Top-down mass spectrometry for characterizing the low molecular weight proteome of canine osteosarcoma cell phenotypes.

Osteosarcoma (OS) is the most common primary malignant tumor of bone, which occupying about 20% of all bone cancers. To increase understanding of the biology of OS, we developed and evaluated a top-down mass spectrometry approach to detect, identify and quantify low molecular weight (MW) proteins (i.e., 1 kDa < MW < 30 kDa) in osteosarcoma cells. Top-down proteomic (TDP) data was acquired using reversed phase nano-liquid chromatography in conjunction with high-resolution mass spectrometry and resulted in the assignment of 328 proteins and 820 proteoforms or degradation products with high confidence. Eight post-translational modifications (PTMs) were identified in the present study, including N-terminal acetylation, lysine acetylation, succinylation, malonylation, serine/tyrosine phosphorylation, histidine methylation and N-acetylleucine. We confirmed that a truncated N-terminal proteoform lost 73 Da of mass through removal of the N-terminal Met (-131 Da), acetylation of the second amino acid (+42 Da), and Met oxidation (+16 Da). The results showed that the levels of proteoforms/biodegradable peptides correlated with the metastatic phenotypes of osteosarcoma cell lines. This study demonstrates the benefits of TDP for the characterization and relative quantification of proteoforms with relevance to OS biology and the potential of small molecular weight proteoforms to serve as a still underappreciated source of biomarkers.

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ.

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 cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH2, 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 GSK3ß 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.

The Ahr2-Dependent wfikkn1 Gene Influences Zebrafish Transcriptome, Proteome, and Behavior.

The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals, including polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48 h postfertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 was highly coexpressed 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 dimethyl sulfoxide or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p < .05, log2FC > 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.

PermaPhos Ser : autonomous synthesis of functional, permanently phosphorylated proteins.

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 "PermaPhos Ser " 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 GSK3ß 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 .

Exosomal proteomic signatures correlate with drug resistance and carboplatin treatment outcome in a spontaneous model of canine osteosarcoma.

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.