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Reversible protein phosphorylation is an important mechanism for regulating (dis)assembly of biomolecular condensates. However, condensate-specific phosphosites remain largely unknown, thereby limiting our understanding of the underlying mechanisms. Here, we combine solubility proteome profiling with phosphoproteomics to quantitatively map several hundred phosphosites enriched in either soluble or condensate-bound protein subpopulations, including a subset of phosphosites modulating protein–RNA interactions. We show that multi-phosphorylation of the C-terminal disordered segment of heteronuclear ribonucleoprotein A1 (HNRNPA1), a key RNA-splicing factor, reduces its ability to locate to nuclear clusters. For nucleophosmin 1 (NPM1), an essential nucleolar protein, we show that phosphorylation of S254 and S260 is crucial for lowering its partitioning to the nucleolus and additional phosphorylation of distal sites enhances its retention in the nucleoplasm. These phosphorylation events decrease RNA and protein interactions of NPM1 to regulate its condensation. Our dataset is a rich resource for systematically uncovering the phosphoregulation of biomolecular condensates.

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, fewer than 5% of thousands of recently discovered phosphosites have been functionally annotated. In this study, we devised a chemical genetic approach to study the functional relevance of phosphosites in Saccharomyces cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion, suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale. ispartof: NATURE BIOTECHNOLOGY vol:40 issue:3 pages:382-+ ispartof: location:United States status: published

SummaryIntracellular bacterial pathogens inject effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32Salmonella entericaserovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effector proteins, and quantified PPIs in macrophages and epithelial cells by Affinity-Purification Quantitative Mass-Spectrometry. Thereby, we identified 25 previously described and 421 novel effector-host PPIs. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. Using reciprocal co-immunoprecipitations, we validated 13 out of 22 new PPIs. We then used this host-pathogen physical interactome resource to demonstrate that SseJ and SseL collaborate in redirecting cholesterol to theSalmonellaContaining Vacuole (SCV) via NPC1, PipB directly recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by directly phosphorylating formin-like proteins.

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS‐CoV‐2 hijacks host cellular machineries on a system‐wide scale so that potential host‐directed therapies can be developed. In situ proteome‐wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS‐CoV‐2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS‐CoV‐2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS‐CoV‐2 infection., Thermal proteome profiling (TPP) measurements of protein state and interaction changes in SARS‐CoV‐2 infection reveal pronounced temporal alterations in host protein thermostability during infection and indicate druggable host targets.

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.

Here we demonstrate that biomolecular contaminants, such as nucleic acid molecules, can seriously interfere with immobilized metal ion affinity chromatography (IMAC)-based phosphopeptide enrichments. We address and largely solve this issue, developing a robust protocol implementing methanol/chloroform protein precipitation and enzymatic digestion using benzonase, which degrades all forms of DNA and RNA, before IMAC-column loading. This simple procedure resulted in a drastic increase of enrichment sensitivity, enabling the identification of around 17,000 unique phosphopeptides and 12,500 unambiguously localized phosphosites in human cell-lines from a single LC-MS/MS run, constituting a 50% increase when compared with the standard protocol. The improved protocol was also applied to bacterial samples, increasing the number of identified bacterial phosphopeptides even more strikingly, by a factor 10, when compared with the standard protocol. For E. coli we detected around 1300 unambiguously localized phosphosites per LC-MS/MS run. The preparation of these ultra-pure phosphopeptide samples only requires marginal extra costs and sample preparation time and should thus be adoptable by every laboratory active in the field of phosphoproteomics.

SummaryReversible protein phosphorylation regulates virtually every cellular process and is arguably the most well-studied post-translational modification. Still, less than 3% of the phosphorylation sites identified in humans have annotated functions. Functionally-relevant phosphorylation sites are known to trigger conformational changes to proteins and/or to regulate their interactions with other proteins, nucleic acids and small molecules - all of which can be reflected in the thermal stability of a protein. Thus, combining thermal proteome profiling (TPP) with phosphoproteomics (phospho-TPP) provides a way to assess the functional relevance of identified phosphorylation sites on a proteome-wide scale by comparing the melting behavior of a protein and its phosphorylated form(s). We performed phospho-TPP experiments in HeLa cells with an optimized protocol, and conclude that phosphorylation does affect protein thermal stability, but to a much lesser extent than previously reported.

The rapid emergence of antimicrobial resistance is a major threat to human health. Antibiotics modulate a wide range of biological processes in bacteria and as such, the study of bacterial cellular signaling could aid the development of urgently needed new antibiotic agents. Due to the advances in bacterial phosphoproteomics, such a systemwide analysis of bacterial signaling in response to antibiotics has recently become feasible. Here we present a dynamic view of differential protein phosphorylation upon antibiotic treatment and antibiotic resistance. Most strikingly, differential phosphorylation was observed on highly conserved residues of resistance regulating transcription factors, implying a previously unanticipated role of phosphorylation mediated regulation. Using the comprehensive phosphoproteomics data presented here as a resource, future research can now focus on deciphering the precise signaling mechanisms contributing to resistance, eventually leading to alternative strategies to combat antimicrobial resistance.

Here we demonstrate that biomolecular contaminants, such as nucleic acid molecules, can seriously interfere with immobilized metal ion affinity chromatography (IMAC)-based phosphopeptide enrichments. We address and largely solve this issue, developing a robust protocol implementing methanol/chloroform protein precipitation and enzymatic digestion using benzonase, which degrades all forms of DNA and RNA, before IMAC-column loading. This simple procedure resulted in a drastic increase of enrichment sensitivity, enabling the identification of around 17,000 unique phosphopeptides and 12,500 unambiguously localized phosphosites in human cell-lines from a single LC-MS/MS run, constituting a 50% increase when compared with the standard protocol. The improved protocol was also applied to bacterial samples, increasing the number of identified bacterial phosphopeptides even more strikingly, by a factor 10, when compared with the standard protocol. For E. coli we detected around 1300 unambiguously localized phosphosites per LC-MS/MS run. The preparation of these ultra-pure phosphopeptide samples only requires marginal extra costs and sample preparation time and should thus be adoptable by every laboratory active in the field of phosphoproteomics.

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, less than 5% of thousands of recently discovered phosphorylation sites have a known function. Here, we devised a chemical genetic approach to study the functional relevance of phosphorylation in S. cerevisiae. We generated 474 phospho-deficient mutants that, along with the gene deletion library, were screened for fitness in 102 conditions. Of these, 42% exhibited growth phenotypes, suggesting these phosphosites are likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. While some phosphomutants showed loss-of-function phenotypes, a higher fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This study provides a roadmap for functionally characterizing phosphorylation in a systematic manner.