the following pages I will argue that this need is unappeasable by scholarly proposals. The much desired revitalization of historical writing lies instead in reconciling ourselves with the dual meaning of the word history, in exploring the necessary interconnection between history understood as the course of events and as historical writing. Despite the general tendency of the last decades to forbid philosophizing about history in the former sense (at least in departments of history and philosophy), I think that to a certain extent we already do so without succumbing to substantive thought. We already have the sprouts of a speculative although only quasi-substantive philosophy of history that nevertheless takes seriously the postwar criticism of the substantive enterprise. In this essay I will first try to outline this quasi-substantive philosophy of history that attests to the historical sensibility of our times; and second, I will try to outline its consequences regarding history as historical writing. Finally, in place of a conclusion I will suggest that historical writing is not as much a contribution to public agendas as it is the very arena in which public life is at stake

Evolutionary Divergence of Duplicate Copies of the Growth Hormone Gene in Suckers (Actinopterygii: Catostomidae)

Catostomid fishes (suckers) have duplicate copies of the growth hormone gene and other nuclear genes, due to a genome duplication event early in the group’s history. Yet, paralogs of GH in suckers are more than 90% conserved in nucleotide (nt) and amino acid (aa) sequence. Within paralogs across species, variation in nt and aa sequence averages 3.33% and 4.46% for GHI, and 3.22% and 2.43% for GHII, respectively. Selection tests suggest that the two GH paralogs are under strong purifying selection. Consensus trees from phylogenetic analysis of GH coding region data for 23 species of suckers, other cypriniform fishes and outgroups resolved cypriniform relationships and relationships among GHI sequences of suckers more or less consistently with analyses based on other molecular data. However, the analysis failed to resolve all sucker GHI and GHII sequences as monophyletic sister groups. This unexpected topology did not differ significantly from topologies constrained to make all GH sequences monophyletic. We attribute this result either to limitations in our GHII data set or convergent adaptive changes in GHII of tribe Catostomini

Analytic Markovian Rates for Generalized Protein Structure Evolution

A general understanding of the complex phenomenon of protein evolution requires the accurate description of the constraints that define the sub-space of proteins with mutations that do not appreciably reduce the fitness of the organism. Such constraints can have multiple origins, in this work we present a model for constrained evolutionary trajectories represented by a Markovian process throughout a set of protein-like structures artificially constructed to be topological intermediates between the structure of two natural occurring proteins. The number and type of intermediate steps defines how constrained the total evolutionary process is. By using a coarse-grained representation for the protein structures, we derive an analytic formulation of the transition rates between each of the intermediate structures. The results indicate that compact structures with a high number of hydrogen bonds are more probable and have a higher likelihood to arise during evolution. Knowledge of the transition rates allows for the study of complex evolutionary pathways represented by trajectories through a set of intermediate structures

Nicotine Overrides DNA Damage-Induced G1/S Restriction in Lung Cells

As an addictive substance, nicotine has been suggested to facilitate pro-survival activities (such as anchorage-independent growth or angiogenesis) and the establishment of drug resistance to anticancer therapy. Tobacco smoking consists of a variety of carcinogens [such as benzopyrene (BP) and nitrosamine derivatives] that are able to cause DNA double strand breaks. However, the effect of nicotine on DNA damage-induced checkpoint response induced by genotoxins remains unknown. In this study, we investigated the events occurred during G1 arrest induced by γ-radiation or BP in nicotine-treated murine or human lung epithelial cells. DNA synthesis was rapidly inhibited after exposure to γ-radiation or BP treatment, accompanied with the activation of DNA damage checkpoint. When these cells were co-treated with nicotine, the growth restriction was compromised, manifested by upregulation of cyclin D and A, and attenuation of Chk2 phosphorylation. Knockdown of cyclin D or Chk2 by the siRNAs blocked nicotine-mediated effect on DNA damage checkpoint activation. However, nicotine treatment appeared to play no role in nocodazole-induced mitotic checkpoint activation. Overall, our study presented a novel observation, in which nicotine is able to override DNA damage checkpoint activated by tobacco-related carcinogen BP or γ-irradiation. The results not only indicates the potentially important role of nicotine in facilitating the establishment of genetic instability to promote lung tumorigenesis, but also warrants a dismal prognosis for cancer patients who are smokers, heavily exposed second-hand smokers or nicotine users