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The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

Transportin 3 (TNPO3) is a nuclear import receptor known for its broad substrate specificity, often recognizing arginine-serine (SR/RS) repeat-rich nuclear localization signals (NLS) in SRSF proteins. While serine phosphorylation or glutamate presence has been associated with these NLSs, recent proteomic studies identified TNPO3 cargoes lacking SR/RS repeats. One such example is the cold-inducible RNA-binding protein (CIRBP), which contains a non-classical RSY-NLS. Using X-ray crystallography, here we investigate the TNPO3-CIRBP interaction and find that tyrosines within the RSY-NLS play a key role in binding, independent of phosphorylation. Surprisingly, serine and tyrosine phosphorylation in CIRBP’s NLS inhibits TNPO3 binding, suggesting a regulatory mechanism for nuclear import. Our study reveals a non-conventional nuclear import mechanism mediated by TNPO3, which may extend to other known or yet undiscovered TNPO3 cargoes. Here, the authors present the crystal structure of Transportin 3 (TNPO3) bound to its cargo cold-inducible RNA-binding protein (CIRBP), uncovering a distinct mechanism of protein nuclear import regulation independent of phosphorylation.

The insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates. However, the mechanism that regulates its assembly into condensates remains unknown. By using proteomics, we demonstrate that phosphorylation of IGF2BP1 at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulate RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impairs IGF2BP1 condensate formation in vitro and in cells, whereas a Y396E mutant in the second linker increases condensate size and dynamics. Structural approaches show that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1’s folded domains and these interactions are partially impaired in the Y396E mutant. Importantly, the phosphomimetic mutants impact IGF2BP1’s interaction with RNAs and remodel the transcriptome in cells. Our data reveal how phosphorylation modulates low-affinity interaction networks in disordered linkers to regulate RNP condensate formation and RNA metabolism. How phosphorylation regulates the RNA-binding protein IGF2BP1 remained largely unclear. Here, the authors discovered that phosphorylation of IGF2BP1 in disordered linkers regulates its assembly into ribonucleoprotein granules and impacts metabolism of its target RNAs.

A central process contributing to the phenotype of aging is cellular senescence. We recently identified the FOXO4 – p53 axis as pivotal in maintaining the viability of senescent cells, and that senescent cells can be targeted selectively with the senolytic peptide FOXO4-DRI. Here, we solve the solution NMR structural models of the p53 transactivation domain in complex with the FOXO4 forkhead domain and in complex with FOXO4-DRI. Strikingly, we find that the disordered FOXO4-DRI binds to the disordered p53 TAD2 and forms a transiently folded complex. In this complex, both, the FOXO4-derived region and the cationic cell permeability peptide contribute to the interaction. Furthermore, we show that p53 phosphorylation enhances the affinity for both FOXO4 and FOXO4-DRI. Summarizing we provide a detailed characterization of the interaction of p53 with FOXO4 and FOXO4-DRI which is the basis for development of p53 inhibitors to treat diseases linked to cellular senescence such as cancers. Cellular senescence drives aging, with the p53–FOXO4 axis sustaining senescent cell viability. This study reveals structural insights into p53 binding to FOXO4/FOXO4-DRI, informing the development of p53-targeted senolytics for age related diseases.

Sepsis biomarkers and potential therapeutic targets are urgently needed. With proton nuclear magnetic resonance (1H NMR) spectroscopy, several metabolites can be assessed simultaneously. Fifty-three adult medical ICU sepsis patients and 25 ICU controls without sepsis were prospectively enrolled. 1H NMR differences between groups and associations with 28-day and ICU mortality were investigated. In multivariate metabolomic analyses, we found separate clustering of ICU controls and sepsis patients, as well as septic shock survivors and non-survivors. Lipoproteins were significantly different between sepsis and control patients. Levels of the branched-chain amino acids (BCAA) valine (median 43.3 [29.0–53.7] vs. 64.3 [47.7–72.3] normalized signal intensity units; p = 0.005), leucine (57.0 [38.4–71.0] vs. 73.0 [54.3–86.3]; p = 0.034) and isoleucine (15.2 [10.9–21.6] vs. 17.9 [16.1–24.4]; p = 0.048) were lower in patients with septic shock compared to those without. Similarly, BCAA were lower in ICU non-survivors compared to survivors, and BCAA were good discriminators for ICU and 28-day mortality. In uni- and multivariable logistic regression analyses, higher BCAA levels were associated with decreased ICU- and 28-day mortality. In conclusion, metabolomics using 1H NMR spectroscopy showed encouraging potential for personalized medicine in sepsis. BCAA was significantly lower in sepsis non-survivors and may be used as early biomarkers for outcome prediction.

Deposition of the nuclear DNA/RNA-binding protein Fused in sarcoma (FUS) in cytosolic inclusions is a common hallmark of some cases of frontotemporal lobar degeneration (FTLD-FUS) and amyotrophic lateral sclerosis (ALS- FUS ). Whether both diseases also share common pathological mechanisms is currently unclear. Based on our previous finding that FUS deposits are hypomethylated in FTLD-FUS but not in ALS- FUS , we have now investigated whether genetic or pharmacological inactivation of Protein arginine methyltransferase 1 (PRMT1) activity results in unmethylated FUS or in alternatively methylated forms of FUS. To do so, we generated FUS-specific monoclonal antibodies that specifically recognize unmethylated arginine (UMA), monomethylated arginine (MMA) or asymmetrically dimethylated arginine (ADMA). Loss of PRMT1 indeed not only results in an increase of UMA FUS and a decrease of ADMA FUS, but also in a significant increase of MMA FUS. Compared to ADMA FUS, UMA and MMA FUS exhibit much higher binding affinities to Transportin-1, the nuclear import receptor of FUS, as measured by pull-down assays and isothermal titration calorimetry. Moreover, we show that MMA FUS occurs exclusively in FTLD-FUS, but not in ALS- FUS . Our findings therefore provide additional evidence that FTLD-FUS and ALS- FUS are caused by distinct disease mechanisms although both share FUS deposits as a common denominator.

A fundamental step in developing a protein drug is the selection of a stable storage formulation that ensures efficacy of the drug and inhibits physiochemical degradation or aggregation. Here, we designed and evaluated a general workflow for screening of protein formulations based on small-angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling, temperature control, and fast data analysis and provides protein particle interaction information. SAXS, together with different methods including turbidity analysis, dynamic light scattering (DLS), and SDS-PAGE measurements, were used to obtain different parameters to provide high throughput screenings. Using a set of model proteins and biopharmaceuticals, we show that SAXS is complementary to dynamic light scattering (DLS), which is widely used in biopharmaceutical research and industry. We found that, compared to DLS, SAXS can provide a more sensitive measure for protein particle interactions, such as protein aggregation and repulsion. Moreover, we show that SAXS is compatible with a broader range of buffers, excipients, and protein concentrations and that in situ SAXS provides a sensitive measure for long-term protein stability. This workflow can enable future high-throughput analysis of proteins and biopharmaceuticals and can be integrated with well-established complementary physicochemical analysis pipelines in (biopharmaceutical) research and industry.

Millions of people worldwide are affected by neurodegenerative diseases (NDs), and to date, no effective treatment has been reported. The hallmark of these diseases is the formation of pathological aggregates and fibrils in neural cells. Many studies have reported that catechins, polyphenolic compounds found in a variety of plants, can directly interact with amyloidogenic proteins, prevent the formation of toxic aggregates, and in turn play neuroprotective roles. Besides harboring amyloidogenic domains, several proteins involved in NDs possess arginine-glycine/arginine-glycine-glycine (RG/RGG) regions that contribute to the formation of protein condensates. Here, we aimed to assess whether epigallocatechin gallate (EGCG) can play a role in neuroprotection via direct interaction with such RG/RGG regions. We show that EGCG directly binds to the RG/RGG region of fused in sarcoma (FUS) and that arginine methylation enhances this interaction. Unexpectedly, we found that low micromolar amounts of EGCG were sufficient to restore RNA-dependent condensate formation of methylated FUS, whereas, in the absence of EGCG, no phase separation could be observed. Our data provide new mechanistic roles of EGCG in the regulation of phase separation of RG/RGG-containing proteins, which will promote understanding of the intricate function of EGCG in cells.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the presence of poly-PR/GR dipeptide repeats, which are encoded by the chromosome 9 open reading frame 72 (C9orf72) gene. Recently, it was shown that poly-PR/GR alters chromatin accessibility, which results in the stabilization and enhancement of transcriptional activity of the tumor suppressor p53 in several neurodegenerative disease models. A reduction in p53 protein levels protects against poly-PR and partially against poly-GR neurotoxicity in cells. Moreover, in model organisms, a reduction of p53 protein levels protects against neurotoxicity of poly-PR. Here, we aimed to study the detailed molecular mechanisms of how p53 contributes to poly-PR/GR-mediated neurodegeneration. Using a combination of biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy, fluorescence polarization, turbidity assays, and differential interference contrast (DIC) microscopy, we found that p53 physically interacts with poly-PR/GR and triggers liquid–liquid phase separation of p53. We identified the p53 transactivation domain 2 (TAD2) as the main binding site for PR25/GR25 and showed that binding of poly-PR/GR to p53 is mediated by a network of electrostatic and/or hydrophobic interactions. Our findings might help to understand the mechanistic role of p53 in poly-PR/GR-associated neurodegeneration.

L-ornithine L-aspartate (LOLA) is administered as a therapeutic and/or preventive strategy against hepatic encephalopathy either intravenously or orally in patients with liver cirrhosis. Here, we analyzed how LOLA influences the microbiome and metabolome of patients with liver cirrhosis. We retrospectively analyzed the stool microbiome, stool, urine and serum metabolome as well as markers for gut permeability, inflammation and muscle metabolism of 15 cirrhosis patients treated orally with LOLA for at least one month and 15 propensity-score-matched cirrhosis patients without LOLA. Results were validated by comparing the LOLA-treated patients to a second set of controls. Patients with and without LOLA were comparable in age, sex, etiology and severity of cirrhosis as well as PPI and laxative use. In the microbiome, Flavonifractor and Oscillospira were more abundant in patients treated with LOLA compared to the control group, while alpha and beta diversity were comparable between groups. Differences in stool and serum metabolomes reflected the pathophysiology of hepatic encephalopathy and confirmed LOLA intake. In the urine metabolome, ethanol to acetic acid ratio was lower in patients treated with LOLA compared to controls. LOLA-treated patients also showed lower serum levels of insulin-like growth factor (IGF) 1 than patients without LOLA. No differences in gut permeability or inflammation markers were found. A higher abundance of Flavonifractor and Oscillospira in LOLA-treated patients could indicate LOLA as a potential microbiome modulating strategy in patients with liver disease. The lower levels of IGF1 in patients treated with LOLA suggest a possible link between the pathophysiology of hepatic encephalopathy and muscle health.