Protein phosphorylation serves as a primary mechanism of signal transduction in the cells of biological organisms. Technical advancements over the last several years in mass spectrometry now allow for the large-scale identification and quantitation of in vivo phosphorylation at unprecedented levels. These developments have occurred in the areas of sample preparation, instrumentation, quantitative methodology, and informatics so that today, ten to twenty thousand phosphorylation sites can be identified and quantified within a few weeks. With the rapid development and widespread availability of such data, its translation into biological insight and knowledge is a current obstacle. Here we present an overview of how this technology came to be and is currently applied, as well as future challenges for the field.

Iron, a major protein cofactor, is essential for most organisms. Despite the well-known effects of O2 on the oxidation state and solubility of iron, the impact of O2 on cellular iron homeostasis is not well understood. Here we report that in Escherichia coli K-12, the lack of O2 dramatically changes expression of genes controlled by the global regulators of iron homeostasis, the transcription factor Fur and the small RNA RyhB. Using chromatin immunoprecipitation sequencing (ChIP-seq), we found anaerobic conditions promote Fur binding to more locations across the genome. However, by expression profiling, we discovered that the major effect of anaerobiosis was to increase the magnitude of Fur regulation, leading to increased expression of iron storage proteins and decreased expression of most iron uptake pathways and several Mn-binding proteins. This change in the pattern of gene expression also correlated with an unanticipated decrease in Mn in anaerobic cells. Changes in the genes posttranscriptionally regulated by RyhB under aerobic and anaerobic conditions could be attributed to O2-dependent changes in transcription of the target genes: aerobic RyhB targets were enriched in iron-containing proteins associated with aerobic energy metabolism, whereas anaerobic RyhB targets were enriched in iron-containing anaerobic respiratory functions. Overall, these studies showed that anaerobiosis has a larger impact on iron homeostasis than previously anticipated, both by expanding the number of direct Fur target genes and the magnitude of their regulation and by altering the expression of genes predicted to be posttranscriptionally regulated by the small RNA RyhB under iron-limiting conditions.

Using a modified ETD-enabled QLT mass spectrometer, we demonstrate the utility of IR activation concomitant with ETD ion-ion reactions (activated-ion ETD, AI-ETD). Analyzing 12 SCX fractions of a LysC digest of human cells (HS) using ETD, CAD, and AI-ETD, we find that AI-ETD generates 13,405 peptide spectral matches (PSMs) at a 1% false-discovery rate (1% FDR), surpassing both ETD (7,968) and CAD (10,904). We also analyze 12 SCX fractions of a tryptic HS digest and find that ETD produces 6,234 PSMs, 130 PSMs, and CAD 15,209 PSMs. Compared to ETcaD, AI-ETD generates ~80% more PSMs for tryptic whole cell lysate and 50% more PSMs for LysC whole cell lysate.

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.

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The ferric-uptake regulator (Fur) is an Fe-responsive transcription factor that coordinates iron homeostasis in many bacteria. Recently, we reported that expression of the Fur regulon is also impacted by O tension. Here, we show that for most of the Fur regulon, Fur binding and transcriptional repression increase under anaerobic conditions, suggesting that Fur is controlled by O availability. We found that the intracellular, labile Fe pool was higher under anaerobic conditions compared with aerobic conditions, suggesting that higher Fe availability drove the formation of more Fe-Fur and, accordingly, more DNA binding. O regulation of Fur activity required the anaerobically induced FeoABC Fe uptake system, linking increased Fur activity to ferrous import under iron-sufficient conditions. The increased activity of Fur under anaerobic conditions led to a decrease in expression of ferric import systems. However, the combined positive regulation of the operon by ArcA and FNR partially antagonized Fur-mediated repression of under anaerobic conditions, allowing ferrous transport to increase even though Fur is more active. This design feature promotes a switch from ferric import to the more physiological relevant ferrous iron under anaerobic conditions. Taken together, we propose that the influence of O availability on the levels of active Fur adds a previously undescribed layer of regulation in maintaining cellular iron homeostasis.

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.

Cohesin proteins help maintain the physical associations between sister chromatids that arise in S-phase and are removed in anaphase. Recent studies found that cohesins also localize to the centrosomes, the organelles that organize the mitotic bipolar spindle. We find that the cohesin protein Rad21 localizes to centrosomes in a manner that is dependent upon known regulators of sister chromatid cohesion as well as regulators of centrosome function. These data suggest that Rad21 functions at the centrosome and that the regulators of Rad21 coordinate the centrosome and chromosomal functions of cohesin.