The WD40 protein Caf4p is a component of the mitochondrial fission machinery and recruits Dnm1p to mitochondria

The mitochondrial division machinery regulates mitochondrial dynamics and consists of Fis1p, Mdv1p, and Dnm1p. Mitochondrial division relies on the recruitment of the dynamin-related protein Dnm1p to mitochondria. Dnm1p recruitment depends on the mitochondrial outer membrane protein Fis1p. Mdv1p interacts with Fis1p and Dnm1p, but is thought to act at a late step during fission because Mdv1p is dispensable for Dnm1p localization. We identify the WD40 repeat protein Caf4p as a Fis1p-associated protein that localizes to mitochondria in a Fis1p-dependent manner. Caf4p interacts with each component of the fission apparatus: with Fis1p and Mdv1p through its NH2-terminal half and with Dnm1p through its COOH-terminal WD40 domain. We demonstrate that mdv1{Delta} yeast contain residual mitochondrial fission due to the redundant activity of Caf4p. Moreover, recruitment of Dnm1p to mitochondria is disrupted in mdv1{Delta} caf4{Delta} yeast, demonstrating that Mdv1p and Caf4p are molecular adaptors that recruit Dnm1p to mitochondrial fission sites. Our studies support a revised model for assembly of the mitochondrial fission apparatus

Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets

The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10 cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10) and identified 54 proteins that were uniquely recovered from rpn10 cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely inabundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS

multipanelfigure: Simple Assembly of Multiple Plots and Images into a Compound Figure

In scholarly publications, multiple graphical elements such as plots or raster images are traditionally combined into a single compound figure using panels arranged in a grid. Package multipanelfigure is a GPL-3 licensed R package that eases assembly of such compound figures, allowing for straightforward integration of R specific plots using base graphics, lattice and ggplot2 plots, as well as grid grobs in general. Also provided are facilities to incorporate R external graphical output stored as SVG, PNG, JPEG, or TIFF formatted files

Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast

A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT

Author Correction: Effect of induced hypoglycemia on inflammation and oxidative stress in type 2 diabetes and control subjects (Scientific Reports, (2020), 10, 1, (4750), 10.1038/s41598-020-61531-z)

© 2020, The Author(s). The original version of this Article contained a typographical error in the spelling of the author Johannes Graumann, which was incorrectly given as Johannes Grauman. This has now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file

Quantitative Profiling of Ubiquitylated Proteins Reveals Proteasome Substrates and the Substrate Repertoire Influenced by the Rpn10 Receptor Pathway

The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10{Delta}) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uim{Delta})). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10{Delta} cells, whereas only one-fifth as many accumulated in uim{Delta} cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS

Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming

SummaryReprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells

Genome-wide identification and functional analysis of Apobec-1-mediated C-to-U RNA editing in mouse small intestine and liver

BackgroundRNA editing encompasses a post-transcriptional process in which the genomically templated sequence is enzymatically altered and introduces a modified base into the edited transcript. Mammalian C-to-U RNA editing represents a distinct subtype of base modification, whose prototype is intestinal apolipoprotein B mRNA, mediated by the catalytic deaminase Apobec-1. However, the genome-wide identification, tissue-specificity and functional implications of Apobec-1-mediated C-to-U RNA editing remain incompletely explored.ResultsDeep sequencing, data filtering and Sanger-sequence validation of intestinal and hepatic RNA from wild-type and Apobec-1-deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs, all within 3' untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites are unique to liver. Changes in RNA editing lead to corresponding changes in intestinal mRNA and protein levels for 11 genes. Analysis of RNA editing in vivo following tissue-specific Apobec-1 adenoviral or transgenic Apobec-1 overexpression reveals that a subset of targets identified in wild-type mice are restored in Apobec-1-deficient mouse intestine and liver following Apobec-1 rescue. We find distinctive polysome profiles for several RNA editing targets and demonstrate novel exonic editing sites in nuclear preparations from intestine but not hepatic apolipoprotein B RNA. RNA editing is validated using cell-free extracts from wild-type but not Apobec-1-deficient mice, demonstrating that Apobec-1 is required.ConclusionsThese studies define selective, tissue-specific targets of Apobec-1-dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific

Plasma proteome association with coronary heart disease and carotid intima media thickness: results from the KORA F4 study

Background and aims Atherosclerosis is the main cause of stroke and coronary heart disease (CHD), both leading mortality causes worldwide. Proteomics, as a high-throughput method, could provide helpful insights into the pathological mechanisms underlying atherosclerosis. In this study, we characterized the associations of plasma protein levels with CHD and with carotid intima-media thickness (CIMT), as a surrogate measure of atherosclerosis. Methods The discovery phase included 1000 participants from the KORA F4 study, whose plasma protein levels were quantified using the aptamer-based SOMAscan proteomics platform. We evaluated the associations of plasma protein levels with CHD using logistic regression, and with CIMT using linear regression. For both outcomes we applied two models: an age-sex adjusted model, and a model additionally adjusted for body mass index, smoking status, physical activity, diabetes status, hypertension status, low density lipoprotein, high density lipoprotein, and triglyceride levels (fully-adjusted model). The replication phase included a matched case-control sample from the independent KORA F3 study, using ELISA-based measurements of galectin-4. Pathway analysis was performed with nominally associated proteins (p-value < 0.05) from the fully-adjusted model. Results In the KORA F4 sample, after Bonferroni correction, we found CHD to be associated with five proteins using the age-sex adjusted model: galectin-4 (LGALS4), renin (REN), cathepsin H (CTSH), and coagulation factors X and Xa (F10). The fully-adjusted model yielded only the positive association of galectin-4 (OR = 1.58, 95% CI = 1.30–1.93), which was successfully replicated in the KORA F3 sample (OR = 1.40, 95% CI = 1.09–1.88). For CIMT, we found four proteins to be associated using the age-sex adjusted model namely: cytoplasmic protein NCK1 (NCK1), insulin-like growth factor-binding protein 2 (IGFBP2), growth hormone receptor (GHR), and GDNF family receptor alpha-1 (GFRA1). After assessing the fully-adjusted model, only NCK1 remained significant (β = 0.017, p-value = 1.39e-06). Upstream regulators of galectin-4 and NCK1 identified from pathway analysis were predicted to be involved in inflammation pathways. Conclusions Our proteome-wide association study identified galectin-4 to be associated with CHD and NCK1 to be associated with CIMT. Inflammatory pathways underlying the identified associations highlight the importance of inflammation in the development and progression of CHD