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We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110a of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF- 10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer. [ABSTRACT FROM AUTHOR]

The article discusses the technique of shotgun/ bottom-up proteomics for the analysis of protein. It states that bottom-up process characterizes protein by analyzing the peptides released from the protein through proteolysis. It discusses several techniques related to shotgun proteomics which include two dimensional polyacrylamide gel electrophoresis (20-PAGE), chromatographic methods and post-translational modification (PTM) analysis by Mass Spectrometry.

To investigate the influence of meteorological conditions on the temperature distribution of the surrounding rock and lining of tunnel in seasonally frozen areas, taking into account phase change, a temperature field model was established based on the principles of continuum mechanics and thermodynamics. The effect of different meteorological conditions on the radial temperature field distribution of the tunnel was explored by varying the annual mean temperature and annual temperature amplitude in the model. Then, the spatio-temporal distribution model of temperature field in the surrounding rock was established. Results show that the latent heat of phase change is not negligible in the modelling process of tunnel in cold region. The annual average temperature has a significant impact on the freezing depth, and the freezing depth of the surrounding rock will gradually increase when the annual average temperature falls below 0°C. The freezing depth of the surrounding rock varies periodically when the average annual temperature is greater than 0°C. The amplitude of the surrounding rock temperature decreases exponentially with the increase of the radial depth of the surrounding rock. After reaching a certain depth, the amplitude of temperature tends to 0, and the average temperature of surrounding rock changes logarithmically. The conclusions provide a significant reference for the frost protection design of tunnel in cold regions. [ABSTRACT FROM AUTHOR]

At present most quantitative proteomics investigations are focused on the analysis of protein expression differences between two or more sample specimens. With each analysis a static snapshot of a cellular state is captured with regard to protein expression. However, any information on protein turnover cannot be obtained using classic methodologies. Protein turnover, the result of protein synthesis and degradation, represents a dynamic process, which is of equal importance to understanding physiological processes. Methods employing isotopic tracers have been developed to measure protein turnover. However, applying these methods to live animals is often complicated by the fact that an assessment of precursor pool relative isotope abundance is required. Also, data analysis becomes difficult in case of low label incorporation, which results in a complex convolution of labeled and unlabeled peptide mass spectrometry signals. Here we present a protein turnover analysis method that circumvents this problem using a 15N-labeled diet as an isotopic tracer. Mice were fed with the labeled diet for limited time periods and the resulting partially labeled proteins digested and subjected to tandem mass spectrometry. For the interpretation of the mass spectrometry data, we have developed the ProTurnyzer software that allows the determination of protein fractional synthesis rates without the need of precursor relative isotope abundance information. We present results validating ProTurnyzer with Escherichia coli protein data and apply the method to mouse brain and plasma proteomes for automated turnover studies. [ABSTRACT FROM AUTHOR]

The adsorption-desorption transition of DNA in DNA-dendrimer solutions is observed when high-valence anions, such as hexavalent anions, are added to the DNA-dendrimer solutions. In the DNA-dendrimer solutions with low-valence anions, dendrimers bind tightly with the V-shaped double-stranded DNA. When high-valence anions, such as pentavalent or hexavalent anions, are added to the DNA-dendrimer solutions, the double-stranded DNA chains can be stretched straightly and the dendrimers are released from the double-stranded DNA chains. In fact, adding high-valence anions to the solutions can change the charge spatial distribution in the DNA-dendrimer solutions, and weaken the electrostatic interactions between the positively charged dendrimers and the oppositely charged DNA chains. Adsorption-desorption transition of DNA is induced by the overcharging of dendrimers. This investigation is capable of helping us understand how to control effectively the release of DNA in gene/drug delivery because an effective gene delivery for dendrimers includes non-covalent DNAdendrimer binding and the effective release of DNA in gene therapy. [ABSTRACT FROM AUTHOR]

Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer's disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD. Notably, phosphorylation of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD. [ABSTRACT FROM AUTHOR]

Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer's disease and Parkinson's disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD+ synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including α-synuclein, Tau (K19), amyloid β, and islet amyloid polypeptide. By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases. [ABSTRACT FROM AUTHOR]

Inflammaging refers to low-grade, chronically activated innate immunity that has deleterious effects on healthy lifespan. However, little is known about the intrinsic signaling pathway that elicits innate immune genes during aging. Here, using Drosophila melanogaster, we profile the microRNA targetomes in young and aged animals, and reveal Dawdle, an activin-like ligand of the TGF-β pathway, as a physiological target of microRNA-252. We show that microRNA-252 cooperates with Forkhead box O, a conserved transcriptional factor implicated in aging, to repress Dawdle. Unopposed Dawdle triggers hyperactivation of innate immune genes coupled with a decline in organismal survival. Using adult muscle tissues, single-cell sequencing analysis describes that Dawdle and its downstream innate immune genes are expressed in distinct cell types, suggesting a cell nonautonomous mode of regulation. We further determine the genetic cascade by which Dawdle signaling leads to increased Kenny/IKKc protein, which in turn activates Relish/NF-jB protein and consequentially innate immune genes. Finally, transgenic increase of microRNA-252 and Forkhead box O pathway factors in wild-type Drosophila extends lifespan and mitigates the induction of innate immune genes in aging. Together, we propose that microRNA-252 and Forkhead box O promote healthy longevity by cooperative inhibition on Dawdle-mediated inflammaging. [ABSTRACT FROM AUTHOR]

To explore the tectonic framework and features of stratigraphic distribution in the Tuolai Sag, Yin-E Basin, a 47-km-long magnetotelluric (MT) sounding measurement was performed around Well MAZD1 in the sag. During field data acquisition, a remote reference technique was used to ensure data quality, with apparent resistivity and phase curves of all measuring points obtained using the Fourier transform, power spectrum selection, robust estimation and other methods. After MT data processing, dimensionality analysis and the degree of two-dimensional deviation indicated that the study area had the dimensionality needed for two-dimensional inversion. The major electrical axis in the sag was determined, using a multipoint–multifrequency point statistical imaging technique, to be in the WNW direction. Within the constraints of the resistivity log data for Well MAZD1, inversion results for TE and TM models were compared, after which the TM model, which corresponded well to geological conditions, was selected for conducting the nonlinear conjugate gradient method inversion and a reliable resistivity model was finally obtained. Based on regional petrophysical properties, resistivity logging, and near-well bathymetric data, the electrical characteristics of different formations within the sag were obtained and a set of low-resistance clastic rock identified in the lower Carboniferous strata. Based on an integrated analysis of the regional surface geology, tectonic setting, and depositional environment, and within the constraints of gravity to fit with electrical structure, a tectonic framework of two subsags sandwiched by an uplift is proposed for the Tuolai Sag. The scale of the northern subsag is large, with development of pre-Carboniferous nappe as well as of Carboniferous–Permian strata within the lower part of the nappe. The southern subsag is small and filled mainly with Carboniferous–Permian strata. [ABSTRACT FROM AUTHOR]

CKS proteins are small (9-kDa) polypeptides that bind to a subset of the cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2, share an overlapping function that is essential for early development. However, both proteins are frequently overexpressed in human malignancy. It has been shown that CKS protein overexpression overrides the replication stress checkpoint, promoting continued origin firing. This finding has led to the proposal that CKS protein-dependent checkpoint override allows premalignant cells to evade oncogene stress barriers, providing a causal link to oncogenesis. Here, we provide mechanistic insight into how overexpression of CKS proteins promotes override of the replication stress checkpoint. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphorylate the key replication initiation protein treslin in vitro. Furthermore, stimulation of treslin phosphorylation does not occur by the canonical adapter mechanism demonstrated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mutants are capable of stimulating treslin phosphorylation. This effect is recapitulated in vivo, where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and overexpression of wild-type or CDK binding-defective Cks2 prevents checkpointdependent dephosphorylation of treslin. Finally, we provide evidence that the role of CKS protein-dependent checkpoint override involves recovery from checkpointmediated arrest of DNA replication. [ABSTRACT FROM AUTHOR]

In eukaryotes, aberrant expression of transposable elements (TEs) is detrimental to the host genome. Piwi-interacting RNAs (piRNAs) of ∼23 to 30 nucleotides bound to PIWI clade Argonaute proteins silence transposons in a manner that is strictly dependent on their sequence complementarity. Hence, a key goal in understanding piRNA pathways is to determine mechanisms that modulate piRNA sequences. Here, we identify a protein-protein interaction between the 3'-to-5' exoribonuclease Nibbler (Nbr) and Piwi that links Nbr activity with piRNA pathways. We show that there is a delicate balance in the interplay between Nbr and Hen1, a methyltransferase involved in 2'-O-methylation at the 3' terminal nucleotides of piRNAs, thus connecting two genes with opposing activities in the biogenesis of piRNA 3' ends. With age, piRNAs become shorter and fewer in number, which is coupled with the derepression of select TEs. We demonstrate that activities of Nbr and Hen1 inherently contribute to TE silencing and age-dependent profiles of piRNAs. We propose that antagonistic roles of Nbr and Hen1 define a mechanism to modulate piRNA 3' ends. [ABSTRACT FROM AUTHOR]

Hexokinase II (HK2), a key enzyme involved in glucose metabolism, is regulated by growth factor signaling and is required for initiation and maintenance of tumors. Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non-acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA). Our data demonstrate that FLT3 is an important sensor of cellular nutritional state and elucidate the role and molecular mechanism of CMA in metabolic regulation and mediating cancer cell death. Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability. We reveal a new mechanism by which excessive activation of CMA may be exploited pharmacologically to eliminate cancer cells by inhibiting both FLT3 and autophagy. Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder characterized by severe intellectual disability and other symptoms including autism. Although caused by the silencing of a single gene, Fmr1 (fragile X mental retardation 1), the complexity of FXS pathogenesis is amplified because the encoded protein, FMRP, regulates the activity-dependent translation of numerous mRNAs. Although the mRNAs that associate with FMRP have been extensively studied, little is known regarding the proteins whose expression levels are altered, directly or indirectly, by loss of FMRP during brain development. Here we systematically measured protein expression in neocortical synaptic fractions from Fmr1 knockout (KO) and wild-type (WT) mice at both adolescent and adult stages. Although hundreds of proteins are up-regulated in the absence of FMRP in young mice, this up-regulation is largely diminished in adulthood. Up-regulated proteins included previously unidentified as well as known targets involved in synapse formation and function and brain development and others linked to intellectual disability and autism. Comparison with putative FMRP target mRNAs and autism susceptibility genes revealed substantial overlap, consistent with the idea that the autism endophenotype of FXS is due to a "multiple hit" effect of FMRP loss, particularly within the PSD95 interactome. Through studies of de novo protein synthesis in primary cortical neurons from KO and WT mice, we found that neurons lacking FMRP produce nascent proteins at higher rates, many of which are synaptic proteins and encoded by FMRP target mRNAs. Our results provide a greatly expanded view of protein changes in FXS and identify age-dependent effects of FMRP in shaping the neuronal proteome. [ABSTRACT FROM AUTHOR]