A Knockout of the Tsg101 Gene Leads to Decreased Expression of ErbB Receptor Tyrosine Kinases and Induction of Autophagy Prior to Cell Death

The Tumor Susceptibility Gene 101 (Tsg101) encodes a multi-domain protein that mediates a variety of molecular and biological processes including the trafficking and lysosomal degradation of cell surface receptors. Conventional and conditional knockout models have demonstrated an essential requirement of this gene for cell cycle progression and cell viability, but the consequences of a complete ablation of Tsg101 on intracellular processes have not been examined to date. In this study, we employed mouse embryonic fibroblasts that carry two Tsg101 conditional knockout alleles to investigate the expression of ErbB receptor tyrosine kinases as well as stress-induced intracellular processes that are known to be associated with a defect in growth and cell survival. The conditional deletion of the Tsg101 gene in this well-controlled experimental model resulted in a significant reduction in the steady-state levels of the EGFR and ErbB2 but a stress-induced elevation in the phosphorylation of mitogen activated protein (MAP) kinases independent of growth factor stimulation. As part of an integrated stress response, Tsg101-deficient cells exhibited extensive remodeling of actin filaments and greatly enlarged lysosomes that were enriched with the autophagy-related protein LC3. The increase in the transcriptional activation and expression of LC3 and its association with Lamp1-positive lysosomes in a PI3K-dependent manner suggest that Tsg101 knockout cells utilize autophagy as a survival mechanism prior to their ultimate death. Collectively, this study shows that a knockout of the Tsg101 gene causes complex intracellular changes associated with stress response and cell death. These multifaceted alterations need to be recognized as they have an impact on defining particular functions for Tsg101 in processes such as signal transduction and lysosomal/endosomal trafficking

Probing the Flexibility of Large Conformational Changes in Protein Structures through Local Perturbations

Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate—the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of α-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen Receptor ligand-binding domain, the RIP deformation map is quite sparse except for one large conformational change involving Helix-12, which is the structural element that allosterically links ligand binding to receptor activation. RIP analysis has the potential to discover sites of functional conformational changes and the linchpin residues critical in determining these conformational states

HMMerThread: Detecting Remote, Functional Conserved Domains in Entire Genomes by Combining Relaxed Sequence-Database Searches with Fold Recognition

Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak sequence similarity. Our predictions open up new avenues for biological and medical studies. Genome-wide HMMerThread domains are available at http://vm1-hmmerthread.age.mpg.de

China's War on Air Pollution: Can Existing Governance Structures Support New Ambitions?

Abstract Unprecedented and highly visible degraded air quality in China's urban centres has prompted a step change in central government control efforts in recent years. This “War on Air Pollution” has included a mixture of administrative controls, regulatory clampdowns, economic incentives and public education campaigns. A critical constraint on how policies are designed and implemented is the central government's capacity to access accurate cost information, and monitor, evaluate and enforce the policies at subordinate levels of government. We examine in detail the directives and arrangements that underpin China's “War on Air Pollution” at the provincial level, taking Hebei province as a case study. Located upwind of Beijing, Hebei's heavy industries have been a particular focus of the environmental policies. The current approach, which requires highly specific and costly local actions, yet allocates funds centrally, suffers from misaligned incentives and does not address longstanding weaknesses in local policy monitoring, evaluation and enforcement

Consumption-Based Adjustment of Emissions-Intensity Targets: An Economic Analysis for China’s Provinces

China’s Twelfth Five-Year Plan (2011–2015) aims to achieve a national carbon intensity reduction of 17 % through differentiated targets at the provincial level. Allocating the national target among China’s provinces is complicated by the fact that more than half of China’s national carbon emissions are embodied in interprovincial trade, with the relatively developed eastern provinces relying on the center and west for energy-intensive imports. This study develops a consistent methodology to adjust regional emissions-intensity targets for trade-related emissions transfers and assesses its economic effects on China’s provinces using a regional computable-general-equilibrium (CGE) model of the Chinese economy. This study finds that in 2007 China’s eastern provinces outsource 14 % of their territorial emissions to the central and western provinces. Adjusting the provincial targets for those emissions transfers increases the reduction burden for the eastern provinces by 60 %, while alleviating the burden for the central and western provinces by 50 % each. The CGE analysis indicates that this adjustment could double China’s national welfare loss compared to the homogenous and politics-based distribution of reduction targets. A shared-responsibility approach that balances production-based and consumption-based emissions responsibilities is found to alleviate those unbalancing effects and lead to a more equal distribution of economic burden among China’s provinces.Massachusetts Institute of Technology. Joint Program on the Science & Policy of Global Change MIT Energy InitiativeChina. Ministry of Science and TechnologyChina. National Development and Reform CommissionRio Tinto (Group

Synergy between Pollution Control and Climate Policy: Comparing China and the United States

We estimate the potential synergy between pollution and climate control in the U.S. and China, summarizing the results as emissions cross-elasticities of control. In both countries, ancillary carbon reductions resulting from SO2 and NOx control tend to rise with the increased stringency of control targets, reflecting the eventual need for wholesale change toward non-fossil technologies when large reductions are required. Under stringent pollution targets, the non-target effects tend to be higher in China than in the U.S., due to China's heavy reliance on coal. This result suggests that China may have greater incentives to reduce SO2 and NOx with locally apparent pollution benefits, but related efforts would at the same time reduce CO2 emissions significantly. We also find strong non-target effects of CO2 abatement in both countries, but the cross effects in this direction depend less on the stringency of control and are stronger in the U.S. than in China. © 2014 Elsevier B.V