Single molecule force spectroscopy reveals that electrostatic interactions affect the mechanical stability of proteins

It is well known that electrostatic interactions play important roles in determining the thermodynamic stability of proteins. However, the investigation into the role of electrostatic interactions in mechanical unfolding of proteins has just begun. Here we used single molecule atomic force microscopy techniques to directly evaluate the effect of electrostatic interactions on the mechanical stability of a small protein GB1. We engineered a bi-histidine motif into the force-bearing region of GB1. By varying the pH, histidine residues can switch between protonated and deprotonated states, leading to the change of the electrostatic interactions between the two histidine residues. We found that the mechanical unfolding force of the engineered protein decreased by ∼34% (from 115 pN to 76 pN) on changing the pH from 8.5 to 3, due to the increased electrostatic repulsion between the two positively charged histidines at acidic pH. Our results demonstrated that electrostatic interactions can significantly affect the mechanical stability of elastomeric proteins, and modulating the electrostatic interactions of key charged residues can become a promising method for regulating the mechanical stability of elastomeric proteins.     

Topological cluster analysis reveals the systemic organization of the Caenorhabditis elegans connectome

The modular organization of networks of individual neurons interwoven through synapses has not been fully explored due to the incredible complexity of the connectivity architecture. Here we use the modularity-based community detection method for directed, weighted networks to examine hierarchically organized modules in the complete wiring diagram (connectome) of Caenorhabditis elegans (C. elegans) and to investigate their topological properties. Incorporating bilateral symmetry of the network as an important cue for proper cluster assignment, we identified anatomical clusters in the C. elegans connectome, including a body-spanning cluster, which correspond to experimentally identified functional circuits. Moreover, the hierarchical organization of the five clusters explains the systemic cooperation (e.g., mechanosensation, chemosensation, and navigation) that occurs among the structurally segregated biological circuits to produce higher-order complex behaviors.     

Fluorescence lifetime imaging reveals that the environment of the ATP binding site of myosin in muscle senses force

Fluorescence lifetime imaging microscopy is used to demonstrate that different loads applied to a muscle fiber change the microenvironment of the nucleotide binding pocket of myosin. Permeabilized skeletal muscle fibers in rigor were labeled with a fluorescent ATP analog, 3′-DEAC-propylenediamine (pda)-ATP (3′-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP), which was hydrolyzed to the diphosphate. Cycles of small-amplitude stretches and releases (<1% of muscle segment length) were synchronized with fluorescence lifetime imaging and force measurements to correlate the effect of force on the lifetime of the ATP analog bound to the actomyosin complex. Analysis of the fluorescence decay resolved two lifetimes, corresponding to the free nucleotide DEAC-pda-ATP (τ₁ = 0.47 ± 0.03 ns; mean ± SD) and nucleotide bound to the actomyosin complex (τ₂ = 2.21 ± 0.06 ns at low strain). Whereas τ₁ did not change with force, τ₂ showed a linear dependence with the force applied to the muscle of 0.43 ± 0.05 ps/kPa. Hence, the molecular environment of the nucleotide binding pocket of myosin is directly affected by a change of length applied at the ends of the fiber segments. These changes may help explain how force modulates the actomyosin ATPase cycle and thus the physiology and energetics of contraction.     

Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling

The oligoadenylate synthetase (OAS) enzymes are cytoplasmic dsRNA sensors belonging to the antiviral innate immune system. Upon binding to viral dsRNA, the OAS enzymes synthesize 2′-5′ linked oligoadenylates (2-5As) that initiate an RNA decay pathway to impair viral replication. The human OAS-like (OASL) protein, however, does not harbor the catalytic activity required for synthesizing 2-5As and differs from the other human OAS family members by having two C-terminal ubiquitin-like domains. In spite of its lack of enzymatic activity, human OASL possesses antiviral activity. It was recently demonstrated that the ubiquitin-like domains of OASL could substitute for K63-linked poly-ubiquitin and interact with the CARDs of RIG-I and thereby enhance RIG-I signaling. However, the role of the OAS-like domain of OASL remains unclear. Here we present the crystal structure of the OAS-like domain, which shows a striking similarity with activated OAS1. Furthermore, the structure of the OAS-like domain shows that OASL has a dsRNA binding groove. We demonstrate that the OAS-like domain can bind dsRNA and that mutating key residues in the dsRNA binding site is detrimental to the RIG-I signaling enhancement. Hence, binding to dsRNA is an important feature of OASL that is required for enhancing RIG-I signaling.     

Comparative kinetic analysis reveals that inducer-specific ion release precedes the mitochondrial permeability transition

Relationships among the multiple events that precede the mitochondrial membrane permeability transition (MPT) are not yet clearly understood. A combination of newly developed instrumental and computational approaches to this problem is described. The instrumental innovation is a high-resolution digital apparatus for the simultaneous, real-time measurement of four mitochondrial parameters as indicators of the respiration rate, membrane potential, calcium ion transport, and mitochondrial swelling. A computational approach is introduced that tracks the fraction of mitochondria that has undergone pore opening. This approach allows multiple comparisons on a single time scale. The validity of the computational approach for studying complex mitochondrial phenomena was evaluated with mitochondria undergoing an MPT induced by Ca²⁺, phenylarsine oxide or alamethicin. Selective ion leaks were observed that precede the permeability transition and that are inducer specific. These results illustrate the occurrence of inducer-specific sequential changes associated with the induction of the permeability transition. Analysis of the temporal relationship among the multiple mitochondrial parameters of isolated mitochondria should provide insights into the mechanisms underlying these responses.     

Comparative kinetic analysis reveals that inducer-specific ion release precedes the mitochondrial permeability transition

Relationships among the multiple events that precede the mitochondrial membrane permeability transition (MPT) are not yet clearly understood. A combination of newly developed instrumental and computational approaches to this problem is described. The instrumental innovation is a high-resolution digital apparatus for the simultaneous, real-time measurement of four mitochondrial parameters as indicators of the respiration rate, membrane potential, calcium ion transport, and mitochondrial swelling. A computational approach is introduced that tracks the fraction of mitochondria that has undergone pore opening. This approach allows multiple comparisons on a single time scale. The validity of the computational approach for studying complex mitochondrial phenomena was evaluated with mitochondria undergoing an MPT induced by Ca(2+), phenylarsine oxide or alamethicin. Selective ion leaks were observed that precede the permeability transition and that are inducer specific. These results illustrate the occurrence of inducer-specific sequential changes associated with the induction of the permeability transition. Analysis of the temporal relationship among the multiple mitochondrial parameters of isolated mitochondria should provide insights into the mechanisms underlying these responses.     

Tethered-function analysis reveals that elF4E can recruit ribosomes independent of its binding to the cap structure

The cap-binding complex elF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: elF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865-4874). Here we exploit this approach to assess which of the other subunits of elF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the lambda phage antiterminator protein N to generate the chimeric proteins lambda4A, lambda4E-102 (abolished cap binding), and lambda4E-73-102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific lambda-N binding site (boxB) in the intercistronic space. We show that lambda4E-102, but neither the double mutant lambda4E-73-102 nor lambda4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all lambda-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, lambda4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.     

Comparative analysis of protein interaction networks reveals that conserved pathways are susceptible to HIV-1 interception

Background

Human immunodeficiency virus type one (HIV-1) is the major pathogen that causes the acquired immune deficiency syndrome (AIDS). With the availability of large-scale protein-protein interaction (PPI) measurements, comparative network analysis can provide a promising way to study the host-virus interactions and their functional significance in the pathogenesis of AIDS. Until now, there have been a large number of HIV studies based on various animal models. In this paper, we present a novel framework for studying the host-HIV interactions through comparative network analysis across different species.

Results

Based on the proposed framework, we test our hypothesis that HIV-1 attacks essential biological pathways that are conserved across species. We selected the Homo sapiens and Mus musculus PPI networks with the largest coverage among the PPI networks that are available from public databases. By using a local network alignment algorithm based on hidden Markov models (HMMs), we first identified the pathways that are conserved in both networks. Next, we analyzed the HIV-1 susceptibility of these pathways, in comparison with random pathways in the human PPI network. Our analysis shows that the conserved pathways have a significantly higher probability of being intercepted by HIV-1. Furthermore, Gene Ontology (GO) enrichment analysis shows that most of the enriched GO terms are related to signal transduction, which has been conjectured to be one of the major mechanisms targeted by HIV-1 for the takeover of the host cell.

Conclusions

This proof-of-concept study clearly shows that the comparative analysis of PPI networks across different species can provide important insights into the host-HIV interactions and the detailed mechanisms of HIV-1. We expect that comparative multiple network analysis of various species that have different levels of susceptibility to similar lentiviruses may provide a very effective framework for generating novel, and experimentally verifiable hypotheses on the mechanisms of HIV-1. We believe that the proposed framework has the potential to expedite the elucidation of the important mechanisms of HIV-1, and ultimately, the discovery of novel anti-HIV drugs.

     

Pan-genomic analysis reveals that the evolution of Dietzia species depends on their living habitats

The bacterial genus Dietzia is widely distributed in various environments. The genomes of 26 diverse strains of Dietzia, including almost all the type strains, were analysed in this study. This analysis revealed a lipid metabolism gene richness, which could explain the ability of Dietzia to live in oil related environments. The pan-genome consists of 83,976 genes assigned into 10,327 gene families, 792 of which are shared by all the genomes of Dietzia. Mathematical extrapolation of the data suggests that the Dietzia pan-genome is open. Both gene duplication and gene loss contributed to the open pan-genome, while horizontal gene transfer was limited. Dietzia strains primarily gained their diverse metabolic capacity through more ancient gene duplications. Phylogenetic analysis of Dietzia isolated from aquatic and terrestrial environments showed two distinct clades from the same ancestor. The genome sizes of Dietzia strains from aquatic environments were significantly larger than those from terrestrial environments, which was mainly due to the occurrence of more gene loss events during the evolutionary progress of the strains from terrestrial environments. The evolutionary history of Dietzia was tightly coupled to environmental conditions, and iron concentrations should be one of the key factors shaping the genomes of the Dietzia lineages.     

Spectral analysis by XANES reveals that GPNMB influences the chemical composition of intact melanosomes

GPNMB is a unique melanosomal protein. Unlike many melanosomal proteins, GPNMB has not been associated with any forms of albinism, and it is unclear whether GPNMB has any direct influence on melanosomes. Here, melanosomes from congenic strains of C57BL/6J mice mutant for Gpnmb are compared to strain-matched controls using standard transmission electron microscopy and synchrotron-based X-ray absorption near-edge structure analysis (XANES). Whereas electron microscopy did not detect any ultrastructural changes in melanosomes lacking functional GPNMB, XANES uncovered multiple spectral phenotypes. These results directly demonstrate that GPNMB influences the chemical composition of melanosomes and more broadly illustrate the potential for using genetic approaches in combination with nano-imaging technologies to study organelle biology.     

Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3β-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response.     

Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint

Yeast cells lacking Ctf18, the major subunit of an alternative Replication Factor C complex, have multiple problems with genome stability. To understand the in vivo function of the Ctf18 complex, we analyzed chromatin composition in a ctf18Δ mutant using the quantitative proteomic technique of stable isotope labeling by amino acids in cell culture. Three hundred and seven of the 491 reported chromosomal proteins were quantitated. The most marked abnormalities occurred when cells were challenged with the replication inhibitor hydroxyurea. Compared with wild type, hydroxyurea-treated ctf18Δ cells exhibited increased chromatin association of replisome progression complex components including Cdc45, Ctf4, and GINS complex subunits, the polymerase processivity clamp PCNA and the single-stranded DNA-binding complex RPA. Chromatin composition abnormalities observed in ctf18Δ cells were very similar to those of an mrc1Δ mutant, which is defective in the activating the Rad53 checkpoint kinase in response to DNA replication stress. We found that ctf18Δ cells are also defective in Rad53 activation, revealing that the Ctf18 complex is required for engagement of the DNA replication checkpoint. Inappropriate initiation of replication at late origins, because of loss of the checkpoint, probably causes the elevated level of chromatin-bound replisome proteins in the ctf18Δ mutant. The role of Ctf18 in checkpoint activation is not shared by all Replication Factor C-like complexes, because proteomic analysis revealed that cells lacking Elg1 (the major subunit of a different Replication Factor C-like complex) display a different spectrum of chromatin abnormalities. Identification of Ctf18 as a checkpoint protein highlights the usefulness of chromatin proteomic analysis for understanding the in vivo function of proteins that mediate chromatin transactions.     

Genome-wide analysis reveals that Smad3 and JMJD3 HDM co-activate the neural developmental program

Neural development requires crosstalk between signaling pathways and chromatin. In this study, we demonstrate that neurogenesis is promoted by an interplay between the TGFβ pathway and the H3K27me3 histone demethylase (HDM) JMJD3. Genome-wide analysis showed that JMJD3 is targeted to gene promoters by Smad3 in neural stem cells (NSCs) and is essential to activate TGFβ-responsive genes. In vivo experiments in chick spinal cord revealed that the generation of neurons promoted by Smad3 is dependent on JMJD3 HDM activity. Overall, these findings indicate that JMJD3 function is required for the TGFβ developmental program to proceed.