Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine live 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate the sequence and detail of shape changes, the tissues and molecular physiology involved, and the control of these movements. Morphometric analysis and targeted perturbation suggest that the movement is driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent canal system. Thermal proteome profiling and quantitative phosphoproteomics confirm the control of cellular relaxation by an Akt/NO/PKG/PKA pathway. Agitation-induced deflation leads to differential phosphorylation of proteins forming epithelial cell junctions, implying their mechanosensitive role. Unexpectedly, untargeted metabolomics detect a concomitant decrease in antioxidant molecules during deflation, reflecting an increase in reactive oxygen species. Together with the secretion of proteinases, cytokines, and granulin, this indicates an inflammation-like state of the deflating sponge reminiscent of vascular endothelial cells experiencing oscillatory shear stress. These results suggest the conservation of an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals and offer a possible mechanism for whole-body coordination through diffusible paracrine signals and mechanotransduction.

Protein-Peptide Turnover Profiling reveals the order of PTM addition and removal during protein maturation.

Post-translational modifications (PTMs) regulate various aspects of protein function, including degradation. Mass spectrometric methods relying on pulsed metabolic labeling are popular to quantify turnover rates on a proteome-wide scale. Such data have traditionally been interpreted in the context of protein proteolytic stability. Here, we combine theoretical kinetic modeling with experimental pulsed stable isotope labeling of amino acids in cell culture (pSILAC) for the study of protein phosphorylation. We demonstrate that metabolic labeling combined with PTM-specific enrichment does not measure effects of PTMs on protein stability. Rather, it reveals the relative order of PTM addition and removal along a protein's lifetime-a fundamentally different metric. This is due to interconversion of the measured proteoform species. Using this framework, we identify temporal phosphorylation sites on cell cycle-specific factors and protein complex assembly intermediates. Our results thus allow tying PTMs to the age of the modified proteins.

Global mapping of Salmonella enterica-host protein-protein interactions during infection.

Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.

Mix and match of the tumor metastasis suppressor Nm23 protein isoforms in vitro and in vivo.

Nm23/NME was identified 30 years ago as the first metastatic gene suppressor family. Despite extensive studies, the mechanism of action behind the observed antimetastatic potential of Nm23 has remained largely unresolved. Human Nm23 is present in various isoforms, of which Nm23-H1 and Nm23-H2 are by far the most dominant. Both isoforms are multifunctional enzymes involved in important cellular processes, through their nucleic acid binding ability, their protein-protein interactions and/or their histidine kinase activity. Although Nm23-H1 and Nm23-H2 exhibit 88% sequence homology, they often are considered to have distinct biological functions. Here, we developed an efficient and robust purification protocol to pull-down Nm23 isoforms in their native state. We applied this protocol to purify both overexpressed isoform pure as well as endogenous Nm23 proteins from several human cell lines and mouse brain tissue. Subsequent native mass spectrometry (MS) analysis revealed that all purified Nm23 samples form hexamers, whereby the endogenous protein assembly is primarily present as heterohexamers formed by statistical association of the Nm23-H1 and Nm23-H2 isoforms. Therefore, we conclude that isoform-pure hexameric Nm23 complexes scarcely exist in vivo. We also used native and top-down MS to investigate the histidine autophosphorylation activity of purified Nm23 assemblies. Our data in fine challenge the biological relevance of studying the genes/proteins Nm23-H1 and Nm23-H2 individually.