Tobias C. Walther

Lysine acetylation targets protein complexes and co-regulates major cellular functions.

Lysine Acetylation Catalog Covalent posttranslational modification is an essential cellular regulatory mechanism by which the activity of proteins can be controlled. Advances in mass spectrometry made it possible for Choudhary et al. (p. 834, published online 16 July) to assess the prevalence of lysine acetylation throughout the whole proteome. Acetylation is much more widespread than previously appreciated and occurs on proteins participating in all sorts of biological functions. Acetylation can influence susceptibility of proteins to phosphorylation and occurs frequently on enzymes that control the modification of other proteins by covalent ubiquitination and on proteins that form large macromolecular complexes. The findings also help to characterize the actions of lysine deacetylase inhibitors, which have shown clinical promise in treatments for cancer. A proteomic-scale analysis of protein acetylation suggests that it is an important biological regulatory mechanism. Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation’s cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Cellular Fatty Acid Metabolism and Cancer

Cancer cells often have characteristic changes in metabolism. Cellular proliferation, a common feature of all cancers, requires fatty acids for synthesis of membranes and signaling molecules. Here, we provide a view of cancer cell metabolism from a lipid perspective, and we summarize evidence that limiting fatty acid availability can control cancer cell proliferation.

Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast

Mass spectrometry is a powerful technology for the analysis of large numbers of endogenous proteins. However, the analytical challenges associated with comprehensive identification and relative quantification of cellular proteomes have so far appeared to be insurmountable. Here, using advances in computational proteomics, instrument performance and sample preparation strategies, we compare protein levels of essentially all endogenous proteins in haploid yeast cells to their diploid counterparts. Our analysis spans more than four orders of magnitude in protein abundance with no discrimination against membrane or low level regulatory proteins. Stable-isotope labelling by amino acids in cell culture (SILAC) quantification was very accurate across the proteome, as demonstrated by one-to-one ratios of most yeast proteins. Key members of the pheromone pathway were specific to haploid yeast but others were unaltered, suggesting an efficient control mechanism of the mating response. Several retrotransposon-associated proteins were specific to haploid yeast. Gene ontology analysis pinpointed a significant change for cell wall components in agreement with geometrical considerations: diploid cells have twice the volume but not twice the surface area of haploid cells. Transcriptome levels agreed poorly with proteome changes overall. However, after filtering out low confidence microarray measurements, messenger RNA changes and SILAC ratios correlated very well for pheromone pathway components. Systems-wide, precise quantification directly at the protein level opens up new perspectives in post-genomics and systems biology.

Lipid Droplets And Cellular Lipid Metabolism

Among organelles, lipid droplets (LDs) uniquely constitute a hydrophobic phase in the aqueous environment of the cytosol. Their hydrophobic core of neutral lipids stores metabolic energy and membrane components, making LDs hubs for lipid metabolism. In addition, LDs are implicated in a number of other cellular functions, ranging from protein storage and degradation to viral replication. These processes are functionally linked to many physiological and pathological conditions, including obesity and related metabolic diseases. Despite their important functions and nearly ubiquitous presence in cells, many aspects of LD biology are unknown. In the past few years, the pace of LD investigation has increased, providing new insights. Here, we review the current knowledge of LD cell biology and its translation to physiology.

Lipid Droplets Finally Get a Little R-E-S-P-E-C-T

Long underappreciated as important cellular organelles, lipid droplets are finally being recognized as dynamic structures with a complex and interesting biology. In light of this newfound respect, we discuss emerging views on lipid droplet biology and speculate on the major advances to come.

Functional genomic screen reveals genes involved in lipid-droplet formation and utilization

Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets enclosed in a monolayer of phospholipids and associated proteins. These dynamic organelles serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. Here we show, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells that about 1.5% of all genes function in lipid-droplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1–COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to our understanding of human diseases involving excessive lipid storage.

The Transcription Factor TFEB Links mTORC1 Signaling to Transcriptional Control of Lysosome Homeostasis

The nuclear translocation of a transcription factor that promotes lysosomal biogenesis is inhibited when lysosomal function is adequate. Sensing Lysosomal Status Lysosomes clear cells of damaged organelles, cellular debris, and internalized materials. Lysosomal biogenesis requires the transcription factor TFEB, and Roczniak-Ferguson et al. investigated the mechanisms by which lysosomal status controlled the activity of TFEB. They found that TFEB interacted with mTOR (mechanistic target of rapamycin), a kinase that localizes to lysosomes. The mTOR-dependent phosphorylation of TFEB caused TFEB to interact with 14-3-3 proteins, which led to the retention of the transcription factor in the cytoplasm. When lysosomal function was inhibited, TFEB no longer interacted with mTOR, was dephosphorylated, and translocated to the nucleus. Thus, the localization (and thus activity) of TFEB is determined by mTOR-mediated phosphorylation, which in turn reflects lysosomal status. Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser211 triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.

The life of lipid droplets

Lipid droplets are the least characterized of cellular organelles. Long considered simple lipid storage depots, these dynamic and remarkable organelles have recently been implicated in many biological processes, and we are only now beginning to gain insights into their fascinating lives in cells. Here we examine what we know of the life of lipid droplets. We review emerging data concerning their cellular biology and present our thoughts on some of the most salient questions for investigation.

Mass spectrometry–based proteomics in cell biology

The global analysis of protein composition, modifications, and dynamics are important goals in cell biology. Mass spectrometry (MS)–based proteomics has matured into an attractive technology for this purpose. Particularly, high resolution MS methods have been extremely successful for quantitative analysis of cellular and organellar proteomes. Rapid advances in all areas of the proteomic workflow, including sample preparation, MS, and computational analysis, should make the technology more easily available to a broad community and turn it into a staple methodology for cell biologists.

The biophysics and cell biology of lipid droplets.

Lipid droplets are intracellular organelles that are found in most cells, where they have fundamental roles in metabolism. They function prominently in storing oil-based reserves of metabolic energy and components of membrane lipids. Lipid droplets are the dispersed phase of an oil-in-water emulsion in the aqueous cytosol of cells, and the importance of basic biophysical principles of emulsions for lipid droplet biology is now being appreciated. Because of their unique architecture, with an interface between the dispersed oil phase and the aqueous cytosol, specific mechanisms underlie their formation, growth and shrinkage. Such mechanisms enable cells to use emulsified oil when the demands for metabolic energy or membrane synthesis change. The regulation of the composition of the phospholipid surfactants at the surface of lipid droplets is crucial for lipid droplet homeostasis and protein targeting to their surfaces.