Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis—a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.     

Association in normal human fibroblasts of elevated levels of adenosine 3':5'-monophosphate with a selective decrease in collagen production.

To evaluate the hypothesis that extracellular mediators may affect collagen production by mesenchymal cells via a cyclic AMP coordinated mechanism, normal human fibroblasts were exposed to a variety of agents (prostaglandin E1, isoproterenol, cholera toxin) which independently elevated intracellular cyclic AMP during a 6-h incubation. Concomitantly, each agent caused an average 47% reduction in the percentage of total protein synthesis represented by collagen, yet little change in other major extracellular proteins. Since no active collagenase was found in these cultures, these findings suggest cyclic AMP levels may modulate the differentiated state of normal fibroblasts with respect to collagen production.     

The deubiquitylase USP9X controls ribosomal stalling

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.     

Distribution and mobility of lead in soils at an outdoor shooting range

The distribution of total Pb in surface and subsurface soil horizons at an outdoor shooting range in southeastern Michigan was determined by single extraction elemental analysis (AAS and ICP‐AES). Significant Pb enrichment of the site's soils coincides closely with Pb vapor and particulate matter produced from shot shell primers and the downfall of Pb/Sb pellets associated with the recreational shooting of skeet and trap. Surface concentrations in these locations are 10 to 100 times greater than the background concentration found on adjacent properties. The distribution of Pb in the subsurface soil horizons corresponds to the distribution of Pb at the surface, which suggests the Pb is mobilizing and migrating downward through the vadose zone. This mobilization appears to be occurring despite the clay‐rich nature of the soils, and may be due to the transformation of metallic Pb into soluble Pb compounds of carbonate and sulfate: Both compounds appear to be present in crust material found coating many of the pellets found at the site. The downward migration of soluble Pb is a potential threat to groundwater that is present at the site at a depth of less than 1 m. The protection of surface water quality is also a concern because Pb pellets from the shooting range have been found in the bed sediments of a nearby stream.