Decoding Post-Translational Modification Crosstalk With Proteomics.

Post-translational modification (PTM) of proteins allows cells to regulate protein functions, transduce signals and respond to perturbations. PTMs expand protein functionality and diversity, which leads to increased proteome complexity. PTM crosstalk describes the combinatorial action of multiple PTMs on the same or on different proteins for higher order regulation. Here we review how recent advances in proteomic technologies, mass spectrometry instrumentation, and bioinformatics spurred the proteome-wide identification of PTM crosstalk through measurements of PTM sites. We provide an overview of the basic modes of PTM crosstalk, the proteomic methods to elucidate PTM crosstalk, and approaches that can inform about the functional consequences of PTM crosstalk.

Proteome and Phosphoproteome Analysis of Brown Adipocytes Reveals That RICTOR Loss Dampens Global Insulin/AKT Signaling.

Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-min time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation because of chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.

The phosphatidic acid-binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3.

Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.

Higher resolution pooled genome-wide CRISPR knockout screening in Drosophila cells using Integration and Anti-CRISPR (IntAC).

CRISPR screens enable systematic, scalable genotype-to-phenotype mapping. We previously developed a pooled CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries, followed by PCR and sequencing of integrated sgRNAs. While effective, the method relies on early constitutive Cas9 activity that potentially can lead to discrepancies between genome edits and sgRNAs detected by PCR, reducing screen accuracy. To address this issue, we introduce a new method to co-transfect a plasmid expressing the anti-CRISPR protein AcrIIa4 to suppress Cas9 activity during early sgRNA expression, which we term "IntAC" (integrase with anti-CRISPR). IntAC allowed us to construct a new CRISPR screening approach driven by the high strength dU6:3 promoter. This new library dramatically improved precision-recall of fitness genes across the genome, retrieving 90-95% of essential gene groups within 5% error, allowing us to generate the most comprehensive list of cell fitness genes yet assembled for Drosophila. Our analysis determined that elevated sgRNA levels, made permissible by the IntAC approach, drove much of the improvement. The Drosophila fitness genes show strong correlation with human fitness genes and underscore the effects of paralogs on gene essentiality. We further demonstrate that IntAC combined with a targeted sgRNA sub-library enabled precise positive selection of a transporter under solute overload. IntAC represents a straightforward enhancement to existing Drosophila CRISPR screening methods, dramatically increasing accuracy, and might also be broadly applicable to virus-free CRISPR screens in other cell types, including mosquito, lepidopteran, tick, and mammalian cells.

PKC downregulation upon rapamycin treatment attenuates mitochondrial disease.

Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.

A comparison of non-integrating reprogramming methods.

Human induced pluripotent stem cells (hiPSCs) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV), episomal (Epi) and mRNA transfection mRNA methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.