14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellularlike environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH 2 , site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3β phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric�but not dimeric�14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest.

Background Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. Methods In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. Results Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of > 300 days, and dogs with < 100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient’s exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. Conclusions The findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this "PermaPhosSer" system — an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins — we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3-β phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli.

BackgroundOsteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. MethodsIn this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. ResultsBoth cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of >300 days, and dogs with <100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient’s exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. ConclusionsThe findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals, including polycyclic aromatic hydrocarbons (PAHs) and TCDD. AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48-hours post fertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 was highly co-expressed with cyp1a (marker for AHR activation). Thus, we hypothesized wfikkn1’s role in AHR signaling, and showed that wfikkn1 expression was Ahr2 (zebrafish ortholog of human AHR)-dependent in developing zebrafish exposed to TCDD. To functionally characterize wfikkn1, we made a CRISPR-Cas9 mutant line with a 16-bp deletion in wfikkn1’s exon, and exposed wildtype and mutants to DMSO or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p &lt; 0.05, log2FC &gt; 1) between each pair of treatment combinations, suggesting an important role for wfikkn1 in altering both the 48-hpf transcriptome and TCDD-induced expression changes. Mass spectrometry-based proteomics of 48-hpf wildtype and mutants revealed 325 significant differentially expressed proteins. Functional enrichment demonstrated wfikkn1 was involved in skeletal muscle development and played a role in neurological pathways after TCDD exposure. Mutant zebrafish appeared morphologically normal but had significant behavior deficiencies at all life stages, and absence of Wfikkn1 did not significantly alter TCDD-induced behavior effects at all life stages. In conclusion, wfikkn1 did not appear to be significantly involved in TCDD’s overt toxicity but is likely a necessary functional member of the AHR signaling cascade.